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Molecular aspects of ageing and the onset of leaf senescence
Schippers, Jozefus Hendrikus Maria
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Download date: 19-06-2017
Molecular aspects of ageing and the
onset of leaf senescence
Jos Schippers
The studies described in this thesis were performed in the Department of Molecular Biology
of Plants, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of
Groningen, Kerklaan 30, 9750 AA Haren, The Netherlands.
Cover design: Jos Schippers
Printed: Gildeprint Drukkerijen BV, Enschede, The Netherlands.
This thesis is also available in electronic format at:
http://dissertations.ub.rug.nl/
ISBN: 978-90-367-3444-8
2
RIJKSUNIVERSITEIT GRONINGEN
Molecular aspects of ageing and the
onset of leaf senescence
Proefschrift
ter verkrijging van het doctoraat in de
Wiskunde en Natuurwetenschappen
aan de Rijksuniversiteit Groningen
op gezag van de
Rector Magnificus, dr. F. Zwarts,
in het openbaar te verdedigen op
vrijdag 13 juni 2008
om 16.15 uur
door
Jozefus Hendrikus Maria Schippers
geboren op 26 juli 1979
te Assen
3
Promotor:
Prof. dr. J. Hille
Copromotor:
dr. P.P. Dijkwel
Beoordelingscommissie:
Prof. dr. L. Dijkhuizen
Prof. dr. J.T.M. Elzenga
Prof. dr. J. Kok
4
Contents
Scope of the thesis
7
Chapter 1
Developmental and hormonal control of leaf senescence
9
Chapter 2
Ethylene-induced leaf senescence depends on
age-related changes and OLD genes in Arabidopsis
45
Early leaf senescence of old13 is partially dependent on
salicylic acid and associated with increased oxidative
stress and altered water balance
67
A role for cytokinin in the onset of leaf senescence by
ethylene in Arabidopsis
89
The Arabidopsis old5 mutation of quinolinate synthase
affects NAD biosynthesis and causes early ageing
121
Summary
153
Samenvatting
157
Acknowledgements
163
Chapter 3
Chapter 4
Chapter 5
5
6
Scope of the thesis
Autumn is renowned for the short moment during which leaves “shine” golden and
red. However, soon after this performance the “curtains close” and the leaves turn
brown, fall down and dry out. Leaf senescence does not occur randomly, but involves
a programmed general degradation of cellular structures, followed by mobilization of
degradation products to other parts of the plant. The mobilization of these products
benefits growth of the next generation or plant growth during the next season and
presents an evolutionary beneficial trait. The onset and progression of leaf
senescence requires molecular genetic mechanisms involving thousands of genes.
Arabidopsis thaliana is a typical monocarpic plant that has a short life cycle, easy to
recognize developmental stages, and a distinct senescence program. Interestingly,
the onset of leaf senescence occurs in an age-dependent manner in Arabidopsis.
Although many environmental and endogenous factors can influence the timing of
senescence, the onset or delay caused by these factors is still age-dependent. To
understand the molecular mechanisms underlying leaf senescence, genes called
senescence-associated genes (SAGs) were isolated and characterized. Despite
these efforts, there is still insufficient information available to unravel the molecular
genetic mechanisms regulating senescence in plants. The aim of this thesis is to
study the onset of leaf senescence and identify genes modulating the age-dependent
timing of leaf senescence in Arabidopsis.
The current understanding of the initiation of leaf senescence is addressed and
discussed from a genome optimisation perspective in the introductory Chapter 1.
Emphasis is paid on the roles that hormones play during developmental ageing and
the initiation and progression of senescence. In Chapter 2 the effect of leaf age and
genotype on the onset of leaf senescence by the phytohormone ethylene was
studied in detail. For this study we used three wild type accessions and 8 onset of
leaf death mutants (old). The 8 old mutants have genetic defects that result in early
leaf senescence. Based on the results from this chapter we selected three old
mutants to address the molecular mechanism of the ageing process that results in
leaf senescence. First old13 was studied in Chapter 3; the mutant has an ethylene
dependent
senescence
phenotype
and
was
characterized in
detail.
By
characterizing the old13 mutant we focussed on the involvement of hormonal
signalling pathways to dissect the old13 phenotype. In Chapter 4 and Chapter 5, a
7
study is presented on the early senescence mutants old9 and old5, respectively. The
nature of these mutants allowed us to characterize the connection between
development and the induction of leaf senescence. More specifically, which changes
on gene expression level are related to leaf age, what is the role of reactive oxygen
species (ROS) in the ageing process of leaves, which changes occur on metabolic
level during the development of a leaf and how do they correlate with the onset of
leaf senescence, what is the molecular nature of the age-related changes that occur
during leaf development, and finally, questions are asked if some of the processes
important for plant ageing are related to those identified for yeast and animal ageing.
The findings and interpretation of the results of this study are highlighted in the
Summary.
8
Chapter
1
Developmental and hormonal control of leaf senescence
Jos H.M. Schippers1, Hai-Chun Jing2, Jacques Hille1 and Paul P. Dijkwel1
1
Molecular Biology of Plants, Groningen Biomolecular Sciences and Biotechnology Institute, University
of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands
2
Wheat Pathogenesis Programme, Rothamsted Research, Plant–Pathogen Interaction Division,
Harpenden, Herts AL5 2JQ, UK
Senescence processes in plants, Gan S, ed. Oxford: Blackwell Publishing. 145–170. (2007)
9
10
Introduction
What controls the length of life is one of the fundamental biological questions that
has been puzzling scientists for centuries. Plants have many life-forms and differ
greatly in the maximal life spans (Thomas, 2003). Annual and biennial plants finish
life cycles in a single season or in 2 years time, respectively. An age of 4600 years
has been recorded for the perennial tree bristlecone pine (Pinus longaeva), while
some clonal plants can live over 10 000 years (Nooden, 1988). Thus, longevity is a
genetically controlled life-history trait.
The phenomenon of leaf senescence can be appreciated by the color changes
among deciduous trees and in the ripening of cereal crops in late summer and
autumn, which can occur at a global scale to transform the appearance of the earth
from space. During leaf senescence, the sum of morphological, physiological and
molecular changes is generally referred to as the senescence syndrome, which
includes the visible color changes, dismantling of chloroplasts, degradation of RNA,
proteins and DNA and translocation of macro/micromolecules from senescing leaves
to other parts of the plant, resulting in the death of the leaf (Bleecker and Patterson,
1997).
We propose to examine the regulation of leaf senescence from a genome
optimization perspective. We critically analyze the proposed developmental cues that
are implicated in initiating leaf senescence. The prominent roles that hormones play
during developmental ageing and the initiation and progression of senescence will be
reviewed from a molecular point of view based partially on transcriptome data. We
discuss the identified potential physiological, biochemical and molecular events
during developmental senescence.
Developmental senescence: a plant genome is optimized for early survival and
reproduction
In general, a genome has evolved to contain three classes of hereditary information:
(1) the basic metabolism and life maintenance program such as photosynthesis,
respiration and DNA replication and damage repair; (2) the defense program that
regulates plant responses to abiotic and biotic stresses; and (3) the growth and
development program that produces an adult plant optimized for reproduction
11
(Gems, 2000; de Magalhaes and Church, 2005). From an evolutionary point of view,
a genome is selected by the force of natural selection if it facilitates the continuous
reproduction. Thus, natural selection optimizes a genome for reproduction and the
aforementioned three classes of genome programs will be operating only to ensure
normal plant growth and development until reproduction. After reproduction, the force
of natural selection declines with age and this leads to the loss of viability and fitness
of the whole plant and/or plant organs. This phenomenon is known as disposal of
soma as stated in the evolutionary theory of ageing, which is developed from animal
ageing studies (Rose, 1991; Kirkwood, 2005). This argues that in the genome there
are no specific genetic programs for life span and that longevity is an indirect
consequence of genome optimization for reproduction.
However, the annual model plant Arabidopsis thaliana has evolved a reproduction
program that runs in parallel with the death of the whole plant. Seeds are being
produced while the plant starts to senesce, and in this way the plant effectively
reutilizes nutrients stored in leaves for the production of seeds. Here, the evolutionary
theory of ageing can explain leaf senescence if the disposal of a leaf is considered as
an indirect selection for nutrient salvage (Bleecker, 1998). Indeed, selective cell
death is well documented during plant development and defense responses. For
instance,
xylogenesis,
early
embryogenesis,
pollen
tube
growth
and
the
hypersensitive response are typical examples. Thus, a common feature of the plant
body plan and architecture is that almost all the structural units are disposable for the
sake of survival and reproduction. Clearly, the recruitment of nutrients from leaf
tissues, which is a prominent feature of the senescence syndrome and results in the
death of the leaf, is part of the genome optimization program. Following this line of
arguments, we consider that leaf senescence, albeit genetically controlled, is a
consequence of natural selection for genome reproduction. Although there are
debates concerning whether ageing occurs in plants, or whether whole-plant
senescence shares similarities with animal ageing (Thomas, 2002), it has been
proposed that developmental ageing resembles animal ageing, especially when
leaves on a plant are scaled up and viewed as equivalent of animal individuals. Leaf
senescence is a typical postmitotic senescence in plants and its onset shares many
similar regulatory strategies with ageing in animals (Gan, 2003; Jing et al., 2003).
We consider it important to view leaf senescence from such an angle. This view
helps to explain and model the molecular genetic mechanisms of leaf senescence.
12
As predicted by the evolutionary theory of ageing, genes with early-life beneficial but
late-life deleterious effects and late-acting mutations with purely deleterious effects
are important for senescence regulation (Kirkwood and Austad, 2000). In the
following part of the chapter, we will show that programs important for life
maintenance, stress responses and development are important for the onset and
regulation of leaf senescence. We further summarize the recent progress in
examining the interactions between leaf development and ethylene as an example to
present the approaches we think are necessary to understand the complex process
of developmental senescence.
Developmental processes that regulate leaf senescence
When a plant is grown in an environment with sufficient nutrition, away from
pathogen attacks and free of abiotic stresses such as darkness, drought, extreme
temperature, UV-B and ozone, leaf senescence is ultimately initiated and progresses
in a leaf age dependent manner (Gan and Amasino, 1997; Quirino et al., 2000). For
monocarpic plants the regulation of senescence is under correlative control and the
onset of whole-plant senescence is initiated by the developing reproductive sink,
which remobilizes nutrients from the vegetative tissues (Nooden, 1988). In soybean
and wheat the removal of reproductive structures usually delays leaf and whole-plant
senescence (Nooden, 1984; Srivalli and Khanna-Chopra, 2004). Thus genomes of
monocarpic plants are optimized for reproduction, which determines the onset of leaf
and whole-plant senescence. Although whole-plant senescence in Arabidopsis is
controlled by the reproductive structures as well (Nooden and Penney, 2001), only a
weak correlation exists between the appearance of reproductive structures and the
onset of leaf senescence. Arabidopsis leaves have a defined life span and senesce
even under ideal growth conditions, which is due to the developmental programs
underlined by the genome (Hensel et al., 1993; Jing et al., 2002). Thus, here the
onset of leaf senescence is governed mainly by age-related changes. Strategies
employed in animals and humans seem to have been equally used in plants, such as
hormonal modulation as discussed in the next section, reactive oxygen species
(ROS), metabolic flux especially sugar and nitrogen signaling and protein
degradation. Readers are advised to refer to several recent reviews for detailed
13
discussion on them (Gan, 2003; Jing et al., 2003; Lim et al., 2003; Lim and Nam,
2005;). In this section, we will only briefly elaborate on those strategies.
Reactive oxygen species
Leaf senescence and the expression of various senescence-associated genes
(SAGs) were promoted in old leaves upon exposure to UV-B, ozone or treatment with
catalase inhibitors (Miller et al., 1999; John et al., 2001; Navabpour et al., 2003). In
contrast to animal ageing and plant hypersensitive responses during plant–pathogen
interactions in which mitochondria are the generator of ROS (Finkel and Holbrook,
2000; Lam et al., 2001; Biesalski, 2002), the main ROS source in a senescing leaf is
chloroplasts (Quirino et al., 2000). This is consistent with the observation that
knockout of a chloroplast genome encoded ndhF gene, one of the components of
Ndh complex involved in chlororespiratory electron transport chain, delayed leaf
senescence in tobacco (Zapata et al., 2005). ROS can also be generated via lipid
oxidation involving membrane-associated NAD(P)H oxidases (Mittler, 2002). This is
in agreement with the observed altered senescence phenotypes of Arabidopsis
antisense-suppressed phospholipase Dα and SAG101 plants (Fan et al., 1997; He
and Gan, 2002) and in plants with defects in fatty acid biosynthesis pathways (Mou et
al., 2000, 2002;Wellesen et al., 2001). Thus, ROS generated from various sources
are involved in leaf senescence. Several delayed senescence mutants exhibited
enhanced tolerance to oxidative stress, indicating that the extended longevity at least
in part is due to the attenuated tolerance to ROS (Woo et al., 2004). Thus, the
damage generated by ROS can be an important age-related change that will
eventually result in the onset of leaf senescence.
Metabolic flux
One of the distinct features during leaf senescence is the clear metabolic shift from
primary catabolism to anabolism (Smart, 1994; Buchanan-Wollaston, 1997). The
number of catabolic genes highly expressed in senescing leaves is almost twofold of
that of anabolic genes (Guo et al., 2004). Carbon and nitrogen supplies are the two
key components that reflect the control of metabolic flux on leaf senescence. An
elevated CO2 level hastened the drop in the photosynthetic activities and induced leaf
senescence (Miller et al., 1997; Ludewig and Sonnewald, 2000), whereas in Rubisco
antisense tobacco plants and Arabidopsis ore4-1 mutants, less dry weight and
14
chlorophyll content were achieved than in the wild type at maturity, resulting in a
prolonged leaf longevity (Miller et al., 2000; Woo et al., 2002). Thus, carbon supply
achieved through photosynthesis is important for the onset of leaf senescence
(Hensel et al., 1993). Carbon supply may directly alter the sugar sensing and
signaling, which has been shown to regulate leaf senescence as envisaged in the
gin2 mutant that has a lesion in a hexokinase gene (Moore et al., 2003). The
cpr5/hys1 mutant that was originally isolated based on altered pathogen resistance
was shown to have sugar hypersensitivity and early leaf senescence (Bowling et al.,
1997; Yoshida et al., 2002b). Furthermore, glucose (carbon supply) was shown to
induce early leaf senescence when combined with low, but not high nitrogen supply
(Wingler et al., 2004), indicating the importance of carbon–nitrogen balance. Nitrogen
starvation can induce premature leaf senescence, perhaps mainly through
modulating the autophagy functions (see below).
Protein degradation
As one of the essential activities in plant life, protein turnover involves selective and
bulk removal of proteins in many processes, such as the degradation of specific
regulatory gene products, the maintenance of free amino acids, the elimination of
malfunctioning proteins and nutrient recycling (Smalle and Vierstra, 2004; Thompson
and Vierstra, 2005). The identification of Arabidopsis ORE9 as an F-box protein (Woo
et al., 2001) and DLS1 as an arginyl-tRNA:protein arginyltransferase (ATATE1),
which is involved in the N-end rule pathway (Yoshida et al., 2002a), demonstrated
the importance of the selective protein removal route mediated by the ubiquitinmediated proteolysis pathway via 26S proteasome in leaf senescence. Mutations in
these two genes resulted in delayed senescence, suggesting that the degraded
products targeted by ORE9 and DLS1 are positive regulators of leaf senescence, or
that the nondegraded products delay senescence. The bulk protein turnover is mainly
achieved through vacuolar autophagy. The analyses of two autophagic mutants apg7
and apg9-1 demonstrated the importance of autophagy in senescence regulation
(Doelling et al., 2002; Hanaoka et al., 2002). Many more components involved in
autophagy formation, conjugation and targeting to vacuoles have been studied
through mutational analyses in Arabidopsis (Surpin et al., 2003; Yoshimoto et al.,
2004; Thompson et al., 2005; Xiong et al., 2005). In general, knockout of these
components affected the survival under carbon and nitrogen starvation conditions
15
and hastened leaf senescence under normal growth conditions. Interestingly, the
mRNA and protein levels of autophagy genes are senescence enhanced, suggesting
that autophagy is an important aspect of the senescence syndrome.
A major group of SAGs encode cysteine proteases (Bhalerao et al., 2003; Guo et al.,
2004). For instance, RD21 remains in the vacuole as inactive aggregate and
becomes active during senescence by the cleavage of its C-terminal granulin domain
(Yamada et al., 2001). Recently, a novel type of senescence-associated vacuole
(SAV) has been observed in Arabidopsis and soybean which contains many
proteolytic enzymes such as SAG12 (Otegui et al., 2005). The development of SAVs
appears to be differentially regulated from vacuole autophagy that is actively involved
in leaf senescence (Doelling et al., 2002; Hanaoka et al., 2002). Thus, different
vacuoles are functioning during senescence and play a prominent role in
macromolecule degradation (Matile, 1997). Furthermore, a chloroplast nucleotide
encoded protein CND41 was shown to be responsible for the degradation of Rubisco
proteins in senescent tobacco leaves (Kato et al., 2004), indicative of the involvement
of chloroplast genome in leaf senescence. The macromolecule degradation and
nutrient recycling are prominent events during senescence. Thus, it is not surprising
that protein degradation, selective or bulk, is important for senescence regulation.
Hormonal control of leaf senescence
The senescence program is the final developmental phase of a leaf, which is
influenced by several phytohormones, with cytokinin and ethylene having the most
extensively documented roles in delaying or inducing leaf senescence, respectively.
In addition, other hormones, such as abscisic acid (ABA), auxin, gibberellic acid
(GA), jasmonic acid (JA) and salicylic acid (SA), also have effect on the senescence
process. In plants, two types of senescence are evident: mitotic senescence and
postmitotic senescence (Gan, 2003). Cells in leaves divide only during early
development, and thus leaf senescence can be considered postmitotic.
Research on the effect of plant hormones on senescence has been started already in
the late fifties. The effect of various hormones has been reported for dozens of plant
species. The regulation of senescence by cytokinin and ethylene is conserved;
however, the action of other hormones varies between plant species. Hormonal
signaling pathways show significant overlap, which makes the study of the effect of
16
single hormones complex. The generally used linear representation of hormonal
signaling pathways controlling specific aspects of plant growth and development is
too simple. In fact, hormones interact with each other and with a whole range of
developmental, environmental and metabolic signals (Beaudoin et al., 2000).
There are three major ways of controlling the responses to hormones in plants:
regulation by hormone biosynthesis, through hormone perception and signaling
pathways and downstream events leading to selective protein turnover and changes
in gene expression. Although Arabidopsis is the model species for plant research for
the last 15 years, almost all the physiological information about hormonal control of
leaf senescence has been generated in other species. Evidence from hormone
mutants in Arabidopsis strongly supports the role of several hormones in leaf
senescence. Lately, exciting advances through transcriptome studies have revealed
expression data for hormone biosynthesis, signaling and response genes during
senescence, and a closer examination revealed a few interesting points. Combination
of the physiological and genetic information will help creating a model for hormonal
and developmental control of leaf senescence.
Here we try to highlight important findings of several studies that we used to present
a model of hormonal regulation of leaf senescence and address remaining questions
and leads for future research.
Hormones that delay leaf senescence
Gibberellic acid
Gibberellins are diterpenes that promote stem and leaf growth. In some species, GAs
also induce seed germination and modulate flowering time and the development of
flowers, fruits and seeds (Sun and Gubler, 2004). A biochemical relation between leaf
senescence and GA was first reported by Fletcher and Osborne (1965) showing that
GA retarded senescence of excised leaf tissue from Taraxacum officinale by
maintaining chlorophyll levels and RNA synthesis. Another study in Rumex by
Goldthwaite and Laetsch (1968) showed that GA could inhibit senescence in leaf
disks for several days. Both protein degradation and chlorophyll degradation were
delayed 4 days. Even when chlorophyll and protein loss is halfway complete, addition
of GA blocks further degradation. A study performed on the leaves of romaine lettuce
showed a clear age-related decline in GA levels and absence in senesced leaves.
17
This decline in GA was caused by the conversion of free GA to a bound inactive
form, probably GA glucoside (Aharoni and Richmond, 1978). Moreover, retardation of
senescence by kinetin also caused a relatively high level of free GA and absence of
bound GA. Mutations in genes controlling GA biosynthesis or perception have no
effect on senescence. However, mutations in the F-box protein SLEEPY1 (SLY1),
which result in a block of GA-responsive genes (Dill et al., 2004), delay senescence
when crossed to abi1 (Richards et al., 2001). Although not extensively described,
several reports point to a retarding effect of GA on leaf senescence.
Auxin
Auxins are a group of molecules that got their name from the Greek word auxein,
which means ‘to grow’. The diversity of the auxin responses is reflected by the
existence of multiple independent auxin perception mechanisms in a plant (Leyser,
2002). For soybean it has been shown that the senescence can be retarded by
application of auxin (Nooden et al., 1979). During abscission, auxin has been
postulated to play a role in reducing the sensitivity of the cells to ethylene (Sexton
and Roberts, 1982).
The endogenous auxin levels within Coleus leaves showed a decline with increasing
age (dela Fuente and Leopold, 1968). However, a relation between endogenous
auxin levels and senescence does not always seem to follow a same pattern
(Nooden, 1988). The change in auxin response during ageing is the result not only of
decreasing auxin levels, but also of a lower responsiveness to auxin with age
(Chatterjee and Leopold, 1965). Since auxin is in general seen as a senescenceretarding compound it was a surprise that increased indoleacetic acid (IAA) levels
could be detected in S3 phase leaves (Quirino et al., 1999). Since leaves do not
senesce uniformly, the authors suggested that auxin levels are selectively increased
only in a certain population of cells corresponding to a particular senescence stage.
These findings actually correlated with earlier studies that show IAA can induce the
production of ethylene which opposes the senescence-retarding effect of IAA in
tobacco leaf discs (Aharoni et al., 1979). Interestingly, auxin effectively decreased
SAG12 expression, a marker for developmental senescence in a very short period of
treatment in detached senescing leaves (Noh and Amasino, 1999).
Research performed on glucose signaling revealed that the HXK1 glucose signaling
18
pathway interacts intimately with the auxin and cytokinin pathways. Glucose
concentration and photorespiration rates are important determinants for the onset of
senescence. Both cytokinin and auxin are part of a regulatory complex for nutritional
status of the plant through HXK1 signaling pathway (Moore et al., 2003). Thus, the
role of auxin in the regulation of leaf senescence might be linked with other
hormones and metabolic flux.
Cytokinins
Cytokinins have the strongest effect of all hormones on the retardation of leaf
senescence. It was reported by Richmond and Lang (1957) that application of
cytokinin could retard leaf senescence by preventing the chlorophyll breakdown.
While increasing cytokinin production could delay leaf senescence (Gan and
Amasino, 1995; Ori et al., 1999), reducing endogenous cytokinin levels resulted in
accelerated senescence (Masferrer et al., 2002). The drop in cytokinin levels before
the onset of senescence is believed to be a key signal for the initiation (Nooden et
al., 1990; Gan and Amasino, 1995). Recently, exciting advances have been achieved
in dissecting the components involved in cytokinin signaling (Hutchison and Kieber,
2002; Hwang et al., 2002). Among the genes characterized, the receptor CKI1
(cytokinin independent 1) and the Arabidopsis response regulator (ARR) 2 appear to
be involved in regulating leaf senescence (Hwang and Sheen, 2001). A more recent
study identified an extracellular invertase whose activity is induced during cytokininmediated delay of senescence (Balibrea Lara et al., 2004). In transgenic tobacco
plants having a SAG12–IPT or SAG12–KN1 construct, cytokinin biosynthesis was
initiated when SAG12 was induced resulting in a block of the senescence syndrome
and delayed leaf senescence significantly (Gan and Amasino, 1995; Ori et al., 1999).
When extracellular invertase activity is inhibited, cytokinin no longer can inhibit leaf
senescence in transgenic SAG12–IPT lines (Balibrea Lara et al., 2004).
Cytokinin signaling genes such as the type-A ARRs and biosynthesis genes show
reduced transcription during leaf senescence (Buchanan-Wollaston et al., 2005).
Several microarray studies have been performed to reveal cytokinin-dependent gene
expression (Hoth et al., 2003; Rashotte et al., 2003; Kiba et al., 2005). Hoth et al.
used an inducible system to assess the effects of endogenous cytokinin levels. The
study
identified
823
up-
and
917
downregulated
genes
after
24
h
of
isopentenyltransferase (IPT) induction. Although for these studies the seedling stage
19
was used, this IPT system offers an attractive system to study the molecular genetics
of how cytokinin can delay and/or reverse the senescence process. The study by
Rashotte et al. (2003) showed that cytokinin-upregulated type-A ARRs, which were
downregulated in senescing leaves (Buchanan-Wollaston et al., 2005), are the
primary response genes for cytokinins. Also a cytokinin oxidase (that degrades
cytokinins) and several transcription factors were upregulated. Furthermore,
cytokinins induce genes encoding ribosomal proteins (Crowell et al., 1990) and
photosynthetic genes (Mok and Mok, 2001). Application of cytokinins downregulated
several peroxidases, kinases and E3 ubiquitin ligases. The regulation by cytokinin is
related to auxin, light and sugar, since application of cytokinin influences the
expression of genes involved in these signaling pathways.
In general it can be said that cytokinin stimulates the photosynthetic phase of a leaf.
How cytokinin can maintain this phase and delay leaf senescence is still unclear.
Nevertheless, the leaves of SAG12–IPT transgenic plants will undergo senescence,
thus cytokinin action is limited to a certain developmental phase.
Hormones that induce leaf senescence
ABA
ABA plays a major role during processes related to seed development and
germination, for instance the induction of seed dormancy, the synthesis of seed
storage proteins and lipids, the acquisition of desiccation tolerance and the inhibition
of the transition from embryonic to vegetative growth (Nambara and Marion-Poll,
2005). In vegetative tissue, ABA plays a role in response to drought to prevent water
loss by stomatal closure and maintenance of vegetative growth by inhibiting the
transition to reproductive growth. Under nonstressful conditions, ABA in plant cells is
maintained at low levels, since ABA inhibits plant growth. In vegetative tissues, ABA
levels increase during drought, salt and cold stress (Xiong and Zhu, 2003). Changes
in gene expression during water-deficit stress are partially induced by ABA and may
promote the ability of a plant to respond and survive or adapt to the stress (Bray,
2002). For long it was thought that ABA inhibits plant growth rather than maintaining
plant growth. But in tomato, maize and Arabidopsis it has been shown that ABA
maintains shoot growth by inhibiting ethylene production (Sharp, 2002). Moreover,
20
this interaction might also play a role in early leaf senescence and leaf, flower and
fruit abscission (Morgan and Drew, 1997).
Young leaves have the highest ABA levels although this is mainly produced and
transported from the older leaves (Zeevaart and Creelman, 1988). During vegetative
growth the ABA levels are in general very low; however, in parallel with a decline in
free cytokinin and GA just before chlorophyll breakdown in lettuce leaves, an
increase in ABA levels has been observed (Aharoni and Richmond, 1978). As soon
as the chlorophyll breakdown is initiated, a second, more dramatic increase in
endogenous ABA levels is observed. The authors suggest that lowering of GA and
cytokinin levels mark the onset of leaf senescence, which results in increased ABA
levels when the process has been started. Application of ethylene to the lettuce
leaves resulted in a quick drop of GA in 1 day after treatment, but the ABA levels did
not show any difference. This might indicate that ABA and ethylene both control
different aspects of the senescence syndrome which are mediated through different
but partially overlapping signaling pathways. The application of ABA to detached
leaves results in a rapid senescence response; however, application to attached
leaves has a less pronounced effect. Under low nitrogen conditions and high sugar
the abi5 mutant shows delayed senescence. This is consistent with a role for sugar
signaling during leaf senescence. ABI5 can be induced by glucose during later
stages of development. Expression analysis of ABI5 shows an increase during
senescence (Buchanan-Wollaston et al., 2005). The ABA signaling mutants abi2-1
and abi1-1 show signs of early leaf senescence when grown on low nitrogen with
glucose and their transcripts increase during senescence (Pourtau et al., 2004;
Buchanan-Wollaston et al., 2005). Furthermore, the enzymes controlling ABA
synthesis are upregulated during senescence. This indicates that the ABA signaling
and biosynthesis pathway is active during leaf senescence. Interestingly the abi4 and
abi5 signaling mutants and the aba1, aba2 and aba3 ABA-deficient mutants all are
glucose insensitive (Arenas-Huertero et al., 2000). It was noted before that sugar
represses
photosynthesis-associated
genes,
which
leads
to
a
decline
in
photosynthesis and eventually in leaf senescence (Bleecker and Patterson, 1997).
Thus the onset of leaf senescence by ABA appears to be coupled to metabolic flux
changes in Arabidopsis.
Brassinosteroids
21
Brassinosteroids (BRs), polyhydroxylated steroid hormones, regulate the growth and
differentiation of plants throughout their life cycle. In recent years great advances
have been made in the understanding of BR signaling (Vert et al., 2005). External
application of BR results in premature leaf senescence for several species, but it has
not been reported for Arabidopsis. The induction of senescence by BRs might be
mediated through ROS (Clouse and Sasse, 1998). BR signal transduction takes
place at the plasma-membrane-localized receptor kinase, BRI1 (Clouse et al., 1996).
In addition to BRI1, three homologues have been characterized. Downstream of the
receptor kinases is BIN2, a negative regulator of the BR pathway. Further
downstream act BES1 and BZR1 transcription factors of which BES1 promotes the
expression of BR-regulated genes and BZR1 represses BR genes; both are
repressed by BIN2 by targeting of BES1 and BZR1 for ubiquitination and subsequent
proteasome-dependent degradation (Vert et al., 2005). Interestingly, BRs can induce
ethylene biosynthesis genes in mung bean (Yi et al., 1999). Whether BRs also induce
ethylene biosynthesis during senescence is a question that remains to be answered.
Mutants in BR biosynthesis and BR signaling do support a role for BR in senescence
in Arabidopsis. The det2 (de-etiolated2) mutant is defective in an early step of BR
biosynthesis. When grown in light the mutant develops two times more rosette leaves
than does the wild type. Chory et al. (1991) observed that wild-type plants showed
senescence after 30 days, whereas the det2 mutant did not show any signs of visible
senescence after 49 days. One could argue that the mutant has a severe
developmental defect that results in a changed senescence syndrome; however,
other severely affected developmental mutants such as ctr1 still show a normal onset
of the senescence program (Kieber et al., 1993). BR mutants can also result in early
leaf senescence as has been shown by the bes1 mutant (Yin et al., 2002).
Looking at the Arabidopsis transcriptome of leaf senescence, none of the BR
signaling components are identified (Guo et al., 2004). This suggests a minor role for
BR during leaf senescence. Transcriptome analysis identified seven genes encoding
cell-wall-associated proteins that are upregulated after BR treatment (Goda et al.,
2002); these genes were not identified in the transcriptome of senescing leaves (Guo
et al., 2004). However, one study revealed the induction of SAGs in Arabidopsis by
BR (He et al., 2001). Out of 125 enhancer-trap lines screened, 4 showed
upregulation of the reporter after BR application.
22
Although BR mutants show an alternative onset of senescence, molecular genetic
evidence of a direct role for BR is still minimal. More studies about the role of BR in
senescence are necessary.
Ethylene
The gaseous plant hormone ethylene plays an important role in plant growth and
development. From seed germination to organ senescence and from cell elongation
to defense responses, ethylene plays its part. The diverse role that ethylene plays in
growth and development suggests that ethylene action involves expression and
interaction of many different genes and their products (Zhong and Burns, 2003).
Ethylene has long been seen as the key hormone in regulating the onset of leaf
senescence (Zacarias and Reid, 1990). The senescence-delaying hormones like
auxin and cytokinin both stimulate ethylene production in romaine lettuce leaves
(Lactuca sativa L.), which might account for their limited stay-green properties. The
author concluded that the effectiveness of exogenously applied hormones in both
enhancing and retarding senescence is greatly affected by the endogenous ethylene
concentration of the treated plant tissue (Aharoni, 1989). The role of the ethylene
pathway in senescence is demonstrated by several studies. Both ethylene insensitive
mutants etr1-1 and ein2/ore3 showed increased leaf longevities (Grbić and Bleecker,
1995;
Oh
et
al.,
1997),
and
antisense
suppression
of
the
tomato
1-
aminocyclopropane-1-carboxylic acid (ACC) oxidase resulted in delayed leaf
senescence (John et al., 1995). In these cases, however, senescence eventually
begins and progresses normally. Exogenously applied ethylene induces premature
leaf senescence in Arabidopsis. However, constitutive application of ethylene does
not change the longevity of the leaves. Both ctr1 (constitutive triple response)
mutants and Arabidopsis plants grown in the continuous presence of exogenous
ethylene did not show premature senescence (Kieber et al., 1993; Grbić and
Bleecker, 1995). These results suggest a dynamic regulation of the timing of leaf
senescence for which the age-dependent effect of ethylene is utilized.
By making use of an ethylene-induced senescence screen, a large collection of onset
of leaf death (old) mutants has been identified (Jing et al., 2002, 2005). These
mutants confirmed that the effect of ethylene is limited to a range of leaf ages, and
that the effect of ethylene on leaf senescence increases with increase in leaf age
(Grbić and Bleecker, 1995; Jing et al., 2002). Another piece of evidence supporting
23
this notion comes from a study treating Arabidopsis plants of an identical 24-day end
age with various lengths of exogenously applied ethylene (Jing et al., 2005). The
results showed that increasing ethylene treatment from 3 to 12 days caused an
increase in leaf senescence. Surprisingly, a drop in the number of yellow leaves
occurred when a 16-day ethylene exposure was applied. Thus, varying ethylene
exposure time can induce different degrees of senescence symptoms in the leaves of
an identical end age, suggesting that ethylene can actively stimulate or repress agerelated changes that control ethylene-induced leaf senescence. This notion is
genetically supported by the altered responses of eight old mutants to the various
ethylene treatments (Jing et al., 2005). Thus, multiple genetic loci are required to
regulate the action of ethylene in leaf senescence.
A transcriptome study of senescent leaves by Guo et al. (2004) identified three
mitogen-activated protein kinases (MAPKs), three MAPKKs, nine MAPKKKs and one
MAPKKKK. In the Arabidopsis genome, 20 MAPKs, 10 MAPKKs, 80 MAPKKKs and
10 MAPKKKKs have been identified. The few components identified of the MAPK
signal cascades led the authors to the conclusion that the three MAPKs and three
MAPKKs may be at the converging/cross talk points of various signal transduction
pathways. One of the identified MAPKs is MPK6, which is a component of the MAPK
pathway that controls ethylene signaling in plants (Ouaked et al., 2003). MPK6 is
upregulated during osmotic stress but also by other abiotic stresses such as low
temperature, low humidity, wounding or oxidative stress, as well as by pathogens
(Droillard, 2002).
Transcriptional analyses of ethylene mutants and ethylene-treated plants revealed
the molecular actions of ethylene. A study by Zhong and Burns (2003) revealed
genes that are regulated by ethylene. They compared treated wild type, etr1 and ctr1
with untreated wild type. Ethylene treatment of 24-day-old wild-type plants for 24 h
changed the expression of 184 genes. Compared to etr1-1, 248 genes were changed
in expression level. Untreated wild type compared to etr1-1 revealed the
downregulation of nine genes and one upregulated gene in etr1-1. The ctr1 mutant
that did not show any signs of early senescence had 109 genes differentially
expressed. Further research on these genes might help understanding the molecular
regulation of ethylene-induced leaf senescence. To further assess the regulation of
senescence by ethylene, expression of SAGs in ein2 was compared with that of wild
type (Buchanan-Wollaston et al., 2005). Nine percent of the genes that are
24
upregulated during senescence are at least twofold reduced in ein2. Seventy-seven
genes are more than twofold up- or downregulated. Four genes showed
upregulation, a lipid transfer protein, a heavy-metal-binding protein and a
transcription factor (HFR1). Downregulated are nine transcription factors, cell-walldegrading proteins and nucleases. Therefore some senescence-related degradation
functions may be dependent on ethylene. The generation of the transcriptome data
revealed that indeed ethylene controls a subset of SAGs during senescence;
however, the importance of the identified genes for the control of leaf senescence
remains elusive.
Endogenous ethylene levels are important for the initiation of senescence. However,
the age-dependent senescence induction by ethylene limits its control to a specific
age range. The transcriptome studies on SAGs induced by ethylene, together with
physiological studies, reveal extensive cross talks between ethylene and the other
hormones that might be utilized to fine-tune the progression of senescence in an
age-dependent way.
Jasmonic acid
Jasmonates include jasmonic acid (JA), methyl jasmonate (MeJA) and related
compounds and are found in fragrant oils. This group of plant regulators is connected
to plant growth and development such as germination and seedling development,
flower development, tuberisation, tendril coiling, leaf senescence and fruit ripening
(Wasternack and Hause, 2002). The promotional effect of MeJA on senescence was
first shown by application to detached oat leaves (Ueda and Kato, 1980).
Exogenously applied JA or MeJA resulted in a decreased expression of
photosynthesis related genes like Rubisco. Moreover, a change in the polypeptide
composition in senescing tissue was observed, which shared similarity with ABAinduced senescence in detached leaves (Weidhase et al., 1987). In plants two JA
biosynthetic pathways have been identified; a chloroplast-localized pathway and a
cytoplasm localized pathway (Creelman and Mullet, 1995). Exogenous application of
JA typically promotes senescence in attached and detached leaves of Arabidopsis
but not in the JA-insensitive mutant coi1. Also the endogenous JA levels in senescing
leaves increased fourfold as compared to no senescing leaves. Besides an increased
JA level during senescence also the enzymes involved in the JA biosynthesis are
differentially regulated during senescence (He et al., 2002). The coi1 mutant, which is
25
impaired in JA signaling, did not show any altered leaf senescence. Also other JArelated mutants do not show any alterations in the senescence program, which
challenges the idea that JA plays a role in senescence. However, a study with
senescence enhancer-trap lines in Arabidopsis showed that JA can induce GUS (βglucuronidase) expression in 14 out of the 125 lines tested (He et al., 2001). The
authors developed a sensitive large-scale screening method and have screened
1300 Arabidopsis enhancer-trap lines, which resulted in the identification of 147 lines
in which the reporter gene GUS is expressed in senescing leaves but not in
nonsenescing ones. Application of senescence-inducing factors showed that only
ethylene induced GUS expression in more lines than JA and that ABA, BR, darkness
and dehydration were less effective. Based on this, JA appears to be an important
senescence-promoting factor.
The identification and cloning of coi1 resulted in the identification of an F-box protein
which shows the involvement of proteasome-dependent protein degradation in JA
signaling (Xie et al., 1998). Interestingly, application of MeJA to Cucurbita pepo
(zucchini) induces senescence in 7-day-old cotyledons. One of the observed effects
was on the concentration of endogenous cytokinin levels, which reduced rapidly after
MeJA treatment (Ananieva, 2004). A drop in cytokinin levels is necessary before
senescence can be initiated; whether MeJA can directly or indirectly antagonize
cytokinin levels remains to be answered.
Transcriptome analyses of MeJA-treated seedlings showed a self-activation of JA
biosynthesis and cross talk with other hormones (Sasaki et al., 2001). Although the
coi1 mutant does not show any visual senescence defects, a transcriptional analysis
showed that 12% of the identified developmental senescence genes are not
expressed during senescence of coi1 (Buchanan-Wollaston et al., 2005). In addition,
certain genes that are downregulated in the coi1 mutant also appear to be
downregulated in the ein2 or nahG mutants. This further demonstrates the
importance of the JA pathway during leaf senescence.
Salicylic acid
SA, a phenolic compound, has been identified as a key-signaling molecule in various
plant responses to stress, like pathogen invasion (Glazebrook, 1999) and exposure
to ozone and UV-B. The endogenous SA levels in senescing stage 2 leaves are four
times higher than in nonsenescing leaves (Morris et al., 2000). This is consistent with
26
a role for SA during later stages of the senescence program. Study of the nahG,
pad4 and npr1 mutants, which are defective in the SA signaling pathway, showed an
altered expression pattern of a number of SAGs. Furthermore, a delay in yellowing
and reduced necrosis were observed in these plants (Morris et al., 2000). The pad4
mutant has a non-necrotic phenotype that has a reduced expression of SAG12, a
well-known SAG. The authors postulated that SAG12 may take part in a regulatory
pathway leading to cell death and that it supports the transition from senescence to
final cell death. Thus the senescence phenotype of pad4 mutant suggests that SA
might regulate the transition from senescence to final cell death.
Besides biochemical and physiological evidence for role of SA in senescence,
genetic evidence has also been generated by a microarray approach (BuchananWollaston et al., 2005). Of 827 genes that were identified as senescence upregulated
genes, 19% showed at least a twofold reduction in the nahG transgenic plants that
are defective in SA signaling. Interestingly SAG12 expression was substantially
reduced compared to that in wild-type plants and SA-treated plants (Morris et al.,
2000, Buchanan-Wollaston et al., 2003). Since SAG12 is generally seen as a marker
for developmental senescence, this further demonstrates the importance of SA in
senescence.
Involvement of genome programs in the regulation of senescence-associated
genes
Developmental senescence is regulated by diverse programs involved in plant life
maintenance, defense responses and growth and development (see above). This is
consistent with the evolutionary theory of senescence and the proposal of genome
optimization for reproduction, which argues that no specific genetic programs for life
span evolve. Since the expression profiles of SAGs are reliable markers for
senescence, examining the regulation of SAG expression may provide evidence to
support that argument. If the evolutionary theory of ageing applies to plants, we
expect that many SAGs encode proteins with functions throughout the life cycle of
the plant, and not only during developmental senescence. This notion, as a matter of
fact, is well supported by the identities and expression profiles of SAGs. Up to now,
almost all the isolated SAGs, including many involved in nutrient salvage, exhibit a
certain basal level of expression prior to the onset of leaf senescence. This indicates
27
that nutrient salvage is a continuous process occurring in plant cells throughout life.
In this sense, leaf senescence is not different from other leaf developmental stages
but is more committed to recruit the last yet important source of nutrients retained in
an ageing leaf.
Recent omics techniques have allowed us to examine the genes that are upregulated
during senescence on a whole-genome basis. In addition to development, leaf
senescence can be induced by biotic and abiotic stresses. It is therefore possible to
compare the SAG expression profiles of various types of senescence using currently
available microarray data, which enables the better understanding of the nature of
the regulation of developmental senescence. In a whole-genome transcriptome
analysis, a total of 827 SAGs were found upregulated during developmental
senescence (Buchanan-Wollaston et al., 2005). However, most of those are induced
by hormones (SA, JA and ethylene) or darkness as well (Buchanan-Wollaston et al.,
2003; Lin and Wu, 2004). Using these data, we deducted the number of SAGs that
were enhanced by darkness-induced senescence and were downregulated during
leaf senescence in nahG, coi1 and ein2 plants. The remaining SAGs are hence
presumably regulated by other developmental cues and/or stress conditions. As
shown in Table 1, this category under the name of ‘development’ includes a total of
209 SAGs, which interestingly spread almost in all the categories. We further
dissected whether these SAGs are upregulated by carbon and nitrogen metabolism.
For this, the array data from ‘Expression patterns of genes induced by sugar
accumulation during early leaf senescence’ provided by Wingler’s laboratory were
used. Analysis was done by GENEVESTIGATOR (Zimmermann et al., 2004). Nearly
half of the 209 SAGs were upregulated after induction of senescence by glucose in
combination with low nitrogen. Again, the remaining 110 SAGs are wide spreading in
all the categories. If this list of SAGs is compared with the profiles of SAGs in
senescence regulated by other developmental cues such as ROS, other hormones
(cytokinins, ABA, GA, etc.) or protein degradation, it will not be surprising that
perhaps nearly all the SAGs are be regulated by one or more of these cues. In other
words, very few SAGs will be solely induced by developmental senescence, which is
in agreement with the evolutionary theory of ageing.
28
Table 1
Comparison of gene expression profiles of age-regulated and developmental
leaf senescence.
Category
Regulatory genes
Putative transcription factors and
nucleic-acid-binding proteins
Putative protein–protein interaction
Putative ubiquitination control
Protein kinase and phosphatases
Signaling
Calcium related
Hormone pathways
Macromolecule degradation and
mobilization
Protein degradation
Amino acid degradation and N
mobilization
Nucleic acid degradation and
phosphate mobilization
Lipid degradation and mobilization
Chlorophyll degradation
Sulfur mobilization
Carbohydrate metabolism
Lignin synthesis
Transport
ATPases
Metal binding
Stress related
Antioxidants
Stress and detoxification
Defense related
Secondary metabolism
Alkaloid biosynthesis
Flavonoid/anthocyanin pathway
Autophagy
Structural
Unclassified enzymes of unknown
role in senescence
Unknown genes
Total
Development
C/N
Others
Total
Dark
Cell
suspension
nahG/coi1/
ein2
96
47
38
23
9
19
9
30
66
17
13
17
7
18
26
9
5
10
3
23
20
8
4
11
1
6
31
5
8
6
1
2
6
0
1
2
1
2
6
5
0
0
29
27
13
14
14
15
6
10
6
3
3
3
14
3
8
5
1
2
29
2
2
63
3
74
7
10
18
1
0
27
1
31
1
5
13
1
0
22
2
26
2
4
5
0
1
32
0
17
3
1
3
1
1
6
1
10
0
3
5
0
0
6
0
14
1
0
11
17
11
1
9
19
5
4
110
7
7
6
0
6
2
5
0
51
6
4
4
0
5
2
3
0
42
4
5
5
1
5
5
1
1
36
2
2
1
0
0
7
0
1
13
0
4
0
0
0
5
0
2
17
132
827
65
385
55
335
34
257
17
99
15
110
Data sources: Buchanan-Wollaston et al. (2005); C/N: carbon and nitrogen supply (Expression
patterns of genes induced by sugar accumulation during early leaf senescence; Zimmerman et al.,
2004).
The most frequently used SAG to monitor developmental senescence, and perhaps
one of the few SAGs which is specifically induced by developmental senescence, is
SAG12. SAG12 transcripts were found to be very low or below the detection level in
young and mature green leaves, contrasting to the levels of the transcripts of SAG13
and SAG14 (Figure 7.1; Lohman et al., 1994). Unlike other SAGs, including SAG13,
SEN1 and SAG14 whose expression could be enhanced in young leaves by a range
of senescence-inducing treatments such as detachment, hormonal exposure,
29
darkness, drought, wounding and pathogen challenge, SAG12 was only occasionally
found to change its expression under these circumstances (Oh et al., 1997; Park et
al., 1998; Weaver et al., 1998; Noh and Amasino, 1999; Brodersen et al., 2002).
Figure 1. SAG12 and SAG13 expression during Arabidopsis development. Expression levels
of both SAG12 and SAG13 are increased during senescence. In contrast to SAG12, basal
SAG13
expression
levels
are
present
throughout
development.
Data
source:
GENEVESTIGATOR (Zimmermann et al., 2004).
Thus, SAG12 is considered the best marker for developmental senescence that
relies on leaf age, whereas SAG13 and SAG14 may represent stress-induced
senescence or general cell-death markers. That the SAG12 promoter has been used
for the autoregulated production of cytokinin to delay senescence in a number of
species including tobacco (Gan and Amasino, 1995; Ori et al., 1999), lettuce
(McCabe et al., 2001), petunia (Chang et al., 2003) and Arabidopsis (Huynh et al.,
2005) suggests that the developmental senescence regulation of SAG12 is
conserved across species. Moreover, a conserved cis-element of the SAG12
promoter was also found in the Asparagus officinalis asparagine synthetase promoter
30
and was responsible for the induction of transcription of this gene by senescence
(Winichayakul et al., 2004). Thus, monocotyledonous and dicotyledonous plants
appear to share this senescence cis-element, further confirming the conservation of
the regulation of developmental senescence across species. Extensive studies on
the expression of SAGs, including SAG12, have provided exciting new insights into
the developmental regulation of senescence, and future research will likely result in a
better understanding of developmental senescence.
Integrating hormonal action into developmental senescence
Reproduction has specific timing and all the programs need to be timely in place to
ensure successful reproduction. The indirect consequence is that the various
strategies embedded in the programs will initiate developmental senescence in an
age-dependent manner. Thus, developmental senescence is the consequence of
time-specific action of genes. Understanding the timing of the various senescence
strategies is a necessary step for elucidating the molecular mechanisms of
developmental senescence. In this section we intend to put together the action of the
hormones that control leaf senescence and thus developmental ageing in
Arabidopsis.
Previously, we proposed a senescence window concept to explain the involvement of
ethylene in leaf senescence (Jing et al., 2002, 2003). Depending on whether and
how senescence can be induced by ethylene, the life span of a leaf can be split into
three distinct phases (Figure 2A). The experimental evidence supporting this view is
briefly summarized as follows. (1) When plants were exposed to a short-pulse (e.g.
1–3 days) ethylene treatment, no senescence symptoms could be induced in young
leaves (Grbić and Bleecker, 1995;Weaver et al., 1998; Jing et al., 2002). (2) Leaf
senescence is not accelerated in the ctr1 mutants (Kieber et al., 1993). This indicated
that there exists a never-senescence phase in which senescence cannot be induced
by ethylene. (3) Furthermore, in a certain range of leaf ages, the effect of ethylene on
leaf senescence increases with the increase in leaf age (Grbić and Bleecker, 1995;
Weaver et al., 1998; Jing et al., 2002), indicative of an ethylene-dependent
senescence phase. (4) Finally, beyond certain leaf ages, senescence will start even
without the participation of ethylene as shown in the etr1 and ein2 mutants in which
31
Figure 2. The senescence window concept. (A) The senescence window concept as
deduced from the effects of ethylene on leaf senescence. At early leaf development,
ethylene is not able to induce leaf senescence. This is the so-called never-senescence
phase in the model. Only after a certain developmental stage, ethylene can induce leaf
senescence, depending on the environmental conditions. Further development of the leaf will
always result in senescence, even in the absence of ethylene. (B) Hormonal action during
leaf development is age dependent. The action of the senescence-promoting hormones is
strongest at late developmental stages, and is antagonistic to that of the stay-green
hormones. The onset of leaf senescence is achieved by a depletion of the stay-green
hormones, concomitant with an increase in ethylene levels followed by JA, ABA and
eventually SA. Hormone levels and sensitivity change during development, as indicated by
the triangles. The age-related changes limit the action of the various plant hormones to their
own specific age window.
the senescence progresses normally once started (Grbić and Bleecker, 1995; Park et
al., 1998; Buchanan-Wollaston et al., 2005), which suggests the existence of an
ethylene-independent senescence phase. This senescence window concept
emphasizes the developmental control of leaf senescence and considers leaf age as
32
an ultimate determinant of senescence progression. Clearly, genes that control the
phase transitions of the senescence window are important for the onset of
developmental senescence, and evidence suggests that many genetic loci are
required (Jing et al., 2002, 2005). Thus, the senescence window concept provides an
explanation why the senescence-promoting effect of ethylene is variable during
development.
The senescence window concept can, perhaps, integrate the action of all plant
hormones involved in leaf senescence. In Arabidopsis the different hormones seem
to control the onset and progression of senescence in an age-related manner. Figure
2B is an extension of the senescence window concept developed from the interaction
between leaf age and ethylene and shows a tentative model illustrating the timing
and action of the different hormones during developmental senescence. In this
model, age-related changes, and thus development, are considered the primary
regulator of leaf senescence. During ageing, developmental cues lead to the
diminished action of the senescence-retarding hormones such as auxin, GA and
cytokinins, as well as the concomitant strengthening of the action of senescence
enhancing hormones such as ethylene, JA, ABA and SA. The action of the different
hormones during the initiation of leaf senescence does not change suddenly but
gradually, allowing a gradual integration of all the hormones controlling the process.
This suggests that the senescence process is partly reversible by fine-tuning
hormone action and hence amenable for modulation.
The model provides a basis for the explanation of experimental data. For instance,
the major senescence-retarding compound cytokinin can delay senescence when its
level is maintained. However, in transgenic SAG12–IPT plants the senescence
process will start eventually and progresses normally (Gan and Amasino, 1995; Ori et
al., 1999), suggesting that cytokinin action is restricted to certain developmental
stages. On the other hand, cytokinin biosynthesis mutants showed a shorter leaf life
span (Masferrer et al., 2002). This might be explained by assuming that the effect of
the senescence-promoting hormones is antagonistic to those blocking senescence;
older leaves may become less sensitive for cytokinin and more sensitive for
senescence-promoting hormones like JA and ABA (Weaver et al., 1998). Similarly,
blocking the ethylene pathway increases leaf longevity. Finally, however, the leaves
go into senescence because the influence of JA, ABA and SA may increase with the
33
age of the leaf. Thus, the age-related changes limit the action of the various
hormones to their own specific window.
Taken together, although plant hormones are almost universally involved in every
aspect of plant life, they may participate into developmental senescence only in very
specific age windows. The proposed senescence window concept and the model for
hormonal action provide a developmental view to examine the modulation of
developmental senescence by hormones, which certainly requires more experimental
evidence for validation.
Outlook and perspectives
Thanks to the availability of cutting-edge technology and the use of model species
with known whole-genome sequences that have enabled senescence studies to be
carried out at a scale that was not imaginable even 15 years ago, our knowledge on
the regulation of developmental senescence has been advanced tremendously. It is
clear that hormonal modulation, metabolic flux, ROS and protein degradation are the
major cellular and molecular processes that are important for senescence regulation.
Strikingly, these processes are embedded in the genome programs that regulate
plant life maintenance, responses to biotic and abiotic stresses, and growth and
development for the sake of successful reproduction. Thus, leaf senescence can be
viewed as an indirect consequence of genome optimization for reproduction. This
perspective is exciting and worthy of further exploitation, since it coincides with the
evolutionary theory of senescence developed from animal ageing studies. In-depth
molecular genetic studies are required to dress the evolutionary basis of leaf
senescence. In particular, identification of regulatory genes with pleiotropic functions
or late-life deleterious effects should be a priority for further senescence studies.
The complexity of leaf senescence is mainly due to the involvement of multiple
components that exhibit overlapping effects. This is particularly true for the action of
hormones. The proposed senescence window concept provides a theoretic
framework to dissect the action of hormones during senescence depending on their
time of action, which is important to separate the effect of hormones on senescence
from their other effects. Using this concept, it is possible to study genetic components
that control the action of hormones during development, which is an essential step for
ultimately understanding the mode of action of hormones during development.
34
Combined with the genetic dissection, whole-genome analysis should be employed
to define the networking of various regulatory circuits.
In conclusion, senescence is one of the biological phenomena with extreme
complexity. In the current postgenome era, we are provided with both opportunities
and the challenge to dissect the molecular genetic mechanisms of leaf senescence.
The findings in the past have enabled us to look at senescence regulation from a
fresh perspective of genome optimization. We have evolutionary and developmental
theories that guard us to define the proper targets. We are also armed with cuttingedge technologies and tools. Thus, a concerted effort will eventually unveil the
mystery of senescence regulation and provide a genetic basis for senescence
manipulation.
35
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44
Chapter
2
Ethylene-induced leaf senescence depends on age-related
changes and OLD genes in Arabidopsis
Hai-Chun Jing*2, Jos H.M. Schippers*1, Jacques Hille1 and Paul P. Dijkwel1
1
Molecular Biology of Plants, Groningen Biomolecular Sciences and Biotechnology Institute, University
of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands
2
Wheat Pathogenesis Programme, Rothamsted Research, Plant–Pathogen Interaction Division,
Harpenden, Herts AL5 2JQ, UK
*These authors contributed equally to this work.
J. Exp. Bot. 56: 2915-2923 (2005)
45
46
Abstract
Ethylene can only induce senescence in leaves that have reached a defined age.
Thus, ethylene-induced senescence depends on age-related changes (ARCs) of
individual leaves. The relationship between ethylene and age in the induction of leaf
senescence was tested in Arabidopsis Ler-0, Col-0, and Ws-0 accessions as well as
in eight old (onset of leaf death) mutants, isolated from the Ler-0 background. Plants
with a constant final age of 24 d were exposed to ethylene for 3–16 d. The wild-type
accessions showed a common response to the ethylene treatment. Increasing
ethylene treatments of 3–12 d caused an increase in the number of yellow leaves.
However, an ethylene exposure time of 16 d resulted in a decrease in the amount of
yellowing. Thus, ethylene can both positively and negatively influence ARCs and the
subsequent induction of leaf senescence, depending on the length of the treatment.
The old mutants showed altered responses to the ethylene treatments. old1 and
old11 were hypersensitive to ethylene in the triple response assay and a 12-d
ethylene exposure resulted in a decrease in the amount of yellow leaves. The other
six mutants did not show a decrease in yellow leaves with an ethylene treatment of
16 d. The results revealed that the effect of ethylene on the induction of senescence
can be modified by at least eight genes.
Key words
Age-related changes, Arabidopsis, ethylene, leaf senescence, natural variation, old
mutants.
47
Introduction
Senescence is the final developmental phase of a leaf which starts with nutrient
salvage and ends with cell death. The first visible event during senescence is leaf
yellowing, which typically starts at the leaf margins and progresses to the interior of
the leaf blade (Quirino et al., 2000). The protein and RNA degradation parallels a
loss in photosynthetic activity and the majority of the senescence processes have
occurred by the time yellowing of the leaf can be seen (Buchanan-Wollaston et al.,
2003). The degradation products are transported out of the leaves to other parts of
the plant. In this sense, the senescing leaf continues to function as a source of
nutrients to the whole plant, but at the expense of its own ability to survive (Bleecker
and Patterson, 1997).
Senescence is under genetic control and requires differential expression of specific
genes. Expression of photosynthesis-associated genes (PAGs) is downregulated,
while many other genes, designated as senescence-associated genes (SAGs), are
upregulated during senescence. Detailed studies on the SAG identities and their
expression suggest a complex regulation of leaf senescence (Hensel et al., 1993;
Buchanan-Wollaston, 1997; Gan and Amasino, 1997; Nam, 1997; Gan, 2003). In
Arabidopsis the age of individual leaves plays a prominent role in determining leaf
longevity (Hensel et al., 1993; Oh et al., 1997; Nooden and Penney, 2001; Jing et al.,
2002), although floral initiation can influence plant longevity and thus whole-plant
senescence (Levey and Wingler, 2005).
Lim et al. (2003) have suggested that a distinction should be made between the
terms senescence and ageing: senescence refers to the process that leads to the
death of the leaf, while ageing itself occurs throughout development, from the
initiation of a leaf primordium to senescence and death. Thus ageing determines
when senescence starts, but not senescence itself. During ageing, age-related
changes (ARCs) occur as a result of the differential regulation of developmental
processes. Before senescence can be initiated, certain ARCs must have taken place
in the leaf. An example of a process dependent on ARCs in Arabidopsis is agerelated resistance (ARR) (Kus et al., 2002). The authors showed that plants become
more resistant to Pseudonomas syringae as they age. It was suggested that the
ability to accumulate SA is necessary for the ARR response and that SA may act as
a signal for the production of the ARR-associated antimicrobial compound(s) and/or it
may possess direct antibacterial activity against P. syringae. Examples of ARCs in
48
other
model-organisms
are
body
movement
and
pharyngeal
pumping
in
Caenorhabditis elegans (Huang et al., 2004) or accumulation of oxygen free radicals,
protein conformational changes, and decline in chaperone functions in the brain of
Homo sapiens (Drachman, 1997).
The components that control ARCs and thus ageing in plants are still unclear. One of
the parameters for ageing is the photosynthetic capacity of the leaf. From the time of
full leaf expansion, CO2 fixation rates drop and the senescence program is initiated
(Thomas and Howarth, 2000), which indicates that metabolic flux shift may serve as
a general signal for the induction of leaf senescence as implicated by the ore4
mutation (Woo et al., 2002). During plant growth light dosage has an effect on
ageing; high light intensity results in premature senescence when compared with
growth under standard light intensities, while low light intensities delay the
senescence process (Nooden et al., 1996). Many other stress-inducing conditions
such as drought, darkness, ozone, and pathogen attack can hasten leaf senescence
as well (Lim and Nam, 2005). Plant hormones, cytokinin and ethylene being the most
conspicuous, are another group of plant endogenous components that play important
roles in the regulation of the onset of senescence. While increasing cytokinin
production could delay leaf senescence (Gan and Amasino, 1995; Ori et al., 1999),
reducing endogenous cytokinin levels resulted in accelerated senescence (Masferrer
et al., 2002). Recently, exciting advances have been achieved in dissecting the
components involved in cytokinin signaling (Hutchison and Kieber, 2002; Hwang et
al., 2002). Among the genes characterized, the receptor CKI1 and the response
regulator ARR2 appear to be involved in regulating leaf senescence (Hwang and
Sheen, 2001).
The plant hormone ethylene has long been seen as a key hormone in regulating the
onset of leaf senescence. Zacarias and Reid (1990) reported that ethylene does
promote senescence, but it is not an essential compound for the senescence
syndrome induced by other factors (e.g. ABA). The role of ethylene in senescence
has been demonstrated by several studies. Both ethylene-insensitive mutants etr1-1
and ein2 show increased leaf longevities (Grbić and Bleecker, 1995; Oh et al., 1997)
and antisense suppression of the tomato ACC oxidase resulted in delayed leaf
senescence (John et al., 1995). In these cases, however, senescence eventually
begins and progresses normally. Exogenously applied ethylene induces premature
leaf senescence in Arabidopsis. However, constitutive application of ethylene does
49
not change the longevity of the leaves. Both ctr1 mutants and Arabidopsis plants
grown in the continuous presence of exogenous ethylene did not show premature
senescence (Kieber et al., 1993; Grbić and Bleecker, 1995). Thus, ethylene is neither
necessary nor sufficient for the occurrence of senescence. These studies suggest
that ethylene does not directly regulate the onset of leaf senescence. It acts to
modulate the timing of leaf senescence. Only when developmental changes
controlled by leaf age are present can ethylene induce senescence (Hensel et al.,
1993; Grbić and Bleecker, 1995; Jing et al., 2002).
In the present study, advantage was taken of natural variation among Arabidopsis
accessions and the availability of Arabidopsis onset of leaf death (old) mutants to
examine genetic loci that are involved in ethylene-induced senescence. The
combined physiological and genetic studies used here showed that Arabidopsis
accessions exhibited common and discrete senescence responses to exogenous
ethylene. It was observed that ethylene displayed a dual function, both as an inducer
and a repressor, in the induction of leaf senescence and that such a role of ethylene
was differentially modulated by multiple genetic loci. These results lay out a basis for
further
molecular
dissection
of
genes
that
regulate
ethylene-induced
leaf
senescence.
Results
Physiological and molecular markers of leaf senescence correlate with visible
yellowing
The effect of ethylene on age-dependent leaf senescence was determined for Ler-0.
Ler-0 was grown for 11, 21, or 27 d after germination (DAG) and subsequently
treated with ethylene for 3 d. The senescence syndrome was studied using
morphological, physiological, and molecular markers (Figure 1). Plants grown for 14,
24, or 30 d in air did not show any signs of visible yellowing, even though in the
cotyledons the chlorophyll content had decreased approximately 2-fold over time
(Jing et al., 2002). Plants grown for 11 d in air and subsequently for 3 d in air
supplemented with ethylene (11+3) did not show any signs of senescence either.
However, those treated for 21+3 and 27+3 showed signs of visible yellowing.
Concomitant with the visible yellowing, the chlorophyll content of the plants was
decreased after the 21+3 and the 27+3 treatments (Figure 1B).
50
Figure 1. Comparison of ethyleneinduced senescence in cotyledons and
rosette leaves of Ler-0. Plants were
grown first in air for 11, 21, or 27 d, and
then exposed to ethylene for 3 d. The
visible yellowing (A) of cotyledons and
the first pair of rosette leaves was
subsequently scored as the percentage
of yellow cotyledons or rosette leaves
versus the total number of cotyledons or
rosette
leaves,
respectively.
The
chlorophyll contents (B) and steadystate mRNA levels (C) of several
senescence
associated
genes
were
subsequently analyzed. For each time
point,
the
observations
on
visible
yellowing were on three sets of 50
plants and the results for chlorophyll is
shown as mean ± SD of four replicates.
Total RNA was isolated from leaf
samples of approximately 100 plants.
Five µg total RNA was used for northern
blotting.
sequentially
The
membrane
hybridized
with
was
cDNA
probes of the indicated SAGs, CAB, and
rRNA.
The chlorophyll content was lower in cotyledons than in the first leaf pair after both
the 21+3 and the 27+3 treatments. The drop in chlorophyll content correlated with a
decrease in the mRNA levels of CAB (Chlorophyll a/b binding protein) in the 21+3
and 27+3 samples (Fig. 1C). Expression of SAG12, SAG13, and SAG14 was
upregulated in the 21+3 and 27+3 samples compared with the 11+3 samples. Low
SAG14 expression, however, was detected in the green cotyledons of 11+3 samples.
The results are in agreement with previous studies, demonstrating that ethylene-
51
induced senescence is dependent on leaf age (Grbić and Bleecker, 1995; Jing et al.,
2002).
Thus the effect of ethylene on inducing visible yellowing correlated well with changes
in chlorophyll content and in the SAG mRNA levels. The senescence syndrome
continued being examined by using visible yellowing as a parameter.
Arabidopsis accessions exhibited similar and discrete responses to the induction of
senescence by different ethylene exposure times
The effect of ethylene exposure time on leaf senescence was assessed in the
Arabidopsis accessions Ler-0, Columbia (Col-0), and Wassilewskija (Ws-0). Plants
were grown until 8, 12, 18, or 21 DAG under standard conditions and were further
grown under continuous light and ethylene conditions until 24 DAG. Thus, the plants
that were treated for the longest time with ethylene received the treatment at a
younger developmental stage. Figure 2 shows that ethylene has major effects on
plant growth and development; the longest ethylene treatment caused the plants to
phenocopy the ctr1-1 mutant.
Figure 2. Representative Ler-0 plants after
growth in air or after ethylene treatment.
Plants were grown either in air for 24 d, or
first in air for 21, 18, 12, or 8 d and then
exposed to ethylene for 3, 6, 12, or 16 d,
respectively. At the end of the treatment
(24 d), representative plants were selected
and photographed. The bar represents 0.5
cm.
The effect of the treatments on leaf
52
yellowing was determined and the results are shown in Figure 3. The overall
response was similar for the three accessions tested; an increase in the duration of
the ethylene treatment from 3 d to 12 d caused an increase in the number of yellow
leaves. Longer ethylene treatments of 16 d resulted in a decrease in the number of
yellow leaves, when compared with the 12 d ethylene treatment.
Figure 3. Visible yellowing of 24-d-old wild-type
plants, exposed to ethylene for various amounts of
time. The number of yellow leaves of three
Arabidopsis
accessions
exposed
to
different
ethylene treatments is indicated. Ler-0, Ws-0, and
Col-0 plants were grown first in air for 21, 18, 12, or
8 d, and then exposed to ethylene for 3, 6, 12, or
16 d, respectively.
The visible yellowing was subsequently scored and
expressed as means ± SD of at least three
replicates of 30 plants each. Plants that were
grown for 24 d in air did not show any sign of
senescence (not shown).
These results are consistent with the phenotype of the ctr1-1 mutant, which does not
show early leaf senescence and the observation that continuous ethylene exposure
does not promote early senescence in Ler-0 (Kieber et al., 1993). There were also
differences in ethylene-induced senescence among the three accessions. Ws-0
plants exhibited the highest amount of yellow leaves. Ler-0, furthermore, had a more
pronounced senescence response than Col-0. Thus, the number of yellow leaves
that can be induced by ethylene is dependent on the genetic background and the
length of the ethylene treatment.
Prolonged ethylene treatments inhibited cell expansion but stimulated plant
development, as judged by leaf emergence. The average total leaf numbers in plants
53
with various ethylene treatments were compared and the results showed that plants
that experienced a longer ethylene exposure time had more leaves (Table 1). The
increase in leaf number did not seem to correlate with the number of senescing
leaves, Col-0 had more leaves than Ws-0 and Ler-0, but exhibited less visible
yellowing. Thus, the effect of ethylene exposure time on leaf senescence is different
from its effects on the inhibition of cell elongation and promotion of development.
Table1. Total leaf numbers of Arabidopsis accessions and old mutant lines after various
ethylene treatments
Lines
21+3
18+6
12+12
8+16
Ler-0
12.7 ± 0.2
13.3 ± 0.2
15.1 ± 0.2
15.1 ± 0.4
Col-0
13.3 ± 0.1
14.1 ± 0.4
15.5 ± 0.1
16.0 ± 0.4
Ws-0
12.1 ± 0.1
12.6 ± 0.3
13.8 ± 0.2
13.6 ± 0.1
ctr1-1
11.8 ± 0.1
11.8 ± 0.2
13.0 ± 0.5
13.0 ± 0.1
old1
11.0 ± 0.5
11.0 ± 0.4
10.9 ± 0.1
11.0 ± 0.1
old5
12.6 ± 0.2
13.3 ± 0.1
14.4 ± 0.3
13.9 ± 0.2
old14
11.5 ± 0.2
11.30 ± 0.0
11.1 ± 0.3
11.4 ± 0.2
old9
12.7 ± 0.3
12.7 ± 0.1
13.7 ± 0.1
14.5 ± 0.1
old11
12.6 ± 0.3
12.9 ± 0.1
13.4 ± 0.2
12.9 ± 0.1
old13
12.1 ± 0.3
12.4 ± 0.3
14.0 ± 0.2
13.8 ± 0.3
old3
4.0 ± 0
4.0 ± 0
4.0 ± 0
4.0 ± 0
old12
11.5 ± 0.0
11.5 ± 0.1
12.1 ± 0.0
12.3 ± 0.4
Plants were grown and treated with ethylene as described in materials and methods. A leaf was
scored when it emerged and was over 1mm in size. Scoring was performed on 24-d-old plants at the
end of the ethylene treatment. Approximatly 30 plants were used for each line.
Arabidopsis old mutants reveal multiple genetic loci involved in the regulation of
ethylene-induced leaf senescence
As shown above, the genotype has an effect on the senescence response to
ethylene. To address the genetic regulation of ethylene-induced leaf senescence
further, use was made of previously isolated old mutants (Jing et al., 2002). The
mutants were identified from an EMS mutagenized population of Ler-0 seeds and
show an early senescence phenotype before and/or after ethylene treatment (Fig. 4).
The old mutants were selected and subgrouped into three different classes. The
54
selected Class I mutants comprises the formerly described mutant old1 (Jing et al.,
2002) and the mutants old5 and old14. The mutants have early leaf senescence
symptoms in air (compare Fig. 4A to C and E) and those symptoms are further
enhanced by ethylene treatment (Fig. 4B, D, F).
Figure 4. Phenotype of old mutants.
Representative 24-d-old Ler-0 airgrown
(A) and 21+3 ethylene-treated Ler-0 (B).
24-d airgrown old5 (C), old14 (E), and
old12 (J). 21+3 ethylene-treated old5 (D),
old14 (F), old9 (G), old11 (H), and old13
(I). The bar represents 0.5 cm.
55
When grown in air, old5 and old14 did not differ markedly from the wild type, except
for their early senescence phenotype which was observed after ~19 d. After 24 d of
growth both mutants have 1–2 yellow leaves (results not shown). Air-grown Class II
mutants were not different from the wild type when grown under the standard
conditions used here, but displayed enhanced senescence symptoms when treated
with ethylene (Fig. 4G, H, I). In old13, ethylene-induced yellowing was associated
with lesion formation. The old12 mutant (Fig. 4J) and the previously described old3
mutant (Jing et al., 2002) belong to the third class of mutants and have advanced
senescence symptoms in air that cannot be further induced by ethylene treatment.
The old12 mutant not only displayed early senescence symptoms but also differed
from the wild type in size, exemplified by reduced leaf expansion, shorter shoot
length, and the formation of thicker siliques (data not shown).
Table 2. Genetic segregation of old mutations
Class
I
a
Male
Female
Generation
Wild-type
Mutant
old5
Ler-0
F1
31
0
F2
151
64
F1
39
0
F2
156
40
F1
23
25
F2
18
73
F1
11
23
F2
19
67
F1
52
0
F2
180
79
F1
24
0
F2
328
87
old14
II
b
old9
old11
old13
a
III
a
old12
Ler-0
Ler-0
Ler-0
Ler-0
Ler-0
χ
2c
2.61
2.20
1.32
0.75
4.18
3.61
The Class I and Class III mutants showed early senescence when grown in standard growth
conditions. Thus, the scoring of phenotypes was performed before ethylene treatment and plants with
clearly visible yellowing cotyledons and/or rosette leaves were scored as mutants.
b
The segregation analysis for Class II mutants was performed after ethylene treatment. The
-1
phenotype scoring was carried out on 21-d-old plants treated with 10µl l ethylene for 3 d. The criteria
were: wild-type plants with up to two yellow cotyledons, Class II mutants with at least 2 yellow
cotyledons and 1 yellow leaf.
c
2
All the χ values were calculated for the 1:3 segregation ratios of mutant:wild type except in the case
of old9 and old11, where a 3:1 ratio of mutant:wild type was calculated
56
Genetic analyses showed that the characterized old alleles segregated as single
monogenic recessive traits except old9 and old11, which segregated as conditional
(ethylene-dependent) co-dominant traits (Table 2). Allelism tests between mutants
within each class revealed that they belong to different complementation groups
(data not shown). Genetic mapping placed the old12 and old9 mutant alleles on
chromosome 2 at ~7 Mb and ~17 Mb, respectively; old14 on chromosome 3 at ~15
Mb; and old11, old5, and old13 on chromosome 5 at ~8 Mb, ~20 Mb, and ~23 Mb,
respectively. The authors are not aware of mutants with similar phenotypes in the
vicinity of these mapped regions.
The response of the selected old mutants to several plant hormones and sugar was
tested by germination assays. Since ethylene was used as the senescence inducer,
the sensitivity of the mutants to ACC was tested by a triple response assay (Table 3).
Both old11 and old1 showed an extremely short hypocotyl. All other old mutants
showed a wild-type response to ACC. Germination on glucose plates (ArenasHuertero et al., 2000) showed that old12 and old1 are hypersensitive to glucose,
moreover germination was completely inhibited by 4% glucose (data not shown).
Table 3. Triple response assay of the wild type Ler-0 and the selected old mutants*.
Plant
MS medium (mm)
10 µM ACC medium (mm)
Ler-0
6.89 ± 0.19
3.25 ± 0.14
old1
6.81 ± 0.10
1.18 ± 0.13
old5
7.01 ± 0.28
3.05 ± 0.19
old14
6.81 ± 0.14
3.03 ± 0.17
old9
6.74 ± 0.12
3.07 ± 0.05
old11
6.82 ± 0.22
1.39 ± 0.06
old13
6.99 ± 0.20
3.17 ± 0.03
old12
7.14 ± 0.23
3.32 ± 0.05
* The average hypocotyl length after 5 days of growth on 10 µM ACC is compared.
For each data point at least 40 seedlings were used.
The glucose phenotype was independent of osmolarity since all mutants developed
like the wild type on mannitol. The response of the old mutants to ABA and jasmonic
acid was not different from the wild type (data not shown).
Thus, the selected old mutants show various senescence phenotypes that are
caused by defects in independent genetic loci.
57
old mutants and ctr1-1 have altered responses to varying ethylene-exposure lengths
The old mutants and ctr1-1 were exposed to ethylene for 3–16 d. Figure 5 shows that
all the old mutants responded to the ethylene treatment in a different way than Ler-0.
The old1 and old11 mutants showed an enhanced response to long ethylene
treatments: a maximum number of visible yellowing leaves occurred after the 18+6
treatment, instead of the 12+12 treatment in the Ler-0 plants. A further increase in
the length of ethylene treatment resulted in a reduced number of yellow leaves.
Remarkably, old1 had fewer yellow leaves than Ler-0 after the 12+12 treatment and
so could be considered a delayed senescence mutant after long ethylene treatments.
These data are consistent with the observation that old1 and old11 are
hypersensitive to ethylene in the triple response. A similar number of yellow leaves,
regardless of the length of the ethylene treatment, was observed in the old3, old9,
old12, and old14 mutants.
Figure 1. Visible yellowing of 24-d-old old mutants and ctr1-1, exposed to ethylene for
various amounts of time. The old mutants were grown first in air for 21, 18, 12, or 8 d, and
then exposed to ethylene for 3, 6, 12, or 16 d. The visible yellowing was subsequently scored
and expressed as means ± SD of at least three replicates of 30 plants each.
58
Both old3 and old12 are Class III mutants, which were determined to senesce
independently of ethylene, and the results shown here are consistent with that
classification. Mutants of old5 and old13 had a constant number of yellow leaves
when the ethylene treatment was longer than 3 d.
The average total leaf numbers of the mutants after various ethylene treatments were
compared and the results showed that the old1, old14, and old11 mutants responded
differently to the ethylene treatments than the wild type (Table 1). In contrast to the
wild type, these mutants did not show an increase in the rate of development, as
judged by the emergence of new leaves. Nevertheless, in cases where the total
number of leaves of the mutants was less than the wild type, the number of yellow
leaves was higher. The one exception is the old1 mutant, where after the 12+12
treatment, a reduced number of total leaves correlated with a reduced number of
yellow leaves. The results suggest that it is unlikely that the differences observed in
the senescence response are caused by different growth and developmental rates
between the mutants and the wild type. Taken together, two mutants (old1 and
old11) became desensitized and showed fewer senescence symptoms upon
extended long ethylene exposure, while the other six mutants show a similar
response for every ethylene treatment.
Compared with the wild-type Col-0 plants, the ctr1-1 mutant showed much lower
numbers of yellow leaves in all the treatments tested, indicating that constitutively
activating the ethylene signaling pathway through knockingout CTR1 has a
pronounced effect in inhibiting the onset of leaf senescence. Interestingly, ethylene
treatment did cause some leaf yellowing that was not observed in mutants that were
not exposed to ethylene. Remarkably, the induction of leaf yellowing followed a
similar response as the wild-type accessions: an increase in ethylene exposure time
resulted in an increase in leaf yellowing up to 12 d of ethylene treatments. A further
increase to 16 d caused a reduction in the number of yellow leaves.
59
Discussion
The effect of ethylene on the induction of leaf senescence was shown to be under
the direct influence of age (Hensel et al., 1993; Grbić and Bleecker, 1995; Jing et al.,
2002). The data presented here confirm these results as senescence was not
induced in young cotyledons or leaves. The relationship between ARCs and
ethylene-induced senescence was further assessed by treating plants of an identical
end age with different ethylene exposure lengths. The three different Arabidopsis
accessions showed a common response to the different treatments implying a
conserved perception mechanism of ethylene-inducible senescence. An increase in
the length of the ethylene treatment, and thus treatment at a younger age, resulted in
an increase in the amount of leaf yellowing. The longest treatment, however, caused
a reduction in the amount of leaf yellowing. In particular, the ctr1-1 mutants showed
much lower numbers of yellow leaves compared with the wild-type plants, suggesting
that constitutive activating ethylene signaling strongly inhibits the induction of leaf
senescence. Thus, the effect of ethylene on leaf senescence relies on the exposure
time. Since ethylene-induced senescence depends on ARCs, the results presented
here suggest that ethylene treatment can influence ARCs. Depending on the length
and start of the treatment, ethylene can stimulate, or suppress ARCs. Ethylene also
caused the inhibition of cell expansion and the stimulation of plant development. In all
accessions the longest ethylene treatment resulted in approximately two additional
leaves. Here, the longest treatment did not cause a reduction in the stimulation of
plant development. This suggests that the effect of the longest ethylene treatment on
leaf yellowing is different from and independent of the effect on plant development.
Besides the common response of the accessions to ethylene, natural variation was
present and the Ws accession showed the strongest senescence symptoms after the
ethylene treatments. These results confirm a previous study on natural variation in
the regulation of leaf senescence by Levey and Wingler (2005). Natural variation has
been observed for a variety of life history traits in Arabidopsis including the control of
flowering time (Koornneef et al., 1998), disease resistance and tolerance (Kover and
Schaal, 2002), and the control of cytosine methylation in the nucleolus organizer
regions (Riddle and Richards, 2002). Remarkably, ethylene treatment of ctr1-1
resulted in a senescence response that was similar to that of the three wild-type
accessions. The ctr1-1 mutant exhibits continuous activation of the ethylene signaling
pathway and has a wild-type timing of senescence under standard growth conditions
60
(Hensel et al., 1993). It was found here that, by applying exogenous ethylene, early
senescence can be induced. Although ctr1-1 loss-of-function mutants display a
severe ethylene phenotype, these mutants remain ethylene responsive (Larsen and
Chang, 2001). Quadruple loss-of-function mutants in the ethylene receptor family
have a more severe phenotype than ctr1-1 (Hua and Meyerowitz, 1998), suggesting
that an alternative mechanism bypassing CTR1 in ethylene signaling exists in
Arabidopsis (Cancel and Larsen, 2002). Since endogenous ethylene levels in ctr1-1
mutants are equal or even lower than in wild-type plants (Ecker and Kieber, 1994) the
observed senescence may be a direct cause of the applied exogenous ethylene
which is signaled through an alternative ethylene pathway. Although the amount of
yellowing was reduced in the ctr1-1 mutants, the induction of senescence was agedependent and ethylene exposure-time dependent. This suggests that the effect of
ethylene on ARCs is maintained in ctr1-1 mutants.
The results imply that ethylene stimulates ARCs in an ethylene treatment-duration
dependent way. However, the relation between treatment length and induction of
ARCs is not linear and there is an optimum treatment length that causes the
strongest senescence response.
Previously, several old mutants were isolated that show an altered senescence
phenotype before and/or after ethylene treatment (Jing et al., 2002). Eight old
mutants were selected and the effect of different lengths of ethylene treatment on
leaf yellowing was measured. Although all the mutants responded to the treatments
in a different way from the wild type, there were two fundamentally different
responses. Both old1 and old11 already showed a decrease in senescence with a
shorter ethylene treatment than the wild type, suggesting that they may respond
more strongly to the ethylene. Likewise, both old1 and old11 were found to be
hypersensitive to ACC in the triple response assay. These results are consistent with
the hypothesis that the mutants have an enhanced effect on ARCs as a result of a
stronger response to ethylene. The second type of response was found in the other
six mutants, where no decrease in leaf yellowing was found with the longest ethylene
treatment. This suggests that the mutants may have lost the ability to suppress ARCs
by long ethylene treatments. Interestingly, the six mutants fall into different classes.
old5 and old14 represent Class I mutants, which already have an early senescence
phenotype in air. The senescence phenotype of Class III mutants old3 and old12 is
not dependent on ethylene and, as expected, a similar response was observed for
61
every ethylene treatment applied. Only old9 and old13, which are Class II mutants,
are fully dependent on ethylene for their senescence phenotype.
In summary, it can be concluded that ethylene has an effect on processes that
regulate ARCs in a treatment-duration dependent way. In eight old mutants this effect
was altered, but the function of the OLD genes in the control of ARCs and ethyleneinduced senescence is at present unclear. Future cloning and analysis of the OLD
genes shall shed new light on the regulation of ethylene-induced leaf senescence
and the role of ethylene on processes that control ARCs.
Methods
Plant material and growth conditions
Arabidopsis
accessions
Landsberg
erecta
(Ler-0),
Columbia
(Col-0),
and
Wassilewskija (Ws-0) were used in this study. Plants were grown in a growth
chamber at 21 °C and 65% relative humidity with a day length of 16 h. The light
intensity was set at 120 µmol cm-2 s-1. An organic-rich soil (TULIP PROFI No.4,
BOGRO B.V., Hardenberg, The Netherlands) was used for the experiments
described in Fig. 1. For the other experiments a γ-ray irradiated soil was used
(Hortimea Groep, Elst, The Netherlands).
Plants for ethylene exposure were treated in a flow-through chamber at 20 °C and a
humidity of 40% under continuous illumination. The ethylene dosage was set at 10 µl
l-1 since it has been shown that a dose ranging from 1–100 µl l-1 was sufficient to
generate similar effects on several ethylene responses, including leaf yellowing
(Chen and Bleecker, 1995; H-C Jing, unpublished data).
For germination studies, seeds were surface-sterilized by soaking in 20% bleach for
5 min after which they were washed twice with sterile water. The sterilized seeds
were plated on Murashige and Skoog medium containing 0.8% agar. The plates were
stored at 4 °C for 4 d after which they were transferred to a growth chamber at 21 °C
and 16 h of light. For the triple-response assay 10 µM filter-sterilized ACC was added
to the plates. Seedlings were allowed to grow for 5 d in the dark before analysis.
Genetic analysis of the old mutants
Generation of M1 and M2 seeds and mutant screening were done as described by
Jing et al. (2002). In this study, eight old mutants with early leaf senescence
phenotypes were selected. Since the mutants were isolated from the Ler-0
62
background, they were crossed to Col-0 plants for mapping. At least 100 F2 plants
with an early senescence phenotype were selected for DNA isolation using the
SHORTY
quick
preparation
method
(http://www.hos.ufl.edu/meteng/Hanson
Webpagecontents/NucleicAcidIsolation.html#Arabidopsis%20Genomic%20DNA).
The linkage analysis was performed by using a subset of simple sequence length
polymorphism markers, making use of the Monsanto database (Jander et al., 2002).
Both old9 and old11 are co-dominant traits and there was some overlap between the
wild type and the heterozygous phenotypes of the mutants. In the F2 populations of
these two mutants backcrossed to wild type, sometimes a wild-type plant was found
that had one yellow true leaf and occasionally a heterozygous mutant was found
without any yellow true leaves following a 3 d ethylene treatment of 21-d-old plants.
Thus, for the segregation analysis of old9 and old11, any plant with at least one
yellow true leaf was counted as a mutant. Plants with up to two yellow cotyledons
were counted as wild types.
Observation of visible yellowing, chlorophyll content measurement, and northern blot
analyses
Cotyledons or rosette leaves with over 5% yellow area of the leaf blade were judged
as yellow as suggested by Lohman et al. (1994). However, in this study’s
experiments, yellowing could be initiated at the leaf tips, at the petiole side of the leaf,
or in the middle of the leaf blade, which did not always resemble the wild-type
developmental yellowing pattern of leaf senescence.
Chlorophyll was extracted in 80% (v/v) acetone overnight at 4 °C in darkness and
quantified spectrophotometrically using the method of Inskeep and Bloom (1985).
The RNA extraction and northern blot analyses was done as reported before by Jing
et al. (2002). DNA from the following genes was used for the hybridization; SAG12
(AT5G45890), SAG13 (AT2G29350), and SAG14 (AT5G20230) as described by
Lohman et al. (1994). CAB gene expression was measured as described by Leutwiler
et al. (1986).
Acknowledgement
We would like to thank Bert Venema and Otto Lip for their excellent technical
support. We also thank the Arabidopsis Biological Resource Centre for providing
Arabidopsis seeds.
63
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66
Chapter
3
Early leaf senescence of old13 is partially dependent on
salicylic acid and associated with increased oxidative stress
and altered water balance
Jos H.M. Schippers1, Renate Ellens1, Jacques Hille1 and Paul P. Dijkwel1
1
Molecular Biology of Plants, Groningen Biomolecular Sciences and Biotechnology Institute, University
of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands
67
68
Abstract
Programmed cell death (PCD) manifests itself during the final stage of leaf
development by leaf senescence. However, leaf senescence can occur prematurely
as response to environmental stress and pathogen attack. The highly coordinated
regulation of leaf senescence can increase plant fitness and survival. Here we report
that the onset of leaf death 13 mutant displays enhanced ethylene-induced
senescence and lesions formation in the absence of pathogen, suggesting that
OLD13 is involved in stress response signaling and cell death regulation. The mutant
has increased oxidative stress as determined by NBT staining and expression of
ROS markers. Double mutant analysis reveals that the lesion formation requires
salicylic acid. The ethylene-induced senescence phenotype was suppressed by a
mutation in EIN2 but not by mutations in PAD4 and ABI4. The mutant has an
increased anion content suggesting an altered water balance. Taken together we
suggest that OLD13 functions as a modulator of leaf senescence upon environmental
stress such as drought, salinity and pathogen attack.
Introduction
In the early sixties of the last century interest in the active onset of leaf senescence
was emerging as depicted by the following citation: The signal for mass hari-kari, so
to speak, for all members of these enormous populations is such a fantastically
dramatic physiological event that it seems most singular that plant physiologists have
not given more attention to the matter (Leopold, 1961). It is argued that senescence
is beneficial for plant fitness and survival and suggests the existence of a mobilizing
force that actively drives the senescence process.
Since that time plant programmed cell death (PCD) has been implicated in the
recycling of nutrients by senescence, the development of the plant body, selfelimination of cells invaded by pathogens and as a response to (a)-biotic stress
(Jones, 2001). PCD is not a passive process resulting in death but requires the active
regulation of thousands of genes which prepare the end phase of a cell. PCD in leaf
senescence has some unique features when compared to other PCD processes.
Leaf senescence occurs at the whole organ or whole plant level while other PCD
processes occur highly localized (Pennel and Lamb, 1997). Next to that senescence
is a slow progressing PCD especially compared to the hypersensitive-response (HR)
to pathogen attack (Lamb and Dixon, 1997). The orderly dismantling of a leaf results
69
in efficient remobilization of nutrients to maintain development of other parts of the
plant (Himelbau and Amasino, 2001). In Arabidopsis the execution of the senescence
program is depending on the developmental age of the leaf (Bleecker, 1998; Jing et
al., 2003). However, leaf senescence is also influenced by diverse internal and
environmental signals that are integrated with the age to determine the onset (Lim et
al., 2003). This integrated senescence response allows plants to obtain optimal
fitness by incorporating the environmental and endogenous status of plants in a
given ecological setting by fine-tuning the initiation timing, progression rate, and
nature of leaf senescence (Lim et al., 2007). Unfavorable environmental factors like
drought (Munné-Bosch and Alegre, 2004), nutrient limitation (Wingler et al., 2006),
temperature (Page et al., 2001), oxidative stress by UV or ozone (Miller et al., 1999),
darkness (Fujiki et al., 2001) and pathogen attack (Tang et al., 2005) can prematurely
induce the senescence program.
The response of plants to environmental factors involves the transcriptional activation
or repression of genes (Zhu, 2002). Several studies on improving stress resistance
have revealed cross-talk and overlap between the different stress pathways and their
activation. Overexpression of the dehydration responsive element binding (DREB) 1A
results in plants more tolerant to drought, salt and cold stress but at the expense of
growth and productivity (Kasuga et al., 1999). However, expression of DREB1A
under the control of a stress-inducible promoter improves stress tolerance without
affecting plant health and thus demonstrating the need for a targeted response. In
another example, overexpression of heat shock transcription factor (Hsf) A2 confers
higher tolerance to temperature, salt and osmotic stress but also results in growth
retardation (Ogawa et al., 2007). Taken together, stress tolerance can delay the
timing of senescence induced by stress and is thus an important factor in controlling
onset of senescence. Interestingly, stress responses and senescence share
overlapping signaling pathways as more than two-thirds of the transcription factors
induced during senescence are also induced after various stress treatments (Chen et
al. 2002).
The integration of age and environmental signals involves, next to transcriptional
factors, plant hormones which act as endogenous factors controlling growth and
development including leaf senescence (Schippers et al., 2007). Ethylene, jasmonic
acid (JA) and salicylic acid (SA) have all been implicated in the onset of senescence
(Jing et al., 2002; Buchanan-Wollaston et al., 2005; Miao et al., 2007) and responses
70
to various stresses (Chen et al., 2002). The ENHANCED DISEASE RESISTANCE 1
(edr1) mutant shows enhanced resistance to pathogens, early ethylene-induced
senescence and spontaneous lesion formation during drought stress. EDR1
functions both in ethylene and SA signaling pathways and regulates senescence and
cell death (Tang et al., 2005). Another mutant that regulates the cross-talk between
several hormones is the onset of leaf death 1 (old1) which shows early leaf
senescence,
constitutive
expression
of
pathogenesis
related
proteins
and
hypersensitivity to sugars (Jing et al., 2007). Thus the response to various stresses is
regulated by stress signaling pathways that are interconnected at several levels by
shared genetic factors between the pathways (Knight and Knight, 2001). One
common class of signaling molecules are the reactive oxygen species (ROS) which
have been implicated in the regulation of development and stress response pathways
(Pitzschke et al., 2006). Among the different ROS species only hydrogen peroxide
can cross membranes and therefore function as a cell to cell signaling molecule.
Hydrogen peroxide is involved in many plant processes including ABA-dependent
stomatal closure (McAinsh et al., 1996), signaling via the ETR1 receptor (Desikan et
al., 2005) and oxidative burst during pathogen attack and subsequent induction of
systemic immunity (Alvarez et al., 1998). While during plant defense accumulation of
ROS is required, ROS is scavenged during abiotic stress to counteract the
accumulation (Pitzschke et al., 2006). The response of the plant to ROS is
determined by the location of the signal in the cell where they are produced or
accumulate. ROS have also been implicated in the onset of leaf senescence as
demonstrated by treatment of Arabidopsis leaves with the herbicide 3-AT which
inhibits catalase activity and causes H202 stress resulting in the expression of SAG
genes (Navapbour et al., 2003). Next to that the senescence-specific transcription
factor WRKY53 is induced by H202 (Miao et al., 2004; Miao and Zentgraf, 2007).
Taken together the integration of signals from the environment with the age of the
leaf involves a complex signaling network of overlapping and interacting pathways
which together determine the onset of leaf senescence.
To gain insight into the pathways and mechanisms underlying leaf senescence the
previously identified class II early leaf senescence mutant old13 (Chapter 2) was
characterized in detail. Here we present evidence for the involvement of OLD13 in
regulating stress induced senescence and lesion formation.
71
Results
Characterization of old13 mutant under standard conditions
Previously, we reported that the old13 mutant is phenotypically normal when grown
under optimal conditions but displays early senescence upon ethylene treatment
(Jing et al., 2005). To characterize the old13-mediated senescence response we
examined plants grown under standard conditions and after stress treatment in detail.
The development of the first leaf pair of old13 was followed and compared to wild
type from day 21 till day 33. old13 plants are identical in size when compared to wild
type, however, the initiation of the primary inflorescence starts 2-4 days earlier than
the wild type (data not shown). A primary marker for early senescence is leaf
yellowing caused by the loss of chlorophyll (Oh et al., 1997). From day 21 till day 33
no chlorophyll degradation and visual senescence is observed for the first leaf pair of
both air-grown wild type and mutant old13 (Figure 1A and B). Next to that the
photochemical efficiency of photosystem II stays at a similar level as wild type during
development (Figure 1C). Thus old13 is a typical class II mutant that has no early
senescence symptoms when grown under optimal conditions (Jing et al., 2002; Jing
et al., 2005).
Figure 1. Characteristics of air-grown old13
mutants. (A) The chlorophyll contents of soilgrown old13 (open circles) and wild type (closed
circles) were quantified every 3 days from day 21
till day 33. (B) Representative first leaves of old13
and wild type were selected and photographed at
day 24. (C) The photochemical efficiency was
determined as described in methods and is
expressed as Fv/Fm. Error bars indicate SD;
results are mean of 3 replicates.
72
A backcross of the old13 mutant revealed that it segregates as a recessive trait both
in the original mutant accession Landsberg (Ler) and the mapping background
Columbia (Col). 1800 F2 plants of the mapping population were phenotypically
selected for positional cloning. The position of the mutated locus was narrowed down
to a region on chromosome V, covering 101kb of sequence on BAC K19B1 and
MRG21. The mapped region contains 32 annotated candidate genes which are listed
in Table 1.
Table 1. Genes annotated for the mapped region that contains the old13 mutation. The 32
genes listed are shown with a putative function and description according to TAIR
annotation. Asterisks indicate that gene has been cloned and sequenced but no mutation
was detected.
TAIR- ID
Name
Description
At5g62410
SMC2
Structural maintenance of chromosome II
At5g62420
Aldo/keto reductase family protein
At5g62430
CDF1
Cycling Dof Factor 1, transition vegetative to reproductive
At5g62440
DOM1
Domino 1, nucleolus formation maintenance
At5g62460
zinc finger (C3HC4-type RING finger) family protein
At5g62470
MYB96
Encodes a R2R3 type Myb transcription factor, ABA dependent
At5g62480
ATDSTU9 Glutathione S-Transferase TAU 9
At5g62490 ATHAV22B Arabidopsis thaliana HVA22 Homologue B
At5g62500
ATEB1B Microtubule End Binding Protein
At5g62510
F-box family protein
At5g62520
SRO5
PARP motif ROS production and salt stress response. siRNA
At5g62530
P5CDH
Delta-pyrroline-5- carboxylate dehydrogenase, siRNA
At5g62540
UBC3
Ubiquitin-Conjugating Enzyme
At5g62550
similar to unknown protein
At5g62560
armadillo/beta-catenin repeat family protein
At5g62570
calmodulin-binding protein
At5g62575
similar to unknown protein
At5g62580*
similar to Os02g0739900
At5g62600
transportin-SR-related
At5g62610
basic helix-loop-helix (bHLH) family protein
At5g62620
Putative galactosyltransferase family protein
At5g62623*
Defensin-Like
At5g62627*
Defensin-Like
At5g62630
HIPL2
HIPL2 PROTEIN PRECURSOR
At5g62640*
ELF5
Early flowering locus 5
At5g62650*
similar to Os01g0225300
At5g62660
F-box family protein
At5g62670*
AHA11
H-ATPasa
At5g62680*
proton-dependent oligopeptide transport
At5g62690*
TUB2
Tubulin beta 2
At5g62700*
TUB3
Tubulin beta 3
At5g62710*
leucine-rich repeat family protein
PubMed
11276426
10467030
16002617
15341625
NF
16463103
12090627
12081371
14557818
NF
16377568
16377568
11019805
NF
NF
NF
NF
NF
NF
NF
17630273
15955924
15955924
12805588
15125772
NF
NF
15821287
NF
1498609
1498609
NF
73
Ethylene and detachment induce early leaf senescence in old13
In Arabidopsis leaves, diverse internal and environmental signals are integrated with
the age to modulate the onset of leaf senescence (Grbic and Bleecker, 1995; Jing et
al., 2003; Lim et al., 2003). Ethylene, detachment and drought are known to induce
premature senescence (Lim et al., 2007). old13 plants were grown under standard
growth conditions for 3 weeks and subsequently treated with ethylene or subjected to
a detachment experiment, to follow the progression of leaf senescence. Mutant
leaves show reduced chlorophyll levels when compared to wild type after 3 days of
ethylene treatment (Figure 2A). Noteworthy, ethylene treatment results in a more
pronounced early flowering phenotype in the mutant, as compared to growth under
standard conditions (results not shown). For the detachment experiment the first leaf
pair was followed for 12 days during incubation in the dark (Figure 2B).
Figure 2. The senescence syndrome of old13 leaves. (A) The chlorophyll contents of old13
(open circles) and wild type (closed circles) were quantified during ethylene treatment of 3
days. Content is shown in mean percentage ±SD of total content at day 21. (B) Detachment
induced senescence of the wild type and old mutants was photographed after 9 days of
treatment. WT, wild type; 5, old5; 9, old9; 11, old11; 13, old13; 14, old14. (C) Chlorophyll
content of old13 (open circles) and wild type (closed circles) during the detachment
experiment are represented as the mean± SD of three replicates of 6 leaves. (D) Nitrogen,
carbon and sulfur content at 21 and 24 days of growth in air and after 3 days of ethylene
treatment for wild type (black bars) and old13 leaves (gray bars). Content is represented as
mean percentage ±SD of total content at day 21.
74
Detachment of old13 leaves results in a slightly enhanced breakdown of chlorophyll
when compared to the wild type (Figure 2C). As a control we tested the effect of
detachment on leaves of other old mutants and demonstrate that the class II mutants
old9 and old11 appear wild type while the class I mutants old5 and old14 senesce
early upon detachment and more severe than old13 (Figure 2B). We observed that
old13 plants are more sensitive than wild-type plants to limited water availability,
resulting in early leaf senescence (data not shown). Taken together these results
suggest that early senescence in old13 plants can be induced by different stress
signals.
We examined the ethylene-induced leaf senescence in more detail by examining
nutrient remobilization and expression of senescence-associated genes (SAGs). A
characteristic feature of leaf senescence is the relocalization of nutrients to other
parts of the plant (Himelbau and Amasino, 2001). Remobilization starts with the
transport of stored compounds, once these reserves have been drained, proteins and
polymers will start to be catabolized (Thomas et al., 2002). Up to 95% of the stored
nitrogen in leaves is relocated during senescence which mainly arises from the
degradation of the chloroplast (Hortensteiner and Feller, 2002). By determining the
elemental composition of dried leaves we estimated the relocalization of nitrogen,
carbon and sulfur. Interestingly, both the wild type and the mutant show a strong
decline in nitrogen (Figure 2D). The significant decrease in the mutant when
compared to the wild type correlates with the start of chlorophyll degradation after 3
days of ethylene. The level of carbon remains at a similar level while sulfur declines
both during air-growth as during ethylene treatment. Thus ethylene treatment induces
remobilization of nitrogen containing compounds in wild type and old13, although
significantly more in the mutant. Analyses of the expression profiles of senescenceassociated genes after 1 day of ethylene treatment showed that old13 mutants
exhibit higher transcription levels of SAG12, SAG13, and SAG21 in comparison with
wild-type plants (Figure 3A). Next to that the expression of CAB was approximately 5fold reduced after ethylene treatment. This result, the increased expression of SAG12
and SAG13 (Weaver et al., 1998) demonstrates that old13 enhances the ethylenedependent senescence process not only on physiological level but also at the
molecular level.
75
Figure 3. SAGs, Hypersensitive response-like
lesions, and oxidative stress. (A) Marker
transcripts levels after ethylene treatment.
SAG12, SAG13, SAG21 and CAB transcript
levels after 1 day of ethylene treatment in the
wild type (black bars) and old13 (gray bars).
The relative expression shown is the mean of
three repeats ± SD (indicated by error bars).
(B) Tryphan blue stain of ethylene treated
leaves
of
old13
and
a
representative
photograph of a first leaf after 3 days of
ethylene (arrow indicates lesions). (C) Staining
for superoxide by NBT of 24 day old leaves.
(D)
Marker
transcript
levels
during
development. DEFL, PAD3 and PR2 relative
transcript abundance after 21 day of growth on
soil for the wild type (black bars) and old13
(gray bars).
Ethylene and drought induce hypersensitive response-like lesions in old13 which are
associated with oxidative stress
In addition to early leaf senescence, old13 plants also developed spontaneous
yellow-brown necrotic lesions under drought conditions or after ethylene treatment
(Figure 3A). The necrotic spots resemble phenotypically the hypersensitive-response
(HR) which is commonly observed during pathogen attack
(Mur et al., 2007).
Staining of ethylene treated mutant leaves with tryphan blue reveals the appearance
76
of randomly localized dead cells after two days of ethylene treatment (Figure 3B).
Interestingly, staining of air-grown old13 plants with NBT for superoxide reveals that
the mutant has an increased level of cellular ROS during unchallenged conditions
(Figure 3C). Moreover, old13 plants have a 6-fold increased expression of a
defensin-like (DEFL) gene which is expressed upon 7 different ROS-inducing agents
(Gadjev et al., 2006) (Figure 3D). Thus old13 plants have increased oxidative stress
when compared to the wild type and develop spontaneous lesions during ethylene
treatment and drought stress.
old13 functions both in biotic and a-biotic plant response pathways
During the hypersensitive response, an oxidative burst through rapid accumulation of
ROS is essential for the local cell death and the activation of a signaling cascade to
induce systemic acquired resistance (Alvarez et al., 1998). Next to that a-biotic
factors that cause salt, osmotic and drought stress all result in increased oxidative
stress (Knight and Knight, 2001). We found that a salicylic acid dependent defense
gene, PAD3 (Glazebrook and Ausebel, 1994) and the PR2 gene which is related to
pathogenesis but also leaf senescence (Gaffney et al., 1993) are 5 to 9 fold
increased in expression in the mutant when compared to the wild type (Figure 3D).
Figure 4. Anion content of wild type and old13 leaves during development. Chloride, nitrate
and sulfate content were measured every 6 days starting from day 21 until day 33. Wild type
is represented as black bars and mutant as gray bars. Content is represented as content
±SD of mean.
We noticed that the timing of watering before, at the start or during the ethylene
treatment could influence the senescence response of old13. Therefore we
anticipated that the mutant might have a defect in its water balance. To test this we
determined the anion concentration during the development of the first leaf pair.
Anions play a role both in the regulation of the membrane potential and in the
77
osmolarity of the cell (Tyerman, 1992). The determination of the anion content by
HPLC revealed that the mutant exhibited elevated levels of Cl- especially at young
age when the levels are 6-fold increased (Figure 4). Next to that the concentration of
nitrate is elevated at 21 days but decreases below wild type levels at 33 days. Sulfate
levels are at day 21 and 27 two-fold increased in comparison to the wild type but
restore to wild type levels at day 33. Taken together, the results suggest that old13
mutant plants grow under constitutive water stress.
HR-like lesions and senescence in old13 are dependent on intact hormone signaling
pathways
We showed before that the early induction of senescence in old13 is not a
consequence of increased sensitivity to ethylene (Chapter 2). To test the role of
hormone signaling pathways in adult plants we constructed double mutants between
Figure 5. Senescence phenotypes of old13 double mutants. (A) Plants were grown in air
until 21 days and subsequently treated with ethylene for 3 days after which representative
plants were selected and photographed. Double mutant abbreviations: o13a4, old13/abi4-1;
o13e2, old13/ein2-1; o13p4, old13/pad4-1. Bars indicate 5 mm. (B) Leaf yellowing after
78
ethylene treatment, yellowing is presented as the mean ± SD of 30 plants. (C) Number of
leaves with lesions after ethylene treatment as mean ± SD of 30 plants.
old13 and mutants impaired in the sensing and/or signaling of ethylene, salicylic acid
and abscisic acid. The double mutants were selected by PCR based identification of
the mutant alleles. Subsequently the double mutants were grown till 21 days in air
and treated for 3 days with ethylene (Figure 5A). The abi4-1 mutation (Finkelstein et
al., 1998) did not affect the old13 senescence symptoms or appearance of lesions
after ethylene treatment (Figure 5B and C). Crossing the old13 mutation with
ethylene insensitive mutant ein2-1 (Guzman and Ecker, 1990) results in absence of
leaf senescence and lesions during ethylene treatment. Interestingly, a cross
between old13 and pad4-1 (Jirage et al., 1999) results in the loss of the lesion
phenotype, while the effect on leaf yellowing was limited.
These data suggest that the old13-mediated ethylene-induced senescence
phenotype is independent of SA signaling, while the lesion phenotype is dependent
on the salicylic acid pathway.
Discussion
Senescence is a developmental event that results in the death of a cell, an organ, or
an organism upon aging. In plants senescence has evolved as a beneficial trait which
increases the survival of the species (Bleecker, 1998).
While the onset of leaf senescence in old13 mutant is similar to wild type under
optimal growth conditions, we observed that old13 mutants senesce early and display
spontaneous lesions formation after ethylene treatment or drought stress. This
suggests that old13 mutants are more sensitive to environmental conditions than wild
type and that OLD13 might be involved in stress-induced growth, senescence and
cell death.
One effect of the old13 mutation is the increased anion content, which might suggest
a defect in water balance. Most strongly increased is the level of chloride, which is 6fold higher at day 21. An increase in chloride content has been observed before
during N limitation experiments in which chloride replaces nitrate as an osmoticum
(White and Broadley, 2001). However, we measured an increased availability of
nitrate in leaves of old13 which argues against the possibility that nitrogen deficiency
plays a role in the mutant. Drought and salt stress have been shown before to induce
senescence. In rice it was shown that NaCl increases the rate of developmental
79
senescence (Lutts et al., 1996) while in Arabidopsis several SAG genes including
ERD1, SAG13, SAG14, SAG21 respond to drought-induced senescence (Weaver et
al., 1998). High saline environments cause cytosolic accumulation of calcium which is
a potent signal for stress responses that can result in either adaptation or death
(Hasegawa et al., 2000). Recently a calcium-dependent protein kinase has been
characterized which plays a role in salt and drought tolerance (Ma and Wu, 2007).
One response to accumulation of Cl- is restoration of the osmotic balance by the
accumulation of a non-harmful compatible solute like sucrose, fructose, glycerol,
trehalose or proline (Hasegawa et al., 2000). Levels of ROS increase rapidly during
salt stress and can result into an oxidative burst (Apel and Hirt, 2004). Tryphan blue
staining of old13 together with the increased expression of a defensin-like gene
indicates increased ROS levels in the mutant. Next to that it was shown for
Arabidopsis that increased H202 levels promote reproductive growth (Zimmermann et
al., 2006), which might explain the early flowering of old13. A possible explanation for
the increased ROS is that during water deficit photorespiration is inhibited due to a
reduced availability of CO2 which results in excess excitation energy and subsequent
increased oxidative stress (Smirnoff, 1993). Interestingly, the assimilation of nitrate
depends on the rate of photorespiration (Rachmilevitch et al., 2004). This observation
which might explain the measured increase in nitrate, although it was only observed
for young leaves. Increased ROS production causes lipid peroxidation and can affect
the integrity of the membrane resulting in permeability to electrolytes and finally
changes in the cellular ionic homeostasis (Kourie, 1998). ROS scavenging is an
important factor in adapting to salt stress as shown by ascorbate-deficient
Arabidopsis plants which have an increased sensitivity to salt-stress (Huang et al.,
2005). The photoautotrophic salt tolerate 1 (pst1) mutant has increased superoxide
dismutase (SOD) and ascorbate peroxidase (APX) activity resulting in increased salt
and drought tolerance (Tsugane et al., 1999). Thus, the rapid detoxification of ROS
during salt or drought stress is an important factor in determining tolerance.
Detoxification of ROS can also be achieved by increasing the level of non-enzymatic
antioxidant compounds. Synthesis of the sulfur-containing anti-oxidant glutathione
(GSH) is upregulated during oxidative stress (Noctor and Foyer, 1998). Ozone is a
potent inducer of oxidative stress which results in accumulation of glutathione and
up-regulation of sulfur assimilation (Bick et al. 2001). Therefore the increased sulfate
content in old13 might be the direct consequence of oxidative stress. However, the
80
observed changes in anion content can be explained as a general change in iontransport (Frachisse et al., 1999) and thus may not be related to salt stress. Excess
nitrate and chloride are stored by the same mechanism in the vacuole and therefore
the OLD13 gene product might function directly in ion transport (Harada et al., 2004).
Although the mechanism might relate to storage or transport of anions, in old13 the
increased anion content might cause the drought sensitive phenotype. Next to
increased cellular ROS and anion concentration the old13 mutant also displays
lesion formation. The physiology of old13 resembles that of the edr1 mutation which
results in early ethylene-induced senescence, spontaneous lesion formation during
drought stress and enhanced resistance to pathogens. Although, pathogen response
of old13 has not been tested we did find that the pathogenesis-related transcripts of
PR2 and PAD3 accumulate in the mutant. EDR1 encodes for a MAPKKK suggesting
that different stress factors might initiate the same signaling cascade (Tang and
Innes, 2005). By double mutant analysis it was shown that the drought-induced
phenotype of edr1 is depending on SA while the senescence phenotype requires
ethylene signaling. Our double mutant analysis shows that lesion formation in old13
is also dependent on SA-signaling pathway while ein2 blocks the senescence
phenotype. In contrast to the role of PAD4 during developmental senescence in
which it modulates the switch to PCD during the final phase (Morris et al., 2000) it is
necessary for the onset of senescence during pathogen attack (Pegadaraju et al.,
2005). This suggests that only a part of the old13 senescence response is related to
defense since the lesion and senescence phenotype are regulated by different
pathways. Interestingly crossing the late senescence mutant ore9 with edr1 results in
suppression of ethylene and drought induced senescence but not plant defense
responses (Tang et al., 2005), confirming that the different phenotypes can be
separated in the edr1 mutant as well and are the result of different signaling
pathways. Previously, the early leaf senescence mutant old1 has been implicated as
an integration node of different stimuli including pathogen response, ethyleneinduced senescence and sugar signaling (Jing et al., 2007). Taken together, our
analysis suggests that OLD13 might represent another OLD protein that regulates
plant development and senescence by monitoring the environment and endogenous
status of plants and adjusts development accordingly.
The characterization of the old13 mutation reveals a novel locus involved in
modulating the developmental program of Arabidopsis during stress conditions. One
81
important concept is that during stress a plant has to decide if a certain organ can be
sacrificed or not. Although most of the current research focuses on delayed
senescence to improve productivity in several crops it is important to note that a rapid
and localized senescence response is essential for stress tolerance and survival.
Therefore, we would like to encourage future research that aims at senescence
modulators that control plant fitness.
Methods
Plant materials and growth conditions
Arabidopsis thaliana ecotype Landsberg erecta (Ler-0) was used in this study. The
old9 mutant was obtained from an EMS mutagenized collection (Jing et al., 2005).
Plants were grown on either soil or half-strength Murashige and Skoog medium at
23°C and 65% relative humidity with a day length of 16 h. The light intensity was set
at 120 µmol·m-2·s-1. An organic-rich γ-ray radiated soil was used (Hortimea Groep,
Elst, The Netherlands). Plants for ethylene exposure were treated in a flow-through
chamber at 20 °C and a humidity of 40% under continuous illumination. The ethylene
dosage was set at 10 µl l–1 as suggested by Chen and Bleecker (1995). Cotyledons
or rosette leaves with over 5% yellow area of the leaf blade were judged as yellow as
suggested by Lohman et al. (1994).
The mutant alleles and transgenic plants used for the double mutants were ein2-1
(Guzman and Ecker, 1990), pad4-1 (Jirage et al., 1999), and abi4-1 (Finkelstein et
al., 1998). The old13 mutation was crossed into the mutant lines that have a Col-0
background, thus the F2 double mutant population contains a mixed Ler-0 and Col-0
background.
NBT staining
The first leaf pair was harvested and placed in an aqueous solution of 0.05%
nitroblue tetrazolium; blue color indicated superoxide (O2−) generation (Flohe and
Otting, 1984). After 2 hours leaves were fixed and decolorized in 96% ethanol
overnight. Decolorized and cleared leaves were mounted in saturated chloral hydrate
and analyzed under a light microscope. Subsequently, photographs were taken of
representative leaves.
82
Tryphan blue staining
Appearance of cell death was studied in whole leaf mounts stained with lactophenoltryphan blue (10 mL of lacticacid, 10 mL of glycerol, 10 g of phenol, 10 mg of tryphan
blue, dissolved in 10 mL of distilled water) (based on Keogh et al.,1980). Whole
leaves were boiled for 1 min in the staining solution and then overnight decolorized in
chloral hydrate. Subsequently the leaves were mounted in chloral hydrate and
viewed under a light microscope.
Pigment determination and measurement of photochemical efficiency
For extraction of chlorophyll and carotenoids samples were incubated overnight with
N,N-dimethylformamide at 4°C in darkness. The chlorophyll content was quantified
spectrophotometrically according to Wellburn (1994) at 647 and 664nm.
Chlorophyll fluorescence emission was measured from the upper surface of the first
leaf, at room temperature (23 °C) with a pulse-amplitude modulation portable
fluorometer (PAM-2000; H. Walz, Effeltrich, Germany) according to Maxwell and
Johnson, 2000. Plants were dark-adapted for 1 to 2 hr before measurements to
ensure complete relaxation of the thylakoid pH gradient. An attached, fully expanded
rosette leaf was placed in the leaf clip, allowing air to circulate freely on both sides of
the leaf. At the start of each experiment, the leaf was exposed to 2 min of far-red
illumination (2 to 4 µmol photons m-2 s-1) for determination of Fo (minimum
fluorescence in the dark-adapted state). Saturating pulses of white light (8000 µmol
photons m-2 s-1) were applied to determine Fm or Fm' values. PSII efficiency was
calculated as (Fm - Fo)/Fm.
RNA-isolation and RT-PCR
Total RNA was isolated using TRIZOL reagent (Sigma) according to the
manufacturer's protocol. Five hundred nanograms of RNA were used as template for
first-strand cDNA synthesis using 200U of RevertAid H-minus MMuLV reverse
transcriptase (Fermentas, USA) and an oligo(dT21) primer. Primer pairs for real-time
PCR were designed with open-source PCR primer design program PerlPrimer
v1.1.10 (Marshall, 2004). The primer sequences are available upon request. Briefly,
real-time PCR amplification was performed with 50 µL of reaction solution, containing
2 µL of 10-fold–diluted cDNA, 0.5 µl of a 10 mM stock of each primer, 1 µl of 25mM
stock MgCl2 (Fermentas), 5 µl PCR buffer +Mg (Roche), 1 µl of a 1000x diluted
83
SYBR-green stock (Sigma), 0.5 µl 100xBSA (New England Biolabs), and 1u of
Roche Taq Polymerase. The PCR program was 2’ at 94, 40x (94-10”/60-10”/72-25”).
Obtained data was analyzed with BioRad software.
CNS measurements
The first leaf pair of wild type and mutant was collected after treatment and oven
dried at 80°C. Subsequently the tissue was weighted and grinded to a fine powder.
For determination of the C, N, and S content we made use of a LECO CHNS 932
(USA) analyzer by combustion at 1200°C.
Detachment-induced senescence
Leaves were incubated in light on two layers of Whatman filter papers saturated with
MES solution (pH 5.7) and collected after 0, 3, 6, 9 and 12 days for chlorophyll
content measurement. Three replicates of 6 pairs of leaves were analyzed for each
data point.
Intracellular anion content
For chloride, nitrate and sulfate analysis, fresh first leaf pairs were harvested and
homogenized in demineralized water, with an Ultra Turrax (T25 Basic Ika
Labortechnik, Staufen, Germany). The homogenate was incubated at 100 °C for 10
min, filtered and centrifuged at 30 000 g for 15 min. Chloride, nitrate and sulfate were
separated by HPLC on an IonoSpher A anion exchange column (Varian/Chrompack
Benelux, Bergen op Zoom, The Netherlands) and determined refractrometrically
according to Buchner et al. (2004).
Acknowledgements
We would like to thank Bert Venema, Margriet Ferwerda and Kitty Philipsen for their
excellent technical support.
84
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White, J.P., and Broadley, M.R. (2001). Chloride in Soils and its Uptake and
Movement within the Plant: A Review. Ann. Bot. 88: 967-988
Wingler, A., Purdy, S., MacLean, J.A., and Pourtau, N. (2006). The role of sugars in integrating
environmental signals during the regulation of leaf senescence. J. Exp. Bot. 57: 391-399.
Zhu, J.K. (2002). Salt and drought stress signal transduction in plants. Annu. Rev. Plant Biol. 53: 247273.
Zimmermann, P., Heinlein, C., Orendi, G., and Zentgraf, U. (2006). Senescence-specific regulation
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88
Chapter
4
A role for cytokinin in the onset of leaf senescence by ethylene in
Arabidopsis
Jos H.M. Schippers1, Emily Breeze2, Vicky Buchanan-Wollaston2, Jacques
Hille1 and Paul P. Dijkwel1
1
Molecular Biology of Plants, Groningen Biomolecular Sciences and Biotechnology Institute, University
of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands
2
Warwick HRI, University of Warwick, Wellesbourne CV35 9EF, UK.
89
90
Abstract
The expression of several Arabidopsis senescence-associated genes (SAGs) was
found to be upregulated during leaf development of the onset of leaf death 9 mutant
under standard growth conditions. Treatment of the mutant with ethylene resulted in
a severe senescence response both phenotypically as well as on transcript level. A
RNAi-based strategy to downregulate the expression of a SAG encoding for a
hydroxyproline-rich glycoprotein, attenuated the ethylene induced senescence
response of old9. Comparison of the old9 transcriptome revealed that 67% of the
differentially expressed genes with wild type are cytokinin responsive. Cytokinins are
well known for their effect to retard leaf senescence. However, by applying cytokinin
early in development the response to ethylene-induced senescence could be
enhanced. We argue that the cytokinin effect we observed is the result of increasing
the sink strength of young tissue while opposite to that ethylene promotes source
strength of old leaves. Therefore we propose that the balance between sink and
source tissue can modulate the progression rate and timing of senescence.
Introduction
The development of a leaf starts with the formation of a leaf primordium from the
shoot apical meristem (Van Lijsenbettens and Clarke, 1998). A subsequent
differentiation and expansion of the different cells gives rise to a mature leaf
consisting of an epidermis, a mesophyll layer and vascular tissue (Pyke et al., 1991).
The determinate program of leaf development ends with the age dependent onset of
senescence resulting in the death of the tissue. However, this final stage of leaf
development is critical for plant fitness as nutrient relocation to young developing
parts is achieved through this process. Leaf senescence is thus an evolutionary
selected developmental process and represents an important phase in the plant life
91
cycle (Bleecker, 1998; Buchanon-Wollaston et al., 2003). The study of leaf
senescence will not only increase our understanding of a fundamental biological
process, but also contributes to means to control leaf senescence to improve traits of
crops and ornamental plants including postharvest storage.
The development of a leaf is as a mitotic event which occurs prior to unfolding and
cell expansion is initiated. The mitotically active cells that form the leaf primordium
arise from the shoot apical meristem (SAM), which contains a pluripotent stem cell
population (Bowman and Eshed, 2000). The initiation of a leaf from this population of
stem cells involves the action of several phytohormones. First the maintenance of the
stem cells requires biosynthesis of cytokinin (Bowman and Eshed, 2000). However,
the initiation of a leaf primordium requires gibberellin (GA), which accumulates due to
the repression of cytokinin biosynthesis (Yanai et al., 2005; Jasinski et al., 2005).
Plants with reduced cytokinin production have less leaves and contain fewer leaf
cells (Werner et al., 2001) which was interestingly also shown in the triple mutant for
a cytokinin receptor arabidopsis histidine kinase2 (ahk2), ahk3, ahk4 (Nishimura et
al., 2004). On the other hand transgenic Arabidopsis plants which overproduce
cytokinin have more leaf cells (Rupp et al., 1999). Thus cytokinin has a regulatory
function in leaf cell formation. The cell division in the leaf primordium of Arabidopsis
sets an age-gradient from “old” cells at the leaf tip and margins to younger cells at the
base of the leaf (Ferreira et al., 1994).
The post mitotic phase of leaf development involves cell expansion until the final size
is reached at maturity. The expansion is controlled in part by the ANGUSTIFOLIA
gene in Arabidopsis which acts through arrangement of cortical microtubules and the
expression of cell-wall loosening genes (Kim et al., 2002). During the expansion of
mesophyll cells, partially differentiated chloroplasts start to divide actively (Pyke and
92
Leech, 1994) to attain photosynthetic competence of a leaf. Arabidopsis grown in
medium containing cytokinin shows an accelerated photomorphogenesis phenotype
coinciding with induced expression of photosynthetic genes (Schmülling et al., 1997;
Brenner et al., 2005). Several effects of cytokinin can also be induced by light,
including leaf expansion, chlorophyll accumulation and inhibition of hypocotyl
elongation. Although both cytokinin and light can induce similar processes the action
of light involves a phytochrome-dependent pathway while cytokinin in part requires
the ethylene response pathway (Cary et al., 1995; Su and Howell, 1995).
Interestingly, cytokinin induces the expression of nitrate reductase in plants
(Samuelson et al., 1995; Taniguchi et al., 1998) and was suggested to have a
regulatory role in nitrate assimilation (Tischner, 2000). Cytokinin affects source-sink
relationships and causes nutrient accumulation in leaves treated with the
phytohormone (Roitsch and Ehness, 2000). The transport of carbon and nitrogen
compounds from source leaves to sink leaves requires glucose, sucrose, and amino
acid transporters which have been identified in the Arabidopsis genome as large
gene families (Lalonde et al., 2004). One of these sucrose transporters AtSUC2 was
shown to be developmentally regulated and is induced during the sink-to-source
transition of leaves (Truernit and Sauer, 1995). The application of cytokinin to
seedlings results in a downregulation of AtSUC1 (Brenner et al, 2005) while
overexpression of ARR22 (Kiba et al., 2004) results in down regulation of both
AtSUC1 and AtSUC2 suggesting that cytokinin can modulate the transition of sourceto-sink. The final stage of leaf development results in a total commitment to source
function and results in massive nutrient remobilisation by the onset of leaf
senescence (Balibrea Lara et al., 2004). In Arabidopsis the onset of leaf senescence
occurs in a leaf-age dependent manner (Gan and Amasino, 1997; Quirino et al.,
93
2000). Ageing of the leaves starts with the initiation of a leaf primordium and occurs
throughout the development resulting in senescence and death (Lim et al., 2003).
The developmental changes during the lifespan of a leaf can be viewed as agerelated changes (ARCs) that set the developmental age and thus determine if a leaf
can undergo senescence (Jing et al., 2005; Schippers et al., 2007). The onset of
senescence can be delayed by the application of cytokinin on leaf senescence
(Richmond and Lang, 1957) which is thus acting as an anti-ageing factor. In addition,
expression of cytokinin biosynthesis genes under the promoter of a senescenceassociated gene (SAG) delays the initiation of leaf senescence in several plant
species (Gan and Amasino, 1995; McCabe et al., 2001; Khodakovskaya et al., 2005).
The SAM gene knotted1 (kn1) regulates cytokinin content and delays leaf
senescence when expressed under the control of a SAG promoter in tobacco (Ori et
al., 1999). The mechanism of cytokinin-mediated delay of senescence is dependent
on an extracellular invertase. Activation of the invertase increases the sink strength of
the tissue and the rate of sugar utilization which results in delayed onset of
senescence (Balibrea Lara et al., 2004). These studies suggest that sugar
metabolism mediated by cytokinin is an important factor in determining the onset of
leaf senescence and that the transition of sink-to-source is an important ARC during
the development of a leaf.
In our attempt to elucidate factors that control the onset of leaf senescence we make
use of the senescence inducing phytohormone ethylene. In the present study, we
selected the Arabidopsis onset of leaf death 9 (old9) mutant. The mutant was shown
in chapter 2 to senescence early upon ethylene treatment. Since the effect of
ethylene is age-dependent the mutant presents a good starting point for determining
regulation of age-related changes (ARCs). Here we present evidence for a role of
94
cytokinin on the timing of ethylene-induced senescence in Arabidopsis. The mutant
old9 shows altered expression of cytokinin responsive genes. Furthermore we
demonstrate that exogenous application of cytokinin can prime a leaf for ethyleneinduced senescence and argue it to be caused by changes in the sink/source
strength of the leaves.
Results
old9 presents a co-dominant locus involved in ethylene-inducible senescence
The old9 mutant of Arabidopsis was isolated initially as an early leaf senescence
mutant upon ethylene treatment (Jing et al., 2005). old9 is classified as a class II
mutant (Jing et al., 2002) and was found to develop as wild type under standard
growth conditions. Since the induction of leaf senescence by ethylene is dependent
on the age of the leaf (Grbić and Bleecker, 1995, Jing et al., 2002) we anticipate that
old9 might be involved in the integration of ARCs and environmental stimuli to
regulate the onset of senescence.
Figure 1. Senescence response of old9 and wild type leaves to ethylene treatment. Wild
type and old9 plants were grown for 21 days in air and were subsequently treated for 2 or 3
days with ethylene. Representative leaves from the first true leaf pair were photographed (A)
or stained with tryphan blue (B) as described in the Methods section. (C). Transcript
accumulation of the senescence-associated genes SAG12 and SAG13 after ethylene
treatment in old9 (gray bars) and wild type (black bars). The values shown are the means of
three repeats ± SD (indicated by error bars).
95
For the characterization of the mutant we first examined the senescence symptoms
during ethylene-induced senescence. The first leaf pair of old9 has reduced leaf
longevity upon ethylene treatment at 21 days as observed visually (Figure 1A). After
2 days the leaves become pale green and after 3 days the entire leaf turned yellow
which indicated the complete execution of senescence (Grbić and Bleecker, 1995). In
contrast, leaves of the wild type show visual senescence symptoms after 4 days of
ethylene treatment (data not shown). After 3 days of treatment cell death is observed
in the mutant but not in the wild type (Figure 1B). The appearance of cell death
occurs in an ordered way starting from the leaf tip. The onset of leaf senescence is
marked by increased expression of senescence associated genes (SAGs) (Nam,
1997). Expression of the SAG12 and SAG13 genes increased dramatically after 3
days of ethylene treatment in old9 mutants (Figure 1C). Taken together, the complete
yellowing of the leaf, appearance of cell death and SAG gene expression upon
ethylene treatment are consistent with the suggested occurrence of early ARCs in the
mutant (Jing et al., 2005).
A backcross of the old9 mutant revealed that it segregates as a co-dominant trait
both in the original mutant accession Landsberg (Ler) and the mapping background
Columbia (Col). The position of the old9 locus was narrowed down to a region on
chromosome II of 146kb on BAC F6E13 and F4I1. The identified region contains 54
annotated genes which are summarized in Table 1. In an attempt to identify the
mutation seven genes have been sequenced but were shown to be identical to the
wild type gene.
Table 1. List of genes in mapped old9 area. Shown is the data from the TAIR annotation and
a Pubmed record when available. Asterisks indicate genes that were sequenced and found
to be identical to the wild type.
TAIR- ID
at2g43960
at2g43970
at2g43980
at2g43990
at2g44000
at2g44010
at2g44020
at2g44030
at2g44040
at2g44050
at2g44060
96
Name
ITPK4
COS1
Description
PubMed
similar to SWAP
La domain-containing protein
inositol 1,3,4,5-tetrakisphosphate isomerase
Unknown protein
Unknown protein
Unknown protein
mitochondrial transcription termination factor-related
Kelch-repeat containing F box
dihydrodipicolinate reductase; lysine biosynthesis
6,7-dimethyl-8-ribityllumazine (DMRL) synthase
late embryogenesis abundant family protein
NF
NF
17698066
NF
NF
NF
NF
NF
15652176
10419541
NF
at2g44065
at2g44070
at2g44080*
at2g44090
at2g44100*
at2g44110*
at2g44120
at2g44130*
at2g44140*
at2g44150*
at2g44160
at2g44170
at2g44175
at2g44180
at2g44190
at2g44195
at2g44200
at2g44210
at2g44220
at2g44230
at2g44240
at2g44250
at2g44260
at2g44270
at2g44280
at2g44290
at2g44300
at2g44310
at2g44320*
at2g44330
at2g44340
at2g44350
at2g44360
at2g44370
at2g44380
at2g44390
at2g44400
at2g44410
at2g44420
at2g44430
at2g44440
at2g44450
at2g44460
at2g44470
at2g44480
ARL
ATGDI1
MLO15
RPL7C
ATG4a
ASHH3
MTHFR2
NMT2
MAP2a
ATCS
Ribosomal protein L2
eukaryotic translation initiation factor 2B family protein
AGROS-like gene involved in cell expansion; BR induced
Unknown protein
Guanoside diphosphate dissociation inhibitor
7 transmembrane (7TM) G-protein-coupled receptor
60S ribosomal protein L7
Kelch-repeat containing F box
Autophagy 4a
Histone-Lysine N methyl transferase
methylenetetrahydrofolate reductase
N-myristoyltransferase
Unknown protein
Methionine Aminopeptidase
Unknown protein
Unknown protein
Unknown protein
Unknown protein
Unknown protein
Unknown protein
Unknown protein
Unknown protein
Unknown protein
Unknown protein
similar to lactose permease-related
protease inhibitor/seed storage/lipid transfer protein
lipid transfer protein-related
calcium-binding EF hand family protein
TRNA serine
zinc finger (C3HC4-type RING finger)
VQ motif-containing protein
Citrate synthase 4
Unknown protein
DC1 domain-containing protein
DC1 domain-containing protein
DC1 domain-containing protein
DC1 domain-containing protein
protein binding / zinc ion binding
protein N-terminal asparagine amidohydrolase family
protein
DNA-binding bromodomain-containing protein
emsy N terminus domain-containing protein
glycosyl hydrolase family 1 protein
putative thioglucosidase gene
glycosyl hydrolase family 1 protein
glycosyl hydrolase family 1 protein
NF
NF
16824178
NF
8953772
16525893
15821981
NF
15178341
17295027
NF
12912986
NF
11060042
NF
NF
NF
NF
NF
NF
NF
NF
NF
NF
NF
NF
12805588
NF
NF
15644464
NF
8979399
NF
NF
NF
NF
NF
NF
NF
NF
NF
16287169
17063378
NF
NF
Transcriptional analysis of air-grown old9
As mentioned before the induction of senescence by ethylene is age-dependent
(Hensel et al., 1993; Jing et al., 2002) in Arabidopsis. Therefore old9 might represent
a locus that monitors the age of a leaf to set the developmental program accordingly.
Since the mutant shows normal sensitivity to ACC (see chapter 2), we suggest that
97
an early age-related change (ARC) occurs in the mutant priming the leaves for
senescence. Micro-array analysis was used to identify differentially expressed
transcripts which could function as a marker for possible ARCs in the mutant.
Transcript levels were assessed in the first leaf pair of old9 and wild type after 21
days of growth under standard conditions thus when no visual difference between the
two genotypes is observed. 89 genes were found to be differentially regulated in the
combined biological and technical replicates of air-grown plants (Table 2).
Table 2. Genes differentially expressed in old9 after 21 days of growth under standard
conditions. The 89 genes listed show statistical significance versus the wild type control (P <
0.05, Student’s t test). Shown are TAIR ID, gene name, description, Pubmed ID and ratio. BA
indicates ratio found during micro-array study on genes responsive to cytokinin (Brenner et
al., 2005). (S). Senescence associated (-, no; +, yes)
TAIR- ID
At1g01090
At1g04820
At1g08200
At1g11840
At1g12840
At1g16850
At1g20510
At1g21312
At1g21520
At1g23040
At1g28380
At1g32470
At1g35710
At1g37130
At1g67105
At1g67740
At1g67865
At1g68190
At1g69935
At1g71030
At1g76990
At1g78710
At1g78995
At1g55490
At2g06850
At2g18700
At2g19620
At2g20120
At2g21130
At2g24850
At2g25490
At2g25950
At2g26560
At2g29630
At2g30230
98
Name
PDH-E1
TUA4
AXS2
AtGLX1
DET3
OPCL1
NSL1
NR2
PSBY
Description
PubMed
Ratio
BA
S
pyruvate dehydrogenase alpha;
Tubulin alphaL-4 chain; cytoskeleton
UDP-D-apiose/UPD-D-xylose synthetase
lactoylglutathione lyase activity
V-ATPase; cell expansion and photomorphogenesis
Role in NaCl and cold stress; anthocyanin synthesis
Jasmonic acid biosynthesis; wound-induced
RNA recognition motif (RRM)-containing protein
Unknown protein
hydroxyproline-rich glycoprotein
Necrotic spotted lesions; plant defense
glycine cleavage system
leucine-rich repeat transmembrane protein kinase
Nitrate reductase
Senescence-associated Gene
PsbY precursor Part of Photosystem II
Unknown protein
zinc finger (B-box type) family protein
Unknown protein
9393637
1498608
12969423
1.61
0.57
0.29
0.30
0.41
0.47
1.66
3.58
0.46
3.01
1.42
0.61
2.61
3.52
19.56
1.72
1.65
1.57
0.61
1.36
1.53
0.31
0.65
0.59
0.60
1.62
0.45
1.54
0.54
6.94
1.93
0.32
1.93
1.66
0.31
1.51
0.71
ND
0.50
1.15
0.22
0.38
1.21
1.50
0.5
0.61
1.23
1.03
1.94
ND
1.48
1.19
1.74
1.13
5.27
1.77
0.56
0.09
0.59
1.08
8.17
0.68
1.58
0.97
1.73
1.73
0.69
0.48
1.27
ND
+
+
+
+
+
+
+
+
+
+
+
MYB transcription factor repressed by BZR1
ACR3
amino acid-binding protein
Stress related; unknown protein
CPN60B
EXGT-1A
TPS11
COV1
CYP1
TAT3
EBF1
PLP2
Unknown protein
beta subunit of the chloroplast chaperonin 60
Endo-xyloglucan transferase; br responsive
Trehalose biosynthesis
Ndr family protein; cell differentiation
Vascular patterning
Cyclophyllin
Tyr amino transferase
F-box binds EIN3; OE ethylene insensitive
Unknown protein
Phospholipase induced by pathogen attack
thiamine biosynthesis family protein
Unknown protein
NF
11604454
17888165
16963437
NF
NF
12805588
16900325
14671022
NF
3393528
NF
9829828
NF
NF
NF
15681342
12481063
NF
NF
12668771
11673616
11520870
17565583
12668628
17473866
9342878
14675533
NF
16297072
NF
NF
At2g31890
At2g32670
At2g40000
At2g40080
At2g40360
At2g43100
At2g46450
At2g47960
At2g29080
At3g01490
At3g06710
At3g10020
At3g10260
At3g11410
At3g16520
At3g18490
At3g20790
At3g22320
At3g23700
At3g23820
At3g48360
At3g55240
At3g57260
At4g12830
At4g14890
At4g17390
At4g18200
At4g19170
At4g19860
At4g28220
At4g32020
At4g34740
At4g35090
At4g36020
At4g36040
At5g02500
At5g03850
At5g08050
At5g08520
At5g10760
At5g15650
At5g16150
At5g17460
At5g18630
At5g19140
At5g22580
At5g24530
At5g35180
At5g37475
At5g40450
At5g43060
At5g47560
At5g55960
At5g57760
Unknown protein; endomembrane system
VAMP275 Vesicle associated membrane protein
Putative nematode resistance gene
ELF4
Photoperiodism; red light response; PhyB signaling
putative WD-40 repeat protein; BOP1 domain
aconitase C-terminal domain-containing protein
CNGC12 Cyclic nucleotide gated channel; fungus response
Transglutaminase, putative
FTSH3
Ftsh protease; repair PSII
Putative MAPKKK, MRK1-like; sucrose phospho
similar to protein binding / zinc ion binding
similar to Os12g0147200
reticulon family protein
AtPP2CA Protein phosphatase 2C, represses ABA
UDP-glucoronosyl/UDP-glucosyl transferase protein
aspartyl protease family protein
oxidoreductase family protein
RPABC24 RNA polymerase I, II and III 24.3 kDa subunit
S1 RNA-binding domain-containing protein
GAE6
UDP-D-glucuronate 4-epimerase; pectin synthesis
BT2
Involved in TAC1-mediated telomerase activation
Unknown protein; OE Pseudo Etiolation in Light
BGL2/PR2 beta 1,3-glucanase
hydrolase, alpha/beta fold family protein
ferredoxin family protein; light protection
60S ribosomal protein L15
AtPUP7
Putative cytokinin and purine transporter
NCED4
Nine-cis-epoxycarotenoid dioxygenase 4
lecithin:cholesterol acyltransferase
NDB1
NAD(P)H DEHYDROGENASE B1
Unknown XRN4 target
ATASE2 de novo purine biosynthesis
CAT2
peroxisomal catalase; photosynthetic active tissue
CSDP1
Encodes a cold shock domain protein
DNAJ heat shock like protein
HSC70
Heat shock cognate 70 kDa protein
40S ribosomal protein S28
Thylakoid protein
MYB family transcription factor
aspartyl protease family protein
RGP2
alpha-1,4-glucan-protein synthase
GLT1
plastidic glucose exporter, trehalose responsive
Unknown mitochondrial protein
triacylglycerol lipase
auxin-responsive genes
Putative monooxygenase
flavanone 3-hydroxylase like protein
EDR2-like (enhanced disease resistance 2)
translation initiation factor-related
Unknown protein
RD21-like cysteine proteinase, sugar responsive
ATTDT
malate transmembrane transporter
Unknown protein
DNA binding protein, MPK4 dependent
NF
14500793
15448178
14605220
NF
NF
16461580
NF
14630971
NF
NF
NF
NF
16361522
NF
NF
NF
NF
NF
15225656
17220202
17227551
1824335
NF
NF
NF
10662864
15862093
NF
17673460
NF
15266056
17080932
17169986
NF
12805626
NF
NF
NF
NF
9536051
7031512
NF
NF
NF
15213437
NF
NF
NF
NF
NF
12947042
NF
16813576
1.86
3.24
1.57
0.59
0.19
2.18
3.61
1.61
0.65
0.64
2.08
1.51
0.08
1.66
1.99
1.85
0.14
0.62
1.35
1.59
0.23
1.53
5.48
0.13
0.65
0.66
1.61
2.02
1.44
1.87
1.70
0.60
1.46
0.63
1.92
4.61
0.62
0.63
1.37
2.07
0.49
1.51
0.53
1.60
1.84
0.43
2.43
1.71
0.30
4.60
1.36
2.12
0.66
0.24
0.82
1.67
4.31
0.13
0.61
0.08
0.78
1.74
0.89
ND
1.24
4.18
0.65
0.58
1.21
0.78
0.54
1.25
1.05
0.82
9.87
ND
0.31
0.60
1.46
1.20
nd
1.88
1.33
1.23
0.45
0.88
1.47
1.44
4.73
1.82
1.37
1.30
1.03
0.91
ND
1.51
0.60
1.32
1.20
1.13
1.27
0.63
ND
0.62
1.20
3.46
0.81
3.68
99
+
+
+
+
+
+
+
+
+
+
+
+
+
-
To confirm the microarray data we performed real-time PCR analysis of several
identified genes as shown in Figure 2. NIA2, TAT3 and EBF1 were confirmed to be
up regulated in air-grown old9 compared to the wild type. Moreover the cell wall Dapiose synthetase (AXS2) is downregulated according to the RT-PCR analysis
confirming the microarray data. Thus the RT-PCR analysis validated the genes
identified by the microarray experiment.
Figure 2. Real-Time PCR validation of transcripts
identified by microarray analysis of old9. Transcript
levels are shown as ratio between wild type and
mutant. The values shown are the means of three
repeats ± SD (indicated by error bars). Experiments
were repeated once and similar results were
obtained. Asterisks indicate statistical significance
versus the wild type control in each case (* P < 0.05,
Student’s t test).
Microarray analysis results in large datasets of differentially expressed genes,
however, these datasets are insignificant without a biological-orientated analysis.
Therefore the identified genes that are differentially expressed in the mutant were
studied by their annotation and classification in the TAIR database and by literature
search. The developmental program of young leaves is regulated and controlled by
many genes, interestingly several early leaf developmental genes are differentially
expressed in old9 plants as compared to the wild type. DET3 (At1g12840), encodes
for a subunit of a vacuolar ATPase involved in cell elongation during leaf
development (Schumacher et al., 1999). The expression of DET3 decreases during
the development of the leaf, and the mutant has a more than 2-fold decreased
expression compared to the wild type. Formation of vascular tissue is repressed by
COV1 (At2g20120) which when mutated results in increased vascular tissue in
Arabidopsis (Parker et al., 1999). In old9 an increased expression of this repressor is
detected at 21 days. Telomerase activity is shut down after mitotic division of cells by
decreasing the expression of its activator BT2 (At3g48360) (Ren et al., 2007), BT2 is
4 fold downregulated in old9 compared to wild type. The altered expression of these
100
transcripts in old9 is consistent with the suggestion that certain ARCs occur earlier in
the mutant.
Next to that transcripts were identified that are involved in genetic pathways
implicated in the biosynthesis or response to phytohormones. Ethylene promotes the
onset of leaf senescence in many plant species and in old9 an increased expression
of the negative regulator of ethylene responses EBF1 (At2g25490) (Potuschak et al.,
2003) was observed. Besides that OPCL1 (At1g20510), involved in the biosynthesis
of Jasmonic acid (Koo et al., 2006) shows increased expression in old9. Furthermore
a negative regulator of ABA signaling, PP2CA (At3g11410), is more abundantly
expressed in the mutant (Kuhn et al., 2006). Thus, the old9 mutation may cause a
partial repression of ABA and ethylene signaling after 21 days of growth in air.
Noteworthy is the observation that several genes encoding for cell-wall modifying
enzymes are differentially expressed in old9. Two genes involved in cross-linking of
the cell wall are differentially expressed; a D-apiose gene is downregulated
(At1g08200) while a hydroxyproline-rich glycoprotein (HRGP) is up regulated
(At1g23040). Next to that a xyloglucan transferase (At2g06850) is down regulated
while a gene involved in pectin synthesis (At3g23820) is up regulated. Furthermore,
an alpha-1,4-glucan synthase (At5g15650) is down regulated while beta-1,3glucanase is more than 5-fold increased in expression. This suggests that the cell
wall of old9 might have a different composition than the wild type. Interesting to note
is the increased expression of a putative cytokinin transporter (At4g18200) and the
up regulation of nitrate reductase (At1g37130).
To gain further insight in the air-grown transcriptome of old9 a comparison with other
microarray experiments was carried out by making use of the GENEVESTIGATOR
tool (Zimmerman et al., 2004). The analysis revealed that 67% of the differentially
expressed genes in old9 also respond to a 2 hour cytokinin treatment in 8 day old
seedlings (Brenner et al., 2005) (Table 2). Since cytokinin prevents senescence this
observation might explain the absence of early senescence under normal growth
conditions. However, it can not be ruled out that altered expression of cytokinin
responsive genes might cause the early onset of senescence by ethylene. Of the 89
transcripts, 24 are senescence associated as shown by Guo et al., (2004). Moreover,
At1g67105 is 19.5 fold increased in old9 and was shown to be upregulated during
leaf senescence (data not show). Also TAT3 and BGL2 are induced during leaf
senescence and are strongly up regulated in old9. These results are consistent with
101
the notion that old9 mutants may experience early ARCs and suggests that old9
leaves may be primed for senescence or that the senescence program may be
partially induced during normal growth conditions.
To determine a common regulation between the differentially expressed genes in
old9, we used coexpression analysis within the ATTED-II database (Obayashi et al.,
2007). The coexpression analysis reveals gene-to-gene relationships and is therefore
a useful tool to identify gene networks. 29 of the 89 identified genes have an
association with one or more other genes. Their association is presented in Figure 3
and shows 3 gene networks containing 3 or more genes and 6 interactions between
2 genes. The largest network contains 8 genes of which 2 are involved in lipid
metabolism (AT4g19860 and At5g18630), 3 chloroplastic genes (NCED4, At4g36040
and At5g19140), a gene involved in trehalose metabolism (TPS11) and a MYB
transcription factor (At1g71030). The second network is built around the BGL2
(At3g57260) which encodes a 1,3-beta-glucosidase involved in hydrolyzing fungal
cell walls (Mauch et al., 1988) and TAT3 gene that encodes for an enzyme
concerned in the synthesis of radical scavengers tocopherols from tyrosine (Hématy
et al., 2007). These 2 networks consist for 50 to 66% out of SAG genes which are
late leaf developmental genes and thus suggest an advanced leaf developmental
program in old9. The third network is built around a sucrose-regulated putative
MAPKKK (Niittylä et al., 2007).
Figure 3. Gene-gene relation between the identified old9 microarray transcripts was
determined by coexpression analysis. Genes are shown as green spheres and a relation
between two genes is represented by lines. Lines are only drawn between genes with
Pearson correlations >0.6. An S indicates that the gene is senescence associated.
102
The other 2 genes encode a putative CONSTANS-like transcription factor
(At1g68190) and an unknown DNA-binding domain-containing protein (At5g57760).
Taken together this analysis suggests that on the transcriptional level the leaf
developmental program of old9 is more progressed. Furthermore it suggests that
cytokinin might be an important factor in controlling the old9 phenotype.
Onset of senescence in old9 after ethylene treatment
The accelerated execution of senescence in old9 by ethylene was investigated by
making use of microarray analysis. We performed the microarray after 8 hours of
ethylene treatment when both wild type and mutant show no sign of visible yellowing.
We prefer this timepoint because ethylene can induce thousands of transcripts within
24 hours and next to that the progression of senescence involves a massive
transcriptional change. We anticipated that by limiting the length of the ethylene
exposure only genes involved in the start of leaf senescence and ethylene signaling
will show up, facilitating the analysis of the dataset. However, after ethylene
treatment hundreds of transcripts were identified that showed altered expression
between the mutant and the wild type. To focus our analysis we only investigated
genes that are more than 3-fold changed in expression (Table 3).
Table 3. Genes differentially expressed in old9 after 8 hours of ethylene treatment. 38
transcripts are more than 3-fold induced (P < 0.05, Student’s t test). Shown are TAIR ID,
gene name, description, Pubmed ID and ratio. E indicates ethylene-inducible ; S indicates
senescence associated; D indicates defense related. Genes less than 1.5-fold change are
considered as not changed represented by 0. +, positive correlation, - negative correlation.
TAIR- ID
Name
Description
At1g03850
At1g06000
At1g16260
At1g19380
At1g33960
At1g56660
At1g73650
At1g74590
At1g75830
At2g29350
At2g29720
At2g30140
At2g32240
At2g32670
At2g43570
At2g43680
Glutaredoxin family protein, response cytokinin
flavonol-7-O-rhamnosyltransferase
WAKL8
Wall-associated kinase like 8
Unknown protein
AIG1
Avirulence induced gene
Similar to wound-inducible KED of tobacco
3-oxo-5-alpha-steroid 4-dehydrogenase like
ATGSTU10 Glutathione S-transferase
PDF1.1
Plant defensin protein
SAG13
Senescence associated; alcohol dehydrogenase
CTF2B
flavoprotein monooxygenase
UDP-glucoronosyl/UDP-glucosyl transferase
similar to kinesin
VAMP275 Vesicle associated membrane protein
Putative endochitinase
IQD14
calmodulin binding, calcium signaling
PubMed
Ratio
E
S
D
16212609
17314094
12068092
3.2
3.1
3.9
3.2
9.4
4.3
3.3
4.3
8.2
3,5
4,6
3,3
3,3
3,6
3,2
3,2
0
0
+
0
0
0
0
+
+
0
+
+
0
0
+
+
+
+
+
+
+
+
+
-
+
0
+
+
0
0
+
+
+
0
+
+
0
+
0
NF
8742710
10945337
NF
12090627
15955924
10444084
NF
NF
NF
14500793
15915637
16368012
103
At3g13950
At3g18250
At3g22060
At3g23550
At3g25882
At3g44700
At3g54920
At3g62770
At4g14450
At4g17500
At4g25110
At4g33050
At4g34390
At4g35180
At4g37290
At5g13080
At5g26690
At5g48540
At5g54610
At5g58300
At5g58350
At5g64120
Ndr1 dependent
Unknown protein
NIMIN-2
PMR6
ATG18
ATERF1
ATMC2
EDA39
XLG2
LHT7
WRKY75
ANK
WNK4
Unknown protein
Multi antimicrobial extrusion protein
a kinase that physically interacts with
NPR1/NIM1
Unknown protein
Powdery mildew resistant 6; pectate lyase-like
autophagosome formation
C terminal similar to hydroxyporline rich
glycoprotein
Ethylene responsive element binding factor 1
Metacaspase 2
calmodulin-binding protein
Extra-large GTP binding protein;
LYS/HIS transporter 7
Unknown protein
WRKY Transcription Factor
Heavy metal transport/detoxification protein
33 kDa secretory protein-related
Ankyrin; SA induced
leucine-rich repeat transmembrane protein
kinase
WNK kinase
cell wall bound peroxidase; oxidative burst
17181774
NF
17761682
NF
9,1
4,8
3,2
3,3
0
+
+
+
+
-
+
+
+
+
15749762
3,7
N
-
N
NF
12215508
15860012
132.0
160.0
5,0
0
0
0
-
0
NF
3,1
N
-
N
10715325
15326173
15634699
10394945
16607029
NF
17322336
NF
16115070
16307367
3,3
4,6
3,4
3,2
5,9
3,2
7,5
7.0
4,1
4,4
+
+
+
0
+
+
+
N
0
0
+
+
+
+
-
+
+
+
0
+
+
+
N
+
+
16280546
3,2
-
-
-
12506983
16551688
3.0
5,7
0
+
+
+
+
+
With these parameters we identified 40 genes whose transcripts are increased in the
mutant compared to the wild type. We evaluated our data to other microarray
experiments and the senescence transcripts identified by Guo et al. (2004). In Figure
4 a VENN diagram illustrates the overlap between the old9 genes and the different
experiments.
Figure 4. A Venn-diagram is used to visualize the overlap
between
the
old9 microarray
dataset and that of other
experiments. E: Microarray dataset of plants treated for 3 hours
with ethylene; P: microarray dataset of plants exposed to
Phytophthora;
S:
EST
dataset
of
senescence
associated
transcripts.
The NASCARRAYS-32 was performed with seedlings treated for 3 hours of ethylene
by F.F. Millenaar. The identified transcripts correlate for 36.8% with the old9
transcriptome data. Next to that, 60% of the genes induced by ethylene in old9 were
previously identified during a plant defense experiment NASCARRAYS-123 by D.
Scheel. Interestingly, the experiments of Millenaar and Scheel share an overlap of
104
65%, indicating that ethylene induces many plant defense associated transcripts.
Importantly, 44.7% of the induced genes in old9 have been implicated before in leaf
senescence (Guo et al., 2004). Thus, as expected we find many ethylene-inducible
genes but the notion that almost 50% of the genes are senescence-associated
suggests early onset of leaf senescence in the mutant.
Ten transcripts are specifically up regulated in the old9 mutant as compared to the
other experiments. One of these encodes a flavonol 7-O-rhamnosyltransferase which
is involved in the biosynthesis of flavonols that function as either cell protectant,
signaling molecules or regulator of auxin transport (Yonekura-Sakakibara et al.,
2007). Next to that the ABA synthesis gene CTF2b is up regulated in old9 (Bilodeau
et al., 1999). ABA was shown to antagonize ethylene induced cell death in maize and
barley (Young and Gallie, 2000; Wang et al., 1999). The microarray analysis of
ethylene treated plants further supports the notion that in old9 cell wall associated
transcripts are differentially expressed as compared to the wild type. In old9 we
observed up regulation of ATVAMP275 (At2g32670) a SNARE-encoding gene
involved in vesicle transport of membrane proteins as well as cell wall materials from
the plasma membrane (Uemera et al., 2004). PMR6 encodes a pectate-lyase gene
which is 160-fold induced after ethylene treatment of the mutant and is involved in
cell-wall modification (Vogel et al., 2002). The degradation products of the cell wall
were shown to trigger a signaling cascade including a putative leucine-rich repeat
transmembrane protein kinase (At5g58300) which is specifically induced in old9
(Moscatiello et al., 2006). Another interesting gene that is up regulated in old9
encodes a G-protein (At4g34390) which serves as a physical coupler between cell
surface, 7 transmembrane (7TM) G-protein-coupled receptors (GPCRs) and
downstream targets (Temple and Jones, 2007). Noteworthy is the up regulation of
IQD14 (At2g43680) which is involved in calcium-signaling. During nutrient starvation
and senescence, autophagy is used to breakdown cellular components and regulate
nutrient relocalization. The autophagy gene ATG18 is 5 fold induced after ethylene
treatment of old9 and was shown before to function in nutrient remobilization during
leaf senescence of Arabidopsis (Xiong et al., 2005). To determine the relation
between old9 transcripts identified during air growth and after ethylene treatment we
performed a coexpression analysis (Figure 5). The analysis revealed that 15 of the
genes identified after ethylene treatment correlate to the genetic networks identified
in air-grown old9 mutant plants. The BGL2 network covers 15 genes of which 9 after
105
ethylene treatment, and contains in total 10 identified SAGs (Guo et al., 2004). The
second group that is built around TPS11 covers 16 genes of which 12 have been
identified as SAG (Guo et al., 2004).
Figure 5. Correlation between transcripts of the microarray on air-grown old9 and ethylene
treated. Genes of the air-grown microarray dataset are represented by green spheres and
those of the ethylene microarray dataset are shown in red. The relation between two is
represented by lines. Lines are only drawn between genes with Pearson correlations >0.6.
An S indicates that the gene is senescence associated.
Taken together this data shows that within 8 hours of ethylene treatment a
senescence program is initiated in old9.
Cytokinin primes leaves for ethylene-induced senescence
Microarray analysis of air-grown old9 mutants revealed that more than two third of
the identified transcripts is responsive to cytokinin (Table 2). However, more than
2000 genes are responsive to cytokinin (Brenner et al., 2005) and only a small
fraction was identified in the microarray. This suggest that only a specific set of
cytokinin-responsive genes are important for the old9 phenotype. Cytokinins are wellknown for their ability to delay leaf senescence when applied exogenously (Lim et al.,
2003) or when overproduced in transgenic plants (Gan and Amasino, 1995).
However, the application or production of cytokinin in these experiments is always
restricted to the last phase of leaf development just before or during the onset of leaf
106
senescence. The observation that long-term ethylene exposures of old9, starting at 8
days result in an early senescence phenotype (Chapter 2) suggests that the role of
cytokinin in the old9 phenotype is related to changes at early leaf development
phase. Therefore we designed an experiment in which we tested the application of
cytokinin at early and late developmental stages of the wild type and old9. If cytokinin
promotes the onset of senescence by ethylene than application to the wild type
should result in a phenocopy of the mutant.
Plants were given a cytokinin treatment at day 10, day 17 or at both days and grown
in air till 21 days and subsequently treated for 3 days with ethylene. The visual
senescence was assessed (Lohman et al., 1994) and presented in Figure 6 for wild
type and old9.
Figure 6. Cytokinin promotes the onset of ethylene-induced leaf senescence. Wild type and
old9 plants were treated with cytokinin at day 17, day 10 or at both days as described in
methods. After 21 days plants were treated with ethylene to induce senescence and 3 days
later the number of yellow leaves per plant was scored. Experiments were repeated 3 times
and similar results were obtained. Asterisks indicate statistical significance versus the mock
treatment in each case (P < 0.05, Student’s t test).
Application of cytokinin at 17 days resulted in a delay in the onset of senescence in
both wild type and old9 when compared to mock treatment, consistent with Lim et al.
(2003) and Gan and Amasino (1995). Interestingly this effect disappeared when
plants were treated at both 10 and 17 days for both wild type and old9. This suggests
that early application of cytokinin results in either insensitivity to the application at day
17 or the application at early stage promotes the onset of senescence by ethylene
which is again counteracted by application at day 17. Application at day 10 resulted
in an increased senescence response of the wild type accession while this had no
107
effect on the senescence of the mutant. Taken together these results demonstrate
that cytokinin can result in a partial phenocopy of the mutant phenotype in wild type
when applied early in leaf development. More importantly, these results suggest that
cytokinin can result in an ARC required for ethylene-inducible senescence when
applied early in development.
Role of a hydroxyproline-rich glycoprotein in old9 response
The old9 mutant plant has induced expression of several SAG genes when grown in
air. The expression of these SAG genes might either prime old9 for the induction of
leaf senescence by ethylene or be a marker for this possible priming. Therefore we
applied RNA interference (RNAi) to down-regulate one of these SAG genes and test
whether this influences the induction of leaf senescence. RNAi constructs obtained
from the Agrikola (Arabidopsis Genomic RNAi Knock-out Line Analysis) resource
(Hilson et al., 2004) were screened and transformed to the mutant. This resulted in a
stable RNAi line for the senescence associated hydroxyproline-rich glycoprotein
(Hrgp) (At1g23040). Homozygous T2 plants of 3 independent lines were grown till 21
days and treated for 3 days with ethylene. The independent lines were obtained by
transforming different copies of the same construct to different old9 plants.
Interestingly, all 3 lines showed a reduced senescence response compared to old9
(Figure 7).
Figure 7. Visible yellowing of old9 plants carrying a
RNAi against HRGP. The number of yellow leaves
of three independent RNAi lines is indicated and
compared to old9 control plants. The visible
yellowing was scored and expressed as means
±SD of at least three replicates of 30 plants each.
Plants that were grown for 24 d in air did not show
any sign of senescence (not shown). Asterisks
indicate statistical significance versus the old9 mutant in each case (* P < 0.05, Student’s t
test). Black bars, RNAi line; Grey bars, old9 mutant.
However, the senescence response is enhanced when compared to the wild type.
These results demonstrate that Hrgp is an important determinant for the senescence
108
response of old9 and supports the notion that at least one of the identified SAGs can
prime a leaf for ethylene-inducible senescence.
Discussion
Plants are continuously interacting with the environment and adapt their growth and
developmental strategy accordingly. During non-stress conditions the leaves of
Arabidopsis undergo age-dependent senescence (Gan and Amasino, 1997; Quirino
et al., 2000; Jing et al., 2002). The progression of senescence occurs in a
coordinated manner starting from the tip of a leaf toward the base. The controlled
breakdown of the leaf during senescence is in part to ensure effective remobilization
of nutrients. Therefore it is not surprising that senescence is a highly regulated
process involving many genetic programs (Buchanon-Wollaston et al., 2005;
Keskitalo et al., 2005; van der Graaff et al., 2006). The two major plant hormones
cytokinin and ethylene have well established opposite effects on leaf senescence.
Here we report on the identification of an early ethylene-inducible senescence mutant
that has increased expression of several SAG genes during air-grown conditions but
no visual differences with the wild type. Interestingly, we demonstrate that cytokinin
application at an early stage of plant development can promote the onset of leaf
senescence by ethylene.
Endogenous levels of cytokinin drop concomitant with the progression of leaf
development (Gan and Amasino, 1996) suggesting that the hormone plays a role
during the development of the young leaf. The retardation of leaf senescence by
cytokinin has been demonstrated by applying the hormone just before or during the
onset of leaf senescence (Gan and Amasino, 1995; Lim et al., 2003). However, in our
study we demonstrate that cytokinin can also have the opposite effect on the onset of
leaf senescence. First the delay of senescence by applying cytokinin at 17 days is
make ineffective when plants are also treated at 10 days with cytokinin. This might
imply that application of cytokinin early in development has an opposite effect to
application late in development. This is evident from the application at 10 days which
is sufficient to induce early senescence after ethylene treatment in the wild type.
Cytokinin is known to delay senescence by increasing the sink strength of the tissue
(Balibrea Lara et al., 2004) and thus reverses the leaf senescence program which
mobilizes nutrients from the senescing leaf to other parts of the plant. Thus by
influencing the sink-to-source transition of a leaf, cytokinin might modulate the onset
109
of senescence. In support to this it was demonstrated in tobacco that application of
cytokinin to leaf blades of decapitated plants is much less effective than application
to intact plants to delay senescence (Singh et al., 1992) which was explained by
suggesting that an increase of the sink strength only is not sufficient to delay
senescence but also remobilization of nutrients to the tissue is required. Interestingly,
detached leaves of old9 senesce at the same time as wild type plants (Chapter 5),
consistent with the idea that a sink tissue might be required for the accelerated
senescence of old9 leaves. Since we sprayed whole plants with cytokinin we
interfered with the sink/source strength of all leaves. Therefore we propose that
cytokinin promotes the onset of leaf senescence both in old9 and treated wild type
plants, by increasing the sink strength of young tissue. Endogenous cytokinin content
and sensitivity to cytokinin regulates the developmental rate of Arabidopsis as
mutants with a reduced cytokinin sensitivity had a slower rate of rosette leaf
emergence and leaf expansion (Smalle et al., 2002), while plants with increased
cytokinin sensitivity showed rapid chloroplast development and proliferation (Kubo
and Kakimoto, 2000). Furthermore plants overproducing cytokinin develop more
rosette leaves (Chaudhury et al., 1993). In old9 several genes involved in different
leaf developmental programs including cell elongation, vascular tissue formation and
telomerase activity are down regulated compared to the wild type at 21 days,
suggesting that the leaf developmental program is in a different phase than the wild
type. Overexpression of the cytokinin receptor AHK3 or a downstream target ARR2
delays leaf senescence in Arabidopsis (Kim et al., 2006). Both transformants have a
constitutive overexpression of cytokinin responsive genes. It would be interesting to
determine the effect of ethylene on leaf senescence in relation to cytokinin with these
mutants. This might reveal whether or not the old9 phenotype is depending on a
transient activation of cytokinin responsive genes or constitutive activation and if the
subset of cytokinin responsive genes is important or all (Brenner et al., 2005).
The orderly visual appearance of cell death is in accordance with the induction of a
senescence program in old9. Interestingly, the senescence starts at the tip of the leaf
and thus the oldest leaf cells in old9 (Ferreira et al., 1994) which is in agreement with
the age-dependent progression of leaf senescence. The first leaf pair of 21 day old9
mutants has increased expression of several SAGs which is not sufficient to induce
early senescence under normal conditions but might prime old9 for ethylene-induced
110
leaf senescence. Since SAG genes are associated with the last developmental
phase they are a marker for an early ARC in the development of old9 leaves. That
these SAGs play a role in the ability of ethylene to induce early senescence is at
least confirmed for one transcript. Transformation of the mutant with RNAi constructs
directed against HRGP transcripts result in a decreased senescence response. Since
the induction of leaf senescence by ethylene is age-dependent this suggests that
reducing the expression of HRGP might interfere with the ageing of the leaf and thus
causes a reduced response. HRGPs constitute one of the most abundant structural
proteins in the plant cell wall and can confer resistance to pathogens in Pearl millet
(Deepak et al., 2007). However, in Arabidopsis they have been implicated in embryo
development (Hall and Cannon, 2002) but also in response to ABA (van Hengel and
Roberts, 2003) suggesting that they play a role in growth and development of
Arabidopsis. The accumulation of HRGP might represent a marker for a
developmental change which is related to the progression towards leaf senescence.
The microarray analysis suggests important changes on the cell wall composition of
old9. First of all a pectin synthesis gene is enhanced during air-growth, however
during ethylene treatment a pectate lyase gene is induced 160 fold suggesting that
the senescence of old9 also involves a rapid degradation of the cell wall. Interestingly
pectate lyase activity has been related to fruit ripening in strawberry and banana
(Medina-Escobar, 1997; Asif and Nath, 2005) and resistance to Powdery Mildew
when knocked-out (Vogel et al., 2002). Recently it has been demonstrated that pectic
fragments of plant cell walls are able to induce defense and developmental
responses (Moscatiello et al., 2006). Therefore the increased expression of the
pectate lyase gene after ethylene treatment of old9 might partially explain the
abundance of plant defense genes after ethylene treatment. However, as shown,
many plant defense genes are ethylene inducible and thus are probably not related
to degradation of pectate. The effect of cell wall composition on the induction of leaf
senescence has not been well described in literature and thus might be an aim for
future research.
One question that remains is why old9 does not show early leaf senescence during
normal development in air. If the mutant is primed to senescence due to ARCs than it
would be logical to conclude that this also affects the onset of developmental
111
senescence. A study by Jing et al. (2002) proposed the existence of a so-called
senescence window. The senescence window is a developmental phase during
which ethylene can induce premature leaf senescence. This model explains the
absence of early developmental senescence in the mutant since OLD9 regulates at
which developmental age a leaf can perceive ethylene to promote leaf senescence
but not when to start to senesce. Since no early developmental senescence occurs
this suggests that old9 is not involved in the direct execution of senescence.
The conferred change by the old9 mutation might be useful for controlling the
ripening of crops or fruits. For example the simultaneous start of fruit ripening will
make it possible to harvest all products at the same time.
Methods
Plant materials and growth conditions
Arabidopsis thaliana ecotype Landsberg erecta (Ler-0) was used in this study. The
old9 mutant was obtained from an EMS mutagenized collection (Jing et al., 2005).
Plants were grown on either soil or half-strength Murashige and Skoog medium at
23°C and 65% relative humidity with a day length of 16 h. The light intensity was set
at 120 µmol·m-2·s-1. An organic-rich γ-ray radiated soil was used (Hortimea Groep,
Elst, The Netherlands). Plants for ethylene exposure were treated in a flow-through
chamber at 20 °C and a humidity of 40% under continuous illumination. The ethylene
dosage was set at ~10 µl l–1 as suggested by Chen and Bleecker (1995). Cotyledons
or rosette leaves with over 5% yellow area of the leaf blade were judged as yellow as
suggested by Lohman et al. (1994).
Tryphan blue staining
Appearance of cell dead were studied in whole leaf mounts stained with lactophenoltryphan blue (10 ml of lacticacid, 10 ml of glycerol, 10 g of phenol, 10 mg of tryphan
blue, dissolved in 10 ml of distilled water) (based on Keogh et al.,1980). Whole
leaves were boiled for 1 min in the staining solution and then overnight decolorized in
saturated solution of chloral hydrate. Subsequently the leaves were mounted in
chloral hydrate and viewed under a light microscope.
112
RNA-isolation and RT-PCR
Total RNA was isolated using TRIZOL reagent (Sigma) according to the
manufacturer's protocol. Five hundred nanograms of RNA were used as template for
first-strand cDNA synthesis using 200U of RevertAid H-minus MMuLV reverse
transcriptase (Fermentas, USA) and an oligo(dT21) primer. Primer pairs for real-time
PCR were designed with open-source PCR primer design program PerlPrimer
v1.1.10 (Marshall, 2004). The primer sequences are available upon request. Briefly,
real-time PCR amplification was performed with 50 µL of reaction solution, containing
2 µL of 10-fold–diluted cDNA, 0.5 µl of a 10 mM stock of each primer, 1 µl of 25mM
stock MgCl2 (Fermentas), 5 µl PCR buffer +Mg (Roche), 1 µl of a 1000x diluted
SYBR-green stock (Sigma), 0.5 µl 100xBSA (New England Biolabs), and 1u of
Roche Taq Polymerase. The PCR program was 2’ at 94, 40x (94-10”/60-10”/72-25”),
meltcurve. Obtained data was analyzed with BioRad software.
Microarray analysis
All the array experiments were carried out using the CATMA2 Arabidopsis GeneChip
microarray
containing
24576
gene-specific
tags
from
Arabidopsis
thaliana
(http://www.catma.org). For each comparison, one technical replication with
fluorochrome reversal was performed for each pool of RNA. cRNA were produced
from 2 µg of total RNA from each sample using the Message Amp aRNA® kit
(Ambion). Then 5 µg of cRNA was reverse transcribed in the presence of 300 units of
SuperscriptTM II DNA polymerase (Invitrogen/Life technologies), cy3-dUTP and cy5dUTP (PerkinElmer Life Sciences) for each slide. Slides were prehybridized for 1 h
and hybridized overnight at 42°C in 25% formamide. Slides were washed in 2× SSC
and 0.1% SDS for 4 min, 1× SSC for 4 min, 0.2× SSC for 4 min, and 0.05× SSC for 1
min and dried by centrifugation. Raw data from each experiment were normalized in
GeneSpring (Silicon Genetics, Redwood City, CA, USA) using the default procedure.
GeneSpring was used to filter out genes detected in only one of the slides and to
identify up- and down regulated genes for further analysis. The identified genes were
compared to 2 experiments available from the Nottingham Arabidopsis Stock Centre
(NASC) microarray database (http://affy.arabidopsis.info).
Coexpression analysis
113
Coexpression analyses were performed by using a Coexpression Gene Search
algorithm on the RIKEN PRIMe web site. The Coexpression Gene Search program is
a web-based application designed to identify correlated genes from gene expression
data produced using Affymetrix Gene-Chip technology by the AtGenExpress
consortium (RIKEN Plant Science Center and the Max-Planck Institute for Molecular
Plant Physiology) deposited in TAIR. The database can be searched by using the
AGI annotation. The ATTED-II release directly gives the correlation coefficients
(Obayashi et al., 2007).
Cytokinin treatment
Plants were grown on soil for 21 days and subsequently exposed to ethylene for 3
days. During the growth of the plants a foliar spray technique was used to apply
cytokinin. The synthetic cytokinin, benzyladenine (BA) was applied exogenously in a
concentration of 50 µM dissolved in 0.5% Tween-20. As a control plants were treated
by spraying with a solution of 0.5% Tween-20. Plants were sprayed in the morning
and approximately 10 ml of solution was applied to a tray containing 104 plants.
RNAi lines
A 175-bp HRGP-specific fragment (clone CATMA1a22110) (from +308 to +482 of
HRGP cDNA) was cloned into a binary vector by the AGRICOLA consortium to
create a hairpin construct (Hilson et al., 2004). The pAGRIKOLA plasmid was verified
according to the validation protocols by Hilson et al. 2004. The binary plasmid was
introduced into Agrobacterium tumefaciens strain GV3101 together with the pSOUP
helper plasmid through electroporation. old9 mutants were subsequently transformed
with the RNAI construct by the floral dip method (Clough and Bent, 1998).
Acknowledgements
We would like to thank Bert Venema and Margriet Ferwerda for their excellent
technical support.
114
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Chapter
5
The Arabidopsis old5 mutation of quinolinate synthase affects NAD
biosynthesis and causes early ageing
Jos H.M. Schippers1, Adriano Nunes-Nesi2, Roxana Apetrei1, Jacques Hille1,
Alisdair R. Fernie2 and Paul P. Dijkwel1.
1
Molecular Biology of Plants, Groningen Biomolecular Sciences and Biotechnology Institute, University
of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands
2
Max Planck Institute for Molecular Plant Physiology, Potsdam-Golm 14766, Germany.
121
122
Abstract
Leaf senescence in Arabidopsis is regulated by a strict, genetically controlled nutrient
recovery program which typically progresses in an age-dependent manner. To date,
several genes altering the onset of senescence have been identified, suggesting that
unigenic factors influence leaf lifespan. That said, the understanding of factors that
control leaf ageing and the onset of senescence remain incomplete. Leaves of the
previously identified Arabidopsis onset-of-leaf-death 5 (old5) mutant exhibit early
developmental senescence. Here, we identify that OLD5 encodes quinolinate
synthase, a key enzyme in the de novo synthesis of NAD. Despite harboring a lesion
in this enzyme, the mutant contains increased NAD steady-state levels likely due to
increased expression of enzymes in the NAD salvage pathway. NAD plays a key role
in cellular redox reactions including those of the TCA cycle. Broad range metabolite
profiling of the old5 mutant revealed that it contains higher levels of TCA cycle
intermediates and nitrogen-containing amino acids. The increased TCA cycle
intermediates coincide with a higher respiration rate for the mutant and increased
expression of oxidative stress markers. Taken together the results of this study
suggest that early ageing of the mutant is a consequence of mitochondrial oxidative
stress. These observations are consistent with the “free-radical” theory of ageing that
has been widely accepted as the mechanism behind animal ageing.
Introduction
In Arabidopsis thaliana the onset of leaf senescence proceeds within a predictable
time-window (Jing et al., 2002). Only when a leaf is under attack or grows in
unfavorable conditions the senescence program will be prematurely initiated
(Schippers et al., 2007). Senescence is a critical component of the plant life cycle
where it contributes to relocation of valuable resources from old, dying tissue to
young developing parts of the plant. The transfer of nutrients supports the filling of
grains in order to afford the plant reproductive success. Recent comprehensive
genomics studies have demonstrated that senescence is under strict genetic control
(Buchanan-Wollaston et al., 2003, 2005; Guo et al., 2004), representing a carefully
maintained developmental program which ultimately results in cell death. Aside from
the fundamental importance to understand senescence, the commercial implications
of increasing crop yield or the longevity of ornamental plants are vast. In this light, a
recently identified transcription factor in wheat, that was shown to affect crop yield by
123
30% (Uauy et al., 2006), underlines the impact of senescence research on future
agriculture.
Despite recent advances in the understanding of senescence itself, little is known
regarding the factors that determine its onset. In Arabidopsis senescence occurs in a
leaf-age dependent manner even when the plant is challenged by (a)-biotic stress
(Gan and Amasino, 1997; Quirino et al., 2000). Whereas in animal and yeast
research the terms senescence and ageing are interchangeable, in plants
senescence refers to the process that leads to the death of the leaf, while ageing
itself occurs throughout the development from initiation of a leaf primordium
throughout senescence and death (Lim et al., 2003).
Given that most animals reproduce early in life and die before they age no specific
ageing program is required (Kirkwood, 2002). Although many genes have been
identified that alter longevity in organisms ranging from yeast, to worms, fruit flies and
mammals (Jing et al., 2003), no combination of genes has been found to abolish
ageing. This can be explained in two ways, either no ageing program exists or many
overlapping programs are present rendering the process highly robust (Kirkwood,
2005). Although ageing of Arabidopsis is influenced by its reproductive strategy there
is a weak correlative control between the appearance of reproductive structures and
leaf senescence. While male and female sterile mutant plants live 20 days longer
(Nooden and Penney, 2001) the longevity of individual rosette leaves of these
mutants is unaltered. Nutrient salvage during leaf senescence is a vital part of
development and it is under evolutional selection (Bleecker, 1998). Thus the agedependent onset of senescence allows to study ageing in plants from an evolutionary
point of view by looking at individual leaves. Processes that lead to ageing, termed,
age-related changes occur as a result of the differential regulation of development
and growth (Jing et al., 2005). In general, ageing is controlled by programs involved
in life maintenance, stress responses and development (Schippers et al., 2007).
From the moment leaves reach their full size they are subjected to post-mitotic
ageing (Gan, 2003). If leaves are considered as independent systems we can
compare their ageing with that of animal systems (Jing et al., 2003). In the mid-1950s
it was postulated that Reactive Oxygen Species (ROS) are the main cause of animal
ageing beginning with cumulative damage that results in loss of viability (Harman,
1956). In keeping with this theory the delayed leaf senescence mutants ore1, ore3
124
and ore9 (Woo et al., 2004) and the late flowering and longevity mutant gigantea of
Arabidopsis (Kurepa et al., 1998) all show higher tolerance to oxidative stress. These
combined data support the notion that ROS influence both the ageing process and
the lifespan of Arabidopsis leaves. Whilst in animals ROS are produced by
mitochondria, the main ROS source in a senescent leaf is the chloroplast (Quirino et
al., 2000). That said plant mitochondria produce considerable ROS during the
hypersensitive response invoked by plant-pathogen interactions (Finkel and
Holbrook, 2000), and under a variety of other cellular circumstances (Sweetlove et
al., 2006).
Increased tissue contents of ROS (Navabpour et al., 2003) and exogenously applied
causal agents of oxidative stress such as UV-B and ozone (John et al., 2001; Miller
et al., 1999) lead to expression of senescence-associated genes (SAGs). SAGs were
previously identified on the basis of their characteristic up-regulation during leaf
senescence. Furthermore, reducing the expression of SAG101 or phopholipase Dα
has been demonstrated to delay the onset of senescence in Arabidopsis (Fan et al.,
1997; He and Gan, 2002), providing further evidence of a link between ROS induced
SAG activation, ageing and the onset of leaf senescence.
An important source of ROS production in animals and plants are the membraneassociated nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidases that
generate ROS via lipid oxidation (Finkel and Holbrook, 2000; Mittler, 2002). NAD(P)H
are well-studied redox molecules which mediate hundreds of reactions and are at the
basis of almost every metabolic pathway in the cell (Noctor et al., 2006). ROS
production in the chloroplast has been clearly demonstrated to be under the influence
of NADP/NADPH ratios in this compartment (Asada, 2006) whereas production of
mitochondrial ROS by the electron transport chain is largely dependent on NAD(P)
status (Dutilleul et al., 2005; Shen et al., 2006, Sweetlove et al., 2006). The turn-over
of anti-oxidants pools such as those of glutathione and ascorbate are also
maintained by NAD(P)H (Noctor et al., 2006). Recently an NAD–dependent histone
deacetylase, SIR2 was found in yeast to control longevity (Lin et al., 2000) and the
NAD-consuming enzyme poly(ADP-ribose)polymerase (PARP) was described to
protect cells from oxidative stress induced DNA damage (De Block et al., 2005).
Pyridine nucleotides are the main redox regulators since they react slowly with
oxygen species in an enzyme dependent way. On the contrary, the small antioxidant
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molecules glutathione and ascorbate react spontaneously with ROS. The
regeneration of these small molecules is operated by a high-capacity system that
depends on the in vivo activities of enzymes that require the reducing power of
NADH or NADPH in order to catalyze the reactions that they mediate (Noctor, 2006).
Thus glutathione and ascorbate are part of the NAD(P)-dependent buffering system
that regulates the cellular targets with which ROS are allowed to interact. This
function, alongside that NAD homeostasis plays in the regulation of central
metabolism (Dutilleul et al., 2005), makes NAD a key player in regulating metabolism
and development.
The availability of senescence mutants has given us the opportunity to discover
which processes play a role in plant ageing and determine the onset of leaf
senescence (Jing et al., 2002; Jing et al., 2005). Here we report a mutation in de
novo pyridine nucleotide synthesis which results in an early onset of developmental
senescence. Despite the fact that the genetic legion of this mutant is in the pathway
of NAD synthesis the mutant exhibits increased content of pyridine nucleotides. The
most likely explanation for this was provided by RT-PCR-based transcript analysis,
which revealed an up regulation of the NAD salvage pathway. Further
characterization of the mutant revealed many changes in the levels of transcripts that
are suggestive of oxidative stress whilst the metabolic profile obtained for the mutant
shows increased levels of metabolites of central metabolism. The results are
discussed in the context of current theories of ageing and senescence that have
been previously documented in the plant and mammalian literature.
Results
Characterization of the old5 mutant during developmental senescence
The old5 mutant was identified as a monogenic recessive trait during a screen of an
EMS treated population of Arabidopsis plants (Jing et al., 2002) and was shown to
display an early onset of senescence and an enhanced senescence response after
ethylene treatment (Jing et al., 2005). The mutant shows yellowing of the first leaf
pair after 30 days (Figure 1A), whereas the wild type shows senescence symptoms
after 40 days (data not shown). The visual yellowing runs parallel with a decline in
chlorophyll content and photochemical efficiency (Figure 1B). Thus the old5 mutant
126
exhibits the physiological hallmarks of leaf senescence. The early senescence of the
mutant results in a decrease in lifespan of approximately 2 weeks (Figure 1C).
Figure 1. The onset of leaf senescence in old5 mutant Arabidopsis plants. (A) Visible leaf
yellowing in the old5 mutant line. The first rosette leaves are shown from 21 till 33 day old
plants of wild type (WT) and old5. (B) Photochemical efficiency (PSII) and chlorophyll levels
of the old5 mutant line decrease at the onset of senescence (old5 open circles, wild type
closed circles). The photochemical efficiency was determined with a PAM-2000. The values
shown are the means of three repeats ± SD (indicated by error bars). Asterisks indicate
statistical significance versus the wild type control in each case (* P < 0.05, Student’s t test).
(C) Six-week-old adult plants. The old5 mutant plants display strong senescence phenotype
compared to wild type.
The expression of two established senescence marker genes was followed by realtime PCR. First, we examined SAG13 whose expression is associated with oxidative
stress and senescence (Weaver et al., 1998). SAG13 expression was detected from
day 24 in both the mutant and the wild type. However, the expression in the mutant
was 10 (day 24) and progresses to a 10000 fold (day 33) higher than in the wild type
(Figure 2). In addition we examined the hallmark of age-induced senescence,
SAG12. SAG12 was detected after 30 days only in old5 but not the wild type. The
SAG12 expression correlates with the first symptoms of yellowing at 30 days implying
together with the expression of the SAG13 marker, that senescence occurs in an
age-dependent way.
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Figure 2. Transcript accumulation of the senescence-associated genes, SAG12 and SAG13,
in old5 mutant plants. The relative levels of SAG12 and SAG13 were examined by semiquantitative RT-PCR analysis, using ACT2 as internal control, of first rosette leaves of 21 to
33 day old plants (old5 gray bars, wild type solid black bars). For SAG13 the relative
transcript levels detected in the wild type plants were arbitrarily assigned a value of 1 after
normalization to the ACT2 samples. For SAG12 the relative transcript level detected at day
30 in old5 was set at 1 and used for comparison with day 33. The values shown are the
means of three repeats ± SD (indicated by error bars). Experiments were repeated and
showed the same results. Asterisks indicate statistical significance versus the wild type
control in each case (* P < 0.05, Student’s t test).
OLD5 encodes a quinolinate synthase
The old5 mutant was identified in the Landsberg (Ler) accession and was crossed to
Columbia (Col) to facilitate mapping. The mapping population segregated in a 1:3
ratio, demonstrating that the mutant phenotype is encoded by a single locus. In a
population of 2500 F2 plants, the location of old5 could be narrowed down to a 60kb
region, containing 19 genes, in the overlap on BACs K6A12 and MXI22 on
chromosome V between markers K6A12-23K and MXI22-12K. From these 19 genes,
eleven candidate genes were selected for sequence comparison between the mutant
and the Ler wild type. In the sequence of At5g50210 a C to T change in the first exon
of the old5 mutant was detected. This gene has previously been annotated as
quinolinate synthase (QS) (Katoh et al., 2006), which is a component of the pathway
of the de novo synthesis of NAD from aspartate. A homozygous T-DNA knock-out of
this enzyme is embryo lethal (Katoh et al., 2006), suggesting that the old5 mutation
does not result in a complete loss of functionality.
The identity of At5g50210 as the OLD5 gene was confirmed by a complementation
test. A 5.5 kb Ler genomic fragment containing the coding sequence, a 2.1-kb 5’
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promoter sequence according to the AGRIS database (Davuluri et al., 2003) and a
0.5 kb 3’ sequence was transformed into old5 mutant plants.
Figure 3. OLD5 encodes for quinolinate synthase. (A) Schematic representation of the
protein domain organization of OLD5. The first domain is a transit peptide that targets OLD5
to the chloroplast. The other 2 domains are a quinolinate synthase domain and a SufE/YgdK
domain. (B) The OLD5 SufE domain shows similarity to SufE proteins from E. coli (SufE and
YgdK). This domain is also present in 2 other Arabidopsis proteins, At4g26500 encoding for
AtSufE, and At1g67810, a putative pollen specific SufE protein. The amino-acid change in
old5 mutant protein is in a proline residue that is conserved between all three Arabidopsis
proteins (indicated by arrow). (C) Modeling by 3D-Jigsaw server predicts an effect of the
mutated proline on the structure of the SufE/YgdK domain (indicated by arrow). Yellow: E.coli
YgdK structure (Liu et al., 2005). Magenta: wild type Arabidopsis OLD5. Cyan: old5 mutant
protein.
129
10 T1 lines were selected on kanamycin, these all showed absence of the old5
phenotype, as expected since the mutation is recessive. By PCR we showed that the
lines contained both the KANr gene and the homozygous parental old5 point
mutation (see methods). The T2 segregated 3:1 (wildtype:mutant) confirming the
identity of the OLD5 mutant gene.
old5 mutants have an amino acid change in the SufE/YgdK domain
The OLD5 protein consists of 3 domains: a chloroplast targeting signal, a SufE/YgdK
domain and a quinolinate synthase domain (Figure 3A). The old5 mutation results in
a proline to serine amino-acid substitution in the SufE/YgdK domain. This domain is
highly homologous to the previously identified AtSufE/CpSufE (Xu and Møller, 2006;
Ye et al., 2006), the Ygdk and SufE proteins of E.coli (Loiseau et al., 2005) and a
putative gene, At1g67810, which was found by a BLAST search of the OLD5
SufE/YgdK domain (residues 84 to 214) against the Arabidopsis genome (Figure 3B).
AtSufE/CpSufE, SufE, and YgdK share functional homology, as acceptors of sulfur
atoms and stimulators of cysteine desulfurase activity. SufE of E.coli (Loiseau et al.,
2003) and CpSufE of Arabidopsis (Ye et al., 2006) stimulate SufS cysteine
desulfurase activity. The Fe-S cluster which is inserted by SufS into QS is absolutely
required for enzyme activity in E.coli (Ollagnier-de Choudens et al., 2005). In the
mutated Arabidopsis old5 protein proline-101 is replaced by a serine. The proline-101
residue is present in all Arabidopsis SufE/YgdK containing proteins, suggesting a
conserved role. Utilizing the Jigsaw 3D protein modeling server (Bates et al., 2001)
the OLD5 protein could be modeled with high confidence on the resolved structure of
YgdK (Liu et al., 2005). The resultant model suggests that the mutation of the proline
residue into serine affects the loop between helix I and II of the protein (Figure 3C).
OLD5 interacts with a SufS-like protein
The YgdK/SufE proteins of E.coli interact with their SufS counterpart (Louiseau et al.,
2003; Loiseau et al., 2005). By analogy we therefore expected the SufE/YgdK
domain of OLD5 to interact with a SufS-like protein. Recent studies show that the
Arabidopsis SufS-like protein, CpNifS interacts with AtSufE (Xu and Møller, 2006; Ye
et al., 2006). Furthermore, it has been suggested that quinolinate synthase might
work in an enzyme complex with aspartate oxidase (Sakuraba et al., 2005) since the
product of aspartate oxidase is unstable (Yang et al., 2003).
130
By a database search we found that the Arabidopsis genome contains 2 other
putative plastidic SufS-like proteins, CpNifS2 and CpNifS3, respectively. In order to
test whether OLD5 can interact with a cysteine desulfurase, a yeast-two hybrid assay
was performed. As a positive control we tested the interaction between CpNifS and
AtSufE. The results of our screen are summarized in Table 1. On selection medium
lacking either histidine or adenine we found that the OLD5 protein interacts with
CpNifS and CpNifS3. These interactions were found for the mutated old5 protein only
on His- selection medium but not on the more stringent Ade- plates. Interestingly, an
interaction with aspartate oxidase was also detected, suggesting that OLD5 works as
part of an enzyme complex.
Table 1. Result of yeast-two-hybrid analysis
Interaction
OLD5 - OLD5
OLD5 - CpNIFS
OLD5 - CpNIFS2
OLD5 - CpNIFS3
OLD5 - NADB
old5 – old5
old5 - CpNIFS
old5 - CpNIFS2
old5 - CpNIFS3
old5 - NADB
AtSufE - CpNIFS
Empty - Empty
His- +2mM 3 AT
++
++
++
++
++
++
++
-
Ade++
++
++
++
-
Taken together these results suggest that OLD5 has a functional SufE/YgdK domain.
Recently, Murthy et al., (2007) demonstrated that the SufE/YgdK domain can indeed
increase the cysteine desulfurase activity of CpNifS by 70-fold.
The old5 mutation affects steady state pyridine nucleotide levels
Since the OLD5 SufE/Ygdk domain is mutated we expect the enzyme to be less
active. Moreover, it has previously been demonstrated that the QS domain is
dependent on the biogenesis of an Fe-S cluster for optimal activity (Gardner and
Fridovich, 1991). Moreover, replacing the cysteine residue of the SufE/YgdK domain
with a serine disables the full length protein from complementing an E. coli ∆NadA
strain (Murthy et al., 2007) indicating the necessity of this domain for QS activity.
Given that a reduced QS activity may be anticipated to result in lower NAD steady
131
state levels we determined the pyridine nucleotide content in the first leaf pair of 21
and 27 day old plants. At these time points the old mutant leaves show no
phenotypical difference in comparison to the wild type (Figure 1A). Measurement of
pyridine nucleotide content revealed, however, that the mutants displayed a
significantly increased NAD, NADH, NADP and NADPH content after 21 and 27 days
(Figure 4A). The values we report for NAD, NADP and NADPH in wild type are
similar to those of previous studies (Wang and Pichersky, 2007; Chai et al., 2005).
Interestingly, the NAD/NADH and NADP/NADPH ratios in mutant do not differ from
the wild type suggesting a similar redox balance for the pyridine nucleotides.
The maintenance of the NAD pool results from the balance of de novo synthesis,
active pyridine salvage and degradation (Wang and Pichersky, 2007). The de novo
synthesis and the pyridine salvage pathway result in the production of Nicotinate
mononucleotide (NaMN) which by 2 enzymatic steps is converted to NAD (Figure
4B). The increased NAD pool suggests an increased expression of enzymes in the
salvage pathway to compensate for insufficient biosynthesis via the de novo
pathway. Such a mechanism was previously observed for the enzymes of the
salvage pathway of pyrimidine nucleotides following the antisense inhibition of an
enzyme of the de novo pathway of pyrimidine biosynthesis (Geigenberger et al.,
2005). The salvage of nicotinamide (NIM), released from NAD, starts with
deamidation by nicotinamidases to nicotinate (Wang and Pichersky, 2007) and
subsequent conversion by nicotinate phosphoribosyltransferase (NaPRT). In
Arabidopsis, AtNIC1 is the best characterized nicotinamidase which, when knockedout, almost completely abolishes the salvage of NIM. Overexpression of AtNIC1
result in an increase in NAD levels, thus the expression level of AtNIC1 correlates
with NAD content (Wang and Pichersky, 2007). The expression of AtNIC1 is
constitutively higher in the old5 mutant (Figure 4B). The product of AtNIC1, nicotinate
is converted by NaPRTases, the Arabidopsis genome encodes 2 homologes, in old5
At2g23420 (NaPRT2) is up regulated when compared to the wild type, while the
other putative NaPRTase (At4g36940) is expressed at similar levels as the wild type
(data not shown). The increase in expression of both AtNIC1 and At2g23420,
together with the increased pyridine levels, suggests an increased activity of the
salvage pathway in the old5 mutant.
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Figure 4. Pyridine nucleotide biosynthesis is increased during leaf development and ageing
of old5. (A) Pyridine nucleotide content in old5 and wild type at day 21 and day 27 (old5 gray
bars, wild type solid black bars). The values shown are the means of six repeats ± SD
(indicated by error bars). Asterisks indicate statistical significance versus the wild type control
(* P < 0.05, Student’s t test). The ratio of pyridine nucleotides is not affected by the old5
mutation. (B) Schematic representation of NAD biosynthetic pathways. Transcripts of AO,
AtNIC1 and NaPRT2 accumulate in old5 mutants (old5 open circles, wild type closed circles).
The values shown are the means of three repeats ± SD (indicated by error bars). Asterisks
indicate statistical significance versus the wild type control (* P < 0.05, Student’s t test).
Aspartate oxidase (AO), Quinolinate Synthase (QS), Quinolinate Phosporibosyltransferase
133
(QPRT), Nicotinamidase (AtNIC1), Nicotinate Phosporibosyltransferase (NaPRT2), NAD
synthase (NAD-S).
In addition to our analysis of the salvage pathway we determined the expression of
genes encoding key enzymes in the pathway of the de novo synthesis (Figure 4B).
The first step in this pathway is the conversion of L-aspartate into iminoaspartate
mediated by L-aspartate oxidase (AO). The transcript level of AO was 3-fold higher in
old5 mutant than in the wild type at all time points measured. The next step, of the de
novo biosynthesis, the synthesis of the pyridine ring is controlled by OLD5/QS. The
expression of QS decreases with age in both the wild type and the mutant. However,
during senescence the expression of QS is upregulated in the mutant, reflecting a
possible increased demand for NAD during the process. Finally, NaMN are produced
by quinolinate phosphoribosyltransferase (QPRT), which for both the wild type and
the mutant gradually increase in expression with age but do not differ between the
two.
Taken together we observe an increased AO expression in de novo synthesis and
induction of expression of key salvage enzymes. This is consistent with a block in the
de novo synthesis in old5 mutants compensated for by the salvage pathway.
old5 is characterized by increased TCA intermediates
Since previous studies have shown that alterations in NAD/NADH levels can have
dramatic consequences on cellular metabolism (Shen et al., 2006; Dutilleul et al.,
2005) we performed a comprehensive metabolite profiling using an established GCMS protocol capable of quantifying the relative levels of over 80 metabolites (Lisec et
al., 2006). For this purpose leaves were harvested, in the middle of the light period,
from both the old5 mutant and the wild type at days 21 and 27. Interestingly although
sugar levels in the old5 mutant were similar to the wild type at 21 days, maltose and
sucrose levels were significantly higher at 27 days (Figure 5A). Increased sugar
levels have been observed previously before the onset of senescence (Diaz et al.,
2005; Wingler et al., 2006), suggesting that the old5 lesion invokes an early change
in metabolism reminiscent of senescence. Sugars are subsequently processed
through glycolysis to form the pyruvate needed to support mitochondrial respiration
through TCA cycle (Fernie et al., 2004). After 21 days we found an increase in the
TCA cycle intermediate succinate in the mutant in comparison to the wild type,
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whereas other intermediates of this cycle were invariant (Figure 5B). However, at this
time point amino acids derived from TCA cycle intermediate α-ketoglutarate
accumulate in old5 to higher levels than in the wild type. These amino acids include
Figure 5. Metabolite profiling of old5 leaves along development. Relative metabolite content
of leaves at day 21 and day 27 is given together with change in time as indicated by ratio of
27/21 (old5 gray bars, wild type solid black bars). Metabolite contents were identified and
quantified by GC-MS, and their relative amounts were calculated as described by RoesnerTunali et al., (2003) relative to wild type control. Histograms show the relative amount of (A)
soluble sugars, (B) TCA-cycle intermediates and (C) amino acids.
glutamate, glutamine, proline and the polyamine spermidine (Figure 5C). Importantly,
aspartate, which is the precursor for the de novo NAD synthesis was also
significantly up regulated in old5 after 21 days. From aspartate several other
compounds are synthesized of which asparagine was observed to accumulate after
21 days in old5. At 27 days all the measured α-ketoglutarate derived compounds
including GABA (4-amino-butyric acid), ornithine, putrescine and β-alanine were
more abundant in old5. Furthermore the branched-chain amino acids isoleucine and
valine, that derive from pyruvate (Binder et al., 2007), are significantly higher after 27
days of growth in the old5 mutant. Isoleucine and valine have previously been
documented to accumulate in mutants of the electron-transfer flavoprotein complex
which display accelerated dark induced senescence (Ishizaki et al., 2005; 2006).
Moreover, aspartate precursor oxaloacetate, as well as succinate and malate are
135
more abundant at 27 days in the mutant. These changes might be indicative for an
increased mitochondrial metabolic rate in old5 mutants (Noctor et al., 2007).
Previously it was shown that redox manipulation by DTT treatment of Arabidopsis
leaves increases the level of succinate, malate and oxaloacetate (Kolbe et al., 2006),
suggesting that the changes in NAD/NADH level maybe the direct cause of the
increase found in TCA cycle intermediates for the old5 mutant.
Comparison of differences in the metabolite levels observed between 21 and 27 days
may reveal differential metabolic regulation in the mutant with respect to the wild
type. Although the levels of the TCA cycle intermediates are higher in old5 than in the
wild type at both time points, the pattern of change (i.e. decrease) between the time
points is conserved across the genotypes (Figure 5B). However, that is not true for
pryridine nucleotide levels which in the wild type decrease with time (Figure 4A) while
in old5 they increase (Figure 4A).
Increased respiration rate and antioxidant accumulation in old5
Both increased amounts of organic acids and higher levels of pyridine nucleotide can
be attributed to an increased rate of respiration (Priault et al., 2007).The metabolite
profiling results suggested that the changed redox state in old5 leads to an altered
respiration rate. To analyze that possibility we determined the oxygen consumption of
Figure 6. Increased respiration of old5 coincides with increased glutathione levels. (A) The
oxygen consumption in the dark was measured and quantified with a clark-type electrode
(old5 gray bars, wild type solid black bars). (B) Elevated levels of glutathione (GSH) are
observed in old5 when compared to the wild type. GSH was extracted from leaf tissues of 21
and 27 days old plants and the analysis was performed using reverse phase (RP)-HPLC.
Asterisks indicate statistical significance versus the wild type control (* P < 0.05, Student’s t
test).
136
whole leaves in the dark. Figure 6A shows oxygen consumption at 21 and 27 days.
At both time points the old5 mutant consumes approximately 2-fold more oxygen
than the wild type demonstrating an altered respiration rate. An over reduction of the
mitochondrial electron transport chain (mETC) gives rise to increased ROS
production which can be alleviated by activation of the alternative oxidase 1 (AOX1)
(Noctor et al., 2007). The transcript abundance of AOX1 is also activated directly by
TCA cycle intermediates (Vanlerberghe et al., 1997; Gray et al., 2004). Figure 7
shows that AOX1 is constitutively higher expressed in old5 further supporting the
notion that the mutant has a higher respiration rate than the wild type. Higher
respiratory rates are generally coupled to an increased production of ROS by
complex I and III of the mETC (Apel and Heribert, 2004; Noctor et al., 2007). The first
Figure 7. Relative expression of marker genes indicative of oxidative stress. The relative
expression of several genes was determined by real time PCR and calculated relative to the
wild type. The alternative oxidase is constitutively higher expressed (old5 gray bars, wild type
solid black bars). Two oxidative stress marker genes were tested and found to be
upregulated in the mutant (At2g23420 and At3g13610). Next to that GDH is more abundant
than in the wild type. The values shown are the means of three repeats ± SD (indicated by
error bars). Experiments were repeated and showed the same results. Asterisks indicate
statistical significance versus the wild type control in each case (* P < 0.05, Student’s t test).
137
indication that old5 suffers from ROS stress is SAG13 expression (Figure 2) which is
associated with oxidative stress (Miller et al., 1999) and low antioxidants levels
(Conklin and Barth, 2004) as well as with senescence. Moreover the mutant showed
a change in expression of two other well-established ROS marker genes: At2g43510
and At3g13610. The defensin-like gene At2g43510 which has previously been shown
to be ubiquitously induced by various ROS (Gadjev et al., 2006) was constitutively
expressed at higher levels in old5 leaves and was strongly upregulated after 30 days
(Figure 7). The second marker, 2-oxoglutarate-dependent dioxygenase At3g13610
which is specifically expressed during H2O2-related stress (Gadjev et al., 2006)
showed a distinct expression after 24 days in old5 which increased rapidly with age.
ROS are continuously generated as a by-product of aerobic metabolism and need to
be rapidly detoxified by either enzymatic or non-enzymatic pathways since they are
highly toxic (Apel and Heribert, 2004). Protection against oxidative stress is partially
provided for by low molecular weight antioxidants such as glutathione and ascorbate.
The thiol glutathione, which is synthesized from its constituent amino acids, L-Glu, LCys, and Gly was more abundant in the mutant. Moreover glutathione levels
increased from 21 to 27 days in old5, but decreased in the wild type (Figure 6B).
Finally ROS stress in Arabidopsis has previously been reported to affect nitrate
assimilation catalyzed by glutamate dehydrogenase (Skopelitis et al., 2006). The
altered metabolite profile and increased expression of ROS marker genes coincide
with an increased expression of GDH from day 27 in old5 mutant (Figure 7). Taken
together, these results show that the old5 mutation results in both a different
metabolite profile as well as oxidative stress.
Discussion
old5 reveals a role for NAD in plant ageing
Plants differ fundamentally from animals in the sense that they do not have a rigid
body plan and their organs can easily be added or removed making them highly
adaptive to the environment. As a result of developmental and adaptive strategies
that resist, avoid and anticipate ageing, it is suggested that plants do not age at all in
any sense recognizable in animals (Thomas, 2002). However, the lifespan of
individual leaves clearly resembles animal ageing (Gan, 2003; Jing et al., 2003; Lim
et al., 2003; Schippers et al., 2007). That is especially visualized in Arabidopsis
138
where the onset of leaf senescence occurs in an age-dependent way (Gan and
Amasino, 1997; Quirino et al., 2000; Jing et al., 2002). Moreover, the recent
identification of genes that affect the onset of leaf senescence (Woo et al., 2001; Jing
et al., 2005; Jing et al, 2007) reveals common mechanisms in the regulation of
ageing between plants and animals.
In this study we observed early leaf senescence in the mutant old5 induced by age,
as indicated by the expression of SAG12. The cloning of old5 identified a mutation in
the gene encoding the quinolinate synthase (QS). QS is an essential enzyme in the
de novo synthesis of the pyridine nucleotide NAD (Katoh et al., 2006). Pyridine
nucleotides have well-characterized roles as redox carriers in processes such as
oxidative
phosphorylation,
the
TCA
cycle,
and
as
electron
acceptors
in
photosynthesis (Hunt et al., 2004). Next to their role in metabolism they play a role
during several stress conditions including oxidative stress (Moller, 2001; Hayashi et
al., 2005), wound-response (Sinclair et al., 2000), ABA-signaling and salt stress
(Shen et al., 2006; Wang and Pichersky, 2007). Recently it was found that the NADdependent histone deacylase SIR2 influences the lifespan for several model
organisms including yeast, C. elegans, mice and human cells (Lin et al., 2000;
Tissenbaum and Guarente, 2001; Lim et al., 2006). Remarkably, SIR2 is involved in
both mitotic and post-mitotic ageing as demonstrated by yeast and human cells
affecting replicative lifespan and C.elegans, respectively. The current view is that
NAD levels serve as a metabolic sensor which is integrated in the developmental
program by the effect of SIR2 on chromatin remodeling (Grewal and Moazed, 2003).
Here we show that a mutation that causes alteration in the level of NAD influences
the lifespan of Arabidopsis suggesting a possible conservation for the role of NAD on
development and ageing in plants.
old5 mutation results in a metabolite profile characteristic for early ageing
Redox reactions are the fundamental metabolic processes through which cells
convert and distribute the energy that is necessary for growth and maintenance
(Noctor, 2006). Changes in the availability of pyridine nucleotide levels were shown
before to have a dramatic effect on the metabolite profile of plants (Dutilleul et al.,
2005; Shen et al., 2006). Next to that, increased levels of NADH affect other
reactions that produce NADH, and disturb the cellular oxide/redox balance to cause
oxidative stress (Shen et al., 2006; Moller, 2001). The increased NAD levels in the
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old mutant coincide with increases in the level of TCA cycle intermediates and the
precursor for NAD biosynthesis aspartate. In addition nitrogen metabolism is strongly
affected since the amino-acids deriving from ketoglutarate are more abundant. A
previous study on the Tobacco nad7 mutant revealed a similar result, in which the
mutant has NAD/NADH levels higher than the wild type and accumulation of nitrogen
containing amino acids (Dutilleul et al., 2005).
Given that the onset of leaf senescence in old5 is age-induced it could be anticipated
that certain metabolites are characteristic for an age-related change (ARC) of the leaf
(Diaz et al., 2005; Jing et al., 2005). The difference in metabolite profile between the
mutant and the wild type is more pronounced with increasing age. Several ARCs of
metabolism specific for the onset of leaf senescence are observed in old5. First the
accumulation of the branched chain amino acids valine and isoleucine in old5
mutants was previously shown to occur before the onset of senescence (Masclaux et
al., 2000; Diaz et al., 2005; Ishizaki et al., 2005). One of the most intriguing
metabolites that shows an age-related accumulation is GABA (4-amino-butyric acid),
which accumulates in old5, but not in the wild type at day 27. Studies on Osl2 a
mitochondrial GABA transaminase in rice (Ansari et al., 2005) suggest a switch in
metabolism at the onset of senescence. Moreover, it seems likely that in plants, as in
animals, the GABA shunt is up regulated in times of high respiratory demand
(Sweetlove et al., 2007). In addition to this it has been found that GABA induces
senescence in sunflower by enhancing the synthesis of ethylene (Kathiresan et al.,
1997). This is in line with a previous study in which we suggested that the ethyleneinduced leaf senescence of old5 is depending on ARCs (Jing et al., 2005). The
identified metabolites might serve as useful biomarkers to determine the viability of
leaves and progression of senescence.
In addition to previously documented markers of ageing we observed the
accumulation over development of the polyamines in the old5 mutant. Spermidine
has the same precursor as aminocyclopropane (ACC), an important source of
ethylene which can induce premature senescence (Imai et al., 2004). However, the
accumulation of the polyamines might not be an ARC perse but may also reflect a
protection against ROS generation and programmed cell death (Papadakis et al.,
2005). Moreover polyamines as cytokinins are well-known for their senescence
retarding effect.
140
Interaction between de novo synthesis and the salvage pathway of NAD and their
role in stress signaling
The analysis of old5 quinolinate synthase mutant protein showed that it weakly
interacts with components of Fe-S biogenesis complex, CpNifS and CpNifS3 in a
yeast two-hybrid experiment. As the QS is dependent on a functional Fe-S
biogenesis complex (Loiseau et al., 2005), the observed weak interaction likely leads
to a lower in vivo activity of the whole protein. Thus we anticipated that the recessive
mutant allele old5 results in a decreased activity of the enzyme and we expected
lower levels of NAD/NADH. However, the analysis revealed an increased NAD/NADH
level for the mutant. The maintenance of the NAD pool is both due to the de novo
synthesis and pyridine salvage pathway. Whereas the de novo synthesis is
absolutely required for plant survival (Katoh et al., 2006) the recycling pathway is
mainly important during stress to recycle the degraded NAD in an energy efficient
manner (Wang and Pichersky, 2007). In apparent response to the retardation of QS,
old5 plants exhibit high expression of aspartate oxidase the first enzyme in de novo
synthesis of NAD (Figure 4). We furthermore observed an increased expression of
the two key enzymes involved in the salvage pathway, AtNiC1 and NaPRT2. The
results point out that old5 compensates for the decreased de novo synthesis of NAD
by up regulation of the salvage pathway of pyridine nucleotides. That is in agreement
with results of Geigenberger et al., (2005) who previously reported that inhibition of
de novo pyrimidine biosynthesis results in a compensatory stimulation of the salvage
pathway. Moreover enhanced expression of the salvage pathway enzyme AtNIC1
has previously been shown to increase the NAD levels in Arabidopsis (Wang and
Pichersky, 2007; Hunt et al., 2007). The increased expression of the NAD salvage
pathway is reminiscent to the need to replenish the NAD pool during stress
conditions (Berglund et al., 1996; Wang and Pichersky, 2007; Hunt et al., 2007).
Therefore the adjustment in NAD metabolism of old5 might result in stress signaling.
One example of a NAD-consuming enzyme induced by oxidative stress is PARP (De
Block et al., 2005) linking increased salvage metabolism to stress. Next to that the
salvage pathway was shown to be activated and necessary for response to osmotic
stress in Arabidopsis (Wang and Pichersky, 2007).
141
Role for mitochondria in early ageing of old5
Longevity is clearly genetically controlled in many species including yeast, C.
elegans, mice and man (Jing et al., 2003). Almost 100 years ago it was postulated
that there is an inverse relationship between metabolic rate and longevity (Rubner,
1908; Pearl, 1928). Nowadays it is clear that caloric restriction (CR) can greatly
extend the maximum lifespan of many species (Sohal and Weindruch, 1996). The
discovery of the first gene (PHA-4) absolutely required for lifespan extension by CR
in C. elegans supports a role for ROS generated by mitochondrial oxidative energy
metabolism in lifespan determination. This is especially evident by the fact that
reduced PHA-4 expression does not suppress the long lifespan of animals with
defective electron transport chains (Panowski et al., 2007). These results fit within the
free-radical theory of ageing, which proposes that ROS produced by respiration
contribute to the ageing of all organisms (Harman, 1956).
In our study we show that the mutant old5 has an increased respiration rate together
with increased oxidative stress. The reducing equivalents that are generated from
TCA-cycle activity are used by the mitochondrial electron transport chain to power
the synthesis of ATP (Fernie et al., 2004). The increased NADH levels in the mutant
might directly influence the rate of mitochondrial electron transport (Hunt et al., 2004).
This results in expression of the alternative oxidase which by-passes the
phosporylation and does not contribute to ATP production but alleviates over
reduction of the chain (Moller, 2001). Transgenic tobacco cells that fail to induce
alternative oxidase go rapidly into programmed cell death (PCD) during oxidative
stress (Vanlerberghe et al., 2002). The increased glutathione levels and increased
expression of H202-induced stress markers in our mutant fit well with previous studies
that during oxidative stress mitochondria produce increased amounts of H202
(Sweetlove et al., 2002; Tiwari et al., 2002; Gadjev et al., 2006). ROS have a role in
the onset of developmental senescence. The natural senescence of pea leaves
coincides with increases in the levels of superoxide and H202 (Pastori and del Rio,
1997). Treatment of Arabidopsis leaves with the herbicide 3-AT inhibits catalase
activity and causes H202 stress resulting in the expression of SAG genes (Navapbour
et al., 2003). Next to that the senescence-specific transcription factor WRKY53 is
induced by H202 (Miao et al., 2004; Miao and Zentgraf, 2007). Taken together this
demonstrates that oxidative stress can function as a signal for the onset of leaf
senescence. Thus the possible accumulation of damage to the mitochondria by
142
increased ROS in old5 might be at the basis of early ageing of the mutant as denoted
by the “free-radical“ theory of ageing (Harman, 1956).
In general it is believed that oxidative stress results in damage to the cell which loses
its viability. To determine if oxidative stress in our mutant results in damage or serves
as a signal for the onset of senescence additional experiments are required.
Making use of Arabidopsis leaves as a model system for plants we have exploited
the opportunity to characterize the mechanisms and genetics of ageing. Our study
supports the idea that an increased mitochondrial respiration rate affects lifespan as
denoted by the “free-radical” theory of ageing. It suggested that factors important for
mammalian ageing appear to be conserved across kingdoms. Since ageing is
manifested in many ways, cloning and physiological characterization of other old
mutants will allow a fuller elucidation of the subtle differences between the various
mechanisms of ageing and senescence.
Methods
Plant materials and growth conditions
Arabidopsis thaliana ecotype Landsberg erecta (Ler-0) was used throughout this
study. The old5 mutant was obtained from an EMS mutaganized collection (Jing et
al., 2005). T-DNA insertion mutant QS-1 (SALK_079205) was obtained from the
Nottingham Arabidopsis Stock Centre (NASC). Complementation of the old5
mutation was completed by making use of the binary vector pGreen029 (Hellens et
al., 2000). Plants were grown on either soil or half-strength Murashige and Skoog
medium at 23°C and 65% relative humidity with a day length of 16 h. The light
intensity was set at 120 µmol·m-2·s-1. An organic-rich γ-ray radiated soil was used
(Hortimea Groep, Elst, The Netherlands).
Pigment determination and measurement of photochemical efficiency
For extraction of chlorophyll and carotenoids samples were incubated overnight with
N,N-dimethylformamide at 4°C in darkness. The chlorophyll content was quantified
spectrophotometrically according to Wellburn (1994) at 647 and 664nm.
Chlorophyll fluorescence emission was measured from the upper surface of the first
leaf, at room temperature (23 °C) with a pulse-amplitude modulation portable
143
fluorometer (PAM-2000; H. Walz, Effeltrich, Germany) according to Maxwell and
Johnson, 2000. Plants were dark-adapted for 1 to 2 hr before measurements to
ensure complete relaxation of the thylakoid pH gradient. An attached, fully expanded
rosette leaf was placed in the leaf clip, allowing air to circulate freely on both sides of
the leaf. At the start of each experiment, the leaf was exposed to 2 min, of far-red
illumination (2 to 4 µmol photons m-2 s-1) for determination of Fo (minimum
fluorescence in the dark-adapted state). Saturating pulses of white light (8000 µmol
photons m-2 s-1) were applied to determine Fm or Fm' values. PSII efficiency was
calculated as (Fm - Fo)/Fm.
RNA-isolation and RT-PCR
Total RNA was isolated using TRIZOL reagent (Sigma) according to the
manufacturer's protocol. Five hundred nanograms of RNA were used as template for
first-strand cDNA synthesis using 200U of RevertAid H-minus MMuLV reverse
transcriptase (Fermentas, USA) and an oligo(dT21) primer. Primer pairs for real-time
PCR were designed with open-source PCR primer design program PerlPrimer
v1.1.10 (Marshall, 2004). The primer sequences are available upon request. Briefly,
real-time PCR amplification was performed with 50 µL of reaction solution, containing
2 µL of 10-fold–diluted cDNA, 0.5 µl of a 10 mM stock of each primer, 1 µl of 25mM
stock MgCl2 (Fermentas), 5 µl PCR buffer +Mg (Roche), 1 µl of a 1000x diluted
SYBR-green stock (Sigma), 0.5 µl 100xBSA (New England Biolabs), and 1u of
Roche Taq Polymerase. The PCR program was 2’ at 94, 40x (94-10”/60-10”/72-25”),
meltcurve. Obtained data was analyzed with BioRad software.
Determination of metabolite levels
Leaf samples were taken at the time points indicated, immediately frozen in liquid
nitrogen and stored at –80°C until further analysis. Extraction was performed by rapid
grinding of tissue in liquid nitrogen and immediate addition of the appropriate
extraction buffer. The relative levels of metabolites were determined using an
established gas chromatography coupled to a time-of-flight mass analyzer (GC-TOFMS) protocol as described by Lisec et al., 2006. Data are presented normalized as
detailed by Roessner et al., 2001. The procedure of extraction and assay of
nicotinamide adenine dinucleotides was performed according to the method
144
described by Gibon and Larher, 1997. The determination of glutathione contents was
performed as described Kreft et al., 2003.
Yeast two-hybrid experiments
The coding sequence of the selected genes were amplified from RNA and cloned in
frame with the GAL4 binding domain of the pGBKT7 and GAL4 activation domain of
the pGAD424 vector (Clonetech). For protein-protein interaction screening, PJ69-4A
(James et al., 1996) was transformed according to the Clonetech yeast protocols
handbook (PR13103) and selected on synthetic dropout (SD) medium lacking Leu
and Trp for transformants. Subsequent colonies were dissolved in SD and spotted on
selection medium for interaction by testing growth on SD lacking either Ade or
medium lacking His with the addition of 2mM 3-amino-1,2,4-triazole. Plates were
incubated at 28 degrees for 2-5 nights.
Respiratory measurements
Assays of oxygen consumption by whole leaves were performed using a Clark-type
oxygen electrode. Leaves were placed on a filter containing CO2 buffer and
subsequently the measuring room was filled after calibration with a 2% oxygen
containing gas before measurement. Measurements were performed in 6 fold and
fresh weight and chlorophyll content of the leaves was determined.
Accesion numbers
The Arabidopsis Genome Initiative locus numbers for the major genes discussed in
this article are At1g08490 for CpNifS1, At1g18490 for CpNifS2, At1g55090 for NADS,
At2g01350 for QPRT, At2g22570 for AtNIC1, At2g23420 for NaPRT2, At2g29350 for
SAG13, At2g43510 for defensin-like, At3g13610 for oxidoreductase, At4g26500 for
AtSufE, At4g36940 for NaPRT1, At5g07440 for GDH, At5g14760 for AO, At5g26600
for CpNifS3, At5g50210 for QS/OLD5 and At5g45890 for SAG12.
Acknowledgements
We would like to thank Bert Venema and Margriet Ferwerda for their excellent
technical support. We also show gratitude to Anne de Jong at the Laboratory of
Molecular Genetics of the University of Groningen for support with RT-PCR.
145
Furthermore we would like to thank Ying Miao and Marcel Proveniers for their
support with the yeast two hybrid experiments.
146
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Summary
Leaf senescence is a genetically regulated process which enables plants to relocate
valuable resources from dying tissue to other parts of the plant. The notion that
thousands of genes are associated with leaf senescence illustrates the complex
biological mechanisms underlying this process. The onset of this program is agedependent in Arabidopsis. Recent studies have led to the isolation of several genes
that alter the timing of leaf senescence. Therefore the leaf is an excellent model to
study the genetic network that underlies the ageing process. The study presented in
this thesis is focussed on isolating genes that regulate the timing of leaf senescence.
In addition, hormonal responses and metabolic processes are characterized to
identify age-related changes that are at the basis of the onset of leaf senescence.
The combination of these results allow for defining important insights into the onset of
leaf senescence.
Hormones are universal regulators of developmental and metabolic processes in all
living organisms. Plant hormones can have senescence retarding or senescence
promoting. One of the best characterized senescence-promoting hormones is the
gaseous molecule ethylene which is involved in several physiological processes
throughout the plant's life cycle. Ethylene promotes the onset of leaf senescence
after a certain developmental stage (Chapter 1). This relation between ethyleneinduced senescence and leaf age was comprehensively studied in Chapter 2.
Interestingly, it was found that an increase in the length of the ethylene exposure
results in a more pronounced senescence response. However, when the exposure to
ethylene is more than 12 days, the senescence response is reduced. Thus the timing
and length of the ethylene treatment influences the senescence response in plants
with the same final age. It is argued that certain age-related changes are necessary
to either promote or reduce the effect of ethylene on the induction of leaf
senescence. The use of eight early senescing onset of leaf death (old) mutants
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indicates that individual genes can modulate the outcome of ethylene-induced
senescence. An important notion here is that the loci influence age-related changes
which constitute molecular checkpoints for the onset of leaf senescence. Thus, the
induction of senescence by ethylene is the result of a complex interaction between
leaf age and ethylene which can be modulated by multiple genetic loci.
Three of the eight mutants presented in chapter 2 were chosen for a detailed
characterization to identify novel components involved in the onset of leaf
senescence. Two of the three selected mutants showed only early leaf senescence
upon ethylene treatment. Since the action of ethylene is age-dependent it was
anticipated that specific mechanisms involved in the interaction between leaf age and
ethylene could be identified. Most senescence mutants described in literature have
additional defects like the delayed senescence mutant ore9, which has a highly
branched shoot. Another example is old1, which is smaller than wild type and is
hypersensitive to sugar and ethylene. Therefore it is unlikely that genes have evolved
that solely have a function in the regulation of leaf senescence. However, the three
mutants were mainly selected for their similar development as the wild type and
might therefore represent loci specific for the onset of leaf senescence
Results obtained with early senescence mutant old13, indicate that the gene product
is involved in several plant programs that can result in senescence. In chapter 3 it is
presented that the mutant displays a hypersensitive response-like phenotype after
treatment with ethylene. Moreover, expression data indicates that the mutant is
involved in plant defense. Next to that the mutant displays early senescence upon
lack of watering and has an increased intracellular anion concentration. Common
signaling molecules involved in biotic and abiotic stress are the reactive oxygen
species (ROS) which are enhanced in the mutant. Taken together, the OLD13 gene
product is suggested to function as a modulator of leaf senescence upon stress in an
age-dependent way. The distinct role of the old13 gene product in senescence
processes is an exciting finding that might indicate that specific regulatory genes
exist.
Chapter 4 reports on the characterization of the early leaf senescence mutant old9.
The mutant displays specifically early ethylene-inducible senescence. The analysis of
the old9 mutant reveals two novel insights in the regulation of the onset of leaf
senescence. Transcriptome analysis of the mutant points at a role for the plant
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hormone cytokinin in the early senescence in the mutant. Cytokinins are known for
their effect to retard the onset of leaf senescence (Chapter 1). However, in this study
it is shown that exogenous application of cytokinin to wild type, early in development
results in an enhanced response to ethylene-induced senescence. Thus, cytokinin
can both induce and delay the timing of leaf senescence depending on the age of the
plant. The cytokinin mediated senescence response is argued to be related to sinkto-source change of the leaf. Another interesting finding is that a cell wall associated
protein can modulate the senescence response of the old9 mutant. A gene encoding
for a hydroxyproline rich glycoprotein is upregulated in air-grown old9 mutants.
Downregulation of the expression level of the transcript by RNA-interference results
in a decreased senescence response of the mutant. The role for cell wall proteins in
the onset of leaf senescence has not been described in literature and represents a
novel pathway involved in the process.
In chapter 5 the characterization and identification of old5 is presented. OLD5
encodes quinolinate synthase, a key enzyme in the de novo synthesis of NAD.
Recently it was found that the NAD-dependent histone deacylase SIR2 influences
the lifespan of several model organisms including yeast, C. elegans, mice, and
human. It is proposed that NAD levels serve as a metabolic sensor which is
integrated in the developmental program by the effect of SIR2. This implies that
ageing in plants can be compared with animal ageing when individual leaves are
used. It has been argued before that plant and animal ageing share similar
strategies; however, this is the first study that reports on a common molecular
pathway. Moreover, it is argued that the early ageing of the mutant is the result of
oxidative stress in the mitochondria, thus, implying that the “free-radical” theory of
ageing can be applicable to plants. The results illustrate that the leaf is an excellent
model system for ageing research in plants that will increase the understanding of
senescence mechanisms in animals as well.
The identification of old5 shows that central metabolism is involved in ageing. The
recent developments in the field of metabolomics add a new tool for the identification
of age-related processes. In chapter 5 it is presented that several metabolites have a
distinct age-related accumulation in leaves of Arabidopsis. These metabolites include
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valine, isoleucine, and GABA. Thus next to transcripts, metabolites appear to mark
age-related changes during development. A combination of metabolite profiling
together with transcriptome data will reveal novel mechanisms and pathways that
underlie ageing processes in plants.
Ten years ago senescence was described as almost too complex and difficult
biological phenomenon to fully comprehend. However, the development of novel
molecular biological techniques has revolutionized the field and the understanding of
the process. Next to that, a continuous increasing number of genes that regulate leaf
senescence are being described. The dedication of more and more research to leaf
senescence will advance the understanding of the process. Therefore the coming
years will emerge as exciting and inspiring when the mechanisms underlying
senescence are being unravelled.
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Samenvatting
De kleurverandering van bladeren is een van de opvallendste kenmerken van de
herfst voordat ze afsterven en afvallen. Tijdens de herfst worden de bouwstoffen die
aanwezig zijn in het blad gerecycled en opgeslagen om na afloop van de winter
gebruikt te worden om nieuwe bladeren te laten uitlopen. Ons woord herfst is
afgeleid van het oud Germaanse woord ‘harbista’, dat via het Hoogduitse ‘herbist’ en
het Middelhoogduitse ‘herbest’ uiteindelijk het Middelnederlandse ‘hervest’ werd. Het
Engelse woord harvest wat oogsten betekent heeft echter niet meer de betekenis
van jaargetijde behouden zoals in Nederland. De herfst is het jaargetijde van het
oogsten en dat doen zowel mensen als planten. Het afsterven van bladeren is niet
passief. Voordat ze afsterven, halen veel planten de voedingsstoffen terug uit hun
bladeren. Ze worden opgeslagen in de wortels en de stengel voor het volgende
voorjaar of worden gebruikt voor de aanmaak van zaden en vruchten. Tijdens mijn
onderzoek heb ik gewerkt met de modelplant Arabidopsis thaliana, oftewel een
onkruid dat vrijwel overal in Nederland is te vinden en ook wel zandraket wordt
genoemd. De zandraket wordt gekenmerkt door een korte levenscyclus wat het
mogelijk maakt om tijdens een promotieonderzoek een bepaalde eigenschap te
volgen in opeenvolgende generaties. Sinds de ontdekking van de structuur van het
DNA zijn wetenschappers in staat om eigenschappen van een organisme te
herleiden tot een gen. Tegenwoordig is de volledige erfelijke informatie die is
opgeslagen in het DNA van de zandraket bekend en weten we dat het voor ongeveer
32000 genen codeert. Een aantal studies heeft aangetoond dat ruim tweeduizend
genen betrokken zijn bij het afsterven van bladeren. Dit toont aan dat bladafsterving
een genetisch gecontroleerd proces is en dus een belangrijk onderdeel vormt van de
levenscyclus van de plant. De bladeren van de zandraket sterven af zodra ze een
bepaalde leeftijd hebben bereikt, daarnaast kan de plant er voor zorgen dat een blad
afsterft zodra deze stress krijgt. Dus een deel van de plant kan worden opgeofferd
voor het behoud van de rest. Een belangrijk plantenhormoon dat het afsterven van
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bladeren bevordert is ethyleen. Daarnaast zorgt ethyleen voor het rijp worden van
bananen en appels maar heeft ook als effect dat snijbloemen snel verwelken.
Daarom mogen vruchten en bloemen nooit samen vervoerd worden. Naast ethyleen
is er een ander plantenhormoon, cytokinine wat juist een anti-verouderings effect op
bladeren heeft en ervoor zorgt dat ze langer groen blijven.
Mijn onderzoek is voornamelijk gericht op veranderingen in bladeren die optreden
tijdens het verouderen. Deze wijzigingen kunnen een signaal zijn voor de plant om
een blad te laten afsterven. Omdat het afsterven van de bladeren van de zandraket
wordt gereguleerd door de leeftijd van het blad vormt het een uitstekend testsysteem.
De studie die hier beschreven is heeft als doel genen te identificeren die betrokken
zijn bij het verouderingsproces van de bladeren. Daarnaast is het effect van het
plantenhormoon ethyleen onderzocht op de ontwikkeling van de zandraket. Naast
het genetisch onderzoek is er ook bepaald hoe het metabolisme van de bladeren
verandert tijdens het ouder worden. De combinatie van de onderzoeksresultaten
heeft nieuwe inzichten opgeleverd over de regulatie van de bladveroudering.
Resultaten van het onderzoek kunnen worden toegepast om uiteindelijk de
opbrengst van gewassen te verbeteren.
Hormonen zijn universeel betrokken bij de ontwikkeling, groei en metabolische
processen van alle organismen. Plantenhormonen kunnen zowel bladafsterving
stimuleren als voorkomen. Het plantenhormoon ethyleen is betrokken bij vele
aspecten van de ontwikkeling van de plant waarvan het aanzetten van bladafsterving
een aspect is. Voor het identificeren van genen die van invloed zijn op de
bladveroudering is er gebruik gemaakt van een mutant analyse. Door zaden van de
zandraket chemisch te behandelen kunnen er mutaties optreden in het DNA. Het
veranderen van een genetische eigenschap op deze manier kan gevolgen hebben
voor de bladveroudering. Met behulp van een ethyleen behandeling is het mogelijk
om mutanten te selecteren die verschillen in hun bladveroudering ten opzichte van
de controle planten. Op deze manier zijn er ruim 100 onafhankelijke mutanten
geïsoleerd. De grote vraag die overblijft is dan, welke van de 32000 genen zijn er
veranderd in deze mutanten? Naast het toepassen van ethyleen voor het
identificeren van genen die mogelijk betrokken zijn bij de bladveroudering is het
effect van ethyleen ook onderzocht tijdens de ontwikkeling van controle planten.
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In hoofdstuk 1 en 2 van dit proefschrift wordt de relatie tussen de leeftijd van een
blad en het effect van ethyleen op de inductie van bladafsterving uitgebreid
beschreven. Ethyleen kan alleen bladafsterving induceren wanneer het blad een
bepaalde leeftijd heeft bereikt, dus jonge bladeren zijn ongevoelig voor het hormoon.
Het onderzoek toonde aan dat het effect van ethyleen op bladveroudering afhankelijk
is van de tijdsduur van blootstelling aan het hormoon. Een langere ethyleen
behandeling resulteert in sterkere verouderingssymptomen, echter een extra lange
behandeling vermindert de effecten van ethyleen op veroudering. Dus het effect van
ethyleen op de inductie van bladveroudering hangt af van de leeftijd van een blad en
de lengte van de behandeling. Naast dit effect van ethyleen op de controle planten
zijn er ook acht mutanten getest. De mutanten worden old (onset of leaf death)
genoemd. De behandeling van de mutanten met ethyleen toont aan dat een enkele
genetische eigenschap een rol kan spelen bij de regulatie van de bladveroudering.
Drie van de acht mutanten die beschreven zijn in hoofdstuk 2 zijn gekozen voor een
gedetailleerde studie om nieuwe genetische componenten te identificeren die
betrokken zijn bij bladveroudering. Twee mutanten, old13 en old9 vertonen alleen
vervroegde bladveroudering tijdens een behandeling met ethyleen. Omdat het effect
van ethyleen leeftijdsafhankelijk is verwachten wij door het bestuderen van deze
mutanten het mechanisme van de interactie tussen leeftijd en ethyleen beter te
begrijpen. Tot nog toe zijn er geen specifieke bladverouderingsgenen gevonden en
hebben de bekende mutanten vaak een veelvoud aan extra defecten. Vanuit het
perspectief van evolutie is het ook minder aannemelijk dat er specifiek genen zijn die
alleen tijdens de laatste ontwikkelingsfase van een blad een rol spelen.
De old13 mutant wordt beschreven in hoofdstuk 3 van dit proefschrift en is betrokken
bij verscheidene processen in de plant. Wanneer een blad wordt aangevallen door
een pathogeen dan kan er een lokale celdood optreden waardoor de invasie kan
worden verhinderd. De old13 mutant vertoont deze eigenschap tijdens de ethyleen
behandeling. Ondanks dat de mutant een normale ontwikkeling vertoont is hij
overgevoelig voor droogte stress. Dit heeft waarschijnlijk te maken met het feit dat de
interne concentratie van anionen (chloride, nitraat en sulfaat) sterk verhoogd is.
Belangrijke signaalmoleculen tijdens zowel pathogeen detectie, droogte stress en
bladafsterving zijn zuurstof radicalen. Tijdens de groei van de mutant is er een
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constante verhoogde aanwezigheid van deze signaalmoleculen. Samenvattend kan
het old13 gen product worden gekarakteriseerd als een belangrijke modulator tussen
omgevingsstress en het aanschakelen van het blad afstervingsproces.
In hoofdstuk 4 wordt de old9 mutant beschreven. De resultaten van het onderzoek
naar de mutant resulteerden in 2 nieuwe inzichten. Ten eerste is er bepaald welke
genen actief zijn in de mutant ten opzichte van de controle planten. Dit heeft
aangetoond dat een grote groep genen die kunnen reageren op het hormoon
cytokinine zich anders gedragen dan in de controle planten. Dit is opmerkelijk omdat
cytokinine is gerelateerd aan het voorkomen van veroudering. Echter, een
experiment waarin controle planten worden behandeld met cytokinine tijdens
verschillende fasen van ontwikkeling toont aan dat het anti-verouderingshormoon
ook veroudering kan veroorzaken. Wanneer planten op jonge leeftijd een
behandeling krijgen met cytokinine dan resulteert dit in een verhoogde bladafsterving
tijdens de ethyleen behandeling. Een ander interessant resultaat is dat een eiwit dat
een onderdeel vormt van de celwand betrokken is bij de vervroegde bladafsterving in
de old9 mutant. In de mutant is het gen dat voor de aanmaak van dit eiwit codeert
sterk actief, echter wanneer dit gen wordt geblokkeerd vermindert de reactie van de
mutant op de ethyleen behandeling. De rol van celwand eiwitten in de regulatie van
de bladveroudering is een nieuw inzicht dat om verder onderzoek vraagt.
In het laatste hoofdstuk van dit proefschrift wordt de old5 mutant gepresenteerd. De
mutant toont vervroegde veroudering tijdens normale groei omstandigheden en heeft
een enkele weken kortere levenscyclus. De mutant wordt veroorzaakt door een
mutatie in een gen dat betrokken is bij de productie van het energiemolecuul NAD
waarvan vitamine B3 een onderdeel is. NAD is belangrijk voor vrijwel alle metabole
processen in de cel. Recent onderzoek in muizen, gist en mensencellen toont aan
dat NAD direct is gekoppeld aan veroudering. Het onderzoek geeft aan dat de
ontwikkeling en groei van cellen wordt aangepast aan de hand van de aanwezige
hoeveelheid NAD. De old5 mutant vertoont vervroegde veroudering die waarschijnlijk
wordt veroorzaakt door vergelijkbare mechanismen die betrekking hebben op
veroudering in dieren. Het was eerder geopperd dat planten en dieren een zelfde
verouderingsstrategie zouden kunnen hebben, echter dit is de eerste studie die dat
inderdaad ook aantoont. De verandering in NAD niveaus in de mutant gaan gepaard
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met een verhoogd cel metabolisme. Metabolisme afhankelijk van zuurstof gaat altijd
hand in hand met oxidatieve stress. Oxidatieve stress ontstaat voornamelijk in de
mitochondriën waar de verbranding plaatsvindt en wordt gezien als de belangrijkste
oorzaak van veroudering in dieren. Deze studie toont aan dat de mitochondriën in
planten wel degelijk een rol spelen bij veroudering in tegenstelling tot de algemeen
aanvaarde opvatting dat de bladgroenkorrels de belangrijkste bron van oxidatieve
stress zijn. Gezien de parallel tussen dieren- en plantenveroudering toont dit
onderzoek aan dat het blad als een uitstekend model kan dienen voor het onderzoek
naar veroudering.
Door het toepassen van een nieuwe techniek waarbij de concentratie van honderden
verschillende verbindingen in de cel tegelijk kan worden bepaald hebben we nieuwe
factoren geïdentificeerd die wellicht de leeftijd van een blad bepalen. Verscheidene
aminozuren vertonen een sterke ophoping vlak voor de start van de bladafsterving.
Deze componenten kunnen dienen als kenmerken voor oude bladeren en dus nuttig
zijn voor het bepalen van de ontwikkelingsfase van een blad. Daarnaast kan de
combinatie van informatie van het metabolisme met die van de genexpressie worden
gebruikt om nieuwe mechanismen te ontdekken die betrokken zijn bij de
bladveroudering.
Ongeveer 10 jaar geleden werd bladveroudering beschreven als een te complex
biologisch proces om volledig te kunnen begrijpen. Echter de ontwikkeling van
nieuwe moleculair biologische technieken heeft een ware revolutie te weeg gebracht
in het onderzoek. Dit heeft geleid tot een continue toename van publicaties over
genen die betrokken zijn bij bladveroudering. De groei in het aantal studies aan
bladveroudering zal zeker leiden tot nieuwe inzichten in het proces. Daarom zullen
de komende jaren opwindend en inspirerend zijn wanneer de processen die ten
grondslag liggen aan bladveroudering worden onthuld.
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Acknowledgements
I don’t really like to thank people from this position but I will do my best:
Firstly, I would like to thank my Supervisor, Dr. Paul Dijkwel. I could not have
imagined having a better advisor for my PhD, and without his common-sense,
knowledge, perceptiveness, rock-n-roll motivation and cracking-of-the-whip I would
never have finished. I would like to say a big 'thank-you' to Prof. Dr. Jacques Hille for
his support and help during the whole period of the study, especially for staying calm
everytime I exceed a deadline.
Of course I would like to acknowledge the colleagues from MBP and Plant
Physiology for creating a pleasurable atmosphere during my Ph.D. research.
Than I can not forget to mention that due to the enthusiasm of Hai-Chun Jing and his
fantastic work I was able to start a wonderfull fascinating project.
Finally, I have to say 'thank-you' to: all my friends and family, wherever they are,
particularly my Mum and Dad; and, most importantly of all, to my Girlfriend, for
everything.
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