Download Physiology and molecular biology of petal senescence

Journal of Experimental Botany, Vol. 59, No. 3, pp. 453–480, 2008
doi:10.1093/jxb/erm356
FOCUS REVIEW PAPER
Physiology and molecular biology of petal senescence*
Wouter G. van Doorn†,‡ and Ernst J. Woltering
Wageningen University and Research Centre, PO Box 17, 6700 AA Wageningen, The Netherlands
Present address: Mann Laboratory, Plant Science Department, University of California, Davis, One Shields
Avenue, Davis, CA 95616, USA
y
Received 7 September 2007; Revised 16 December 2007; Accepted 17 December 2007
Abstract
Petal senescence is reviewed, with the main emphasis
on gene expression in relation to physiological functions. Autophagy seems to be the major mechanism for
large-scale degradation of macromolecules, but it is still
unclear if it contributes to cell death. Depending on the
species, petal senescence is controlled by ethylene or
is independent of this hormone. EIN3-like (EIL) transcription factors are crucial in ethylene-regulated senescence. The presence of adequate sugar levels in the
cell delays senescence and prevents an increase in the
levels of EIL mRNA and the subsequent up-regulation
of numerous senescence-associated genes. A range of
other transcription factors and regulators are differentially expressed in ethylene-sensitive and ethyleneinsensitive petal senescence. Ethylene-independent
senescence is often delayed by cytokinins, but it is still
unknown whether these are natural regulators. A role
for caspase-like enzymes or metacaspases has as yet
not been established in petal senescence, and a role
for proteins released by organelles such as the
mitochondrion has not been shown. The synthesis of
sugars, amino acids, and fatty acids, and the degradation of nucleic acids, proteins, lipids, fatty acids, and
cell wall components are discussed. It is claimed that
there is not enough experimental support for the
widely held view that a gradual increase in cell
leakiness, resulting from gradual plasma membrane
degradation, is an important event in petal senescence.
Rather, rupture of the vacuolar membrane and subsequent rapid, complete degradation of the plasma
membrane seems to occur. This review recommends
that more detailed analysis be carried out at the level of
cells and organelles rather than at that of whole petals.
Key words: Autophagy, cell death, cell wall, gene expression,
hormones, lipids, nucleic acids, pathogens, proteins, reactive
oxygen species, regulation, remobilization, senescence,
transcriptions factors, ultrastucture.
Introduction
This review is intended to give an update on the literature on
petal senescence. It takes information from a number of
recent papers on changes in gene expression, and relates it to
the events that one might anticipate, in particular those that
have been suggested by previous physiological and biochemical studies. This review will first discuss a model of
petal senescence, then consider the underlying data regarding
ultrastructure, the remobilization of macromolecules, transport of mobile compounds out of the petal, and the role of
autophagy in cellular degradation. This review also pays
attention to some issues of contention such as the localization
of membrane degradation and the possibility that reactive
oxygen species (ROS) contribute to cellular death.
There has been some discussion in the literature about the
distinction between senescence and programmed cell death
(PCD). At least three main categories of PCD might be
distinguished in plants: developmental, pathogen induced,
and abiotic stress induced. Petal senescence is considered to
be a subset of developmental PCD, and in the case of petal
senescence the term ‘senescence’ is considered to be a
synonym of ‘PCD’. The term ‘senescence’ will here be used,
except when emphasis is placed on the actual death of cells.
Most flowers have a whorl of green sepals and another
whorl of variously coloured petals. In contrast, flowers from
genera such as Alstroemeria, Hemerocallis, and Iris have two
whorls of variously coloured flower leaves, called tepals. For
the sake of easy reading, the tepals of these flowers will here
* This paper is dedicated to the memory of our colleague Abraham Halevy, who passed away in 2006. Abe kindly suggested one of us (WvD) that we
should produce the present review.
z
To whom correspondence should be addressed. E-mail: [email protected]
ª The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For permissions, please e-mail: [email protected]
454 van Doorn and Woltering
also be called petals, although sometimes reference will be
made to the whorl from which they originated.
Relationship between petal senescence,
abscission, and ethylene
Depending on the species, petal senescence is visibly
shown by wilting or withering. Petal wilting is due to loss
of turgor. Withering is a colour change and slow dehydration. There seems to be no difference in the
mechanisms underlying wilting and withering.
In most species with flowers showing visible senescence
symptoms, the petals will abscise, whereby the time
between visible symptoms and abscission is quite variable. In some species, for example carnation, abscission
occurs much later than visible senescence. In other
flowers, for example Tulipa and Alstroemeria, visible
senescence and abscission occur at about the same time.
The onset of senescence occurs long before the time of
wilting and abscission of Alstroemeria petals (Breeze
et al., 2004). Petals of many other species abscise when
still fully turgid. It depends on the species whether
senescence processes in these abscising petals have started
or not (Yamada et al., 2007b).
Another category consists of flowers with petals that
show wilting because of water stress in the entire cut
flowering stem. In many rose cultivars, for example, the
petals of uncut flowers abscise without senescence
symptoms. In contrast, the petals of these flowers often
wilt due to an occlusion in the xylem, which blocks the
flow of water to the petals. Such petal wilting is not due to
senescence and will not be discussed here.
In one group of flowers, petal senescence is highly
ethylene sensitive, and regulated by ethylene, whilst in
another group it is ethylene insensitive, and not regulated by
this molecule. This difference was found to correlate with
plant families or subfamilies (Woltering and van Doorn,
1988; van Doorn, 2001). The role of ethylene in hastening
petal senescence seems to have evolved naturally as
a mechanism to allow pollination-induced changes. Rapid
petal wilting (but also petal abscission and changes in petal
colour, insofar as advanced by ethylene) seem to direct
pollinating animals to virgin flowers, thus increasing
pollinator efficiency (van Doorn, 1997).
Species often used in studies on petal senescence
include carnation (Dianthus caryophyllus), Petunia
hybrida, and orchids (ethylene-sensitive senescence), as
well as Mirabilis jalapa, Hemerocallis hybrids (closely
related to H. fulva), and Iris3hollandica (ethyleneinsensitive senescence).
A model of petal senescence
Based on our interpretation of the literature, the following
picture of petal senescence is constructed.
Numerous genes become (transiently) up-regulated during
senescence, whilst many other genes are down-regulated.
Most of the changes in gene expression are related to
remobilization of macromolecules, and transport of the mobile
compounds out of the petal. A considerable part of differential
gene expression is related to plant defence. No genes have
as yet been identified that are specific for cellular death.
Degradation of macromolecules in the senescent cell is
mainly due to autophagic processes in the vacuole, in
addition to protein degradation in mitochondria, nuclei,
and the cytoplasm, fatty acid breakdown in peroxisomes,
and degradation of nucleic acids in the nuclei.
It is as yet unknown how the onset of senescence is
regulated. This is true for the onset of the increase in
ethylene production in systems that are controlled by
ethylene as well as for the regulation in species where
senescence is not under the control of ethylene. Although
many transcription factors and signal transduction factors
are differentially expressed during petal senescence, as yet
the functional role is known of only a few (e.g. EIL3 and
Aux/IAA).
It is also not clear how the petal cells die. Death is
preceded by the disappearance of most of the cellular
contents, and might therefore be due to ongoing remobilization, but might also occur independently of the
remobilization process. Several factors that control programmed death of animal cells, such as caspases, proteins
released by mitochondria, and proteins that control the
release of compounds through membranes, have no
apparent counterpart in plant cells, indicating that the
regulation of cell death during petal senescence is quite
different from the regulation of cell death in animal cells.
Tonoplast rupture (or lack of tonoplast semi-permeability)
is widely held to be the immediate cause of developmental
cell death in plants, but the data that support this
contention in petals are still weak. The mechanism that
accounts for the rupture is completely unknown.
The ovary is central in the regulation of ethylene-sensitive
senescence, at least in carnation flowers. It mediates the
hastening effect of pollination on senescence. After pollination the ovary produces ethylene which induces a rapid
increase in petal ethylene production. In the absence of
pollination, the ovary is also causal in petal senescence,
although the mechanism by which ethylene is then produced in the ovary is not clear.
This picture shows the many lacunae in our present
understanding. Some testable hypotheses are put forward (in
the Discussion section) regarding these gaps: (i) the
regulation of the onset of senescence; (ii) the determination
of the species-dependent petal life span; (iii) the mechanism
whereby the rise in ethylene synthesis is produced; (iv) the
role of hormones in the regulation of the onset of ethyleneinsensitive senescence; (iv) the relationship between remobilization and collapse of the tonoplast; and (vi) the mechanism
of tonoplast collapse.
Petal senescence 455
The data underlying the contentions of the above model
of petal senescence will be discussed in the remainder of
this review.
Morphology and ultrastructure of
dying petal cells
One of the earliest changes in cell ultrastructure, at least
during senescence of Iris petals, is the closure of the
plasmodesmata. Plasmodesmata, if open, allow transfer of
relatively small molecules, such as sugars, hormones, and
RNA molecules, between neighbouring cells. If the
plasmodesmata are closed, this transport is halted. In the
outer petals of Iris the plasmodesmata of mesophyll cells
close 2 d before those of the epidermis cells. The
mesophyll cells also die 2 d earlier than the epidermal
cells. Compared with epidermis cells, the mesophyll cells
also exhibited all other ultrastructural changes, indicative
of processes leading to cell death, ;2 d earlier (van
Doorn et al., 2003). Although these data are suggestive of
a role for plasmodesmata in the early regulation of
senescence, the data thus far only show a correlation.
Invaginations have been noted at the tonoplast of
Ipomoea (¼ Convulvulus) tricolor petal cells; numerous
vesicles were found in the vacuole. These vesicles are
probably due to microautophagy and/or macroautophagy.
The vacuoles also contained structures reminiscent of
mitochondria. The vacuole therefore seems a main site of
membrane and organelle degradation (Winkenbach,
1970b; Matile and Winkenbach 1971; Phillips and Kende,
1980; Smith et al., 1992).
Petal cells in Ipomoea (Winkenbach, 1970a), carnation
(Smith et al., 1992), Hemerocallis (Stead and van Doorn,
1994), and Iris (van Doorn et al., 2003) exhibit a decrease
in the number of small vacuoles, and an increase in
vacuolar size. The ongoing increase in vacuole volume in
Iris was accompanied by the loss of a considerable part of
the cytoplasm and with the disappearance of most
organelles. In Iris, one of the earliest ultrastructural
senescence symptoms (after closure of the plasmodesmata) was the loss of most of the endoplasmic reticulum
(ER) and attached ribosomes. Several other organelles,
such as Golgi bodies, then also become less frequent.
Many mitochondria became degraded, but some remained
until vacuolar collapse. Similar changes have been
reported in carnation (Smith et al., 1992).
The nucleus has been found to remain until a late stage
of senescence, but shows several ultrastructural changes. In
tobacco petals the nuclei showed nuclear blebbing, similar
to that observed during apoptosis in animal cells (SerafiniFracassini et al., 2002). Nuclei in young cells have a rather
even distribution of DNA (chromatin). During cell senescence, the chromatin clumps into patches that are found
either throughout the nucleus or mainly at the nuclear
periphery. In several species the nucleus showed brighter
fluorescence after staining the DNA with a fluoroprobe, indicating DNA condensation. This is often accompanied by
a decrease in nuclear diameter (Yamada et al., 2006a, b).
In the petals of Ipomoea, a doubling of the number of
DNA masses (‘nuclei’) per petal was found, together with
a continuing decrease in the diameter of these masses
(Yamada et al., 2006a). As the DNA masses contained an
outer membrane, the results indicate nuclear fragmentation. Evidence for nuclear fragmentation was also found
in the petals of Petunia hybrida and Argyranthemum
frutescens, but not in petals of Antirrhinum majus
(Yamada et al., 2006b). These data, although still limited,
indicate nuclear fragmentation in the senescence of petals
both of some ethylene-sensitive plants (Ipomoea; Petunia)
and of an ethylene-insensitive plant (Argyranthemum).
Other organelles also disappear and the volume of
cytoplasm becomes very low, leaving only a small strip
next to the cell wall. At least some mitochondria, and in
carnation some plastids, stayed until a late phase of
senescence (Winkenbach, 1970b; Smith et al., 1992; van
Doorn et al., 2003).
When the cytoplasm of the cell is almost or completely
empty, electron microscopy (EM) work shows that the
vacuolar membrane (tonoplast) collapses and then disappears. This occurs earlier than the collapse and
disappearance of the plasma membrane (Winkenbach,
1970b; Smith et al., 1992; van Doorn et al., 2003).
Although it cannot be excluded that this is an artefact of
EM preparation, the earlier collapse of the tonoplast might
be a real effect, similar to that in other plant cells, for
example during the PCD of tracheal elements (Obara
et al., 2001), and in several other examples of PCD/
senescence (Jones, 2001; Jones and Dangl, 2004). It will
here be assumed that the earlier tonoplast collapse in petal
cells is real, and the direct cause of cellular death, in line
with the analysis of such effects in several other systems.
In several species tested, the petal mesophyll cells died
considerably earlier than the epidermis cells. This was
observed in the dicotyledonous Ipomoea (Winkenbach,
1970b), and in the monocotyledonous Iris reichenbachii
(Bancher, 1941), Iris3hollandica (van Doorn et al.,
2003), Hemerocallis (Stead and van Doorn, 1994),
Alstroemeria (Wagstaff et al., 2003), and Sandersonia
(O’Donoghue et al., 2002). In contrast, no clear evidence
for earlier death in the mesophyll, compared with the
epidermis, was found in Gypsophila (Hoeberichts et al.,
2005). In some species, cell death starts at the apical part
and gradually moves towards the petal base (Iris, van
Doorn et al., 2003; Alstroemeria, Wagstaff et al., 2003),
but again no evidence for such a difference was found in
Gypsophila (Hoeberichts et al., 2005) and it was also
apparently not observed in carnation (Smith et al., 1992).
Visible petal wilting coincided with collapse of the
epidermis cells (carnation, Smith et al., 1992; Iris, van
Doorn et al., 2003; Gypsophila, Hoeberichts et al., 2005).
456 van Doorn and Woltering
Collectively, the data indicate that petal senescence is
accompanied by large-scale autophagy. A considerable
part of the cytoplasm is lost, and most organelles
disappear, including the ER and ribosomes. Some mitochondria and the nucleus remain until late in the process
but the nucleus usually changes in morphology relatively
early during senescence. The evidence also indicates that
the tonoplast collapses earlier than the plasma membrane,
and that the collapse of the tonoplast is the actual moment
of cellular death. The ultrastructure of petal senescence is
similar in petals where the process is regulated by
ethylene and in petals where it is not.
Is petal death due to autophagy?
The literature indicates an upsurge of autophagy both
during starvation and during senescence. This follows from
ultrastructural evidence, biochemical data, and gene expression data (Thompson and Vierstra, 2005). Autophagy
during senescence seems to be a main process responsible
for cell dismantling and remobilization of compounds that
can be used in other parts of the plants. Autophagy may
not necessarily be required for cell death. In PCD in
animals, a few examples are now known where death was
apparently due to autophagy (Verheye et al., 2007), but
such evidence has not as yet been reported for plants.
In Arabidopsis, a number of autophagy genes (ATG)
have been knocked out, which resulted in hastened leaf
yellowing (Hanaoka et al., 2002; Yoshimoto et al., 2004;
Thompson et al., 2005; Xiong et al., 2005). This might be
interpreted to indicate that death is not due to autophagy,
but is rather delayed by autophagy. However, such an
interpretation might be too hasty. The experiments in which
autophagy genes were silenced showed pleiotropic effects,
such as a small root system. Autophagy occurs at all stages
of cell development. It is a mechanism to dispose of
organelles that are no longer functional. The lack of normal
baseline autophagy can lead to stress, perhaps shown as
precocious leaf yellowing. Similarly, the presence of many
dysfunctional mitochondria resulted in hastened petal
senescence, probably because of stress (Chen et al., 2005).
Since the root system of the transgenic Arabidopsis plants
was smaller, it is possible that nutrient uptake was lower, or
that the root produced fewer cytokinins and/or gibberellins.
The experiments on ATG silencing, as carried out thus far,
thus provide no answer to the question of what the
relationship is between the upsurge in autophagy and leaf
yellowing. Only experiments in which autophagy becomes
attenuated during senescence, for example by silencing
under the control of a senescence-specific promoter, can
provide the necessary information.
Autophagy, as discussed above, is a combination of at
least micro- and macroautophagy (Thompson and
Vierstra, 2005). Microautophagy entails invagination of
the vacuolar membrane, transferring parts of the cyto-
plasm, including organelles, to the vacuole. Macroautophagy is due to double membrane structures formed inside
the cytosol, sequestering parts of the cytosol, including
organelles. Autophagosomes merge with the vacuole.
Prior to delivery of their contents to the vacuole,
autophagosomes are also considered to merge with
vesicles containing hydrolytic enzymes, thus the onset of
degradation can already take place inside the autophagosomes (Inoue and Morioyasu, 2006). In addition to micro/
macroautophagy, the cell death programme induces the
collapse of the tonoplast. Hydrolytic enzymes in the
vacuole are released into the cell and will rapidly degrade
most of what is left by that time, including the plasma
membrane. After the collapse of the tonoplast, the cell can
no longer regulate its degradation. Tonoplast collapse,
therefore, seems to be the moment of cell death. The term
mega-autophagy has been suggested for tonoplast collapse
and its consequences (van Doorn and Woltering, 2005).
Micro- and macroautophagy might progress, theoretically, to a stage whereby essential components disappear.
Tonoplast collapse might be the result of such ongoing
micro-/macroautophagy, although this event might also be
regulated independently of autophagy. Even if tonoplast
collapse is not due to micro/macroautophagy, the two
processes must be closely co-ordinated because remobilization and nutrient transport out of the petals will be
severely compromised if death occurs too early.
Regulation of senescence at the transcriptional,
translational, and post-translational level
Senescence is regulated by levels of mRNA and proteins,
and by activation of proteins. mRNA levels can be
regulated by short interference RNA (siRNA) and microRNA (miRNA). A limiting factor can also be mRNA
transport out of the nucleus. Proteins can be modified after
translation. The eukaryotic translation initiation factor 5A
(eIF-5A) is an example of post-translational modification.
eIF-5A is apparently involved in translocation of specific
mRNAs from the nucleus to the cytoplasm. During
carnation petal senescence, the RNA abundance of eIF5A increased, as well as the mRNA abundance of a gene
involved in post-translational modification of eIF-5A,
deoxyhypusine synthase. eIF-5A is unique among translation initiation factors because it contains hypusine [N(4-amino-2-hydroxybutyl) lysine], which is formed
post-translationally via the transfer and hydroxylation of
a butylamino group to a specific lysine residue. These
events activate eIF-5A. Deoxyhypusine synthase mediates
the first step in the synthesis of hypusine in eIF-5A
(Hopkins et al., 2007). Antisense suppression of deoxyhypusine synthase delayed senescence in Arabidopsis
leaves (Wang et al., 2003) and in carnation petals
(Hopkins et al., 2007). These results are reminiscent of the
delay of petal senescence by inhibitors of translation such
Petal senescence 457
as cycloheximide and narciclasine (van Doorn et al., 2004),
showing the importance of de novo protein synthesis in
senescence. This example illustrates the potential complexity of transcriptional, post-transcriptional, translational, and
post-translational control of senescence.
Post-translational modifications also occur, for example,
in signal transduction (protein phosphorylation by
kinases), during autocatalytic activation of cysteine proteases in the vacuole, and prior to protein degradation in
proteasomes. Targeting for breakdown occurs through the
transfer of several ubiquitin moieties to a protein, by
a ubiquitin ligase complex. Senescence, therefore, can be
regulated at the level of protein synthesis, protein
activation, and protein degradation.
Transduction of the death signal: comparison
with animal cells
In animal cells, several elements of the cell death signal
transduction routes have been characterized. The various
elements can be grouped, roughly, into caspase signal
transduction, and the release of signal chemicals from
mitochondria, the ER, and the lysosome (Cheng et al.,
2006; Hail et al., 2006).
Caspases are cysteine endoproteases that cleave at
aspartate residues. Although several caspases are part of
the signal transduction pathway, some are executioner
proteins, responsible for the degradation of cellular
substrates. Thus far, no caspase homologues have been
found in plant genomic databases, but some plant caspaselike proteins such as the vacuolar processing enzyme
(VPE) have been found. Although there is limited overall
sequence identity between VPE and caspase-1, they share
several structural properties. The activity of isolated VPEs
was blocked by caspase-1 inhibitors (Rojo et al., 2003).
Arabidopsis has four VPE genes (a to d). A VPEc was
found to localize to vesicles that merged with vacuoles. In
the vacuole, this VPEc was autocatalytically converted
into the active form, which regulated maturation/activation
of vacuolar proteases. A VPEc mutant showed 20-fold
increased levels of cystatin, a cystein protease inhibitor.
Homologues of cystatin suppress hypersensitive cell death
in plant and animal systems (Rojo et al., 2003, 2004).
Additionally, in a VPEd-deficient mutant, cell death of
the two layers of the seed coat was delayed (Nakaune
et al., 2005).
The mRNA abundance of two VPE genes increased
during petal senescence in Narcissus. One of the two was
previously described as an asparaginyl endopeptidase, also
known as legumain (Hunter et al., 2004a). A gene
encoding a VPE was also up-regulated during carnation
petal senescence (Hoeberichts et al., 2007).
In addition to caspase-like enzymes, a class of cysteine
proteases in plants are called metacaspases (HaraNishimura et al., 2005). A metacaspase was required for
programmed death in suspensor cells (Bozhkov et al.,
2004), and a plant metacaspase has been reported to induce
cell death in yeast (Watanabe and Lam, 2005). Michelle
Jones (personal communication, 2006) found an increase in
mRNA abundance of a metacaspase in Petunia petals.
However, no effect was observed in petal senescence of
a Petunia line harbouring a metacaspase antisense gene.
Another type of caspase-like protease is localized to the
plant cell wall. Its activity was inhibited by chemicals that
block either serine proteases or cysteine proteases. To
distinguish this proteinase from caspases, which are not
inhibited by serine protease inhibitors, it was called
saspase. A saspase has been implicated in cell death of
plants treated with the fungal toxin victorin (Coffeen and
Wolpert, 2004). Saspase activity has apparently not been
reported in relation to plant senescence.
In animal cells, signals for cell death can be released
from various organelles, such as the ER, the lysosome
(vacuole), and mitochondria. It is still unclear whether
release of factors from these organelles is causal in plant
cell death. Release of cytochrome c from mitochondria,
which is important in many examples of animal PCD, was
not found during senescence in Petunia petals (Xu and
Hanson, 2000). In animal cells, several other signal
factors are released from mitochondria, but their possible
role has not yet been reported in petals.
In animal cells, Bax is one of the pro-apoptotic proteins
of the Bcl-2 family. These proteins insert into membranes.
This results in the release of cell death signal molecules.
Bax-inhibitor-1 is also implicated in the regulation of
animal cell death. A homologue of Bax-inhibitor-1 is
present in plants. As no homologues of Bcl-2 proteins
have been found in plants, the role of the plant Baxinhibitor-1 homologue is unclear.
In animal cells, the mRNA abundance of a gene called
DAD1 (defender against apoptotic cell death) substantially decreased prior to death. Overexpression of the gene
delayed the death of these cells. The function of DAD1 is
unclear; it has been suggested that the cell dies by lack of
DAD1 activity because DAD1 is a household enzyme
involved in glycosylation of proteins at ER membranes
(van der Kop et al., 2003). DAD1 was also shown to
interact with the Bcl-2 family proteins in animal cells
(Makishima et al., 2000), which might suggest a regulatory
role in cell death. In petals, thus far, only correlative
evidence is available between senescence symptoms and
DAD1 expression. The decrease in DAD1 abundance
occurred relatively early during senescence in some
species, but very late in other species (Table 1). The role
of DAD1 in senescence, if any, is therefore as yet unclear.
Free calcium ions may be a factor that is common to the
regulation of cell death in plants and animals. In animals,
a death signal from outside the cell can be transduced
through an increase in inositol-1,4,5-triphosphate (IP3)
levels. This molecule can induce an increase in
458 van Doorn and Woltering
Table 1. Decrease of DAD1 transcript abundance during petal
senescence
Species
Comments
Reference
Alstroemeria
pelegrina
Dianthus
caryophyllus
Gladiolus3
grandiflora
Iris3hollandica
Pisum sativum
Decrease relatively
early
Decrease very late
Wagstaff et al. (2003)
van der Kop et al. (2003)
Decrease occurs late
Yamada et al. (2004)
Decrease very late
Treatments hastening
or delaying senescence
also hastened or
delayed decrease in
mRNA abundance,
respectively
van der Kop et al. (2003)
Orzaez and Granell (1997)
intracellular Ca2+ concentration. Cell death in pollen tubes
depended on an increase in calcium ion levels, which
resulted in a decrease of actin polymerization. Inducing
a major decrease of actin polymerization, similar to the
one occurring prior to normal death, was adequate to
induce death. It was concluded that calcium-induced actin
depolymerization is an early regulator of death in these
cells (Thomas et al., 2006).
A role for calcium ions in petal senescence has thus far
not been established. A possible connection with actin
might follow from work on senescence in Ipomoea petals,
where the mRNA abundance of a gene encoding ataxin-2
transiently increased (Yamada et al., 2007a). In yeast,
increased mRNA abundance of ataxin-2 caused premature
cell death due to defects in actin filament formation
(Satterfield et al., 2002). During senescence in carnation
petals, an early rise occurred in transcript levels of
a putative CBL-interacting protein kinase (CIPK), a regulator of phosphorylation cascades that transmit calcium
signals. This rise was advanced by ethylene and delayed
by silver thiosulphate. It was hypothesized, therefore, that
a calcium signal is involved in carnation petal senescence,
and that this signal is regulated by endogenous ethylene
(Hoeberichts et al., 2007). In Iris petals, the transcript
abundance of a gene encoding a cyclic nucleotide-gated ion
channel (CNGC) increased (van Doorn et al., 2003). Some
CNGCs are involved in calcium signal transduction (Arazi
et al., 2000) and in increasing the internal calcium ion
concentration (Köhler et al., 2001). In Arabidopsis,
a GNGC has been suggested to be involved in developmental cell death (Köhler et al., 2001) and was required for
ozone-induced cell death (Overmyer et al., 2005). The Iris
transcript shows very high homology with this gene.
During Iris petal senescence an increase was seen in the
transcript abundance of a gene encoding a casein kinase
(i.e. CK-type protein kinase), most of which are apparently signal transduction factors (van Doorn et al., 2003).
Some CK proteins have been suggested to be regulators of
the cell survival pathway (Ahmed et al., 2002).
Taken together, it has presently not been established
that petal senescence is regulated by any of the signal
transduction mechanisms that are known from animal
PCD. Although some of the genes found in petals might
be involved in the regulation of senescence, their function
is as yet unknown.
Transcription factors and transcriptional
regulators
Transcription factors are usually classified according to
specific functional amino acid sequences (domains). More
than one type of such domains can be present in a protein.
Table 2 gives an overview of transcription factors that
have hitherto been found to be differentially expressed
during petal senescence. It is to be expected that several
more transcription factors will be discovered that are
associated with petal senescence, similar to the long list of
known transcription factors related to leaf senescence
(Guo et al., 2004).
With the exception of EIL, AP2, Aux/IAAs, NAC,
knotted1, and GCIP, little is known about the role, in
senescence, of the transcription factors shown in Table 2.
EIN3 and EIN3-like (EIL) transcription factors are crucial
in the ethylene signal transduction chain. Thus far, four
EIL genes have been isolated in carnation petals. When
carnation flowers were treated with ethylene, the petal
mRNA abundance of Dc-EIL1-2 and Dc-EIL4 slightly
decreased, whereas the mRNA abundance of Dc-EIL3
drastically increased. Ethylene promoted and STS delayed
the increase in Dc-EIL3 mRNA, and had no effect on
Dc-EIL1. These data indicate that Dc-EIL3 is an important
regulator of gene expression during carnation petal
senescence. It might act as a master switch of ethyleneinduced gene expression (Iordachescu and Verlinden,
2005; Hoeberichts et al. 2007).
Targets of EIN/EIL transcription factors include genes
with an ethylene-responsive element (ERE) in their
promoter. Amongst these is a gene encoding the ethyleneresponsive transcription factor 2 (ERF2), which belong to
the AP2 transcription factor family. In senescing daffodil
petals, a gene (DAFSAG9) encoding an AP2 protein with
very high homology to ERF2 was highly up-regulated
(Hunter et al., 2002).
Aux/IAA genes are transcriptional suppressors, also
often considered transcription factors. Aux/IAA is central
in the regulation of auxin responses. In the absence of
auxin, Aux/IAA inhibits all auxin-responsive genes. When
auxin is present, it binds to its receptor, located on an
F-protein in a nuclear-localized ubiquitin ligase. This
ligase then targets Aux/IAA for degradation in the
proteasome. This results in the expression of auxinresponsive genes (Bishopp et al., 2006).
The protein sequence of the NAC in carnation petals was
highly homologous to that of NAC2 of Arabidopsis. NAC
Petal senescence 459
Table 2. Differential expression, during petal senescence, of genes putatively encoding transcription factors or transcriptional regulators
bHLH, basic helix–loop–helix; bZip, basic-leucine zipper; GCIP, Grap2- and cylin-interacting protein; HD, homeodomain; HLH-Zip, helix–loop–
helix leucine zipper
Factor
Transcription factors related
to ethylene signalling
Dc-EIL1
Dc-EIL1-2
Dc-EIL3
Dc-EIL4
AP2/ERF
Transcription factors and
transcriptional regulators not directly
related to ethylene signalling
Aux/IAA
bHLH/MYC
bZIP
C2H2
HLH-Zip
HD
HD-Zip
HMG box
JUMONJI
MADS
MYB
NAC
a
Species
Comments
Reference
Dianthus caryophyllus
Dianthus caryophyllus
Dianthus caryophyllus
Dianthus caryophyllus
Narcissus pseudonarcissus
Waki et al. (2001)
Iordachescu and Verlinden (2005)
Iordachescu and Verlinden (2005)
Iordachescu and Verlinden (2005)
Hunter et al. (2002)
Dianthus caryophyllus
Dianthus caryophyllus
Dianthus caryophyllus
Mirabilis jalapa
Alstroemeria pelegrina
Mirabilis jalapa
Iris3hollandica
Dianthus caryophyllus
Mirabilis jalapa
Alstroemeria pelegrina
Mirabilis jalapa
Iris3hollandica
Dianthus caryophyllus
Mirabilis jalapa
Dianthus caryophyllus
Mirabilis jalapa
Dianthus caryophyllus
Narcissus pseudonarcissus
Hoeberichts et al. (2007)
Hoeberichts et al. (2007)
Hoeberichts et al. (2007)
Xu et al. (2007)
Breeze et al. (2004)
Xu et al. (2007)
van Doorn et al. (2003)
Hoeberichts et al. (2007)
Xu et al. (2007)
Breeze et al. (2004)a
Xu et al. (2007)
van Doorn et al. (2003)
Hoeberichts et al. (2007)
Xu et al. (2007)
Hoeberichts et al. (2007)
Xu et al. (2007)
Hoeberichts et al. (2007)
Hunter et al. (2002)
GCIP
Knotted-1 type
Data from Supplement 3, for function see Martı́nez-Garcı́a and Quail (1999).
factors (>100 in Arabidopsis) have diverse functions, such
as organ separation, but a role for a NAC in senescence has
not yet been described. AtNAC2 mRNA abundance was
increased after treatment with the ethylene precursor 1aminocyclopropane-1-carboxylic-acid (ACC), by abscisic
acid (ABA), and by an auxin (He et al., 2005).
Knotted1 is a homeobox gene found in apical meristems. Overexpression of this gene yielded phenotypes
similar to those in which the cytokinin gene ipt is
overexpressed. Knotted1 mRNA was not found in tobacco
leaves undergoing senescence, but when knotted1 was
overexpressed, driven by the promoter of a senescencespecific cysteine protease (SAG12), it delayed leaf yellowing. This was associated with a 15-fold increase in
cytokinin levels (Ori et al., 1999). These results suggest
that the knotted1 found in carnation petals (Hoeberichts
et al., 2007) is related to cytokinin metabolism.
In senescing Iris petals, an up-regulated gene encoded
a plant homologue of the animal Grap2- and cylininteracting protein (GCIP; van Doorn et al., 2003). GCIP
is a helix–loop–helix leucine zipper family transcription
factor that inhibits E2F1-mediated transcriptional activity
The transcription factor E2F1 is a member of a family
involved in cell cycle progression, differentiation, and cell
death (Chang et al., 2000; Ma et al., 2007).
In addition to the transcription factors mentioned
(Table 2), WRKY factors might also be regulators of petal
senescence. The mRNA abundance of a gene encoding
a RING domain ankyrin repeat protein, probably (part of)
a ubiquitin ligase, drastically increased during senescence
in Mirabilis petals. Silencing of the gene in Petunia petals
delayed petal wilting by a few days. The promoter of this
gene had putative binding sites for bZip, HD-Zip, Myb,
MADS, and also for WRKY (Xu et al., 2008).
The data show that many transcriptional regulators
become differentially expressed during petal senescence.
However, these factors have as yet not been shown, for
example by gene silencing or overexpression, to function
as regulators of petal senescence.
Ethylene receptors and signal transduction
factors
Receptors of the ethylene signal
In Arabidopsis, ethylene is perceived by a family of five
receptors (ETR1, ETR2, ERS1, ERS2, and EIN4; Mason
and Schaller, 2005). The expression of genes encoding
receptor proteins has been reported in petals of only a few
species. In carnation, for instance, considerable amounts
460 van Doorn and Woltering
of ETR1 and ERS2 mRNA were present in petals at the
time of harvest. Exogenously applied ethylene hastened
senescence but did not clearly change the mRNA
abundance of ETR1 or ERS2 (Shibuya et al., 2002).
Interestingly, two homologues of Arabidopsis ERS1 were
isolated from the petals of Gladiolus which show
ethylene-insensitive senescence. The mRNA abundance
of one of these genes changed during petal senescence,
whereas the other was highly and constitutively expressed
(Arora et al., 2006).
Overexpression of mutated ethylene receptor genes in
petals showed the importance of these receptors in senescence (Table 3). The data show that senescence is delayed
when a single class of ethylene receptors is expressed at
low levels or is absent.
CTR1 and EIN2
Signal transduction is usually taken to comprise the
pathway from a receptor to a change in the levels of
transcription factors. Part of the signal transduction
pathway of ethylene has by now been elucidated (Bishopp
et al., 2006; Etheridge et al., 2006). The pathway is
relatively complex due to the presence of negative
regulators. First, the ethylene receptors are negative
regulators because ethylene binding stops their action. In
the absence of ethylene, the receptors activate a downstream CTR1 protein. Secondly, active CTR1 is a negative
regulator as it inhibits EIN2 and thereby prevents ethylene’s effects. In the presence of ethylene the receptors do
not activate CTR1, which thus cannot block EIN2. This
results in ethylene-related gene expression. The importance of EIN2 in petal senescence was shown in knockout
experiments in which reduced EIN2 mRNA abundance
produced a longer life in (attached) Petunia flowers
(Shibuya et al., 2004). Active EIN2 regulates the levels
of EIN3 and EIL transcription factors, by inhibiting their
degradation in proteasomes (Bishopp et al., 2006).
Ethylene regulation of petal senescence: beyond
EIL
Induction of the rise in ethylene production
The onset of visible wilting in ethylene-sensitive petal
senescence is accompanied by a sudden, transient increase
Table 3. Delay of petal senescence in transgenic plants with
mutated ethylene receptor genes
Inserted gene
Species
Reference
Mutated
Mutated
Mutated
Mutated
Petunia hybrida
Dianthus caryophyllus
Nemesia strumona
Petunia hybrida
Shaw et al. (2002)
Bovy et al. (1999)
Cui et al. (2004)
Wilkinson et al. (1997);
Jones et al. (2005)
ERS
ETR1
ETR1
ETR1
of respiration, associated with a transient upsurge in
ethylene production. Pollination hastens these events.
A signal related to pollination (reviewed elsewhere:
O’Neill, 1997) causes the surge in ethylene production. A
short treatment with ethylene can replace pollination
(Nichols, 1966). It is not known what induces the rise in
ethylene production in unpollinated flowers.
The ethylene synthesis pathway starts with Sadenosylmethionine (SAM), which is converted to ACC,
the direct precursor of ethylene. Genes in this pathway,
such as SAM synthase, ACC synthase, and ACC oxidase,
are up-regulated during ethylene-sensitive petal senescence (Jones, 2004; Hoeberichts et al., 2007). Preventing
the conversion of ACC to ethylene, by decreasing the
level of ACC oxidase mRNA through antisense techniques, prolonged the life of cut carnation flowers (Savin
et al., 1995; Kosugi et al., 2002) and increased the life
span of plant-attached flowers in Torenia fournieri (Aida
et al., 1998). Extended flower longevity has also been
reported in transgenic carnation flowers after decreasing
the abundance of ACC synthase mRNA, using cosuppression (Michael et al., 1993).
ten Have and Woltering (1997) excised petals and other
organs of unpollinated carnation flowers at various times
during vase life. The expression of genes encoding ACC
synthase and ACC oxidase occurred first in the ovary,
then in the stigma plus style (which cannot easily be
separated in this species), and only last in the petals. This
indicated (but did not show) that the ovary is the trigger of
petal senescence. The mobile compound that brought the
signal from the ovary to the petals was apparently
ethylene (ten Have and Woltering, 1997).
Strong evidence for an important role for the gynoecium
(comprising stigma, style, and ovary) in carnation petal
senescence was obtained after finding a method for careful
gynoecium removal. In previous experiments, the gynoecium had been cut off. This apparently induced enough
wounding ethylene to confound the gynoecium effect, and
hasten senescence. Removal of the gynoecium by hand, in
contrast, delayed senescence. In the absence of the
gynoecium, petal ethylene did not reach normal high
levels. Application of ACC, the ethylene precursor, in the
vase water induced substantial ethylene production and
early petal wilting in flowers from which the gynoecia had
not been removed, but the treatment had little effect on
ethylene production and did not induce petal wilting in
flowers without gynoecia. This shows that ethylene
generated in the gynoecium is the trigger for the onset of
the increase in ethylene production in carnation petals
(Shibuya et al., 2000; Nukui et al., 2004).
It has been suggested that the rise in ethylene production in unpollinated flowers with ethylene-sensitive
petal senescence occurs because the cells become more
sensitive to their basal ethylene production, which in turn
might relate to a decrease in cytokinin activity in the
Petal senescence 461
petals. In a line of transgenic Petunia flowers, petal life
was extended by overexpression of a gene encoding
isopentenyltransferase (ipt), which results in high levels
of several cytokinins. The mRNA abundance of a cysteine
protease gene, indicative of ongoing senescence, remained
very low for a considerable period of time when detached
ipt-overexpressing flowers were treated with ethylene
(Chang et al., 2003). These data strongly suggest that
cytokinins regulate petal life span in vivo.
In carnation, a relationship was found between petal
cytokinin levels and the time to senescence, when
comparing the shorter-lived detached flowers with flowers
that remained uncut (van Staden and Dimalla, 1980).
However, cytokinin levels in petals of unpollinated
carnation flowers did correlate well with the increase in
ethylene sensitivity, occurring at later stages of flower
development (van Staden and Dimalla, 1980; van Staden
et al., 1987). Cytokinins in petals therefore seem not to
account fully for the rise in petal ethylene production. As
the gynoecium has been shown to regulate senescence of
carnation petals, the cytokinin levels in the gynoecium
might be more important than the levels in petals.
A detailed analysis of cytokinin activity in the gynoecium
has apparently not been published.
It is concluded that it is not yet known what signal starts
the rise in ethylene production in unpollinated petals with
ethylene-sensitive senescence. The following mechanism
is here tentatively suggested: according to a default
pathway the ovary will undergo senescence, after a
species-specific period of time, if no pollen becomes
deposited on the stigma. In the absence of pollination,
ovary senescence induces petal senescence. If pollination
occurs, ovary senescence is prevented. The pollination
signal nonetheless induces a rapid rise in ovary ethylene
levels, which induces petal senescence. The question as to
what regulates the initial rise in ethylene production, in
flowers that are not pollinated or treated with exogenous
ethylene, is thereby not answered, but only shifts from the
petals to the ovary.
Carnation cultivars with a long vase life: relationship to
ethylene
Some carnation cultivars have an unusually long vase life.
The long life in some of these cultivars was related to
ethylene insensitivity. In other cultivars it was due to
absence of ethylene production, combined with normal
sensitivity to ethylene.
Examples of the first type are a group of related
cultivars (Chinera, Epomeo, Ginevra) derived from
a long-lived non-commercial breeding line. The vase life
of these flowers was highly correlated with their degree of
ethylene insensitivity (van Doorn et al., 1993). During the
vase life of Chinera flowers, the increase in the activities
of ACC synthase and ACC oxidase, and the increase in
the rate of ethylene production, occurred later than in
normal, relatively short-lived, cultivars. Ethylene treatment stimulated the activity of the ethylene biosynthetic
pathway but stimulation was less than in a relatively
short-lived cultivar (Wu et al., 1991a, b; Woltering et al.,
1993). The low ethylene sensitivity in these cultivars may
relate to their abnormal ovary. In Chinera flowers, the
ovary contained style-like and leaf-like structures rather
than the normal reproductive organs. This may explain
why the ovary does not produce the ethylene required to
induce petal senescence (EJW, unpublished results).
Examples of the second group are the cultivars Sandra
(Wu et al., 1991a, b), Sandrosa (which is very similar to
Sandra; Mayak and Tirosh, 1993), and White Candle
(Nukui et al., 2004). Compared with cultivars with
a shorter vase life, White Candle flowers showed lower
expression of ACC synthase genes in the gynoecium, and
repressed gynoecium ethylene production. The low ethylene production in the gynoecium was the apparent cause
of the lack of transcription of ACC synthase genes in the
petals and thus of low ethylene synthesis in the petals
(Nukui et al., 2004).
The data in this section support the idea that petal
senescence in carnation requires a normal ovary, which
determines both ethylene sensitivity of petal senescence
and ethylene production in the petals.
Hormones other than ethylene, in petals with
ethylene-sensitive senescence
As described above, cytokinins have been implicated in
the onset of the rise in ethylene production, in unpollinated, ethylene-sensitive petal senescence. The importance
of cytokinin levels also follows from other observations.
Treatment with cytokinins delayed carnation petal senescence. It delayed the rise in ethylene production, possibly
through a decrease in ethylene sensitivity (Eisinger, 1977;
van Staden, 1995). Cytokinin levels can be regulated by
synthesis, inactivation, and degradation. During Petunia
petal senescence, cytokinins were inactivated by
O-glucosylation. Cytokinin levels were also decreased by
degradation, mainly through the activity of cytokinin
oxidase/dehydrogenase. Feeding carnation flowers with
6-methylpurine, an inhibitor of cytokinin oxidase/dehydrogenase, resulted in a much longer petal life span (Taverner
et al., 2000). The increase in mRNA abundance of two
genes encoding cytokinin oxidase/dehydrogenase, during
carnation petal senescence (Hoeberichts et al., 2007),
might also indicate accelerated cytokinin breakdown.
The role of ABA during ethylene-regulated petal
senescence is still unclear. In cut carnations, a large
increase in ABA levels was observed in the gynoecium,
and a small increase in petals, prior to or concomitant with
the ethylene upsurge (Nowak and Veen, 1982; Eze et al.,
1986; Hanley and Bramlage, 1989; Onoue et al., 2000).
The increase in ABA levels was completely prevented in
carnations pre-treated with silver thiosulphate (Nowak and
462 van Doorn and Woltering
Veen, 1982), showing that ethylene perception was
required. Application of ABA to carnation flowers
advanced the large rise in ethylene production and
hastened petal wilting (Mayak and Dilley, 1976). If the
gynoecia were removed, exogenous ABA no longer
induced ethylene production and early petal wilting.
It was therefore concluded that exogenous ABA acts
through induction of ethylene synthesis in the gynoecium
(Shibuya et al., 2000; Nukui et al., 2004).
Auxins can reduce ethylene sensitivity in some tissues
such as abscission zones, but in other tissues they can
induce ethylene production. Application of IAA hastened
the rise in ethylene production and petal wilting in cut
carnation flowers (van Staden, 1995). The IAA effect was
absent if the gynoecium was removed (Shibuya et al.,
2000). A synthetic auxin (2,4-dichlorophenoxyacetic acid;
2,4-D) induced the expression of ACC synthase genes in
the styles, ovaries, and petals (Jones and Woodson, 1999).
In carnation petals, a transient increase was observed in
the mRNA abundance of an Aux/IAA gene (Hoeberichts
et al., 2007). The central role of the encoded Aux/IAA
proteins in auxin responses has been described above.
Application of GA3 to cut carnation flowers delayed the
rise in ethylene production and postponed petal wilting
(Saks and van Staden, 1993). Data on GA levels in carnation flowers have thus far apparently not been reported.
Treatment with methyl-jasmonate hastened senescence
in Petunia hybrida, Dendrobium, and Phalaenopsis (Porat
et al., 1993, 1995). However, Xu et al. (2006) found only
an earlier colour change and no promotion of petal wilting
after treatment of Petunia inflata with methyl-jasmonate.
It is concluded that there is ample evidence in ethylenesensitive petal senescence for a role for cytokinin in
modulating responsiveness to ethylene. ABA and auxin
also affect ethylene production, but their role during
senescence is still unclear.
Hormonal control of petal senescence that is
insensitive to ethylene
Remarkably little is known about the early regulation of
ethylene-insensitive petal senescence. The required signal
might be endogenous, from the petal cells themselves, and
may not require hormones as an intermediate signal.
Treatment with benzyladenine, a cytokinin, delayed
petal senescence by a few days in Iris flowers (Wang and
Baker, 1979; van der Kop et al., 2003), but data on
cytokinin levels during senescence are lacking.
In contrast to the effect of cytokinins, ABA treatment of
Hemerocallis (Panavas et al., 1998b) or isolated Iris petals
(Pak and van Doorn, unpublished results) hastened the
time to visible senescence. ABA levels in petals of cut
daylily flowers increased before flower opening and
continued to increase during vase life (Panavas et al.,
1998b). During petal senescence of non-cut and cut
daffodil flowers, the petal ABA content also increased.
However, the rise in ABA level occurred after an increase
in the mRNA abundance of senescence-associated genes,
indicating that ABA was not an early regulator. Attempts
to extend floral longevity by using putative inhibitors of
ABA biosynthesis were unsuccessful (Hunter et al.,
2004b). The petal life span of cut Iris flowers was
increased by about half a day (controls had a life of 4 d)
after a pulse treatment with GA3 (Harkema and van
Doorn, unpublished results). The life of cut daffodil
flowers was also extended when they were held in vase
solutions containing GA3 (Hunter et al., 2004b).
Taken together, there are as yet no convincing data
showing that any hormone controls the onset of ethyleneinsensitive petal senescence. Treatment with cytokinins
caused a considerably delay of senescence, but its
functional role has not been established.
Regulation of senescence by sugars
Effects of exogenous sugars, relationship with
internal carbohydrate levels
Sugars in the vase solution extend the life of many cut
flowers. Sugars might serve to improve water relations
(as they increase the levels of osmotic solutes), and
provide the energy for maintaining cellular homeostasis,
but other effects, such as on ethylene sensitivity (further
discussed below), may be even more important.
An apparent paradox in carbohydrate relations of cut
flowers has still not been solved. In some species, such as
carnation, the petal sugar levels remain rather high, and
thus do not seem to become limiting. In apparent
contradiction, sugar feeding extended the life of these
petals. There may be several reasons for this paradox. One
is the inaccurate separation of tissues and cells at various
stages of senescence, which may result in overlooking
a temporary decrease in sugar levels as a trigger of
senescence. Often whole petals or petal parts are sampled,
but the mesophyll cells often show earlier senescence than
the epidermis cells, and the phloem cells, where sugars
accumulate during senescence, show still later senescence.
Another reason for the paradox may be inadequate
separation of organelles in one type of cell. The onset of
senescence may relate to low carbohydrate levels in one
cellular compartment, such as the cytosol, while there is
no such low level in another compartment, for example
the vacuole.
Inclusion of other sugars such as trehalose, mannitol,
and inositol in the vase water also delayed senescence in
some species. Examples are tulips (Iwaya-Inoue and
Nonami, 2003) and Gladiolus (Otsubo and Iwaya-Inoue,
2000; Yamada et al., 2003; Arora and Singh, 2006).
These sugars may delay senescence in a way similar to
sucrose or glucose.
Petal senescence 463
Competition for carbohydrates
In cut lily inflorescences, the life span of individual
flowers was shorter if they were left on the inflorescence
rather than severed shortly after opening. The presence of
flower buds and opening flowers apparently induced
earlier senescence in the older flowers. A sharp decrease
was observed in total carbohydrate content (starch,
glucose, fructose, sucrose, and glycerol glucosides) in the
petals of flowers that remained attached to the inflorescence. This was in contrast to a much smaller decrease in
these compounds in the petals of detached individual
flowers (van der Meulen-Muisers et al., 1995; van der
Meulen-Muisers, 2000).
Using three lily cultivars with a different petal life span,
the time to visible withering of non-cut flowers coincided
with the time until carbohydrate content had been reduced
to about half. This indicates a correlation between the
onset of senescence and lack of carbohydrates, in lily
petals. The senescence of these petals is ethylene insensitive (van der Meulen-Muisers, 2000; van der
Meulen-Muisers et al., 2001). These data are in contrast
to the situation in carnation, where sugar levels did not
decrease prior to visible senescence (van Doorn, 2004).
Effect of sugars on ethylene sensitivity
Exogenous sugars usually considerably delay visible
senescence in flowers with ethylene-sensitive petal senescence, whereas sugars have only a small effect in flowers
with ethylene-insensitive petal senescence (van Doorn and
Stead, 1994; van Doorn, 2004). Flowers in which sugars
have a large effect also generally show much earlier
visible senescence when detached from the plant and
placed in water, compared with flowers where sugars have
only a small effect. This suggests that sugars overcome
some senescence-enhancing effect of detachment, in
ethylene-sensitive senescence. In carnation, feeding with
exogenous sugars decreased the effect of exogenous
ethylene (Mayak and Dilley, 1976). Part of such an effect
might be due to the suppression of ethylene synthesis by
the petals, following sugar treatment.
In carnation flowers, sucrose (and an adequate antimicrobial compound) delayed the increase in mRNA
abundance of almost all senescence-associated genes, very
similarly to the effect of STS. At least part of the sugar
effect could be explained by the abundance of EIL3
mRNA (Hoeberichts et al., 2007) and by lower levels of
EIL3 protein. The presence of high sugar levels was
observed to promote proteasomal degradation of EIN3
(Yanagisawa et al., 2003). As described above, EIL3 is
a key transcription factor that translates the ethylene signal
into the expression of numerous ethylene-responsive
genes. How the sugar signal is translated into an antiethylene signal is not known. Possible sites of interaction
are EIN2 and other regulators of proteasomal degradation.
Sugars can also delay senescence in ethylene-insensitive
petals. In Sandersonia, the effect of exogenous sugars on
senescence was accompanied by a delay in the expression
of genes involved in fatty acid and protein remobilization
(Eason et al., 2002).
Down-regulation of the tricarboxylic acid cycle?
In senescing Alstroemeria petals, Breeze et al. (2004)
found a steady, 7-fold decrease of the transcript
abundance of 2-oxoglutarate dehydrogenase (i.e. aketoglutarate dehydrogenase), an enzyme involved in the
production of succinyl-CoA in the tricarboxylic acid
(TCA) cycle (Brugière et al., 2004). This decrease in
transcript abundance might be associated with a decrease
in enzyme activity, and thus with down-regulation of the
TCA cycle. If true, this would be indicative of a major
shift from anabolism to catabolism. Nonetheless, specific
compounds such as sucrose and some amino acids (see
below) are synthesized until a late stage of senescence.
Remobilization, transport of mineral ions
In cut flowers of which the petals show wilting or
withering as a final symptom of senescence, macromolecules become degraded to mobile compounds. These
are transported, through the phloem, out of the petal. For
example, Hew et al. (1989) noted movement of phosphorus and soluble amino nitrogen out of the petals of
Arachnis orchids.
Macromolecules that are degraded include starch, lipids,
proteins, nucleic acids, and, often, the molecules that form
the cell wall. Carbohydrates are mainly transported in the
phloem as sucrose, whilst hexoses are also found.
Nitrogen is often mainly transported as glutamine and
asparagine, and phosphorus is transferred as the inorganic
anion (Eason et al., 2000). The phloem also carries
cations such as potassium, calcium, and magnesium.
These compounds can become loaded to the phloem
during the remobilization phase of the living cells, but
also after vacuolar collapse.
By the time of visible senescence, the content of soluble
phosphates in Ipomoea petals was reduced by 75%
(Matile and Winkenbach, 1971). Inorganic phosphate
seems to be mainly derived from degradation of proteins,
nucleic acids, and phospholipids. In the vacuole, phosphates are remobilized by enzymes such as acid phosphatase and phosphodiesterase (Matile, 1997). How
phosphates are remobilized from nucleic acids in the
nucleus is unknown, but acid phosphatases are also
reportedly present in nuclei.
Inorganic cations also are removed from petals during
senescence. About a quarter of the K+ content, ;12% of
the Mg2+ content, but not more than 6% of the Ca2+
464 van Doorn and Woltering
content was withdrawn from cotton (Gossypium) petals
during senescence (Phillis and Mason, 1936). In Ipomoea
petals, ;60% of the K+ content, about half of the Mg2+
content, and about a quarter of the Ca2+ content was
transferred to other plant parts (Winkenbach, 1970a). In
Petunia, a large decrease was found in the content of
potassium, but no significant changes were detected in the
contents of calcium, magnesium, and micronutrients
(Verlinden 2003). These data indicate that the pattern of
mineral ion salvage depends on the species.
Many enzymes contain metals, such as copper and zinc,
which are probably released during protein degradation.
Metallothioneins are known to bind copper and zinc ions
(Hall, 2002). The increase in the expression of genes
encoding metallothioneins during petal senescence
(Hunter et al., 2002; Breeze et al., 2004) might therefore
relate to metal transport, but a number of alternative roles
for metallothioneins, for example the detoxification of
ROS (Kiningham and Kasarskis, 1998), cannot be
excluded. The two functions might also be connected.
Intracellular free copper, for example, has to be strictly
limited due to its toxic effects, in particular the generation
of ROS. The expression of genes encoding metallothioneins and other proteins involved in heavy metal
homeostasis is tightly regulated. It prevents toxic levels
of ionic copper (Balamurugan and Schaffner, 2006).
Several genes encoding proteins that transport inorganic
cations or anions through membranes become differentially expressed during petal senescence (Table 4).
Syntaxins are required for the fusion of transport vesicles
to target membranes (Sanderfoot et al., 2001). ABC
proteins are found in the tonoplast. The proteins carry
Table 4. Differential expression, during petal senescence, of
genes putatively involved in membrane transport
Species
Alstroemeria
pelegrina
Dianthus
caryophyllus
Iris3hollandica
Narcissus
pseudonarcissus
a
Gene
Reference
Potassium transporter
ATP-binding cassette
(ABC) transporter
Sugar transporter
Syntaxina
Breeze et al. (2004)
Amino acid permease
Copper transporter
Golgi nucleotide sugar
transporter
MATE efflux transporter
Metal nicotianamide
transporter
Monosaccharide transporter
Na+/myo-inositol transporter
Oligopeptide transporter
Cyclic nucleotide-gated
ion channel
Hoeberichts
et al. (2007)
Nitrate transporter
Hunter et al. (2002)
Data from Supplement 3.
van Doorn
et al. (2003)
glutathione-S-conjugated molecules as well as other
compounds (Lu et al., 1998). Conjugation with glutathione, most probably carried out by a glutathione-Stransferase (GST), is important for movement of various
compounds over membranes. The transcript abundance of
GST increased during carnation petal senescence (Itzhaki
et al., 1994; Hoeberichts et al., 2007). However, GST can
also be involved in detoxification, for example of
peroxidized fatty acids (see below).
The syntaxin that was up-regulated in Alstroemeria
shows high homology with Arabidopsis SYP81, which is
used as a marker of the ER (Uemura et al., 2004). CNGCs
conduct K+ and Ca2+ ions (Köhler et al., 2001).
Sugar synthesis and transport
During petal senescence in Alstroemeria (Supplement 3 of
Breeze et al., 2004) and Iris (van Doorn et al., 2003), the
transcript abundance of a gene encoding a triose phosphate isomerase and that of genes encoding sucrose
synthase increased. In Alstroemeria, the transcripts of
a gene encoding a cell wall invertase also became more
abundant. These data indicate an increase in sucrose
synthesis. Nonetheless, in Iris, some sucrose synthase
genes became down-regulated, which may indicate a shift
of sucrose synthesis to other parts of the cells or tissues.
Petal senescence is accompanied by a high sugar
concentration in the phloem. In Ipomoea and Hemerocallis petals, the concentration of sugars in the phloem
exudate increased by a factor of 15 and 14, respectively,
during the period between the closed floral bud and the
beginning of petal wilting (Wiemken et al., 1976; Bieleski
and Reid, 1992). Sucrose is the main sugar in the phloem
of senescent Ipomoea (Wiemken et al., 1976) and
Hemerocallis (Bieleski, 1995) petals. In order to reach the
phloem, the sugars must be transferred, at some point,
through a membrane. Several genes encoding sugar
transporters were up-regulated during Alstroemeria and
carnation petal senescence (Table 4).
Sugar transport from Ipomoea petal cells to the phloem
was correlated with inactivation of an invertase, probably
localized to the cell walls. It was suggested that this
decrease was instrumental in preventing the cleavage of
apoplastic sucrose, released from the cells during remobilization (Winkenbach, 1970a). Later work confirmed the
increase of an invertase inhibitor protein in several flowers
undergoing senescence, such as carnation, Alstroemeria,
Dahlia, Gladiolus, and Petunia (Halaba and Rudnicki,
1989). As there are also invertases, as well as invertase
inhibitors, in the vacuole (Greiner et al., 2000), it is not
yet clear which invertases decrease in activity during
senescence and in which compartments the invertase
inhibitors become active. Additionally, invertase activity
can be reduced because of invertase protein degradation.
During leaf senescence, a vacuolar invertase was degraded
Petal senescence 465
by proteases, which required the presence of a VPEc
(Rojo et al., 2003).
The data in this section show that sugar metabolism is
active in senescent cells as many carbon skeletons that are
remobilized from macromolecules are transported out of
the petal, mainly as sucrose.
Nucleic acid synthesis and breakdown
Numerous mRNAs are produced during senescence. Such
production requires intact mechanisms of both translation
and post-translational RNA modification. It also requires
an intact nucleus. RNA is often grouped into five classes:
mRNA, tRNA, rRNA, siRNA, and miRNAs. Whether
DNA and the RNAs other than mRNA are (still) produced
during senescence is apparently not known.
RNA and DNA are both degraded during senescence. In
petals, DNA and RNA are present in the nucleus,
mitochondria, chloroplasts (if present), and plastids.
Degradation of nucleic acids in these organelles can take
place before vacuolar collapse, by local enzymes. Breakdown of nucleic acids can also take place after vacuolar
collapse, as vacuoles of cells undergoing senescence
contain several DNases and RNases (Matile, 1997). No
data have as yet been reported to indicate that nucleic
acids are transported to the vacuole and are degraded
there.
RNA degradation
RNA can be degraded by RNases and by some DNases.
Winkenbach (1970a) found that RNA levels had already
decreased by the time of onset of Ipomoea flower
opening. RNA levels then further decreased as the flower
opened, accompanied by a dramatic upsurge of RNase
activity (Winkenbach, 1970b). RNase activity in Hemerocallis petals also increased during senescence (Panavas
et al., 2000). In Petunia inflata, the initial decrease in
mRNA levels was due to the increased activity of
constitutive RNases. Novel RNase activities appeared
rather late (Xu and Hanson, 2000). Overexpression of
a specific RNase inhibitor prevented senescence in an
animal system, indicating that RNA degradation is an
important regulator (Castelli et al., 1998). Whether this
also applies to plants remains to be evaluated.
DNA degradation
Degradation of DNA can be assessed in situ using the
TUNEL (TdT-mediated dUTP-biotin nick end labelling)
method, which labels DNA breaks in individual nuclei.
The TUNEL method preferentially labels double-stranded
breaks (Gravrieli et al., 1992). TUNEL-positive nuclei
were found in petals of Pisum (Orzaez and Granell, 1997),
Petunia (Langston et al., 2005), and Gypsophila. In
Gypsophila, TUNEL-positive nuclei appeared in all cells,
well before visible senescence symptoms and well before
the increase in ethylene production (Hoeberichts et al.,
2004).
Nuclear DNA is often degraded in two phases. During
the first stage, breakdown occurs into large fragments
(50 000–200 000 bp). The second stage consists of
breakdown into multiples of ;180 bp. When the DNA at
this second stage is placed on a gel, the 180 bp multiple
fragments often show a pattern reminiscent of a ladder;
hence the name DNA laddering (Nagata et al., 2003).
Accumulation of DNA fragments with multiples of
180 bp was found during the petal senescence of some
species, but it was not observed in several other species
(Table 5), indicating that it is not a good indicator of
senescence/PCD in petals. In Ipomoea, the onset of the
decrease in DNA content was accompanied by a large
increase in DNase activity (Winkenbach, 1970a, b). In
petals of Hemerocallis (Panavas et al., 1999), narcissus
(Hunter et al., 2004a), and Petunia (Langston et al.,
2005), the mRNA abundance of DNase (endonuclease)
genes increased during senescence.
In conclusion, RNA and DNA breakdown is often
related to an increase in the expression of some genes
encoding DNases, and is in some species accompanied by
a dramatic increase in RNase and DNase activities. In
some species, DNA degradation resulted in accumulation
of oligosomal 180 bp fragments, but in many other
species such accumulation was not observed.
Amino acid synthesis, protein synthesis, and
protein degradation
Amino acid synthesis
During senescence, amino acids are transferred to the
phloem. In Ipomoea petals, Wiemken et al. (1976) noted
a 4-fold increase of amino acids in the phloem exudate,
during the period from flower opening to petal wilting.
The Ipomoea exudate mainly contained glutamine, asparagine, and lysine, but also, at low concentrations,
c-aminobutyric acid and serine, and a range of other amino
acids. In Hemerocallis, glutamine and hydroxyproline
Table 5. Observation, on agarose gels, of clear DNA laddering
or no clear laddering, during petal senescence
Species
DNA
laddering
Reference
Gladiolus hybrid
Pisum sativum
Alstroemeria pelegrina
Antirrhinum majus
Argyranthemum frutescens
Ipomoea nil
Iris3hollandica
Petunia hybrida
+
+
–
–
–
–
–
–
Yamada et al. (2003)
Orzaez and Granell (1997)
Wagstaff et al. (2003)
Yamada et al. (2006b)
Yamada et al. (2006b)
Yamada et al. (2006a)
van der Kop et al. (2003)
Yamada et al. (2006b)
466 van Doorn and Woltering
were the main transport amino acids (Bieleski, 1995), and
in Sandersonia petals asparagine and glutamine were most
prominent (Eason et al., 2002).
The mRNAs of several genes encoding amino acid
synthetases were differentially expressed (Table 6). The
data support the idea of increased synthesis of at least
glutamine, asparagine, and cysteine. Table 6 also shows
increased transcript abundance of several other genes
involved in amino acid metabolism. The isovaleryl-CoA
oxidase, for example, is involved in valine and leucine
catabolism (Däschner et al., 2001), and serine hydroxymethyltransferase converts two glycine molecules to one
serine (Oliver, 1994).
In conclusion, the data in this section show considerable
synthesis of specific amino acids in cells undergoing
senescence, and accumulation of these amino acids in the
phloem.
Protein synthesis
Treatment with compounds that inhibit translation or
transcription delayed the visible symptoms of petal
senescence, showing that protein synthesis is required
(Xu et al., 2007). Down-regulation of eIF-5A delayed
carnation senescence (Hopkins et al., 2007), which leads
to the same conclusion.
In petals of Iris (van Doorn et al., 2003) and carnation
(Hoeberichts et al., 2007), the mRNA abundance of a gene
encoding a plant homologue of the animal translationally
controlled tumour protein (TCTP) increased. In animal
cells, the encoded protein has at least two functions.
It blocks protein synthesis by interacting with a eukaryotic
elongation factor (eEF). It also binds to pro-apoptotic
members of the Bcl-2 family, and thus induces death
(Rinnerthaler et al., 2006). It is not clear what the function
of the protein is in plant cells. It is discussed here under
protein synthesis, as the hypothesis may be put forward
that in plant cells it also interacts with an eEF.
Several genes related to protein synthesis are differentially expressed during petal senescence (Table 6). Both in
daffodil (Hunter et al., 2002) and in Alstroemeria
(Supplement 3 of Breeze et al., 2004), a DEAD/DEAH
domain helicase was up-regulated. These genes have high
homology to eIF-4A.
Protein degradation
Due to various rates of degradation, proteins have
a specific half-life. Protein breakdown occurs in the
proteasomes, vacuoles, mitochondria, the nucleus, and
plastids. Bulk degradation mainly occurs in vacuoles.
Prior to visible senescence symptoms, protein levels in
petals decrease, sometimes drastically as in Ipomoea
(Winkenbach, 1970a, b) and Petunia (Jones et al., 2005).
A decrease in overall protein levels can be due to
a decrease in synthesis as well as an increase in
degradation. In Ipomoea, no upsurge of in vitro protease
activity was detected during petal senescence
(Winkenbach, 1970a, b), but in other species, such as
Hemerocallis (Stephenson and Rubinstein, 1998), Iris
(Pak and van Doorn, 2005), and Petunia (Jones et al.,
2005), a massive increase in protease activity was found.
Table 6. Differential expression, during petal senescence, of genes putatively involved in amino acid metabolism and protein
synthesis
Gene
Amino acid metabolism
Asparagine synthase
Cysteine synthase
Glutamine synthase
Isovaleryl-CoA dehydrogenase
Serine hydroxymethyl transferaseb
Tryptophan synthase
Tyrosine aminotransferase
Protein synthesis
Ribosomal protein L2
Ribosomal protein L23
Ribosomal protein L32
Ribosomal protein S7
Ribosomal protein S12
Ribosomal structural RNAc
Translation initiation factor 4Ad
Translation initiation factor 5A
a
b
c
d
Species
Comment
Reference
Sandersonia aurantiaca
Alstroemeria pelegrina
Alstroemeria pelegrina
Sandersonia aurantiaca
Dianthus caryophyllus
Iris3hollandica
Alstroemeria pelegrina
Dianthus caryophyllus
Up-regulated
Up-regulated
Up-regulated
Highly expressed
Up-regulated
Down-regulated
Down-regulated
Up-regulated
Eason et al. (2000)
Breeze et al. (2004)a
Breeze et al. (2004)
Eason et al. (2000)
Hoeberichts et al. (2007)
van Doorn et al. (2003)
Breeze et al. (2004)
Hoeberichts et al. (2007)
Alstroemeria pelegrina
Alstroemeria pelegrina
Dianthus caryophyllus
Alstroemeria pelegrina
Alstroemeria pelegrina
Iris3hollandica
Alstroemeria pelegrina
Narcissus pseudonarcissus
Narcissus pseudonarcissus
Up-regulated
Up-regulated
Up-regulated
Up-regulated
Up-regulated
Down-regulated
Up-regulated
Up-regulated
Downregulated
Breeze et al. (2004)a
Breeze et al. (2004)
Hoeberichts et al. (2007)
Breeze et al. (2004)
Breeze et al. (2004)
van Doorn et al. (2003)
Breeze et al. (2004)a
Hunter et al. (2002)
Hunter et al. (2002)
Data from Supplement 3.
Accession CF227016, previously described to be homologous to a glycine hydroxymethyltransferase.
Accession CF226940, previous described as senescence-associated protein.
These proteins were described as DEAD/DEAH box helicases.
Petal senescence 467
Protein degradation in proteasomes
Proteasomes are involved in degradation of specific
proteins. They degrade misfolded proteins and can determine the level of normally folded proteins. As
discussed above, proteasomes are important regulators of
ethylene action, as they regulate the level of EIN3/EIL
transcription factors. The level of other important transcriptional regulators, such as those involved in auxin and
gibberellin responses, are also determined by proteasomal
action (Bishopp et al., 2006). The proteasome system was
apparently up-regulated during petal senescence in daylily
(Courtney et al., 1994; Müller et al., 2004), daffodil
(Hunter et al., 2002), and carnation. In carnation, the
mRNA abundance increased of three genes encoding
subunits of the 19S regulatory particle, one of the two
large complexes of the 26S proteasome (Hoeberichts
et al., 2007). Feeding isolated Iris petals with Z-Leu-LeuNva-H, an inhibitor of proteasome activity, produced
a small but significant delay in the time to visible
senescence (Pak and van Doorn, 2005), indicating that
proteasome action is limiting senescence.
Several monomers of ubiquitin, a 76 amino acid polypeptide, become attached to proteins targeted for degradation in the proteasome. Ubiquitin attachment involves
three enzymes referred to as E1, E2, and E3. The cascade
starts with E1 (ubiquitin-activating enzyme) which binds
to ubiquitin and transfers it to E2 (ubiquitin-conjugating
enzyme). Ubiquitin ligase (E3) binds to E2 and to the
target protein, and transfers the ubiquitin moiety from E2
to the target protein. E3 can be monomeric or form
a protein complex. Some types of multimeric E3 complexes contain an F-protein, which determines which
specific protein will be ubiquitinated. During senescence,
the mRNA abundance of several genes related to
ubiquitination increased (Table 7).
These results strongly indicate that petal senescence
is associated with enhanced activity of the ubiquitin–
proteasome route of protein degradation. The delay of
visible senescence symptoms after chemical inhibition of
proteasomes in Iris and after silencing a RING domain E3
protein in Petunia (Xu et al., 2008) even indicate that
proteasome action is required for senescence.
Non-proteasomal protein breakdown
Proteasome-independent protease activity markedly
increases prior to visible senescence (Stephenson and
Rubinstein, 1998; Pak and van Doorn, 2005). Nonproteasome proteases are often divided into exo- and
endoproteases, referring to the position in the target
protein where the cleavage takes place. Endoproteases
include cysteine-, serine-, aspartic-, and metalloproteases,
named after the amino acid residues or the metals that are
required for the cleavage reaction. The activities of these
protease classes have been assessed by using inhibitors
and in vitro measurements whereby a common protein is
Table 7. Increase in the expression of genes putatively involved
in protein ubiquitination (leading to proteasomal protein
degradation), and genes encoding proteins in the 19S subunit
of the proteasome, during petal senescence
Gene group
Species
Reference
Breeze et al. (2004)
Hunter et al. (2002)
E3 HECT domain
Alstroemeria pelegrina
Narcissus
pseudonarcissus
Ipomoea nil
E3 U-box (arm)
Dianthus caryophyllus
E3 RING domain
Dianthus caryophyllus
F-box
Mirabilis jalapa
Petunia hybrida
Dianthus caryophyllus
Proteasomal subunits
19S particle
Dianthus caryophyllus
Ubiquitination
E2
Yamada et al.
(2007a)
Hoeberichts
et al. (2007)
Hoeberichts
et al. (2007)
Xu et al. (2008)
Xu et al. (2008)
Hoeberichts
et al. (2007)
Hoeberichts
et al. (2007)
used as a substrate for all proteases extracted from a tissue.
However, most protease inhibitors are not specific. Only
E-64—and probably antipain—are specific inhibitors,
both affecting cysteine proteases. Using E-64 in vitro,
total protease activity was reduced by about half in petals
of Hemerocallis (Stephenson and Rubinstein, 1998) and
Iris (Pak and van Doorn, 2003) and by >90% in Petunia
(Jones et al., 2005). Antipain reduced protease activity of
Sandersonia petals by ;30% (Eason et al., 2002).
Feeding Iris petals with a membrane-permeable form of
E-64 also indicated that about half of the peak protease
activity was due to cysteine proteases (Pak and van
Doorn, 2003). The results of the other (non-specific)
inhibitors might suggest a small amount of metalloprotease activity at the peak of protease activity in Hemerocallis, Iris, and Petunia, considerable serine protease
activity in Hemerocallis and Iris, but almost no activity in
Petunia, and little or no activity of aspartic proteases in
these species (Stephenson and Rubinstein, 1998; Pak and
van Doorn, 2003; Jones et al., 2005), but these data need
verification by means other than inhibitors.
Treatment of Iris flowers, at the onset of flower life,
with AEBSF and DFP prevented the normal visible
senescence symptoms. These compounds are general
serine protease inhibitors, but AEBSF is also a general
NADPH-oxidase inhibitor. Other effects of both compounds
cannot be excluded. AEBSF and DFP delayed the onset of
bulk protease activity during senescence, thus preventeing
both cysteine and non-cysteine protease activity (Pak and
van Doorn, 2005). Interestingly, AEBSF has also been
reported to inhibit apoptotic PCD in animals (Solovyan
and Keski-Oja, 2006).
AEBSF, which completely suppressed cysteine protease
activity in Iris, delayed senescence, but feeding Iris petals
468 van Doorn and Woltering
with the membrane-permeable form of E-64, which
reduced peak protease activity to about half, had no effect
on senescence (Pak and van Doorn, 2003). The cysteine
protease inhibitor 2,2#-dipyridyl delayed the time to wilting
in Sandersonia petals and prevented the senescenceassociated rise in endoprotease activity (Eason et al.,
2002). This effect might relate to cysteine protease
inhibition, but side effects of 2,2#-dipyridyl cannot be
excluded.
Table 8 summarizes the present data on the increase in
the expression of genes encoding non-proteasomal proteases, most of which are probably vacuole localized.
Genes encoding metalloproteases have not yet been
reported. Some of the encoded cysteine proteases contain
the C-terminal KDEL domain, which is an ER retention
motif (Eason et al., 2002; Wagstaff et al., 2002). KDEL
domain cysteine proteases are generally found in vesicles
that bud from the ER and merge with the vacuole. One
type of vesicles containing KDEL cysteine proteases have
been suggested not to merge with the vacuole but remain
until they are destroyed after vacuolar collapse
(Greenwood et al., 2005). These vesicles, called ricinosomes, were reportedly present in petals of Hemerocallis
undergoing senescence (Schmid et al., 1999).
As many as nine cysteine proteases have been isolated
from senescing Petunia petals. In a transgenic Petunia
line that was ethylene insensitive, the time to petal wilting
was delayed by ;8 d. In this line, the increase in
Table 8. Increase, during petal senescence, in the expression
of genes putatively involved in non-proteasomal protein
degradation
Gene group
Species
Proteases in vacuoles
and vesicles
Aspartic proteases
Alstroemeria pelegrina
Hemerocallis hybrid
Cysteine proteases
Alstroemeria pelegrina
Dianthus caryophyllus
Hemerocallis hybrid
Iris3hollandica
Serine proteases
Proteases in
mitochondria and
plastids
Clp protease
Reference
Breeze et al. (2004)
Panavas et al. (1999)
Wagstaff
et al. (2002)
Hoeberichts
et al. (2007)
Valpuesta
et al. (1995)
van Doorn
et al. (2003)
Hunter et al. (2002)
Narcissus
pseudonarcissus
Petunia hybrida
Jones et al. (1995)
Sandersonia aurantiaca Eason et al. (2002)
Narcissus
Hunter et al. (2002)
pseudonarcissus
Alstroemeria pelegrina
Iris3hollandica
expression of four cysteine protease genes occurred later,
corresponding to the delay of petal wilting (Jones et al.,
2005).
In carnation petals, a gene encoding a cysteine protease
inhibitor, abundant at the time of flower opening, became
gradually down-regulated. Its mRNA had disappeared by
the time of the increase in cysteine protease expression
and petal wilting (Sugawara et al., 2002). This cysteine
protease inhibitor showed high homology with cystatin.
Hoeberichts et al. (2007) found a transient increase in the
expression of another cystatin gene in carnation. Since in
other systems cystatins suppressed PCD (Rojo et al.,
2003, 2004), these vacuolar proteins might be regulators
of petal senescence.
Chloroplasts, non-chloroplast plastids, and mitochondria
usually contain several families of ATP-dependent proteases (called Clp, Lon, and FtsH), and one family of
ATP-independent proteases (called DegP). The Clp, Lon,
and DegP families are serine proteases, and the FtsH
family are metalloproteases (Sinvany-Villalobo et al.,
2004). The transcript abundance of Clp proteases increased prior to visible senescence (Table 8). Plant nuclei
also contain non-proteasomal proteases, for example
a serine protease (Domı́nguez and Cejudo, 2006), which
might be involved in degradation of nuclear proteins
during senescence.
In conclusion, petal senescence is accompanied by bulk
non-proteasomal protein breakdown, mainly in vacuoles
but possibly also in mitochondria, plastids, and nuclei.
Many genes encoding proteases have been identified.
Protease activity in the vacuole seems to be regulated by
enzyme activation and by enzyme inhibition, in which
VPEc seems to be involved. The level of cystatins,
cysteine protease inhibitors, in vacuoles might be a regulator of petal senescence.
Breeze et al. (2004)
van Doorn
et al. (2003)
Lipid synthesis and breakdown
Lipid synthesis
Bieleski and Reid (1992) found evidence for rapid cessation
of overall phospholipid synthesis during Hemerocallis petal
senescence. Nonetheless, several genes that are apparently
involved in lipid synthesis become up-regulated during petal
senescence (Table 9). b-Ketoacyl-CoA synthase is the first
enzyme of fatty acid elongation, whilst acyl thioesterase is
involved in the last step. Acyl-CoA reductase may be
associated with the increase in transcript abundance of
a gene encoding CER1, and be involved in steryl/wax ester
synthesis in membranes (Madey et al., 2001). Fatty acid
hydroxylases are involved in various pathways, including
those producing isoprenoids, carotenoids, and oxylipins.
The fatty acid hydrolases CYP86, CYP94, and CYP96 are
classified in the CYP86 clan, involved in fatty acid
metabolism (Nelson et al., 2004).
Petal senescence 469
Table 9. Differential expression, during petal senescence, of genes putatively involved in fatty acid synthesis
Gene
Species
Comment
Reference
Acyl carrier protein (ACP) thioesterase
ACP b-ketoacyl-CoA synthase
Dianthus caryophyllus
Hemerocallis hybrid
Iris3hollandica
Alstroemeria pelegrina
Ipomoea nil
Alstroemeria pelegrina
Up-regulated
Up-regulated
Down-regulated
Down-regulated
Up-regulated
–b
Hoeberichts et al. (2007)
Pananas et al. (1999)
van Doorn et al. (2003)
Breeze et al. (2004)
Yamada et al. (2007a)
Breeze et al. (2004)
Acyl-CoA reductase
Fatty acid hydroxylase (CYP96/CYP86)a
Fatty acid hydroxylase (CYP94/CYP96)
a
b
Accession AB267814. Previously described (more generally) as a cytochrome P450 protein.
Two genes were up-regulated, whilst one was down-regulated. The encoded proteins were previously described as cytochrome P450 containing.
The data indicate that mass lipid degradation during
senescence is accompanied by the synthesis of some fatty
acids and by some compounds, such as wax esters,
derived from fatty acids.
Table 10. Up-regulation, during petal senescence, of genes
putatively involved in degradation of phospholipids and fatty
acids
Fatty acid degradation
Phospholipid
degradation
Allene oxide
synthase
During senescence, degradation of free fatty acids mainly
occurs by b-oxidation (Pistelli et al., 1992). This pathway,
which repeatedly removes C2 units from the fatty acid
chain, is present in peroxisomes. Several genes encoding
enzymes that degrade fatty acids were reportedly upregulated during petal senescence (Table 10). Acyl-CoA
oxidase isozymes catalyse the first step in peroxisomal
fatty acid b-oxidation. The transcript abundance of a gene
encoding an acyl-CoA synthase increased during senescence. The mRNA abundance of an acyl-CoA dehydrogenase, also part of the b-oxidation chain, also increased.
Thiolases are often localized to peroxisomes, and involved
in b-oxidation, but some isozymes are localized to
mitochondria where they are also involved in fatty acid
degradation.
The end-product of b-oxidation (acetyl-CoA) is fed into
the glyoxylate cycle, also localized to the peroxisomes.
Isocitrate lyase and malate synthase are key enzymes in
this cycle. Both genes were up-regulated during senescence
in cucumber petals (Graham et al., 1994; McLaughlin and
Smith, 1994). The glyoxylate pathway produces organic
acids which can be converted to sugars (gluconeogenesis)
in the cytosol (Chen et al., 2000; Cornah et al., 2004) or
be used for the synthesis of amino acids.
Phospholipid degradation and membrane leakiness
All enzymes required for phospholipid degradation are
apparently always present in the membranes, even in
those of young cells, and are involved in continuous
phospholipid turnover (Fobel et al., 1987; Brown et al.,
1990). During senescence, a massive loss of phospholipids is often found, concomitant with an increase in
phospholipid degradation products (Suttle and Kende,
1980; Thompson et al., 1998; Hopkins et al., 2007).
Isolated plasma membranes from Petunia petals showed
an increase in the levels of free polar lipid head groups,
phosphatidic acid, and phosphatidylinositol monophos-
Gene
GDSL lipase/
hydroxylase
Lipolytic acyl
hydrolase
Unspecific lipases
Fatty acid
degradation
Acyl-CoA
dehydrogenase
Acyl-CoA oxidase
Acyl-CoA
synthasea
Isocitrate lyase
3-Ketoacyl-CoA
thiolase
Malate synthase
Species
Reference
Hemerocallis hybrid
Panavas et al. (1999)
Petunia hybrida
Iris3hollandica
Xu et al. (2006)
van Doorn et al. (2003)
Dianthus caryophyllus
Thompson et al. (2000)
Iris3hollandica
Dianthus caryophyllus
van Doorn et al. (2003)
Hoeberichts et al. (2007)
Iris3hollandica
van Doorn et al. (2003)
Dianthus caryophyllus
Phalaenopsis orchids
Iris3hollandica
Hoeberichts et al. (2007)
Do and Huang (1997)
van Doorn et al. (2003)
Cucumis sativa
Dianthus caryophyllus
Graham et al. (1994)
Hoeberichts et al. (2007)
Cucumis sativa
Graham et al. (1994)
a
Some other contigs with high homology to acyl-CoA synthase were
down-regulated during Iris petal senescence.
phate (Borochov et al., 1994). This is indicative of
phospholipid breakdown.
There is considerable debate about the main sites of
phospholipid degradation. The issue is to what degree the
increase in phospholipid degradation in membranes during
senescence occurs inside the autophagosomes and
vacuoles and to what degree in the membrane before
being sequestered in these organelles. All sides of the
debate accept that some membrane degradation occurs
inside chloroplasts and plastids (Hopkins et al., 2007).
Many apparently intact membranes are observed inside
vacuoles, suggesting that at least part of phospholipid
degradation occurs there. In yeast and animal cells, there
is compelling evidence for vacuolar/lysosomal degradation of phospholipids, but such firm evidence is still
lacking in the case of plants (Inoue and Moriyasu, 2006).
470 van Doorn and Woltering
At least one of the enzymes involved in mass phospholipid degradation, lipolytic acyl hydrolase, seemed to be
mainly present in the vacuole and, under experimental
conditions, able to degrade all phospholipids of the cell in
a very short period. This enzyme might be involved in
vacuolar degradation of phospholipids during senescence,
and will probably rapidly degrade any membranes left by
the time of vacuolar collapse (Travnicek et al., 1999).
An argument against the role of autophagy in phospholipid degradation might be found in the finding that
treatment with 3-methyladenine (3-MA), an inhibitor of
autophagy, did not affect the degradation (Inoue and
Moryasu, 2006). However, this inference might be false
since 3-MA not only inhibits autophagy, but also has
several side effects (Xue et al., 1999).
A second issue of contention is the idea that the plasma
membrane becomes permeable prior to vacuolar collapse,
due to phospholipid degradation inside the membrane
(Hopkins et al., 2007). Ions—and in some coloured petals
also anthocyanins—are increasingly released by petals,
during senescence (Suttle and Kende, 1980; Celikel and
van Doorn, 1995). The release of ions into the apoplast
might, theoretically, be passive (as a result of tonoplast
rupture or as a result of increased plasma membrane
leakiness), or active, regulated as part of remobilization
and export through the phloem. These possibilities have
not been experimentally disentangled, thus far.
In contrast to ions, the release of (vacuole-located)
anthocyanins from epidermis cells is most probably not
active, and thus must be a result of loss of semipermeability in both the tonoplast and the plasma
membrane. The amount of anthocyanin leakage from
petals seems to be a reflection of the number of dead cells.
Hopkins et al. (2007) defended the idea that the plasma
membrane loses its semi-permeability before the cell dies.
They referred to the work of Eze et al. (1986), who noted
increased release of ions from carnation flowers, cut at the
base of all petals and placed in water. In these experiments, however, the ion release was not shown to be due
to membrane leakage. It might have been due to active
efflux of ions into the apoplast. These tests also involved
flowers under severe water stress, indicated by the
considerable loss of fresh weight of the flower stems. The
experiment needs to be repeated with attached flowers,
and has to separate active and passive transport of ions
into the apoplast. The data presently available, therefore,
provide no decisive argument in favour of the hypothesis
that the plasma membrane loses its semi-permeability
before the cell dies.
A change from the fluid to gel phase in membranes, and
an increase in membrane fluidity (microviscosity) have
been suggested to be due to enzyme-induced degradation
processes inside the membranes. These changes were
hypothesized to be the cause of a gradual increase in
membrane leakage in senescent petals (Borochov and
Woodson, 1989). However, as noted, increased leakage of
otherwise intact membranes has not been demonstrated in
such petals. Freeze-fracture EM of senescing carnation
petals indicated the presence of gel phase lipid in the
plasma membrane, ER, and tonoplast (Hopkins et al.,
2007), but it is not clear how much this occurred above
control levels and at what stage of senescence it took
place (before or after vacuolar collapse). The data seem to
be inadequate evidence for the idea of a gradual increase
in membrane leakiness, during senescence.
Biochemistry of phospholipid degradation
Membranes usually consist of phospholipids, proteins, and
sterols. Phospholipid breakdown during petal senescence
has been studied mainly by using reconstituted membranes (microsomes) from the whole tissue. Such an
approach is only a first approximation. The microsomes
are a mixture of all membranes, which in one cell can be
at various stages of degradation. It is also a mixture of the
membranes of a large number of cells that may be at
various stages of senescence. The work on microsomes,
therefore, will not be considered here in detail. Only a few
studies used specific membranes. The plasma membrane
of Petunia petals, for example, showed a decrease in
the phospholipid and protein levels during senescence
(Borochov et al., 1994), but it is not clear if this was due
to vacuolar collapse in some petal cells, or if it occurred
prior to this collapse.
The main phospholipid-degrading enzymes are
(i) phospholipase D (PLD); (ii) phosphatidic acid phosphatase, also called phospholipase C (PLC); (iii) lipolytic
acyl hydrolase (PLA1 and PLA2); and (iv) lipoxygenase.
Phospholipid breakdown is apparently usually initiated
by PLD, which removes the head groups, producing
phosphatidic acid. Phosphatidic acid phosphatase then
removes the phosphate moiety, and thus converts phosphatidic acid to diacylglycerol. Lipolytic acid hydrolases
and other lipases remove the fatty acids from the glycerol
backbone, or from phospholipids. As described, fatty
acids are converted—mainly by b-oxidation—to smaller
molecules.
PLD activity, measured in vitro, decreased rapidly
during senescence of Tradescantia petals (Suttle and
Kende, 1980). In vivo membrane-bound PLD activity
increased at least 2-fold early on during senescence in the
tip of Iris petals (van Doorn et al., 2003). A purported
inhibitor of PLD, lysophosphatidyl-ethanolamine, was
found to delay the onset of visible petal senescence in
Antirrhinum (Kaur and Palta, 1997). If this inhibitor is
PLD specific, the result suggests that PLD activity is
important in determining the time to the visible senescence symptoms. However, confirmation of this effect in
other species has not been reported. In vitro lipolytic acyl
hydrolase activity has thus far been shown only in
Petal senescence 471
Tradescantia petals. It did not change during senescence
(Suttle and Kende, 1980).
Lipoxygenase oxidizes double bonds in fatty acids. Its
activity in Alstroemeria petals decreased almost linearly
from the time of harvest until the senescence symptoms
(Leverentz et al., 2002). A small increase in lipoxygenase
activity was found in Hemerocallis, but only by the time
of visible senescence (Panavas and Rubinstein, 1988). No
increase in lipoxygenase activity was observed during
petal senescence in Phalaenopsis and Dendrobium orchids
(Porat et al., 1995). Lipoxygenases in membranes produce
hydroperoxides, which are the substrate of allene oxidases
(Xu et al., 2006). As inhibitors of lipoxygenase had no
effect on the time to visible senescence in Phalaenopsis
and Dendrobium orchids (Porat et al., 1995), it is
questionable whether lipoxygenases are important in
determining the time to visible effects.
In these experiments, it is not clear whether the in vitro
measurements reflect in vivo changes. If they do reflect
real changes, the results may still conceal what happens in
specific cellular compartments.
The present data regarding the expression of genes
putatively involved in phospholipid degradation are
summarized in Table 10. Genes encoding PLD or PLC
have thus far not been isolated from petals undergoing
senescence. Except for one gene, the functional significance of the phospholipid- and fatty acid-degrading
enzymes has as yet not been reported. Decreased
expression in Arabidopsis of a lipolytic acyl hydrolase
found to be up-regulated by the time of visible senescence
in carnation petals delayed leaf yellowing (Thompson
et al., 2000). The gene might therefore also be important
in the control of petal senescence.
It is concluded that membrane degradation may be
a central step in the programme that leads to cell death, as
collapse of the tonoplast seems to execute the death
sentence. A gradual loss in membrane permeability, rather
than membrane collapse, has thus far not been unequivocally shown, for any membrane that remains functional
until the end of senescence, such as the plasma membrane
and the tonoplast. Already at a relatively early phase of
senescence, membranes are degraded en masse, but it is
not clear where this occurs: in the membranes in situ,
prior to their uptake in autophagosomes and delivery in
the vacuole, or after this uptake.
Do reactive oxygen species contribute to
petal cell death?
ROS include both free radicals and hydrogen peroxide.
They are produced during normal metabolism, in many
organelles. There is good evidence that ROS contribute to
the PCD that is induced by plant pathogens (Apel and
Hirt, 2004; Van Breusegem and Dat, 2005). In contrast,
there is as yet no evidence that an increase in ROS is part
of the causal chain leading to developmental senescence,
either as an early regulator of the onset of senescence or,
because of the destructive nature of ROS, as a direct cause
of cellular death.
ROS might well be increasingly produced during
senescence, but most of the ROS produced might be
rendered innocuous. For example, ROS are probably
formed when lipoxygenase peroxidizes double bonds in
membrane C18–C22 fatty acids. The resulting fatty acid
hydroperoxides are free radicals, able to react with other
fatty acids in a non-enzymatic chain reaction. In Alstroemeria, fatty acid hydroperoxides accumulated in the outer
whorl of petals, whereas no accumulation was found in
the inner whorl. As these parts exhibited visible senescence symptoms at the same time, the fatty acid
hydroperoxide levels were apparently of little importance
for the production of the visible senescence symptoms
(Leverentz et al., 2002).
Evidence for polyunsaturated fatty acid oxidation also
follows from accumulation of some of its end-products:
malondialdehyde and other thiobarbituric acid (TBA)reactive compounds. In the inner Alstroemeria tepals
(homologous to petals), the levels of TBA-reactive
compounds remained almost constant, but in the outer
whorl tepals (sepals) the levels peaked at an early stage
and then declined throughout senescence. It was concluded that lipid peroxidation was ;2–3 times higher in
the outer whorl compared with the inner one, but that
these differences were irrelevant to the onset of senescence
(Leverentz et al., 2002).
It has been concluded that the activity of the main free
radical-scavenging enzymes, during leaf senescence, such
as superoxide dismutase (SOD), catalase, and ascorbate
peroxidase (APX), tend to increase. This might indicate
that an increase in free radical production is counteracted,
at least in part, by an increase in their degradation
(Buchanan-Wollaston, 1997). In petals, the activity of
anti-oxidative enzymes such as SOD, catalase, APX, and
glutathione reductase (GR) have thus far been assessed
only crudely, that is only in vitro and only in whole petals
(Droillard et al., 1987; Bartoli et al., 1995; Panavas and
Rubinstein, 1998; Bailly et al., 2001). These data are of
limited value, as they might not represent in vivo activity.
Moreover, different isoforms of these enzymes are present
in various cell compartments. These have to be separated
before the role of anti-oxidative enzymes can be properly
evaluated.
Levels of non-enzymatic antioxidants, such as ascorbate
and a-tocopherol, have also been determined only in
whole petals (Bartoli et al., 1997; Panavas and Rubinstein,
1998), but must also be determined in each compartment
separately.
Nonetheless, application of anti-oxidants can be used as
a method of preliminary assessment of the role of free
radicals in senescence. In Dianthus (Paulin et al., 1986;
472 van Doorn and Woltering
Droillard et al., 1987) and Hemerocallis (Panavas and
Rubinstein, 1998), anti-oxidants delayed the time to
visible senescence, but in Iris such compounds were
ineffective (Celikel and van Doorn, 1995; Bailly et al.,
2001).
Taking the data on ROS together, it is concluded that in
some species the end-products of lipid peroxidation
accumulate during petal senescence, indicating that ROS
are involved in membrane degradation. This process may
be localized in a specific organelle (such as the vacuole)
where it can be part of ongoing remobilization and not be
harmful to the cell. In other organelles, the production of
ROS may similarly increase. However, there is at present
no conclusive evidence that ROS are either an early signal
or a direct cause of cellular death during petal senescence.
Nevertheless, it cannot at present be excluded that ROS
contribute to cell death. The disruption of the tonoplast
can, theoretically, come about by processes that involve
ROS, causing membrane destabilization.
How does tonoplast collapse come about?
It is still an enigma how the rupture of the tonoplast
occurs. Several hypotheses have been suggested. One is
a mechanism whereby phospholipid synthesis is turned
off. During senescence in daylily petals, the rate of
phospholipid synthesis became lower than the rate of
degradation (Bieleski and Reid, 1992). Tonoplast rupture
may therefore be the result of a change in the equilibrium
between phospholipid breakdown and synthesis.
Since many lipid-degrading enzymes seem to be
localized to the vacuole, it is remarkable that the tonoplast
is not degraded very early. It has been suggested that the
access of the degrading enzyme to the tonoplast is
prevented by a coating of carbohydrates. Membrane
proteins can be glycolysated at the vacuolar side of the
membrane, with the carbohydrate parts sticking out,
preventing access of hydrolases (Travnicek et al., 1999).
This would require, however, a large amount of membrane proteins or very large glycosylation moieties
covering the inner side of the membrane.
Other hypotheses include the idea of membrane degradation in situ. Lipid de-esterification might result in the
accumulation of free fatty acids, which can destabilize
and perturb the membrane due a detergent-like action
(Hopkins et al., 2007). Alternatively, real detergent-like
molecules might accumulate in the vacuole and then insert
in the tonoplast. Furthermore, as knockout of a VPEc
delayed tonoplast rupture and cell death, induced by
a virus (Hara-Nishimura et al., 2005), the activation of
proteases, or other enzymes, by VPEs might be important
as a regulator of tonoplast rupture.
The data in this section show that if tonoplast rupture is
the direct cause of cell death during petal senescence, it is
as yet completely unknown how it comes about. Several
testable hypotheses can be put forward. It is suggested
here that future work on petal senescence requires
considerably more focus on the processes related to the
maintenance and collapse of tonoplast structure.
Degradation of cell wall compounds
Some types of PCD do not result in much cell wall
degradation, for example PCD that leads to the formation
of xylem vessels and tracheids. In petals, the degree of
cell wall degradation seems to be dependent on the
species. In Sandersonia, for example, relatively little wall
degradation takes place. The desiccated flower, after
senescence, still has the same bell shape as the nonsenescent flower. In other species, for example Ipomoea
and Iris, considerable parts of the walls of mesophyll cells
become degraded.
During petal senescence, cell wall components are
broken down both before and after vacuolar collapse.
Ultrastructural work in Iris showed that the walls of
mesophyll cells became inflated and much less electron
dense, indicating degradation. This started prior to
vacuolar collapse (van Doorn et al., 2003). In Ipomoea,
the content of cellulose and hemicellulose even decreased
from the moment of flower opening (Winkenbach, 1970a;
Wiemken-Gehrig et al., 1974).
Visible senescence symptoms in carnation petals coincided with a lower cell wall dry weight, due largely to
a loss of neutral sugars, primarily galactose (45%) and
arabinose (23%). Soluble pectin levels in the walls
decreased. The amount of high molecular weight hemicelluloses decreased, associated with a decrease in xylose
and galactose content (de Vetten and Huber, 1990). In
Sandersonia, in contrast, levels of total pectin, cellulose,
and high molecular weight hemicelluloses increased only
after the first visible senescence symptoms, and despite
the previous death of numerous mesophyll cells
(O’Donoghue et al., 2002, 2005).
Senescence was associated with an increase in the
activities of b-galactosidase (Ipomoea, Winkenbach,
1970a; Wiemken-Gehrig et al., 1974; Sandersonia,
O’Donoghue et al., 2002, 2005; Hemerocallis, Panavas
et al., 1998a), a- and b-glucosidase (Ipomoea,
Winkenbach, 1970a; Wiemken-Gehrig et al., 1974), and
polygalacturonase (Hemerocallis, Panavas et al., 1998a).
In carnation petals undergoing senescence, polygalacturonase activity was not detected (de Vetten and Huber,
1990). In Sandersonia, the activity of xyloglucan endotransglycolase/hydrolose (XET/XTH) was high and declined only by the time of flower wilting (O’Donoghue
et al., 2002, 2005). An increase has been found in the
transcript abundance of several genes that encode
enzymes putatively involved in cell wall degradation
(Table 11). These data tend to confirm the biochemical
data on enzyme activity.
Petal senescence 473
Table 11. Increase in transcript abundance, during petal
senescence, of genes encoding enzymes that are putatively
involved in cell wall degradation
Table 12. Increase in transcript abundance, during petal
senescence, of genes encoding enzymes that are putatively
involved in defence
Gene
Species
Reference
Gene
Species
Reference
b-Galactosidase
Iris3hollandica
Sandersonia aurantiaca
Dianthus caryophyllus
Dianthus caryophyllus
Alstroemeria pelegrina
van Doorn et al. (2003)
O’Donoghue et al. (2005)
Hoeberichts et al. (2007)
Hoeberichts et al. (2007)
Breeze et al. (2004)b
Avr9/Cf-9 rapidly
elicited protein 146
Chitinase IIa
Dianthus caryophyllus
Class III peroxidase
Dianthus caryophyllus
Dianthus caryophyllus
Iris3hollandica
Iris3hollandica
Alstroemeria pelegrina
Hoeberichts et al. (2007)
van Doorn et al. (2003)
van Doorn et al. (2003)
Breeze et al. (2004)b
Heat shock potein 201
Dianthus caryophyllus
Polygalacturonase
inhibitor-like leucine
rich repeat protein
Ribosome-inactivating
proteins
Salicylic acid-binding
protein 2
Thaumatin/PR5 protein
Dianthus caryophyllus
Hoeberichts
et al. (2007)
Breeze et al.
(2004)a
Hoeberichts
et al. (2007)
Hoeberichts
et al. (2007)
Hoeberichts
et al. (2007)
Tropinone reductases
Iris3hollandica
b-Glucosidase
b-Xylosidasesa
Endoxyloglucan
transferase
Expansin
Pectinesterase
XET
Xylose isomerase
a
b
Previously described as glycosyl hydrolase family 3 proteins.
Data from Supplement 3.
The data discussed in this section indicate that cell wall
degradation is part of petal senescence. In most species at
least part of the cell walls becomes remobilized.
a
Expression of genes putatively involved in
defence
Several putative pathogen defence genes were upregulated during petal senescence (Table 12). Tropinone
reductases are involved in tropane-alkaloid synthesis,
which is stimulated by insect herbivory. They defend
against this herbivory. In Iris, many genes encoding
ribosome-inactivating proteins were up-regulated (van
Doorn et al., 2003). It is not clear if these proteins
function as antimicrobial agents or are involved in
inhibiting ribosomes in the cells undergoing senescence.
The transcript abundance of a putative class III peroxidase
(cell wall peroxidase) decreased in Iris petals (van Doorn
et al., 2003), but in carnation petals several genes were
up-regulated (Hoeberichts et al., 2007). The enzymes may
be involved in ROS-mediated defence against pathogens.
Plant defence genes seem not to be causally related to
remobilization or cell death. They might nonetheless be an
integral part of senescence, as numerous cells collapse.
Water, containing nutrients such as sugars and amino
acids, is then released into the cell walls. The senescent
petal might thus be an ideal substrate for micro-organisms,
which from there can enter the remainder of the plant.
Conclusions
The present review points to a number of weaknesses in
the present work on petal senescence. Analysis thus far
often occurs at the level of whole petals, which always
comprise cells that are at various stages of senescence. In
the petals of all species, the cells around the phloem
remain for a considerably longer time than the other cells.
In several species the mesophyll cells die considerably
earlier than the epidermis cells. Moreover, visible wilting
Alstroemeria pelegrina
Iris3hollandica
Dianthus caryophyllus
Dianthus caryophyllus
van Doorn et al.
(2003)
Hoeberichts et al.
(2007)
Hoeberichts et al.
(2007)
van Doorn et al.
(2003)
Data from Supplement 3.
(which tends to reflect the collapse of the epidermis cells)
and other symptoms of senescence often start at the petal
tips and progress toward the base. The level of precision
will probably gain considerably by analysis of cells that
are at the same stage of senescence.
Moreover, detailed analysis has to include the level of
organelles. Topics such as membrane composition, carbohydrate distribution, and ROS clearly need to be investigated at
the organelle level, rather than at the level of whole cells.
During the last decade, some progress has been made in
understanding the physiological and molecular background of senescence in general. Ethylene receptors have
been discovered in other plants, and several receptor
genes have been isolated from petals. In addition, proteins
involved in the ethylene signal transduction pathway have
been discovered, and similar proteins have been found in
senescing petals. One of the findings in (carnation) petals
is the apparent large effect of EIL3.
Numerous genes were found to be differentially
expressed during petal senescence, and the putative
functions of many of the sequenced genes have been
suggested. The main processes suggested by these
changes are remobilization and transport of mobile
compounds out of the petal. The work suggests the
importance of proteasomal protein degradation, and
the apparent role of sugars in preventing the up-regulation
of numerous genes that are controlled by ethylene. In
addition, the gene expression studies revealed upregulation of several factors associated with plant defence.
However, thus far, the work on gene expression has
answered questions neither regarding the onset of the
senescence program, nor about the way the cells die.
Although several signal transduction and transcription
474 van Doorn and Woltering
factors have been found, their role is as yet unknown.
Functional analysis is likely to reveal some of the
regulatory networks controlling the onset and the orderly
progress of senescence.
A main discovery has been the requirement of the ovary
for carnation petal senescence. This requirement might
also be true for other species with ethylene-sensitive petal
senescence, at least those where the pollination signal has
to pass the ovary before it can reach the petals.
There remain several major gaps in our present understanding. These will be discussed here in the temporal
order of the senescence process, from its initiation to the
execution of cellular death. Some testable hypotheses will
also be put forward.
Some of the main questions that are still outstanding are
as follows.
flowers, this rise in ethylene production also occurs.
It is hypothesized that it is due to a decrease in
cytokinin levels, localized, at least in a species like
carnation, in the ovary. If so, the question changes
to what induces the decrease in cytokinin levels.
(iv) What hormonal factors, if any, determine the time to
visible ethylene-independent senescence? It is hypothesized here that this type of senescence, although
it can be affected by hormones, is not naturally
regulated by any hormone, but by gene expression in
the petal cells. It is further hypothesized that
ethylene-insensitive senescence is the basal pathway,
and ethylene-sensitive senescence an additional level
of control allowing pollination to advance the time to
senescence.
(v)
(i) What determines the onset of the senescence process? In several species tested, changes in cellular
ultrastructure, typical for senescence, occur prior to
or during flower opening (e.g. Alstroemeria; Wagstaff et al., 2003; Iris, van Doorn et al., 2003). It is
hypothesized here that, as in other developmental
changes, a regulatory network determines the
change from the non-senescent to the senescent
stage. It is further hypothesized that the initiation of
these changes is due to a time-controlled expression
of genes within each cell. As the cells are at this
stage connected by plasmodesmata, whereby the
mesophyll cells and the epidermis cells belong to
different multicellular domains (van Doorn et al.,
2003), the earlier onset of senescence in mesophyll
cells, found in several species, can be explained by
a difference in the timing of the senescence signal in
different domains.
(ii)
(iii)
What determines the life span of petals? The time
from flower opening to petal senescence (in pollinated as well as unpollinated flowers) is, in each
species, highly constant, which indicates tight
temporal control. It is hypothesized that this control
is due to differences in the timing of the normal
sequence of senescence events, and thus to differences in the changes in gene expression. In several
species a circadian clock is involved in the timing
of flower opening (van Doorn and van Meeteren,
2004). It might also be involved in the regulation of
the timing of senescence events.
What induces the rise in ethylene production in
pollinated and unpollinated ethylene-sensitive flowers? It is clear that the pollination signal (which by
itself is still not fully known; O’Neill, 1997)
promotes the expression of genes in the ethylene
synthesis pathway, thereby producing the rise in
ethylene production in petals. In unpollinated
Is cell death due to remobilization? Death at the end
of the senescence process might be due to ongoing
remobilization or might occur independently of
remobilization. The data on cell death in Zinnia
tracheal elements indicate tonoplast rupture long
before most of the cell has been remobilized (Obara
et al., 2001). This suggests that tonoplast rupture is
not due to a degree of remobilization whereby the
cells die of lack of energy and structure. It is
hypothesized here that tonoplast rupture is the
immediate cause of cell death during petal senescence,
and that it occurs independently of remobilization.
(vi) What, if tonoplast rupture is the immediate cause of
death, induces it? Theoretically, numerous processes
might be responsible. One is the activation of
membrane-bound phospholipid-degrading enzymes;
another is the disappearance of a mechanism that
protects the tonoplast from being attacked by lipases in
the vacuole. Focus on the role of the tonoplast in cell
death, and the cause of its rupture, is one of the several
challenges for future research on petal senescence.
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