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University of Groningen Molecular aspects of ageing and the onset of leaf senescence Schippers, Jozefus Hendrikus Maria IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2008 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Schippers, J. H. M. (2008). Molecular aspects of ageing and the onset of leaf senescence s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 19-06-2017 Molecular aspects of ageing and the onset of leaf senescence Jos Schippers The studies described in this thesis were performed in the Department of Molecular Biology of Plants, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9750 AA Haren, The Netherlands. Cover design: Jos Schippers Printed: Gildeprint Drukkerijen BV, Enschede, The Netherlands. This thesis is also available in electronic format at: http://dissertations.ub.rug.nl/ ISBN: 978-90-367-3444-8 2 RIJKSUNIVERSITEIT GRONINGEN Molecular aspects of ageing and the onset of leaf senescence Proefschrift ter verkrijging van het doctoraat in de Wiskunde en Natuurwetenschappen aan de Rijksuniversiteit Groningen op gezag van de Rector Magnificus, dr. F. Zwarts, in het openbaar te verdedigen op vrijdag 13 juni 2008 om 16.15 uur door Jozefus Hendrikus Maria Schippers geboren op 26 juli 1979 te Assen 3 Promotor: Prof. dr. J. Hille Copromotor: dr. P.P. Dijkwel Beoordelingscommissie: Prof. dr. L. Dijkhuizen Prof. dr. J.T.M. Elzenga Prof. dr. J. Kok 4 Contents Scope of the thesis 7 Chapter 1 Developmental and hormonal control of leaf senescence 9 Chapter 2 Ethylene-induced leaf senescence depends on age-related changes and OLD genes in Arabidopsis 45 Early leaf senescence of old13 is partially dependent on salicylic acid and associated with increased oxidative stress and altered water balance 67 A role for cytokinin in the onset of leaf senescence by ethylene in Arabidopsis 89 The Arabidopsis old5 mutation of quinolinate synthase affects NAD biosynthesis and causes early ageing 121 Summary 153 Samenvatting 157 Acknowledgements 163 Chapter 3 Chapter 4 Chapter 5 5 6 Scope of the thesis Autumn is renowned for the short moment during which leaves “shine” golden and red. However, soon after this performance the “curtains close” and the leaves turn brown, fall down and dry out. Leaf senescence does not occur randomly, but involves a programmed general degradation of cellular structures, followed by mobilization of degradation products to other parts of the plant. The mobilization of these products benefits growth of the next generation or plant growth during the next season and presents an evolutionary beneficial trait. The onset and progression of leaf senescence requires molecular genetic mechanisms involving thousands of genes. Arabidopsis thaliana is a typical monocarpic plant that has a short life cycle, easy to recognize developmental stages, and a distinct senescence program. Interestingly, the onset of leaf senescence occurs in an age-dependent manner in Arabidopsis. Although many environmental and endogenous factors can influence the timing of senescence, the onset or delay caused by these factors is still age-dependent. To understand the molecular mechanisms underlying leaf senescence, genes called senescence-associated genes (SAGs) were isolated and characterized. Despite these efforts, there is still insufficient information available to unravel the molecular genetic mechanisms regulating senescence in plants. The aim of this thesis is to study the onset of leaf senescence and identify genes modulating the age-dependent timing of leaf senescence in Arabidopsis. The current understanding of the initiation of leaf senescence is addressed and discussed from a genome optimisation perspective in the introductory Chapter 1. Emphasis is paid on the roles that hormones play during developmental ageing and the initiation and progression of senescence. In Chapter 2 the effect of leaf age and genotype on the onset of leaf senescence by the phytohormone ethylene was studied in detail. For this study we used three wild type accessions and 8 onset of leaf death mutants (old). The 8 old mutants have genetic defects that result in early leaf senescence. Based on the results from this chapter we selected three old mutants to address the molecular mechanism of the ageing process that results in leaf senescence. First old13 was studied in Chapter 3; the mutant has an ethylene dependent senescence phenotype and was characterized in detail. By characterizing the old13 mutant we focussed on the involvement of hormonal signalling pathways to dissect the old13 phenotype. In Chapter 4 and Chapter 5, a 7 study is presented on the early senescence mutants old9 and old5, respectively. The nature of these mutants allowed us to characterize the connection between development and the induction of leaf senescence. More specifically, which changes on gene expression level are related to leaf age, what is the role of reactive oxygen species (ROS) in the ageing process of leaves, which changes occur on metabolic level during the development of a leaf and how do they correlate with the onset of leaf senescence, what is the molecular nature of the age-related changes that occur during leaf development, and finally, questions are asked if some of the processes important for plant ageing are related to those identified for yeast and animal ageing. The findings and interpretation of the results of this study are highlighted in the Summary. 8 Chapter 1 Developmental and hormonal control of leaf senescence Jos H.M. Schippers1, Hai-Chun Jing2, Jacques Hille1 and Paul P. Dijkwel1 1 Molecular Biology of Plants, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands 2 Wheat Pathogenesis Programme, Rothamsted Research, Plant–Pathogen Interaction Division, Harpenden, Herts AL5 2JQ, UK Senescence processes in plants, Gan S, ed. Oxford: Blackwell Publishing. 145–170. (2007) 9 10 Introduction What controls the length of life is one of the fundamental biological questions that has been puzzling scientists for centuries. Plants have many life-forms and differ greatly in the maximal life spans (Thomas, 2003). Annual and biennial plants finish life cycles in a single season or in 2 years time, respectively. An age of 4600 years has been recorded for the perennial tree bristlecone pine (Pinus longaeva), while some clonal plants can live over 10 000 years (Nooden, 1988). Thus, longevity is a genetically controlled life-history trait. The phenomenon of leaf senescence can be appreciated by the color changes among deciduous trees and in the ripening of cereal crops in late summer and autumn, which can occur at a global scale to transform the appearance of the earth from space. During leaf senescence, the sum of morphological, physiological and molecular changes is generally referred to as the senescence syndrome, which includes the visible color changes, dismantling of chloroplasts, degradation of RNA, proteins and DNA and translocation of macro/micromolecules from senescing leaves to other parts of the plant, resulting in the death of the leaf (Bleecker and Patterson, 1997). We propose to examine the regulation of leaf senescence from a genome optimization perspective. We critically analyze the proposed developmental cues that are implicated in initiating leaf senescence. The prominent roles that hormones play during developmental ageing and the initiation and progression of senescence will be reviewed from a molecular point of view based partially on transcriptome data. We discuss the identified potential physiological, biochemical and molecular events during developmental senescence. Developmental senescence: a plant genome is optimized for early survival and reproduction In general, a genome has evolved to contain three classes of hereditary information: (1) the basic metabolism and life maintenance program such as photosynthesis, respiration and DNA replication and damage repair; (2) the defense program that regulates plant responses to abiotic and biotic stresses; and (3) the growth and development program that produces an adult plant optimized for reproduction 11 (Gems, 2000; de Magalhaes and Church, 2005). From an evolutionary point of view, a genome is selected by the force of natural selection if it facilitates the continuous reproduction. Thus, natural selection optimizes a genome for reproduction and the aforementioned three classes of genome programs will be operating only to ensure normal plant growth and development until reproduction. After reproduction, the force of natural selection declines with age and this leads to the loss of viability and fitness of the whole plant and/or plant organs. This phenomenon is known as disposal of soma as stated in the evolutionary theory of ageing, which is developed from animal ageing studies (Rose, 1991; Kirkwood, 2005). This argues that in the genome there are no specific genetic programs for life span and that longevity is an indirect consequence of genome optimization for reproduction. However, the annual model plant Arabidopsis thaliana has evolved a reproduction program that runs in parallel with the death of the whole plant. Seeds are being produced while the plant starts to senesce, and in this way the plant effectively reutilizes nutrients stored in leaves for the production of seeds. Here, the evolutionary theory of ageing can explain leaf senescence if the disposal of a leaf is considered as an indirect selection for nutrient salvage (Bleecker, 1998). Indeed, selective cell death is well documented during plant development and defense responses. For instance, xylogenesis, early embryogenesis, pollen tube growth and the hypersensitive response are typical examples. Thus, a common feature of the plant body plan and architecture is that almost all the structural units are disposable for the sake of survival and reproduction. Clearly, the recruitment of nutrients from leaf tissues, which is a prominent feature of the senescence syndrome and results in the death of the leaf, is part of the genome optimization program. Following this line of arguments, we consider that leaf senescence, albeit genetically controlled, is a consequence of natural selection for genome reproduction. Although there are debates concerning whether ageing occurs in plants, or whether whole-plant senescence shares similarities with animal ageing (Thomas, 2002), it has been proposed that developmental ageing resembles animal ageing, especially when leaves on a plant are scaled up and viewed as equivalent of animal individuals. Leaf senescence is a typical postmitotic senescence in plants and its onset shares many similar regulatory strategies with ageing in animals (Gan, 2003; Jing et al., 2003). We consider it important to view leaf senescence from such an angle. This view helps to explain and model the molecular genetic mechanisms of leaf senescence. 12 As predicted by the evolutionary theory of ageing, genes with early-life beneficial but late-life deleterious effects and late-acting mutations with purely deleterious effects are important for senescence regulation (Kirkwood and Austad, 2000). In the following part of the chapter, we will show that programs important for life maintenance, stress responses and development are important for the onset and regulation of leaf senescence. We further summarize the recent progress in examining the interactions between leaf development and ethylene as an example to present the approaches we think are necessary to understand the complex process of developmental senescence. Developmental processes that regulate leaf senescence When a plant is grown in an environment with sufficient nutrition, away from pathogen attacks and free of abiotic stresses such as darkness, drought, extreme temperature, UV-B and ozone, leaf senescence is ultimately initiated and progresses in a leaf age dependent manner (Gan and Amasino, 1997; Quirino et al., 2000). For monocarpic plants the regulation of senescence is under correlative control and the onset of whole-plant senescence is initiated by the developing reproductive sink, which remobilizes nutrients from the vegetative tissues (Nooden, 1988). In soybean and wheat the removal of reproductive structures usually delays leaf and whole-plant senescence (Nooden, 1984; Srivalli and Khanna-Chopra, 2004). Thus genomes of monocarpic plants are optimized for reproduction, which determines the onset of leaf and whole-plant senescence. Although whole-plant senescence in Arabidopsis is controlled by the reproductive structures as well (Nooden and Penney, 2001), only a weak correlation exists between the appearance of reproductive structures and the onset of leaf senescence. Arabidopsis leaves have a defined life span and senesce even under ideal growth conditions, which is due to the developmental programs underlined by the genome (Hensel et al., 1993; Jing et al., 2002). Thus, here the onset of leaf senescence is governed mainly by age-related changes. Strategies employed in animals and humans seem to have been equally used in plants, such as hormonal modulation as discussed in the next section, reactive oxygen species (ROS), metabolic flux especially sugar and nitrogen signaling and protein degradation. Readers are advised to refer to several recent reviews for detailed 13 discussion on them (Gan, 2003; Jing et al., 2003; Lim et al., 2003; Lim and Nam, 2005;). In this section, we will only briefly elaborate on those strategies. Reactive oxygen species Leaf senescence and the expression of various senescence-associated genes (SAGs) were promoted in old leaves upon exposure to UV-B, ozone or treatment with catalase inhibitors (Miller et al., 1999; John et al., 2001; Navabpour et al., 2003). In contrast to animal ageing and plant hypersensitive responses during plant–pathogen interactions in which mitochondria are the generator of ROS (Finkel and Holbrook, 2000; Lam et al., 2001; Biesalski, 2002), the main ROS source in a senescing leaf is chloroplasts (Quirino et al., 2000). This is consistent with the observation that knockout of a chloroplast genome encoded ndhF gene, one of the components of Ndh complex involved in chlororespiratory electron transport chain, delayed leaf senescence in tobacco (Zapata et al., 2005). ROS can also be generated via lipid oxidation involving membrane-associated NAD(P)H oxidases (Mittler, 2002). This is in agreement with the observed altered senescence phenotypes of Arabidopsis antisense-suppressed phospholipase Dα and SAG101 plants (Fan et al., 1997; He and Gan, 2002) and in plants with defects in fatty acid biosynthesis pathways (Mou et al., 2000, 2002;Wellesen et al., 2001). Thus, ROS generated from various sources are involved in leaf senescence. Several delayed senescence mutants exhibited enhanced tolerance to oxidative stress, indicating that the extended longevity at least in part is due to the attenuated tolerance to ROS (Woo et al., 2004). Thus, the damage generated by ROS can be an important age-related change that will eventually result in the onset of leaf senescence. Metabolic flux One of the distinct features during leaf senescence is the clear metabolic shift from primary catabolism to anabolism (Smart, 1994; Buchanan-Wollaston, 1997). The number of catabolic genes highly expressed in senescing leaves is almost twofold of that of anabolic genes (Guo et al., 2004). Carbon and nitrogen supplies are the two key components that reflect the control of metabolic flux on leaf senescence. An elevated CO2 level hastened the drop in the photosynthetic activities and induced leaf senescence (Miller et al., 1997; Ludewig and Sonnewald, 2000), whereas in Rubisco antisense tobacco plants and Arabidopsis ore4-1 mutants, less dry weight and 14 chlorophyll content were achieved than in the wild type at maturity, resulting in a prolonged leaf longevity (Miller et al., 2000; Woo et al., 2002). Thus, carbon supply achieved through photosynthesis is important for the onset of leaf senescence (Hensel et al., 1993). Carbon supply may directly alter the sugar sensing and signaling, which has been shown to regulate leaf senescence as envisaged in the gin2 mutant that has a lesion in a hexokinase gene (Moore et al., 2003). The cpr5/hys1 mutant that was originally isolated based on altered pathogen resistance was shown to have sugar hypersensitivity and early leaf senescence (Bowling et al., 1997; Yoshida et al., 2002b). Furthermore, glucose (carbon supply) was shown to induce early leaf senescence when combined with low, but not high nitrogen supply (Wingler et al., 2004), indicating the importance of carbon–nitrogen balance. Nitrogen starvation can induce premature leaf senescence, perhaps mainly through modulating the autophagy functions (see below). Protein degradation As one of the essential activities in plant life, protein turnover involves selective and bulk removal of proteins in many processes, such as the degradation of specific regulatory gene products, the maintenance of free amino acids, the elimination of malfunctioning proteins and nutrient recycling (Smalle and Vierstra, 2004; Thompson and Vierstra, 2005). The identification of Arabidopsis ORE9 as an F-box protein (Woo et al., 2001) and DLS1 as an arginyl-tRNA:protein arginyltransferase (ATATE1), which is involved in the N-end rule pathway (Yoshida et al., 2002a), demonstrated the importance of the selective protein removal route mediated by the ubiquitinmediated proteolysis pathway via 26S proteasome in leaf senescence. Mutations in these two genes resulted in delayed senescence, suggesting that the degraded products targeted by ORE9 and DLS1 are positive regulators of leaf senescence, or that the nondegraded products delay senescence. The bulk protein turnover is mainly achieved through vacuolar autophagy. The analyses of two autophagic mutants apg7 and apg9-1 demonstrated the importance of autophagy in senescence regulation (Doelling et al., 2002; Hanaoka et al., 2002). Many more components involved in autophagy formation, conjugation and targeting to vacuoles have been studied through mutational analyses in Arabidopsis (Surpin et al., 2003; Yoshimoto et al., 2004; Thompson et al., 2005; Xiong et al., 2005). In general, knockout of these components affected the survival under carbon and nitrogen starvation conditions 15 and hastened leaf senescence under normal growth conditions. Interestingly, the mRNA and protein levels of autophagy genes are senescence enhanced, suggesting that autophagy is an important aspect of the senescence syndrome. A major group of SAGs encode cysteine proteases (Bhalerao et al., 2003; Guo et al., 2004). For instance, RD21 remains in the vacuole as inactive aggregate and becomes active during senescence by the cleavage of its C-terminal granulin domain (Yamada et al., 2001). Recently, a novel type of senescence-associated vacuole (SAV) has been observed in Arabidopsis and soybean which contains many proteolytic enzymes such as SAG12 (Otegui et al., 2005). The development of SAVs appears to be differentially regulated from vacuole autophagy that is actively involved in leaf senescence (Doelling et al., 2002; Hanaoka et al., 2002). Thus, different vacuoles are functioning during senescence and play a prominent role in macromolecule degradation (Matile, 1997). Furthermore, a chloroplast nucleotide encoded protein CND41 was shown to be responsible for the degradation of Rubisco proteins in senescent tobacco leaves (Kato et al., 2004), indicative of the involvement of chloroplast genome in leaf senescence. The macromolecule degradation and nutrient recycling are prominent events during senescence. Thus, it is not surprising that protein degradation, selective or bulk, is important for senescence regulation. Hormonal control of leaf senescence The senescence program is the final developmental phase of a leaf, which is influenced by several phytohormones, with cytokinin and ethylene having the most extensively documented roles in delaying or inducing leaf senescence, respectively. In addition, other hormones, such as abscisic acid (ABA), auxin, gibberellic acid (GA), jasmonic acid (JA) and salicylic acid (SA), also have effect on the senescence process. In plants, two types of senescence are evident: mitotic senescence and postmitotic senescence (Gan, 2003). Cells in leaves divide only during early development, and thus leaf senescence can be considered postmitotic. Research on the effect of plant hormones on senescence has been started already in the late fifties. The effect of various hormones has been reported for dozens of plant species. The regulation of senescence by cytokinin and ethylene is conserved; however, the action of other hormones varies between plant species. Hormonal signaling pathways show significant overlap, which makes the study of the effect of 16 single hormones complex. The generally used linear representation of hormonal signaling pathways controlling specific aspects of plant growth and development is too simple. In fact, hormones interact with each other and with a whole range of developmental, environmental and metabolic signals (Beaudoin et al., 2000). There are three major ways of controlling the responses to hormones in plants: regulation by hormone biosynthesis, through hormone perception and signaling pathways and downstream events leading to selective protein turnover and changes in gene expression. Although Arabidopsis is the model species for plant research for the last 15 years, almost all the physiological information about hormonal control of leaf senescence has been generated in other species. Evidence from hormone mutants in Arabidopsis strongly supports the role of several hormones in leaf senescence. Lately, exciting advances through transcriptome studies have revealed expression data for hormone biosynthesis, signaling and response genes during senescence, and a closer examination revealed a few interesting points. Combination of the physiological and genetic information will help creating a model for hormonal and developmental control of leaf senescence. Here we try to highlight important findings of several studies that we used to present a model of hormonal regulation of leaf senescence and address remaining questions and leads for future research. Hormones that delay leaf senescence Gibberellic acid Gibberellins are diterpenes that promote stem and leaf growth. In some species, GAs also induce seed germination and modulate flowering time and the development of flowers, fruits and seeds (Sun and Gubler, 2004). A biochemical relation between leaf senescence and GA was first reported by Fletcher and Osborne (1965) showing that GA retarded senescence of excised leaf tissue from Taraxacum officinale by maintaining chlorophyll levels and RNA synthesis. Another study in Rumex by Goldthwaite and Laetsch (1968) showed that GA could inhibit senescence in leaf disks for several days. Both protein degradation and chlorophyll degradation were delayed 4 days. Even when chlorophyll and protein loss is halfway complete, addition of GA blocks further degradation. A study performed on the leaves of romaine lettuce showed a clear age-related decline in GA levels and absence in senesced leaves. 17 This decline in GA was caused by the conversion of free GA to a bound inactive form, probably GA glucoside (Aharoni and Richmond, 1978). Moreover, retardation of senescence by kinetin also caused a relatively high level of free GA and absence of bound GA. Mutations in genes controlling GA biosynthesis or perception have no effect on senescence. However, mutations in the F-box protein SLEEPY1 (SLY1), which result in a block of GA-responsive genes (Dill et al., 2004), delay senescence when crossed to abi1 (Richards et al., 2001). Although not extensively described, several reports point to a retarding effect of GA on leaf senescence. Auxin Auxins are a group of molecules that got their name from the Greek word auxein, which means ‘to grow’. The diversity of the auxin responses is reflected by the existence of multiple independent auxin perception mechanisms in a plant (Leyser, 2002). For soybean it has been shown that the senescence can be retarded by application of auxin (Nooden et al., 1979). During abscission, auxin has been postulated to play a role in reducing the sensitivity of the cells to ethylene (Sexton and Roberts, 1982). The endogenous auxin levels within Coleus leaves showed a decline with increasing age (dela Fuente and Leopold, 1968). However, a relation between endogenous auxin levels and senescence does not always seem to follow a same pattern (Nooden, 1988). The change in auxin response during ageing is the result not only of decreasing auxin levels, but also of a lower responsiveness to auxin with age (Chatterjee and Leopold, 1965). Since auxin is in general seen as a senescenceretarding compound it was a surprise that increased indoleacetic acid (IAA) levels could be detected in S3 phase leaves (Quirino et al., 1999). Since leaves do not senesce uniformly, the authors suggested that auxin levels are selectively increased only in a certain population of cells corresponding to a particular senescence stage. These findings actually correlated with earlier studies that show IAA can induce the production of ethylene which opposes the senescence-retarding effect of IAA in tobacco leaf discs (Aharoni et al., 1979). Interestingly, auxin effectively decreased SAG12 expression, a marker for developmental senescence in a very short period of treatment in detached senescing leaves (Noh and Amasino, 1999). Research performed on glucose signaling revealed that the HXK1 glucose signaling 18 pathway interacts intimately with the auxin and cytokinin pathways. Glucose concentration and photorespiration rates are important determinants for the onset of senescence. Both cytokinin and auxin are part of a regulatory complex for nutritional status of the plant through HXK1 signaling pathway (Moore et al., 2003). Thus, the role of auxin in the regulation of leaf senescence might be linked with other hormones and metabolic flux. Cytokinins Cytokinins have the strongest effect of all hormones on the retardation of leaf senescence. It was reported by Richmond and Lang (1957) that application of cytokinin could retard leaf senescence by preventing the chlorophyll breakdown. While increasing cytokinin production could delay leaf senescence (Gan and Amasino, 1995; Ori et al., 1999), reducing endogenous cytokinin levels resulted in accelerated senescence (Masferrer et al., 2002). The drop in cytokinin levels before the onset of senescence is believed to be a key signal for the initiation (Nooden et al., 1990; Gan and Amasino, 1995). Recently, exciting advances have been achieved in dissecting the components involved in cytokinin signaling (Hutchison and Kieber, 2002; Hwang et al., 2002). Among the genes characterized, the receptor CKI1 (cytokinin independent 1) and the Arabidopsis response regulator (ARR) 2 appear to be involved in regulating leaf senescence (Hwang and Sheen, 2001). A more recent study identified an extracellular invertase whose activity is induced during cytokininmediated delay of senescence (Balibrea Lara et al., 2004). In transgenic tobacco plants having a SAG12–IPT or SAG12–KN1 construct, cytokinin biosynthesis was initiated when SAG12 was induced resulting in a block of the senescence syndrome and delayed leaf senescence significantly (Gan and Amasino, 1995; Ori et al., 1999). When extracellular invertase activity is inhibited, cytokinin no longer can inhibit leaf senescence in transgenic SAG12–IPT lines (Balibrea Lara et al., 2004). Cytokinin signaling genes such as the type-A ARRs and biosynthesis genes show reduced transcription during leaf senescence (Buchanan-Wollaston et al., 2005). Several microarray studies have been performed to reveal cytokinin-dependent gene expression (Hoth et al., 2003; Rashotte et al., 2003; Kiba et al., 2005). Hoth et al. used an inducible system to assess the effects of endogenous cytokinin levels. The study identified 823 up- and 917 downregulated genes after 24 h of isopentenyltransferase (IPT) induction. Although for these studies the seedling stage 19 was used, this IPT system offers an attractive system to study the molecular genetics of how cytokinin can delay and/or reverse the senescence process. The study by Rashotte et al. (2003) showed that cytokinin-upregulated type-A ARRs, which were downregulated in senescing leaves (Buchanan-Wollaston et al., 2005), are the primary response genes for cytokinins. Also a cytokinin oxidase (that degrades cytokinins) and several transcription factors were upregulated. Furthermore, cytokinins induce genes encoding ribosomal proteins (Crowell et al., 1990) and photosynthetic genes (Mok and Mok, 2001). Application of cytokinins downregulated several peroxidases, kinases and E3 ubiquitin ligases. The regulation by cytokinin is related to auxin, light and sugar, since application of cytokinin influences the expression of genes involved in these signaling pathways. In general it can be said that cytokinin stimulates the photosynthetic phase of a leaf. How cytokinin can maintain this phase and delay leaf senescence is still unclear. Nevertheless, the leaves of SAG12–IPT transgenic plants will undergo senescence, thus cytokinin action is limited to a certain developmental phase. Hormones that induce leaf senescence ABA ABA plays a major role during processes related to seed development and germination, for instance the induction of seed dormancy, the synthesis of seed storage proteins and lipids, the acquisition of desiccation tolerance and the inhibition of the transition from embryonic to vegetative growth (Nambara and Marion-Poll, 2005). In vegetative tissue, ABA plays a role in response to drought to prevent water loss by stomatal closure and maintenance of vegetative growth by inhibiting the transition to reproductive growth. Under nonstressful conditions, ABA in plant cells is maintained at low levels, since ABA inhibits plant growth. In vegetative tissues, ABA levels increase during drought, salt and cold stress (Xiong and Zhu, 2003). Changes in gene expression during water-deficit stress are partially induced by ABA and may promote the ability of a plant to respond and survive or adapt to the stress (Bray, 2002). For long it was thought that ABA inhibits plant growth rather than maintaining plant growth. But in tomato, maize and Arabidopsis it has been shown that ABA maintains shoot growth by inhibiting ethylene production (Sharp, 2002). Moreover, 20 this interaction might also play a role in early leaf senescence and leaf, flower and fruit abscission (Morgan and Drew, 1997). Young leaves have the highest ABA levels although this is mainly produced and transported from the older leaves (Zeevaart and Creelman, 1988). During vegetative growth the ABA levels are in general very low; however, in parallel with a decline in free cytokinin and GA just before chlorophyll breakdown in lettuce leaves, an increase in ABA levels has been observed (Aharoni and Richmond, 1978). As soon as the chlorophyll breakdown is initiated, a second, more dramatic increase in endogenous ABA levels is observed. The authors suggest that lowering of GA and cytokinin levels mark the onset of leaf senescence, which results in increased ABA levels when the process has been started. Application of ethylene to the lettuce leaves resulted in a quick drop of GA in 1 day after treatment, but the ABA levels did not show any difference. This might indicate that ABA and ethylene both control different aspects of the senescence syndrome which are mediated through different but partially overlapping signaling pathways. The application of ABA to detached leaves results in a rapid senescence response; however, application to attached leaves has a less pronounced effect. Under low nitrogen conditions and high sugar the abi5 mutant shows delayed senescence. This is consistent with a role for sugar signaling during leaf senescence. ABI5 can be induced by glucose during later stages of development. Expression analysis of ABI5 shows an increase during senescence (Buchanan-Wollaston et al., 2005). The ABA signaling mutants abi2-1 and abi1-1 show signs of early leaf senescence when grown on low nitrogen with glucose and their transcripts increase during senescence (Pourtau et al., 2004; Buchanan-Wollaston et al., 2005). Furthermore, the enzymes controlling ABA synthesis are upregulated during senescence. This indicates that the ABA signaling and biosynthesis pathway is active during leaf senescence. Interestingly the abi4 and abi5 signaling mutants and the aba1, aba2 and aba3 ABA-deficient mutants all are glucose insensitive (Arenas-Huertero et al., 2000). It was noted before that sugar represses photosynthesis-associated genes, which leads to a decline in photosynthesis and eventually in leaf senescence (Bleecker and Patterson, 1997). Thus the onset of leaf senescence by ABA appears to be coupled to metabolic flux changes in Arabidopsis. Brassinosteroids 21 Brassinosteroids (BRs), polyhydroxylated steroid hormones, regulate the growth and differentiation of plants throughout their life cycle. In recent years great advances have been made in the understanding of BR signaling (Vert et al., 2005). External application of BR results in premature leaf senescence for several species, but it has not been reported for Arabidopsis. The induction of senescence by BRs might be mediated through ROS (Clouse and Sasse, 1998). BR signal transduction takes place at the plasma-membrane-localized receptor kinase, BRI1 (Clouse et al., 1996). In addition to BRI1, three homologues have been characterized. Downstream of the receptor kinases is BIN2, a negative regulator of the BR pathway. Further downstream act BES1 and BZR1 transcription factors of which BES1 promotes the expression of BR-regulated genes and BZR1 represses BR genes; both are repressed by BIN2 by targeting of BES1 and BZR1 for ubiquitination and subsequent proteasome-dependent degradation (Vert et al., 2005). Interestingly, BRs can induce ethylene biosynthesis genes in mung bean (Yi et al., 1999). Whether BRs also induce ethylene biosynthesis during senescence is a question that remains to be answered. Mutants in BR biosynthesis and BR signaling do support a role for BR in senescence in Arabidopsis. The det2 (de-etiolated2) mutant is defective in an early step of BR biosynthesis. When grown in light the mutant develops two times more rosette leaves than does the wild type. Chory et al. (1991) observed that wild-type plants showed senescence after 30 days, whereas the det2 mutant did not show any signs of visible senescence after 49 days. One could argue that the mutant has a severe developmental defect that results in a changed senescence syndrome; however, other severely affected developmental mutants such as ctr1 still show a normal onset of the senescence program (Kieber et al., 1993). BR mutants can also result in early leaf senescence as has been shown by the bes1 mutant (Yin et al., 2002). Looking at the Arabidopsis transcriptome of leaf senescence, none of the BR signaling components are identified (Guo et al., 2004). This suggests a minor role for BR during leaf senescence. Transcriptome analysis identified seven genes encoding cell-wall-associated proteins that are upregulated after BR treatment (Goda et al., 2002); these genes were not identified in the transcriptome of senescing leaves (Guo et al., 2004). However, one study revealed the induction of SAGs in Arabidopsis by BR (He et al., 2001). Out of 125 enhancer-trap lines screened, 4 showed upregulation of the reporter after BR application. 22 Although BR mutants show an alternative onset of senescence, molecular genetic evidence of a direct role for BR is still minimal. More studies about the role of BR in senescence are necessary. Ethylene The gaseous plant hormone ethylene plays an important role in plant growth and development. From seed germination to organ senescence and from cell elongation to defense responses, ethylene plays its part. The diverse role that ethylene plays in growth and development suggests that ethylene action involves expression and interaction of many different genes and their products (Zhong and Burns, 2003). Ethylene has long been seen as the key hormone in regulating the onset of leaf senescence (Zacarias and Reid, 1990). The senescence-delaying hormones like auxin and cytokinin both stimulate ethylene production in romaine lettuce leaves (Lactuca sativa L.), which might account for their limited stay-green properties. The author concluded that the effectiveness of exogenously applied hormones in both enhancing and retarding senescence is greatly affected by the endogenous ethylene concentration of the treated plant tissue (Aharoni, 1989). The role of the ethylene pathway in senescence is demonstrated by several studies. Both ethylene insensitive mutants etr1-1 and ein2/ore3 showed increased leaf longevities (Grbić and Bleecker, 1995; Oh et al., 1997), and antisense suppression of the tomato 1- aminocyclopropane-1-carboxylic acid (ACC) oxidase resulted in delayed leaf senescence (John et al., 1995). In these cases, however, senescence eventually begins and progresses normally. Exogenously applied ethylene induces premature leaf senescence in Arabidopsis. However, constitutive application of ethylene does not change the longevity of the leaves. Both ctr1 (constitutive triple response) mutants and Arabidopsis plants grown in the continuous presence of exogenous ethylene did not show premature senescence (Kieber et al., 1993; Grbić and Bleecker, 1995). These results suggest a dynamic regulation of the timing of leaf senescence for which the age-dependent effect of ethylene is utilized. By making use of an ethylene-induced senescence screen, a large collection of onset of leaf death (old) mutants has been identified (Jing et al., 2002, 2005). These mutants confirmed that the effect of ethylene is limited to a range of leaf ages, and that the effect of ethylene on leaf senescence increases with increase in leaf age (Grbić and Bleecker, 1995; Jing et al., 2002). Another piece of evidence supporting 23 this notion comes from a study treating Arabidopsis plants of an identical 24-day end age with various lengths of exogenously applied ethylene (Jing et al., 2005). The results showed that increasing ethylene treatment from 3 to 12 days caused an increase in leaf senescence. Surprisingly, a drop in the number of yellow leaves occurred when a 16-day ethylene exposure was applied. Thus, varying ethylene exposure time can induce different degrees of senescence symptoms in the leaves of an identical end age, suggesting that ethylene can actively stimulate or repress agerelated changes that control ethylene-induced leaf senescence. This notion is genetically supported by the altered responses of eight old mutants to the various ethylene treatments (Jing et al., 2005). Thus, multiple genetic loci are required to regulate the action of ethylene in leaf senescence. A transcriptome study of senescent leaves by Guo et al. (2004) identified three mitogen-activated protein kinases (MAPKs), three MAPKKs, nine MAPKKKs and one MAPKKKK. In the Arabidopsis genome, 20 MAPKs, 10 MAPKKs, 80 MAPKKKs and 10 MAPKKKKs have been identified. The few components identified of the MAPK signal cascades led the authors to the conclusion that the three MAPKs and three MAPKKs may be at the converging/cross talk points of various signal transduction pathways. One of the identified MAPKs is MPK6, which is a component of the MAPK pathway that controls ethylene signaling in plants (Ouaked et al., 2003). MPK6 is upregulated during osmotic stress but also by other abiotic stresses such as low temperature, low humidity, wounding or oxidative stress, as well as by pathogens (Droillard, 2002). Transcriptional analyses of ethylene mutants and ethylene-treated plants revealed the molecular actions of ethylene. A study by Zhong and Burns (2003) revealed genes that are regulated by ethylene. They compared treated wild type, etr1 and ctr1 with untreated wild type. Ethylene treatment of 24-day-old wild-type plants for 24 h changed the expression of 184 genes. Compared to etr1-1, 248 genes were changed in expression level. Untreated wild type compared to etr1-1 revealed the downregulation of nine genes and one upregulated gene in etr1-1. The ctr1 mutant that did not show any signs of early senescence had 109 genes differentially expressed. Further research on these genes might help understanding the molecular regulation of ethylene-induced leaf senescence. To further assess the regulation of senescence by ethylene, expression of SAGs in ein2 was compared with that of wild type (Buchanan-Wollaston et al., 2005). Nine percent of the genes that are 24 upregulated during senescence are at least twofold reduced in ein2. Seventy-seven genes are more than twofold up- or downregulated. Four genes showed upregulation, a lipid transfer protein, a heavy-metal-binding protein and a transcription factor (HFR1). Downregulated are nine transcription factors, cell-walldegrading proteins and nucleases. Therefore some senescence-related degradation functions may be dependent on ethylene. The generation of the transcriptome data revealed that indeed ethylene controls a subset of SAGs during senescence; however, the importance of the identified genes for the control of leaf senescence remains elusive. Endogenous ethylene levels are important for the initiation of senescence. However, the age-dependent senescence induction by ethylene limits its control to a specific age range. The transcriptome studies on SAGs induced by ethylene, together with physiological studies, reveal extensive cross talks between ethylene and the other hormones that might be utilized to fine-tune the progression of senescence in an age-dependent way. Jasmonic acid Jasmonates include jasmonic acid (JA), methyl jasmonate (MeJA) and related compounds and are found in fragrant oils. This group of plant regulators is connected to plant growth and development such as germination and seedling development, flower development, tuberisation, tendril coiling, leaf senescence and fruit ripening (Wasternack and Hause, 2002). The promotional effect of MeJA on senescence was first shown by application to detached oat leaves (Ueda and Kato, 1980). Exogenously applied JA or MeJA resulted in a decreased expression of photosynthesis related genes like Rubisco. Moreover, a change in the polypeptide composition in senescing tissue was observed, which shared similarity with ABAinduced senescence in detached leaves (Weidhase et al., 1987). In plants two JA biosynthetic pathways have been identified; a chloroplast-localized pathway and a cytoplasm localized pathway (Creelman and Mullet, 1995). Exogenous application of JA typically promotes senescence in attached and detached leaves of Arabidopsis but not in the JA-insensitive mutant coi1. Also the endogenous JA levels in senescing leaves increased fourfold as compared to no senescing leaves. Besides an increased JA level during senescence also the enzymes involved in the JA biosynthesis are differentially regulated during senescence (He et al., 2002). The coi1 mutant, which is 25 impaired in JA signaling, did not show any altered leaf senescence. Also other JArelated mutants do not show any alterations in the senescence program, which challenges the idea that JA plays a role in senescence. However, a study with senescence enhancer-trap lines in Arabidopsis showed that JA can induce GUS (βglucuronidase) expression in 14 out of the 125 lines tested (He et al., 2001). The authors developed a sensitive large-scale screening method and have screened 1300 Arabidopsis enhancer-trap lines, which resulted in the identification of 147 lines in which the reporter gene GUS is expressed in senescing leaves but not in nonsenescing ones. Application of senescence-inducing factors showed that only ethylene induced GUS expression in more lines than JA and that ABA, BR, darkness and dehydration were less effective. Based on this, JA appears to be an important senescence-promoting factor. The identification and cloning of coi1 resulted in the identification of an F-box protein which shows the involvement of proteasome-dependent protein degradation in JA signaling (Xie et al., 1998). Interestingly, application of MeJA to Cucurbita pepo (zucchini) induces senescence in 7-day-old cotyledons. One of the observed effects was on the concentration of endogenous cytokinin levels, which reduced rapidly after MeJA treatment (Ananieva, 2004). A drop in cytokinin levels is necessary before senescence can be initiated; whether MeJA can directly or indirectly antagonize cytokinin levels remains to be answered. Transcriptome analyses of MeJA-treated seedlings showed a self-activation of JA biosynthesis and cross talk with other hormones (Sasaki et al., 2001). Although the coi1 mutant does not show any visual senescence defects, a transcriptional analysis showed that 12% of the identified developmental senescence genes are not expressed during senescence of coi1 (Buchanan-Wollaston et al., 2005). In addition, certain genes that are downregulated in the coi1 mutant also appear to be downregulated in the ein2 or nahG mutants. This further demonstrates the importance of the JA pathway during leaf senescence. Salicylic acid SA, a phenolic compound, has been identified as a key-signaling molecule in various plant responses to stress, like pathogen invasion (Glazebrook, 1999) and exposure to ozone and UV-B. The endogenous SA levels in senescing stage 2 leaves are four times higher than in nonsenescing leaves (Morris et al., 2000). This is consistent with 26 a role for SA during later stages of the senescence program. Study of the nahG, pad4 and npr1 mutants, which are defective in the SA signaling pathway, showed an altered expression pattern of a number of SAGs. Furthermore, a delay in yellowing and reduced necrosis were observed in these plants (Morris et al., 2000). The pad4 mutant has a non-necrotic phenotype that has a reduced expression of SAG12, a well-known SAG. The authors postulated that SAG12 may take part in a regulatory pathway leading to cell death and that it supports the transition from senescence to final cell death. Thus the senescence phenotype of pad4 mutant suggests that SA might regulate the transition from senescence to final cell death. Besides biochemical and physiological evidence for role of SA in senescence, genetic evidence has also been generated by a microarray approach (BuchananWollaston et al., 2005). Of 827 genes that were identified as senescence upregulated genes, 19% showed at least a twofold reduction in the nahG transgenic plants that are defective in SA signaling. Interestingly SAG12 expression was substantially reduced compared to that in wild-type plants and SA-treated plants (Morris et al., 2000, Buchanan-Wollaston et al., 2003). Since SAG12 is generally seen as a marker for developmental senescence, this further demonstrates the importance of SA in senescence. Involvement of genome programs in the regulation of senescence-associated genes Developmental senescence is regulated by diverse programs involved in plant life maintenance, defense responses and growth and development (see above). This is consistent with the evolutionary theory of senescence and the proposal of genome optimization for reproduction, which argues that no specific genetic programs for life span evolve. Since the expression profiles of SAGs are reliable markers for senescence, examining the regulation of SAG expression may provide evidence to support that argument. If the evolutionary theory of ageing applies to plants, we expect that many SAGs encode proteins with functions throughout the life cycle of the plant, and not only during developmental senescence. This notion, as a matter of fact, is well supported by the identities and expression profiles of SAGs. Up to now, almost all the isolated SAGs, including many involved in nutrient salvage, exhibit a certain basal level of expression prior to the onset of leaf senescence. This indicates 27 that nutrient salvage is a continuous process occurring in plant cells throughout life. In this sense, leaf senescence is not different from other leaf developmental stages but is more committed to recruit the last yet important source of nutrients retained in an ageing leaf. Recent omics techniques have allowed us to examine the genes that are upregulated during senescence on a whole-genome basis. In addition to development, leaf senescence can be induced by biotic and abiotic stresses. It is therefore possible to compare the SAG expression profiles of various types of senescence using currently available microarray data, which enables the better understanding of the nature of the regulation of developmental senescence. In a whole-genome transcriptome analysis, a total of 827 SAGs were found upregulated during developmental senescence (Buchanan-Wollaston et al., 2005). However, most of those are induced by hormones (SA, JA and ethylene) or darkness as well (Buchanan-Wollaston et al., 2003; Lin and Wu, 2004). Using these data, we deducted the number of SAGs that were enhanced by darkness-induced senescence and were downregulated during leaf senescence in nahG, coi1 and ein2 plants. The remaining SAGs are hence presumably regulated by other developmental cues and/or stress conditions. As shown in Table 1, this category under the name of ‘development’ includes a total of 209 SAGs, which interestingly spread almost in all the categories. We further dissected whether these SAGs are upregulated by carbon and nitrogen metabolism. For this, the array data from ‘Expression patterns of genes induced by sugar accumulation during early leaf senescence’ provided by Wingler’s laboratory were used. Analysis was done by GENEVESTIGATOR (Zimmermann et al., 2004). Nearly half of the 209 SAGs were upregulated after induction of senescence by glucose in combination with low nitrogen. Again, the remaining 110 SAGs are wide spreading in all the categories. If this list of SAGs is compared with the profiles of SAGs in senescence regulated by other developmental cues such as ROS, other hormones (cytokinins, ABA, GA, etc.) or protein degradation, it will not be surprising that perhaps nearly all the SAGs are be regulated by one or more of these cues. In other words, very few SAGs will be solely induced by developmental senescence, which is in agreement with the evolutionary theory of ageing. 28 Table 1 Comparison of gene expression profiles of age-regulated and developmental leaf senescence. Category Regulatory genes Putative transcription factors and nucleic-acid-binding proteins Putative protein–protein interaction Putative ubiquitination control Protein kinase and phosphatases Signaling Calcium related Hormone pathways Macromolecule degradation and mobilization Protein degradation Amino acid degradation and N mobilization Nucleic acid degradation and phosphate mobilization Lipid degradation and mobilization Chlorophyll degradation Sulfur mobilization Carbohydrate metabolism Lignin synthesis Transport ATPases Metal binding Stress related Antioxidants Stress and detoxification Defense related Secondary metabolism Alkaloid biosynthesis Flavonoid/anthocyanin pathway Autophagy Structural Unclassified enzymes of unknown role in senescence Unknown genes Total Development C/N Others Total Dark Cell suspension nahG/coi1/ ein2 96 47 38 23 9 19 9 30 66 17 13 17 7 18 26 9 5 10 3 23 20 8 4 11 1 6 31 5 8 6 1 2 6 0 1 2 1 2 6 5 0 0 29 27 13 14 14 15 6 10 6 3 3 3 14 3 8 5 1 2 29 2 2 63 3 74 7 10 18 1 0 27 1 31 1 5 13 1 0 22 2 26 2 4 5 0 1 32 0 17 3 1 3 1 1 6 1 10 0 3 5 0 0 6 0 14 1 0 11 17 11 1 9 19 5 4 110 7 7 6 0 6 2 5 0 51 6 4 4 0 5 2 3 0 42 4 5 5 1 5 5 1 1 36 2 2 1 0 0 7 0 1 13 0 4 0 0 0 5 0 2 17 132 827 65 385 55 335 34 257 17 99 15 110 Data sources: Buchanan-Wollaston et al. (2005); C/N: carbon and nitrogen supply (Expression patterns of genes induced by sugar accumulation during early leaf senescence; Zimmerman et al., 2004). The most frequently used SAG to monitor developmental senescence, and perhaps one of the few SAGs which is specifically induced by developmental senescence, is SAG12. SAG12 transcripts were found to be very low or below the detection level in young and mature green leaves, contrasting to the levels of the transcripts of SAG13 and SAG14 (Figure 7.1; Lohman et al., 1994). Unlike other SAGs, including SAG13, SEN1 and SAG14 whose expression could be enhanced in young leaves by a range of senescence-inducing treatments such as detachment, hormonal exposure, 29 darkness, drought, wounding and pathogen challenge, SAG12 was only occasionally found to change its expression under these circumstances (Oh et al., 1997; Park et al., 1998; Weaver et al., 1998; Noh and Amasino, 1999; Brodersen et al., 2002). Figure 1. SAG12 and SAG13 expression during Arabidopsis development. Expression levels of both SAG12 and SAG13 are increased during senescence. In contrast to SAG12, basal SAG13 expression levels are present throughout development. Data source: GENEVESTIGATOR (Zimmermann et al., 2004). Thus, SAG12 is considered the best marker for developmental senescence that relies on leaf age, whereas SAG13 and SAG14 may represent stress-induced senescence or general cell-death markers. That the SAG12 promoter has been used for the autoregulated production of cytokinin to delay senescence in a number of species including tobacco (Gan and Amasino, 1995; Ori et al., 1999), lettuce (McCabe et al., 2001), petunia (Chang et al., 2003) and Arabidopsis (Huynh et al., 2005) suggests that the developmental senescence regulation of SAG12 is conserved across species. Moreover, a conserved cis-element of the SAG12 promoter was also found in the Asparagus officinalis asparagine synthetase promoter 30 and was responsible for the induction of transcription of this gene by senescence (Winichayakul et al., 2004). Thus, monocotyledonous and dicotyledonous plants appear to share this senescence cis-element, further confirming the conservation of the regulation of developmental senescence across species. Extensive studies on the expression of SAGs, including SAG12, have provided exciting new insights into the developmental regulation of senescence, and future research will likely result in a better understanding of developmental senescence. Integrating hormonal action into developmental senescence Reproduction has specific timing and all the programs need to be timely in place to ensure successful reproduction. The indirect consequence is that the various strategies embedded in the programs will initiate developmental senescence in an age-dependent manner. Thus, developmental senescence is the consequence of time-specific action of genes. Understanding the timing of the various senescence strategies is a necessary step for elucidating the molecular mechanisms of developmental senescence. In this section we intend to put together the action of the hormones that control leaf senescence and thus developmental ageing in Arabidopsis. Previously, we proposed a senescence window concept to explain the involvement of ethylene in leaf senescence (Jing et al., 2002, 2003). Depending on whether and how senescence can be induced by ethylene, the life span of a leaf can be split into three distinct phases (Figure 2A). The experimental evidence supporting this view is briefly summarized as follows. (1) When plants were exposed to a short-pulse (e.g. 1–3 days) ethylene treatment, no senescence symptoms could be induced in young leaves (Grbić and Bleecker, 1995;Weaver et al., 1998; Jing et al., 2002). (2) Leaf senescence is not accelerated in the ctr1 mutants (Kieber et al., 1993). This indicated that there exists a never-senescence phase in which senescence cannot be induced by ethylene. (3) Furthermore, in a certain range of leaf ages, the effect of ethylene on leaf senescence increases with the increase in leaf age (Grbić and Bleecker, 1995; Weaver et al., 1998; Jing et al., 2002), indicative of an ethylene-dependent senescence phase. (4) Finally, beyond certain leaf ages, senescence will start even without the participation of ethylene as shown in the etr1 and ein2 mutants in which 31 Figure 2. The senescence window concept. (A) The senescence window concept as deduced from the effects of ethylene on leaf senescence. At early leaf development, ethylene is not able to induce leaf senescence. This is the so-called never-senescence phase in the model. Only after a certain developmental stage, ethylene can induce leaf senescence, depending on the environmental conditions. Further development of the leaf will always result in senescence, even in the absence of ethylene. (B) Hormonal action during leaf development is age dependent. The action of the senescence-promoting hormones is strongest at late developmental stages, and is antagonistic to that of the stay-green hormones. The onset of leaf senescence is achieved by a depletion of the stay-green hormones, concomitant with an increase in ethylene levels followed by JA, ABA and eventually SA. Hormone levels and sensitivity change during development, as indicated by the triangles. The age-related changes limit the action of the various plant hormones to their own specific age window. the senescence progresses normally once started (Grbić and Bleecker, 1995; Park et al., 1998; Buchanan-Wollaston et al., 2005), which suggests the existence of an ethylene-independent senescence phase. This senescence window concept emphasizes the developmental control of leaf senescence and considers leaf age as 32 an ultimate determinant of senescence progression. Clearly, genes that control the phase transitions of the senescence window are important for the onset of developmental senescence, and evidence suggests that many genetic loci are required (Jing et al., 2002, 2005). Thus, the senescence window concept provides an explanation why the senescence-promoting effect of ethylene is variable during development. The senescence window concept can, perhaps, integrate the action of all plant hormones involved in leaf senescence. In Arabidopsis the different hormones seem to control the onset and progression of senescence in an age-related manner. Figure 2B is an extension of the senescence window concept developed from the interaction between leaf age and ethylene and shows a tentative model illustrating the timing and action of the different hormones during developmental senescence. In this model, age-related changes, and thus development, are considered the primary regulator of leaf senescence. During ageing, developmental cues lead to the diminished action of the senescence-retarding hormones such as auxin, GA and cytokinins, as well as the concomitant strengthening of the action of senescence enhancing hormones such as ethylene, JA, ABA and SA. The action of the different hormones during the initiation of leaf senescence does not change suddenly but gradually, allowing a gradual integration of all the hormones controlling the process. This suggests that the senescence process is partly reversible by fine-tuning hormone action and hence amenable for modulation. The model provides a basis for the explanation of experimental data. For instance, the major senescence-retarding compound cytokinin can delay senescence when its level is maintained. However, in transgenic SAG12–IPT plants the senescence process will start eventually and progresses normally (Gan and Amasino, 1995; Ori et al., 1999), suggesting that cytokinin action is restricted to certain developmental stages. On the other hand, cytokinin biosynthesis mutants showed a shorter leaf life span (Masferrer et al., 2002). This might be explained by assuming that the effect of the senescence-promoting hormones is antagonistic to those blocking senescence; older leaves may become less sensitive for cytokinin and more sensitive for senescence-promoting hormones like JA and ABA (Weaver et al., 1998). Similarly, blocking the ethylene pathway increases leaf longevity. Finally, however, the leaves go into senescence because the influence of JA, ABA and SA may increase with the 33 age of the leaf. Thus, the age-related changes limit the action of the various hormones to their own specific window. Taken together, although plant hormones are almost universally involved in every aspect of plant life, they may participate into developmental senescence only in very specific age windows. The proposed senescence window concept and the model for hormonal action provide a developmental view to examine the modulation of developmental senescence by hormones, which certainly requires more experimental evidence for validation. Outlook and perspectives Thanks to the availability of cutting-edge technology and the use of model species with known whole-genome sequences that have enabled senescence studies to be carried out at a scale that was not imaginable even 15 years ago, our knowledge on the regulation of developmental senescence has been advanced tremendously. It is clear that hormonal modulation, metabolic flux, ROS and protein degradation are the major cellular and molecular processes that are important for senescence regulation. Strikingly, these processes are embedded in the genome programs that regulate plant life maintenance, responses to biotic and abiotic stresses, and growth and development for the sake of successful reproduction. Thus, leaf senescence can be viewed as an indirect consequence of genome optimization for reproduction. This perspective is exciting and worthy of further exploitation, since it coincides with the evolutionary theory of senescence developed from animal ageing studies. In-depth molecular genetic studies are required to dress the evolutionary basis of leaf senescence. In particular, identification of regulatory genes with pleiotropic functions or late-life deleterious effects should be a priority for further senescence studies. The complexity of leaf senescence is mainly due to the involvement of multiple components that exhibit overlapping effects. This is particularly true for the action of hormones. The proposed senescence window concept provides a theoretic framework to dissect the action of hormones during senescence depending on their time of action, which is important to separate the effect of hormones on senescence from their other effects. Using this concept, it is possible to study genetic components that control the action of hormones during development, which is an essential step for ultimately understanding the mode of action of hormones during development. 34 Combined with the genetic dissection, whole-genome analysis should be employed to define the networking of various regulatory circuits. In conclusion, senescence is one of the biological phenomena with extreme complexity. 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Dijkwel1 1 Molecular Biology of Plants, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands 2 Wheat Pathogenesis Programme, Rothamsted Research, Plant–Pathogen Interaction Division, Harpenden, Herts AL5 2JQ, UK *These authors contributed equally to this work. J. Exp. Bot. 56: 2915-2923 (2005) 45 46 Abstract Ethylene can only induce senescence in leaves that have reached a defined age. Thus, ethylene-induced senescence depends on age-related changes (ARCs) of individual leaves. The relationship between ethylene and age in the induction of leaf senescence was tested in Arabidopsis Ler-0, Col-0, and Ws-0 accessions as well as in eight old (onset of leaf death) mutants, isolated from the Ler-0 background. Plants with a constant final age of 24 d were exposed to ethylene for 3–16 d. The wild-type accessions showed a common response to the ethylene treatment. Increasing ethylene treatments of 3–12 d caused an increase in the number of yellow leaves. However, an ethylene exposure time of 16 d resulted in a decrease in the amount of yellowing. Thus, ethylene can both positively and negatively influence ARCs and the subsequent induction of leaf senescence, depending on the length of the treatment. The old mutants showed altered responses to the ethylene treatments. old1 and old11 were hypersensitive to ethylene in the triple response assay and a 12-d ethylene exposure resulted in a decrease in the amount of yellow leaves. The other six mutants did not show a decrease in yellow leaves with an ethylene treatment of 16 d. The results revealed that the effect of ethylene on the induction of senescence can be modified by at least eight genes. Key words Age-related changes, Arabidopsis, ethylene, leaf senescence, natural variation, old mutants. 47 Introduction Senescence is the final developmental phase of a leaf which starts with nutrient salvage and ends with cell death. The first visible event during senescence is leaf yellowing, which typically starts at the leaf margins and progresses to the interior of the leaf blade (Quirino et al., 2000). The protein and RNA degradation parallels a loss in photosynthetic activity and the majority of the senescence processes have occurred by the time yellowing of the leaf can be seen (Buchanan-Wollaston et al., 2003). The degradation products are transported out of the leaves to other parts of the plant. In this sense, the senescing leaf continues to function as a source of nutrients to the whole plant, but at the expense of its own ability to survive (Bleecker and Patterson, 1997). Senescence is under genetic control and requires differential expression of specific genes. Expression of photosynthesis-associated genes (PAGs) is downregulated, while many other genes, designated as senescence-associated genes (SAGs), are upregulated during senescence. Detailed studies on the SAG identities and their expression suggest a complex regulation of leaf senescence (Hensel et al., 1993; Buchanan-Wollaston, 1997; Gan and Amasino, 1997; Nam, 1997; Gan, 2003). In Arabidopsis the age of individual leaves plays a prominent role in determining leaf longevity (Hensel et al., 1993; Oh et al., 1997; Nooden and Penney, 2001; Jing et al., 2002), although floral initiation can influence plant longevity and thus whole-plant senescence (Levey and Wingler, 2005). Lim et al. (2003) have suggested that a distinction should be made between the terms senescence and ageing: senescence refers to the process that leads to the death of the leaf, while ageing itself occurs throughout development, from the initiation of a leaf primordium to senescence and death. Thus ageing determines when senescence starts, but not senescence itself. During ageing, age-related changes (ARCs) occur as a result of the differential regulation of developmental processes. Before senescence can be initiated, certain ARCs must have taken place in the leaf. An example of a process dependent on ARCs in Arabidopsis is agerelated resistance (ARR) (Kus et al., 2002). The authors showed that plants become more resistant to Pseudonomas syringae as they age. It was suggested that the ability to accumulate SA is necessary for the ARR response and that SA may act as a signal for the production of the ARR-associated antimicrobial compound(s) and/or it may possess direct antibacterial activity against P. syringae. Examples of ARCs in 48 other model-organisms are body movement and pharyngeal pumping in Caenorhabditis elegans (Huang et al., 2004) or accumulation of oxygen free radicals, protein conformational changes, and decline in chaperone functions in the brain of Homo sapiens (Drachman, 1997). The components that control ARCs and thus ageing in plants are still unclear. One of the parameters for ageing is the photosynthetic capacity of the leaf. From the time of full leaf expansion, CO2 fixation rates drop and the senescence program is initiated (Thomas and Howarth, 2000), which indicates that metabolic flux shift may serve as a general signal for the induction of leaf senescence as implicated by the ore4 mutation (Woo et al., 2002). During plant growth light dosage has an effect on ageing; high light intensity results in premature senescence when compared with growth under standard light intensities, while low light intensities delay the senescence process (Nooden et al., 1996). Many other stress-inducing conditions such as drought, darkness, ozone, and pathogen attack can hasten leaf senescence as well (Lim and Nam, 2005). Plant hormones, cytokinin and ethylene being the most conspicuous, are another group of plant endogenous components that play important roles in the regulation of the onset of senescence. While increasing cytokinin production could delay leaf senescence (Gan and Amasino, 1995; Ori et al., 1999), reducing endogenous cytokinin levels resulted in accelerated senescence (Masferrer et al., 2002). Recently, exciting advances have been achieved in dissecting the components involved in cytokinin signaling (Hutchison and Kieber, 2002; Hwang et al., 2002). Among the genes characterized, the receptor CKI1 and the response regulator ARR2 appear to be involved in regulating leaf senescence (Hwang and Sheen, 2001). The plant hormone ethylene has long been seen as a key hormone in regulating the onset of leaf senescence. Zacarias and Reid (1990) reported that ethylene does promote senescence, but it is not an essential compound for the senescence syndrome induced by other factors (e.g. ABA). The role of ethylene in senescence has been demonstrated by several studies. Both ethylene-insensitive mutants etr1-1 and ein2 show increased leaf longevities (Grbić and Bleecker, 1995; Oh et al., 1997) and antisense suppression of the tomato ACC oxidase resulted in delayed leaf senescence (John et al., 1995). In these cases, however, senescence eventually begins and progresses normally. Exogenously applied ethylene induces premature leaf senescence in Arabidopsis. However, constitutive application of ethylene does 49 not change the longevity of the leaves. Both ctr1 mutants and Arabidopsis plants grown in the continuous presence of exogenous ethylene did not show premature senescence (Kieber et al., 1993; Grbić and Bleecker, 1995). Thus, ethylene is neither necessary nor sufficient for the occurrence of senescence. These studies suggest that ethylene does not directly regulate the onset of leaf senescence. It acts to modulate the timing of leaf senescence. Only when developmental changes controlled by leaf age are present can ethylene induce senescence (Hensel et al., 1993; Grbić and Bleecker, 1995; Jing et al., 2002). In the present study, advantage was taken of natural variation among Arabidopsis accessions and the availability of Arabidopsis onset of leaf death (old) mutants to examine genetic loci that are involved in ethylene-induced senescence. The combined physiological and genetic studies used here showed that Arabidopsis accessions exhibited common and discrete senescence responses to exogenous ethylene. It was observed that ethylene displayed a dual function, both as an inducer and a repressor, in the induction of leaf senescence and that such a role of ethylene was differentially modulated by multiple genetic loci. These results lay out a basis for further molecular dissection of genes that regulate ethylene-induced leaf senescence. Results Physiological and molecular markers of leaf senescence correlate with visible yellowing The effect of ethylene on age-dependent leaf senescence was determined for Ler-0. Ler-0 was grown for 11, 21, or 27 d after germination (DAG) and subsequently treated with ethylene for 3 d. The senescence syndrome was studied using morphological, physiological, and molecular markers (Figure 1). Plants grown for 14, 24, or 30 d in air did not show any signs of visible yellowing, even though in the cotyledons the chlorophyll content had decreased approximately 2-fold over time (Jing et al., 2002). Plants grown for 11 d in air and subsequently for 3 d in air supplemented with ethylene (11+3) did not show any signs of senescence either. However, those treated for 21+3 and 27+3 showed signs of visible yellowing. Concomitant with the visible yellowing, the chlorophyll content of the plants was decreased after the 21+3 and the 27+3 treatments (Figure 1B). 50 Figure 1. Comparison of ethyleneinduced senescence in cotyledons and rosette leaves of Ler-0. Plants were grown first in air for 11, 21, or 27 d, and then exposed to ethylene for 3 d. The visible yellowing (A) of cotyledons and the first pair of rosette leaves was subsequently scored as the percentage of yellow cotyledons or rosette leaves versus the total number of cotyledons or rosette leaves, respectively. The chlorophyll contents (B) and steadystate mRNA levels (C) of several senescence associated genes were subsequently analyzed. For each time point, the observations on visible yellowing were on three sets of 50 plants and the results for chlorophyll is shown as mean ± SD of four replicates. Total RNA was isolated from leaf samples of approximately 100 plants. Five µg total RNA was used for northern blotting. sequentially The membrane hybridized with was cDNA probes of the indicated SAGs, CAB, and rRNA. The chlorophyll content was lower in cotyledons than in the first leaf pair after both the 21+3 and the 27+3 treatments. The drop in chlorophyll content correlated with a decrease in the mRNA levels of CAB (Chlorophyll a/b binding protein) in the 21+3 and 27+3 samples (Fig. 1C). Expression of SAG12, SAG13, and SAG14 was upregulated in the 21+3 and 27+3 samples compared with the 11+3 samples. Low SAG14 expression, however, was detected in the green cotyledons of 11+3 samples. The results are in agreement with previous studies, demonstrating that ethylene- 51 induced senescence is dependent on leaf age (Grbić and Bleecker, 1995; Jing et al., 2002). Thus the effect of ethylene on inducing visible yellowing correlated well with changes in chlorophyll content and in the SAG mRNA levels. The senescence syndrome continued being examined by using visible yellowing as a parameter. Arabidopsis accessions exhibited similar and discrete responses to the induction of senescence by different ethylene exposure times The effect of ethylene exposure time on leaf senescence was assessed in the Arabidopsis accessions Ler-0, Columbia (Col-0), and Wassilewskija (Ws-0). Plants were grown until 8, 12, 18, or 21 DAG under standard conditions and were further grown under continuous light and ethylene conditions until 24 DAG. Thus, the plants that were treated for the longest time with ethylene received the treatment at a younger developmental stage. Figure 2 shows that ethylene has major effects on plant growth and development; the longest ethylene treatment caused the plants to phenocopy the ctr1-1 mutant. Figure 2. Representative Ler-0 plants after growth in air or after ethylene treatment. Plants were grown either in air for 24 d, or first in air for 21, 18, 12, or 8 d and then exposed to ethylene for 3, 6, 12, or 16 d, respectively. At the end of the treatment (24 d), representative plants were selected and photographed. The bar represents 0.5 cm. The effect of the treatments on leaf 52 yellowing was determined and the results are shown in Figure 3. The overall response was similar for the three accessions tested; an increase in the duration of the ethylene treatment from 3 d to 12 d caused an increase in the number of yellow leaves. Longer ethylene treatments of 16 d resulted in a decrease in the number of yellow leaves, when compared with the 12 d ethylene treatment. Figure 3. Visible yellowing of 24-d-old wild-type plants, exposed to ethylene for various amounts of time. The number of yellow leaves of three Arabidopsis accessions exposed to different ethylene treatments is indicated. Ler-0, Ws-0, and Col-0 plants were grown first in air for 21, 18, 12, or 8 d, and then exposed to ethylene for 3, 6, 12, or 16 d, respectively. The visible yellowing was subsequently scored and expressed as means ± SD of at least three replicates of 30 plants each. Plants that were grown for 24 d in air did not show any sign of senescence (not shown). These results are consistent with the phenotype of the ctr1-1 mutant, which does not show early leaf senescence and the observation that continuous ethylene exposure does not promote early senescence in Ler-0 (Kieber et al., 1993). There were also differences in ethylene-induced senescence among the three accessions. Ws-0 plants exhibited the highest amount of yellow leaves. Ler-0, furthermore, had a more pronounced senescence response than Col-0. Thus, the number of yellow leaves that can be induced by ethylene is dependent on the genetic background and the length of the ethylene treatment. Prolonged ethylene treatments inhibited cell expansion but stimulated plant development, as judged by leaf emergence. The average total leaf numbers in plants 53 with various ethylene treatments were compared and the results showed that plants that experienced a longer ethylene exposure time had more leaves (Table 1). The increase in leaf number did not seem to correlate with the number of senescing leaves, Col-0 had more leaves than Ws-0 and Ler-0, but exhibited less visible yellowing. Thus, the effect of ethylene exposure time on leaf senescence is different from its effects on the inhibition of cell elongation and promotion of development. Table1. Total leaf numbers of Arabidopsis accessions and old mutant lines after various ethylene treatments Lines 21+3 18+6 12+12 8+16 Ler-0 12.7 ± 0.2 13.3 ± 0.2 15.1 ± 0.2 15.1 ± 0.4 Col-0 13.3 ± 0.1 14.1 ± 0.4 15.5 ± 0.1 16.0 ± 0.4 Ws-0 12.1 ± 0.1 12.6 ± 0.3 13.8 ± 0.2 13.6 ± 0.1 ctr1-1 11.8 ± 0.1 11.8 ± 0.2 13.0 ± 0.5 13.0 ± 0.1 old1 11.0 ± 0.5 11.0 ± 0.4 10.9 ± 0.1 11.0 ± 0.1 old5 12.6 ± 0.2 13.3 ± 0.1 14.4 ± 0.3 13.9 ± 0.2 old14 11.5 ± 0.2 11.30 ± 0.0 11.1 ± 0.3 11.4 ± 0.2 old9 12.7 ± 0.3 12.7 ± 0.1 13.7 ± 0.1 14.5 ± 0.1 old11 12.6 ± 0.3 12.9 ± 0.1 13.4 ± 0.2 12.9 ± 0.1 old13 12.1 ± 0.3 12.4 ± 0.3 14.0 ± 0.2 13.8 ± 0.3 old3 4.0 ± 0 4.0 ± 0 4.0 ± 0 4.0 ± 0 old12 11.5 ± 0.0 11.5 ± 0.1 12.1 ± 0.0 12.3 ± 0.4 Plants were grown and treated with ethylene as described in materials and methods. A leaf was scored when it emerged and was over 1mm in size. Scoring was performed on 24-d-old plants at the end of the ethylene treatment. Approximatly 30 plants were used for each line. Arabidopsis old mutants reveal multiple genetic loci involved in the regulation of ethylene-induced leaf senescence As shown above, the genotype has an effect on the senescence response to ethylene. To address the genetic regulation of ethylene-induced leaf senescence further, use was made of previously isolated old mutants (Jing et al., 2002). The mutants were identified from an EMS mutagenized population of Ler-0 seeds and show an early senescence phenotype before and/or after ethylene treatment (Fig. 4). The old mutants were selected and subgrouped into three different classes. The 54 selected Class I mutants comprises the formerly described mutant old1 (Jing et al., 2002) and the mutants old5 and old14. The mutants have early leaf senescence symptoms in air (compare Fig. 4A to C and E) and those symptoms are further enhanced by ethylene treatment (Fig. 4B, D, F). Figure 4. Phenotype of old mutants. Representative 24-d-old Ler-0 airgrown (A) and 21+3 ethylene-treated Ler-0 (B). 24-d airgrown old5 (C), old14 (E), and old12 (J). 21+3 ethylene-treated old5 (D), old14 (F), old9 (G), old11 (H), and old13 (I). The bar represents 0.5 cm. 55 When grown in air, old5 and old14 did not differ markedly from the wild type, except for their early senescence phenotype which was observed after ~19 d. After 24 d of growth both mutants have 1–2 yellow leaves (results not shown). Air-grown Class II mutants were not different from the wild type when grown under the standard conditions used here, but displayed enhanced senescence symptoms when treated with ethylene (Fig. 4G, H, I). In old13, ethylene-induced yellowing was associated with lesion formation. The old12 mutant (Fig. 4J) and the previously described old3 mutant (Jing et al., 2002) belong to the third class of mutants and have advanced senescence symptoms in air that cannot be further induced by ethylene treatment. The old12 mutant not only displayed early senescence symptoms but also differed from the wild type in size, exemplified by reduced leaf expansion, shorter shoot length, and the formation of thicker siliques (data not shown). Table 2. Genetic segregation of old mutations Class I a Male Female Generation Wild-type Mutant old5 Ler-0 F1 31 0 F2 151 64 F1 39 0 F2 156 40 F1 23 25 F2 18 73 F1 11 23 F2 19 67 F1 52 0 F2 180 79 F1 24 0 F2 328 87 old14 II b old9 old11 old13 a III a old12 Ler-0 Ler-0 Ler-0 Ler-0 Ler-0 χ 2c 2.61 2.20 1.32 0.75 4.18 3.61 The Class I and Class III mutants showed early senescence when grown in standard growth conditions. Thus, the scoring of phenotypes was performed before ethylene treatment and plants with clearly visible yellowing cotyledons and/or rosette leaves were scored as mutants. b The segregation analysis for Class II mutants was performed after ethylene treatment. The -1 phenotype scoring was carried out on 21-d-old plants treated with 10µl l ethylene for 3 d. The criteria were: wild-type plants with up to two yellow cotyledons, Class II mutants with at least 2 yellow cotyledons and 1 yellow leaf. c 2 All the χ values were calculated for the 1:3 segregation ratios of mutant:wild type except in the case of old9 and old11, where a 3:1 ratio of mutant:wild type was calculated 56 Genetic analyses showed that the characterized old alleles segregated as single monogenic recessive traits except old9 and old11, which segregated as conditional (ethylene-dependent) co-dominant traits (Table 2). Allelism tests between mutants within each class revealed that they belong to different complementation groups (data not shown). Genetic mapping placed the old12 and old9 mutant alleles on chromosome 2 at ~7 Mb and ~17 Mb, respectively; old14 on chromosome 3 at ~15 Mb; and old11, old5, and old13 on chromosome 5 at ~8 Mb, ~20 Mb, and ~23 Mb, respectively. The authors are not aware of mutants with similar phenotypes in the vicinity of these mapped regions. The response of the selected old mutants to several plant hormones and sugar was tested by germination assays. Since ethylene was used as the senescence inducer, the sensitivity of the mutants to ACC was tested by a triple response assay (Table 3). Both old11 and old1 showed an extremely short hypocotyl. All other old mutants showed a wild-type response to ACC. Germination on glucose plates (ArenasHuertero et al., 2000) showed that old12 and old1 are hypersensitive to glucose, moreover germination was completely inhibited by 4% glucose (data not shown). Table 3. Triple response assay of the wild type Ler-0 and the selected old mutants*. Plant MS medium (mm) 10 µM ACC medium (mm) Ler-0 6.89 ± 0.19 3.25 ± 0.14 old1 6.81 ± 0.10 1.18 ± 0.13 old5 7.01 ± 0.28 3.05 ± 0.19 old14 6.81 ± 0.14 3.03 ± 0.17 old9 6.74 ± 0.12 3.07 ± 0.05 old11 6.82 ± 0.22 1.39 ± 0.06 old13 6.99 ± 0.20 3.17 ± 0.03 old12 7.14 ± 0.23 3.32 ± 0.05 * The average hypocotyl length after 5 days of growth on 10 µM ACC is compared. For each data point at least 40 seedlings were used. The glucose phenotype was independent of osmolarity since all mutants developed like the wild type on mannitol. The response of the old mutants to ABA and jasmonic acid was not different from the wild type (data not shown). Thus, the selected old mutants show various senescence phenotypes that are caused by defects in independent genetic loci. 57 old mutants and ctr1-1 have altered responses to varying ethylene-exposure lengths The old mutants and ctr1-1 were exposed to ethylene for 3–16 d. Figure 5 shows that all the old mutants responded to the ethylene treatment in a different way than Ler-0. The old1 and old11 mutants showed an enhanced response to long ethylene treatments: a maximum number of visible yellowing leaves occurred after the 18+6 treatment, instead of the 12+12 treatment in the Ler-0 plants. A further increase in the length of ethylene treatment resulted in a reduced number of yellow leaves. Remarkably, old1 had fewer yellow leaves than Ler-0 after the 12+12 treatment and so could be considered a delayed senescence mutant after long ethylene treatments. These data are consistent with the observation that old1 and old11 are hypersensitive to ethylene in the triple response. A similar number of yellow leaves, regardless of the length of the ethylene treatment, was observed in the old3, old9, old12, and old14 mutants. Figure 1. Visible yellowing of 24-d-old old mutants and ctr1-1, exposed to ethylene for various amounts of time. The old mutants were grown first in air for 21, 18, 12, or 8 d, and then exposed to ethylene for 3, 6, 12, or 16 d. The visible yellowing was subsequently scored and expressed as means ± SD of at least three replicates of 30 plants each. 58 Both old3 and old12 are Class III mutants, which were determined to senesce independently of ethylene, and the results shown here are consistent with that classification. Mutants of old5 and old13 had a constant number of yellow leaves when the ethylene treatment was longer than 3 d. The average total leaf numbers of the mutants after various ethylene treatments were compared and the results showed that the old1, old14, and old11 mutants responded differently to the ethylene treatments than the wild type (Table 1). In contrast to the wild type, these mutants did not show an increase in the rate of development, as judged by the emergence of new leaves. Nevertheless, in cases where the total number of leaves of the mutants was less than the wild type, the number of yellow leaves was higher. The one exception is the old1 mutant, where after the 12+12 treatment, a reduced number of total leaves correlated with a reduced number of yellow leaves. The results suggest that it is unlikely that the differences observed in the senescence response are caused by different growth and developmental rates between the mutants and the wild type. Taken together, two mutants (old1 and old11) became desensitized and showed fewer senescence symptoms upon extended long ethylene exposure, while the other six mutants show a similar response for every ethylene treatment. Compared with the wild-type Col-0 plants, the ctr1-1 mutant showed much lower numbers of yellow leaves in all the treatments tested, indicating that constitutively activating the ethylene signaling pathway through knockingout CTR1 has a pronounced effect in inhibiting the onset of leaf senescence. Interestingly, ethylene treatment did cause some leaf yellowing that was not observed in mutants that were not exposed to ethylene. Remarkably, the induction of leaf yellowing followed a similar response as the wild-type accessions: an increase in ethylene exposure time resulted in an increase in leaf yellowing up to 12 d of ethylene treatments. A further increase to 16 d caused a reduction in the number of yellow leaves. 59 Discussion The effect of ethylene on the induction of leaf senescence was shown to be under the direct influence of age (Hensel et al., 1993; Grbić and Bleecker, 1995; Jing et al., 2002). The data presented here confirm these results as senescence was not induced in young cotyledons or leaves. The relationship between ARCs and ethylene-induced senescence was further assessed by treating plants of an identical end age with different ethylene exposure lengths. The three different Arabidopsis accessions showed a common response to the different treatments implying a conserved perception mechanism of ethylene-inducible senescence. An increase in the length of the ethylene treatment, and thus treatment at a younger age, resulted in an increase in the amount of leaf yellowing. The longest treatment, however, caused a reduction in the amount of leaf yellowing. In particular, the ctr1-1 mutants showed much lower numbers of yellow leaves compared with the wild-type plants, suggesting that constitutive activating ethylene signaling strongly inhibits the induction of leaf senescence. Thus, the effect of ethylene on leaf senescence relies on the exposure time. Since ethylene-induced senescence depends on ARCs, the results presented here suggest that ethylene treatment can influence ARCs. Depending on the length and start of the treatment, ethylene can stimulate, or suppress ARCs. Ethylene also caused the inhibition of cell expansion and the stimulation of plant development. In all accessions the longest ethylene treatment resulted in approximately two additional leaves. Here, the longest treatment did not cause a reduction in the stimulation of plant development. This suggests that the effect of the longest ethylene treatment on leaf yellowing is different from and independent of the effect on plant development. Besides the common response of the accessions to ethylene, natural variation was present and the Ws accession showed the strongest senescence symptoms after the ethylene treatments. These results confirm a previous study on natural variation in the regulation of leaf senescence by Levey and Wingler (2005). Natural variation has been observed for a variety of life history traits in Arabidopsis including the control of flowering time (Koornneef et al., 1998), disease resistance and tolerance (Kover and Schaal, 2002), and the control of cytosine methylation in the nucleolus organizer regions (Riddle and Richards, 2002). Remarkably, ethylene treatment of ctr1-1 resulted in a senescence response that was similar to that of the three wild-type accessions. The ctr1-1 mutant exhibits continuous activation of the ethylene signaling pathway and has a wild-type timing of senescence under standard growth conditions 60 (Hensel et al., 1993). It was found here that, by applying exogenous ethylene, early senescence can be induced. Although ctr1-1 loss-of-function mutants display a severe ethylene phenotype, these mutants remain ethylene responsive (Larsen and Chang, 2001). Quadruple loss-of-function mutants in the ethylene receptor family have a more severe phenotype than ctr1-1 (Hua and Meyerowitz, 1998), suggesting that an alternative mechanism bypassing CTR1 in ethylene signaling exists in Arabidopsis (Cancel and Larsen, 2002). Since endogenous ethylene levels in ctr1-1 mutants are equal or even lower than in wild-type plants (Ecker and Kieber, 1994) the observed senescence may be a direct cause of the applied exogenous ethylene which is signaled through an alternative ethylene pathway. Although the amount of yellowing was reduced in the ctr1-1 mutants, the induction of senescence was agedependent and ethylene exposure-time dependent. This suggests that the effect of ethylene on ARCs is maintained in ctr1-1 mutants. The results imply that ethylene stimulates ARCs in an ethylene treatment-duration dependent way. However, the relation between treatment length and induction of ARCs is not linear and there is an optimum treatment length that causes the strongest senescence response. Previously, several old mutants were isolated that show an altered senescence phenotype before and/or after ethylene treatment (Jing et al., 2002). Eight old mutants were selected and the effect of different lengths of ethylene treatment on leaf yellowing was measured. Although all the mutants responded to the treatments in a different way from the wild type, there were two fundamentally different responses. Both old1 and old11 already showed a decrease in senescence with a shorter ethylene treatment than the wild type, suggesting that they may respond more strongly to the ethylene. Likewise, both old1 and old11 were found to be hypersensitive to ACC in the triple response assay. These results are consistent with the hypothesis that the mutants have an enhanced effect on ARCs as a result of a stronger response to ethylene. The second type of response was found in the other six mutants, where no decrease in leaf yellowing was found with the longest ethylene treatment. This suggests that the mutants may have lost the ability to suppress ARCs by long ethylene treatments. Interestingly, the six mutants fall into different classes. old5 and old14 represent Class I mutants, which already have an early senescence phenotype in air. The senescence phenotype of Class III mutants old3 and old12 is not dependent on ethylene and, as expected, a similar response was observed for 61 every ethylene treatment applied. Only old9 and old13, which are Class II mutants, are fully dependent on ethylene for their senescence phenotype. In summary, it can be concluded that ethylene has an effect on processes that regulate ARCs in a treatment-duration dependent way. In eight old mutants this effect was altered, but the function of the OLD genes in the control of ARCs and ethyleneinduced senescence is at present unclear. Future cloning and analysis of the OLD genes shall shed new light on the regulation of ethylene-induced leaf senescence and the role of ethylene on processes that control ARCs. Methods Plant material and growth conditions Arabidopsis accessions Landsberg erecta (Ler-0), Columbia (Col-0), and Wassilewskija (Ws-0) were used in this study. Plants were grown in a growth chamber at 21 °C and 65% relative humidity with a day length of 16 h. The light intensity was set at 120 µmol cm-2 s-1. An organic-rich soil (TULIP PROFI No.4, BOGRO B.V., Hardenberg, The Netherlands) was used for the experiments described in Fig. 1. For the other experiments a γ-ray irradiated soil was used (Hortimea Groep, Elst, The Netherlands). Plants for ethylene exposure were treated in a flow-through chamber at 20 °C and a humidity of 40% under continuous illumination. The ethylene dosage was set at 10 µl l-1 since it has been shown that a dose ranging from 1–100 µl l-1 was sufficient to generate similar effects on several ethylene responses, including leaf yellowing (Chen and Bleecker, 1995; H-C Jing, unpublished data). For germination studies, seeds were surface-sterilized by soaking in 20% bleach for 5 min after which they were washed twice with sterile water. The sterilized seeds were plated on Murashige and Skoog medium containing 0.8% agar. The plates were stored at 4 °C for 4 d after which they were transferred to a growth chamber at 21 °C and 16 h of light. For the triple-response assay 10 µM filter-sterilized ACC was added to the plates. Seedlings were allowed to grow for 5 d in the dark before analysis. Genetic analysis of the old mutants Generation of M1 and M2 seeds and mutant screening were done as described by Jing et al. (2002). In this study, eight old mutants with early leaf senescence phenotypes were selected. Since the mutants were isolated from the Ler-0 62 background, they were crossed to Col-0 plants for mapping. At least 100 F2 plants with an early senescence phenotype were selected for DNA isolation using the SHORTY quick preparation method (http://www.hos.ufl.edu/meteng/Hanson Webpagecontents/NucleicAcidIsolation.html#Arabidopsis%20Genomic%20DNA). The linkage analysis was performed by using a subset of simple sequence length polymorphism markers, making use of the Monsanto database (Jander et al., 2002). Both old9 and old11 are co-dominant traits and there was some overlap between the wild type and the heterozygous phenotypes of the mutants. In the F2 populations of these two mutants backcrossed to wild type, sometimes a wild-type plant was found that had one yellow true leaf and occasionally a heterozygous mutant was found without any yellow true leaves following a 3 d ethylene treatment of 21-d-old plants. Thus, for the segregation analysis of old9 and old11, any plant with at least one yellow true leaf was counted as a mutant. Plants with up to two yellow cotyledons were counted as wild types. Observation of visible yellowing, chlorophyll content measurement, and northern blot analyses Cotyledons or rosette leaves with over 5% yellow area of the leaf blade were judged as yellow as suggested by Lohman et al. (1994). However, in this study’s experiments, yellowing could be initiated at the leaf tips, at the petiole side of the leaf, or in the middle of the leaf blade, which did not always resemble the wild-type developmental yellowing pattern of leaf senescence. 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Extended leaf longevity in the ore4-1 mutant of Arabidopsis with a reduced expression of a plastid ribosomal protein gene. Plant J. 31: 331-340. Zacarias, L., and Reid, M.S. (1990). Role of growth regulators in the senescence of Arabidopsis thaliana leaves. Physiol. Plant. 80: 549–554. 66 Chapter 3 Early leaf senescence of old13 is partially dependent on salicylic acid and associated with increased oxidative stress and altered water balance Jos H.M. Schippers1, Renate Ellens1, Jacques Hille1 and Paul P. Dijkwel1 1 Molecular Biology of Plants, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands 67 68 Abstract Programmed cell death (PCD) manifests itself during the final stage of leaf development by leaf senescence. However, leaf senescence can occur prematurely as response to environmental stress and pathogen attack. The highly coordinated regulation of leaf senescence can increase plant fitness and survival. Here we report that the onset of leaf death 13 mutant displays enhanced ethylene-induced senescence and lesions formation in the absence of pathogen, suggesting that OLD13 is involved in stress response signaling and cell death regulation. The mutant has increased oxidative stress as determined by NBT staining and expression of ROS markers. Double mutant analysis reveals that the lesion formation requires salicylic acid. The ethylene-induced senescence phenotype was suppressed by a mutation in EIN2 but not by mutations in PAD4 and ABI4. The mutant has an increased anion content suggesting an altered water balance. Taken together we suggest that OLD13 functions as a modulator of leaf senescence upon environmental stress such as drought, salinity and pathogen attack. Introduction In the early sixties of the last century interest in the active onset of leaf senescence was emerging as depicted by the following citation: The signal for mass hari-kari, so to speak, for all members of these enormous populations is such a fantastically dramatic physiological event that it seems most singular that plant physiologists have not given more attention to the matter (Leopold, 1961). It is argued that senescence is beneficial for plant fitness and survival and suggests the existence of a mobilizing force that actively drives the senescence process. Since that time plant programmed cell death (PCD) has been implicated in the recycling of nutrients by senescence, the development of the plant body, selfelimination of cells invaded by pathogens and as a response to (a)-biotic stress (Jones, 2001). PCD is not a passive process resulting in death but requires the active regulation of thousands of genes which prepare the end phase of a cell. PCD in leaf senescence has some unique features when compared to other PCD processes. Leaf senescence occurs at the whole organ or whole plant level while other PCD processes occur highly localized (Pennel and Lamb, 1997). Next to that senescence is a slow progressing PCD especially compared to the hypersensitive-response (HR) to pathogen attack (Lamb and Dixon, 1997). The orderly dismantling of a leaf results 69 in efficient remobilization of nutrients to maintain development of other parts of the plant (Himelbau and Amasino, 2001). In Arabidopsis the execution of the senescence program is depending on the developmental age of the leaf (Bleecker, 1998; Jing et al., 2003). However, leaf senescence is also influenced by diverse internal and environmental signals that are integrated with the age to determine the onset (Lim et al., 2003). This integrated senescence response allows plants to obtain optimal fitness by incorporating the environmental and endogenous status of plants in a given ecological setting by fine-tuning the initiation timing, progression rate, and nature of leaf senescence (Lim et al., 2007). Unfavorable environmental factors like drought (Munné-Bosch and Alegre, 2004), nutrient limitation (Wingler et al., 2006), temperature (Page et al., 2001), oxidative stress by UV or ozone (Miller et al., 1999), darkness (Fujiki et al., 2001) and pathogen attack (Tang et al., 2005) can prematurely induce the senescence program. The response of plants to environmental factors involves the transcriptional activation or repression of genes (Zhu, 2002). Several studies on improving stress resistance have revealed cross-talk and overlap between the different stress pathways and their activation. Overexpression of the dehydration responsive element binding (DREB) 1A results in plants more tolerant to drought, salt and cold stress but at the expense of growth and productivity (Kasuga et al., 1999). However, expression of DREB1A under the control of a stress-inducible promoter improves stress tolerance without affecting plant health and thus demonstrating the need for a targeted response. In another example, overexpression of heat shock transcription factor (Hsf) A2 confers higher tolerance to temperature, salt and osmotic stress but also results in growth retardation (Ogawa et al., 2007). Taken together, stress tolerance can delay the timing of senescence induced by stress and is thus an important factor in controlling onset of senescence. Interestingly, stress responses and senescence share overlapping signaling pathways as more than two-thirds of the transcription factors induced during senescence are also induced after various stress treatments (Chen et al. 2002). The integration of age and environmental signals involves, next to transcriptional factors, plant hormones which act as endogenous factors controlling growth and development including leaf senescence (Schippers et al., 2007). Ethylene, jasmonic acid (JA) and salicylic acid (SA) have all been implicated in the onset of senescence (Jing et al., 2002; Buchanan-Wollaston et al., 2005; Miao et al., 2007) and responses 70 to various stresses (Chen et al., 2002). The ENHANCED DISEASE RESISTANCE 1 (edr1) mutant shows enhanced resistance to pathogens, early ethylene-induced senescence and spontaneous lesion formation during drought stress. EDR1 functions both in ethylene and SA signaling pathways and regulates senescence and cell death (Tang et al., 2005). Another mutant that regulates the cross-talk between several hormones is the onset of leaf death 1 (old1) which shows early leaf senescence, constitutive expression of pathogenesis related proteins and hypersensitivity to sugars (Jing et al., 2007). Thus the response to various stresses is regulated by stress signaling pathways that are interconnected at several levels by shared genetic factors between the pathways (Knight and Knight, 2001). One common class of signaling molecules are the reactive oxygen species (ROS) which have been implicated in the regulation of development and stress response pathways (Pitzschke et al., 2006). Among the different ROS species only hydrogen peroxide can cross membranes and therefore function as a cell to cell signaling molecule. Hydrogen peroxide is involved in many plant processes including ABA-dependent stomatal closure (McAinsh et al., 1996), signaling via the ETR1 receptor (Desikan et al., 2005) and oxidative burst during pathogen attack and subsequent induction of systemic immunity (Alvarez et al., 1998). While during plant defense accumulation of ROS is required, ROS is scavenged during abiotic stress to counteract the accumulation (Pitzschke et al., 2006). The response of the plant to ROS is determined by the location of the signal in the cell where they are produced or accumulate. ROS have also been implicated in the onset of leaf senescence as demonstrated by treatment of Arabidopsis leaves with the herbicide 3-AT which inhibits catalase activity and causes H202 stress resulting in the expression of SAG genes (Navapbour et al., 2003). Next to that the senescence-specific transcription factor WRKY53 is induced by H202 (Miao et al., 2004; Miao and Zentgraf, 2007). Taken together the integration of signals from the environment with the age of the leaf involves a complex signaling network of overlapping and interacting pathways which together determine the onset of leaf senescence. To gain insight into the pathways and mechanisms underlying leaf senescence the previously identified class II early leaf senescence mutant old13 (Chapter 2) was characterized in detail. Here we present evidence for the involvement of OLD13 in regulating stress induced senescence and lesion formation. 71 Results Characterization of old13 mutant under standard conditions Previously, we reported that the old13 mutant is phenotypically normal when grown under optimal conditions but displays early senescence upon ethylene treatment (Jing et al., 2005). To characterize the old13-mediated senescence response we examined plants grown under standard conditions and after stress treatment in detail. The development of the first leaf pair of old13 was followed and compared to wild type from day 21 till day 33. old13 plants are identical in size when compared to wild type, however, the initiation of the primary inflorescence starts 2-4 days earlier than the wild type (data not shown). A primary marker for early senescence is leaf yellowing caused by the loss of chlorophyll (Oh et al., 1997). From day 21 till day 33 no chlorophyll degradation and visual senescence is observed for the first leaf pair of both air-grown wild type and mutant old13 (Figure 1A and B). Next to that the photochemical efficiency of photosystem II stays at a similar level as wild type during development (Figure 1C). Thus old13 is a typical class II mutant that has no early senescence symptoms when grown under optimal conditions (Jing et al., 2002; Jing et al., 2005). Figure 1. Characteristics of air-grown old13 mutants. (A) The chlorophyll contents of soilgrown old13 (open circles) and wild type (closed circles) were quantified every 3 days from day 21 till day 33. (B) Representative first leaves of old13 and wild type were selected and photographed at day 24. (C) The photochemical efficiency was determined as described in methods and is expressed as Fv/Fm. Error bars indicate SD; results are mean of 3 replicates. 72 A backcross of the old13 mutant revealed that it segregates as a recessive trait both in the original mutant accession Landsberg (Ler) and the mapping background Columbia (Col). 1800 F2 plants of the mapping population were phenotypically selected for positional cloning. The position of the mutated locus was narrowed down to a region on chromosome V, covering 101kb of sequence on BAC K19B1 and MRG21. The mapped region contains 32 annotated candidate genes which are listed in Table 1. Table 1. Genes annotated for the mapped region that contains the old13 mutation. The 32 genes listed are shown with a putative function and description according to TAIR annotation. Asterisks indicate that gene has been cloned and sequenced but no mutation was detected. TAIR- ID Name Description At5g62410 SMC2 Structural maintenance of chromosome II At5g62420 Aldo/keto reductase family protein At5g62430 CDF1 Cycling Dof Factor 1, transition vegetative to reproductive At5g62440 DOM1 Domino 1, nucleolus formation maintenance At5g62460 zinc finger (C3HC4-type RING finger) family protein At5g62470 MYB96 Encodes a R2R3 type Myb transcription factor, ABA dependent At5g62480 ATDSTU9 Glutathione S-Transferase TAU 9 At5g62490 ATHAV22B Arabidopsis thaliana HVA22 Homologue B At5g62500 ATEB1B Microtubule End Binding Protein At5g62510 F-box family protein At5g62520 SRO5 PARP motif ROS production and salt stress response. siRNA At5g62530 P5CDH Delta-pyrroline-5- carboxylate dehydrogenase, siRNA At5g62540 UBC3 Ubiquitin-Conjugating Enzyme At5g62550 similar to unknown protein At5g62560 armadillo/beta-catenin repeat family protein At5g62570 calmodulin-binding protein At5g62575 similar to unknown protein At5g62580* similar to Os02g0739900 At5g62600 transportin-SR-related At5g62610 basic helix-loop-helix (bHLH) family protein At5g62620 Putative galactosyltransferase family protein At5g62623* Defensin-Like At5g62627* Defensin-Like At5g62630 HIPL2 HIPL2 PROTEIN PRECURSOR At5g62640* ELF5 Early flowering locus 5 At5g62650* similar to Os01g0225300 At5g62660 F-box family protein At5g62670* AHA11 H-ATPasa At5g62680* proton-dependent oligopeptide transport At5g62690* TUB2 Tubulin beta 2 At5g62700* TUB3 Tubulin beta 3 At5g62710* leucine-rich repeat family protein PubMed 11276426 10467030 16002617 15341625 NF 16463103 12090627 12081371 14557818 NF 16377568 16377568 11019805 NF NF NF NF NF NF NF 17630273 15955924 15955924 12805588 15125772 NF NF 15821287 NF 1498609 1498609 NF 73 Ethylene and detachment induce early leaf senescence in old13 In Arabidopsis leaves, diverse internal and environmental signals are integrated with the age to modulate the onset of leaf senescence (Grbic and Bleecker, 1995; Jing et al., 2003; Lim et al., 2003). Ethylene, detachment and drought are known to induce premature senescence (Lim et al., 2007). old13 plants were grown under standard growth conditions for 3 weeks and subsequently treated with ethylene or subjected to a detachment experiment, to follow the progression of leaf senescence. Mutant leaves show reduced chlorophyll levels when compared to wild type after 3 days of ethylene treatment (Figure 2A). Noteworthy, ethylene treatment results in a more pronounced early flowering phenotype in the mutant, as compared to growth under standard conditions (results not shown). For the detachment experiment the first leaf pair was followed for 12 days during incubation in the dark (Figure 2B). Figure 2. The senescence syndrome of old13 leaves. (A) The chlorophyll contents of old13 (open circles) and wild type (closed circles) were quantified during ethylene treatment of 3 days. Content is shown in mean percentage ±SD of total content at day 21. (B) Detachment induced senescence of the wild type and old mutants was photographed after 9 days of treatment. WT, wild type; 5, old5; 9, old9; 11, old11; 13, old13; 14, old14. (C) Chlorophyll content of old13 (open circles) and wild type (closed circles) during the detachment experiment are represented as the mean± SD of three replicates of 6 leaves. (D) Nitrogen, carbon and sulfur content at 21 and 24 days of growth in air and after 3 days of ethylene treatment for wild type (black bars) and old13 leaves (gray bars). Content is represented as mean percentage ±SD of total content at day 21. 74 Detachment of old13 leaves results in a slightly enhanced breakdown of chlorophyll when compared to the wild type (Figure 2C). As a control we tested the effect of detachment on leaves of other old mutants and demonstrate that the class II mutants old9 and old11 appear wild type while the class I mutants old5 and old14 senesce early upon detachment and more severe than old13 (Figure 2B). We observed that old13 plants are more sensitive than wild-type plants to limited water availability, resulting in early leaf senescence (data not shown). Taken together these results suggest that early senescence in old13 plants can be induced by different stress signals. We examined the ethylene-induced leaf senescence in more detail by examining nutrient remobilization and expression of senescence-associated genes (SAGs). A characteristic feature of leaf senescence is the relocalization of nutrients to other parts of the plant (Himelbau and Amasino, 2001). Remobilization starts with the transport of stored compounds, once these reserves have been drained, proteins and polymers will start to be catabolized (Thomas et al., 2002). Up to 95% of the stored nitrogen in leaves is relocated during senescence which mainly arises from the degradation of the chloroplast (Hortensteiner and Feller, 2002). By determining the elemental composition of dried leaves we estimated the relocalization of nitrogen, carbon and sulfur. Interestingly, both the wild type and the mutant show a strong decline in nitrogen (Figure 2D). The significant decrease in the mutant when compared to the wild type correlates with the start of chlorophyll degradation after 3 days of ethylene. The level of carbon remains at a similar level while sulfur declines both during air-growth as during ethylene treatment. Thus ethylene treatment induces remobilization of nitrogen containing compounds in wild type and old13, although significantly more in the mutant. Analyses of the expression profiles of senescenceassociated genes after 1 day of ethylene treatment showed that old13 mutants exhibit higher transcription levels of SAG12, SAG13, and SAG21 in comparison with wild-type plants (Figure 3A). Next to that the expression of CAB was approximately 5fold reduced after ethylene treatment. This result, the increased expression of SAG12 and SAG13 (Weaver et al., 1998) demonstrates that old13 enhances the ethylenedependent senescence process not only on physiological level but also at the molecular level. 75 Figure 3. SAGs, Hypersensitive response-like lesions, and oxidative stress. (A) Marker transcripts levels after ethylene treatment. SAG12, SAG13, SAG21 and CAB transcript levels after 1 day of ethylene treatment in the wild type (black bars) and old13 (gray bars). The relative expression shown is the mean of three repeats ± SD (indicated by error bars). (B) Tryphan blue stain of ethylene treated leaves of old13 and a representative photograph of a first leaf after 3 days of ethylene (arrow indicates lesions). (C) Staining for superoxide by NBT of 24 day old leaves. (D) Marker transcript levels during development. DEFL, PAD3 and PR2 relative transcript abundance after 21 day of growth on soil for the wild type (black bars) and old13 (gray bars). Ethylene and drought induce hypersensitive response-like lesions in old13 which are associated with oxidative stress In addition to early leaf senescence, old13 plants also developed spontaneous yellow-brown necrotic lesions under drought conditions or after ethylene treatment (Figure 3A). The necrotic spots resemble phenotypically the hypersensitive-response (HR) which is commonly observed during pathogen attack (Mur et al., 2007). Staining of ethylene treated mutant leaves with tryphan blue reveals the appearance 76 of randomly localized dead cells after two days of ethylene treatment (Figure 3B). Interestingly, staining of air-grown old13 plants with NBT for superoxide reveals that the mutant has an increased level of cellular ROS during unchallenged conditions (Figure 3C). Moreover, old13 plants have a 6-fold increased expression of a defensin-like (DEFL) gene which is expressed upon 7 different ROS-inducing agents (Gadjev et al., 2006) (Figure 3D). Thus old13 plants have increased oxidative stress when compared to the wild type and develop spontaneous lesions during ethylene treatment and drought stress. old13 functions both in biotic and a-biotic plant response pathways During the hypersensitive response, an oxidative burst through rapid accumulation of ROS is essential for the local cell death and the activation of a signaling cascade to induce systemic acquired resistance (Alvarez et al., 1998). Next to that a-biotic factors that cause salt, osmotic and drought stress all result in increased oxidative stress (Knight and Knight, 2001). We found that a salicylic acid dependent defense gene, PAD3 (Glazebrook and Ausebel, 1994) and the PR2 gene which is related to pathogenesis but also leaf senescence (Gaffney et al., 1993) are 5 to 9 fold increased in expression in the mutant when compared to the wild type (Figure 3D). Figure 4. Anion content of wild type and old13 leaves during development. Chloride, nitrate and sulfate content were measured every 6 days starting from day 21 until day 33. Wild type is represented as black bars and mutant as gray bars. Content is represented as content ±SD of mean. We noticed that the timing of watering before, at the start or during the ethylene treatment could influence the senescence response of old13. Therefore we anticipated that the mutant might have a defect in its water balance. To test this we determined the anion concentration during the development of the first leaf pair. Anions play a role both in the regulation of the membrane potential and in the 77 osmolarity of the cell (Tyerman, 1992). The determination of the anion content by HPLC revealed that the mutant exhibited elevated levels of Cl- especially at young age when the levels are 6-fold increased (Figure 4). Next to that the concentration of nitrate is elevated at 21 days but decreases below wild type levels at 33 days. Sulfate levels are at day 21 and 27 two-fold increased in comparison to the wild type but restore to wild type levels at day 33. Taken together, the results suggest that old13 mutant plants grow under constitutive water stress. HR-like lesions and senescence in old13 are dependent on intact hormone signaling pathways We showed before that the early induction of senescence in old13 is not a consequence of increased sensitivity to ethylene (Chapter 2). To test the role of hormone signaling pathways in adult plants we constructed double mutants between Figure 5. Senescence phenotypes of old13 double mutants. (A) Plants were grown in air until 21 days and subsequently treated with ethylene for 3 days after which representative plants were selected and photographed. Double mutant abbreviations: o13a4, old13/abi4-1; o13e2, old13/ein2-1; o13p4, old13/pad4-1. Bars indicate 5 mm. (B) Leaf yellowing after 78 ethylene treatment, yellowing is presented as the mean ± SD of 30 plants. (C) Number of leaves with lesions after ethylene treatment as mean ± SD of 30 plants. old13 and mutants impaired in the sensing and/or signaling of ethylene, salicylic acid and abscisic acid. The double mutants were selected by PCR based identification of the mutant alleles. Subsequently the double mutants were grown till 21 days in air and treated for 3 days with ethylene (Figure 5A). The abi4-1 mutation (Finkelstein et al., 1998) did not affect the old13 senescence symptoms or appearance of lesions after ethylene treatment (Figure 5B and C). Crossing the old13 mutation with ethylene insensitive mutant ein2-1 (Guzman and Ecker, 1990) results in absence of leaf senescence and lesions during ethylene treatment. Interestingly, a cross between old13 and pad4-1 (Jirage et al., 1999) results in the loss of the lesion phenotype, while the effect on leaf yellowing was limited. These data suggest that the old13-mediated ethylene-induced senescence phenotype is independent of SA signaling, while the lesion phenotype is dependent on the salicylic acid pathway. Discussion Senescence is a developmental event that results in the death of a cell, an organ, or an organism upon aging. In plants senescence has evolved as a beneficial trait which increases the survival of the species (Bleecker, 1998). While the onset of leaf senescence in old13 mutant is similar to wild type under optimal growth conditions, we observed that old13 mutants senesce early and display spontaneous lesions formation after ethylene treatment or drought stress. This suggests that old13 mutants are more sensitive to environmental conditions than wild type and that OLD13 might be involved in stress-induced growth, senescence and cell death. One effect of the old13 mutation is the increased anion content, which might suggest a defect in water balance. Most strongly increased is the level of chloride, which is 6fold higher at day 21. An increase in chloride content has been observed before during N limitation experiments in which chloride replaces nitrate as an osmoticum (White and Broadley, 2001). However, we measured an increased availability of nitrate in leaves of old13 which argues against the possibility that nitrogen deficiency plays a role in the mutant. Drought and salt stress have been shown before to induce senescence. In rice it was shown that NaCl increases the rate of developmental 79 senescence (Lutts et al., 1996) while in Arabidopsis several SAG genes including ERD1, SAG13, SAG14, SAG21 respond to drought-induced senescence (Weaver et al., 1998). High saline environments cause cytosolic accumulation of calcium which is a potent signal for stress responses that can result in either adaptation or death (Hasegawa et al., 2000). Recently a calcium-dependent protein kinase has been characterized which plays a role in salt and drought tolerance (Ma and Wu, 2007). One response to accumulation of Cl- is restoration of the osmotic balance by the accumulation of a non-harmful compatible solute like sucrose, fructose, glycerol, trehalose or proline (Hasegawa et al., 2000). Levels of ROS increase rapidly during salt stress and can result into an oxidative burst (Apel and Hirt, 2004). Tryphan blue staining of old13 together with the increased expression of a defensin-like gene indicates increased ROS levels in the mutant. Next to that it was shown for Arabidopsis that increased H202 levels promote reproductive growth (Zimmermann et al., 2006), which might explain the early flowering of old13. A possible explanation for the increased ROS is that during water deficit photorespiration is inhibited due to a reduced availability of CO2 which results in excess excitation energy and subsequent increased oxidative stress (Smirnoff, 1993). Interestingly, the assimilation of nitrate depends on the rate of photorespiration (Rachmilevitch et al., 2004). This observation which might explain the measured increase in nitrate, although it was only observed for young leaves. Increased ROS production causes lipid peroxidation and can affect the integrity of the membrane resulting in permeability to electrolytes and finally changes in the cellular ionic homeostasis (Kourie, 1998). ROS scavenging is an important factor in adapting to salt stress as shown by ascorbate-deficient Arabidopsis plants which have an increased sensitivity to salt-stress (Huang et al., 2005). The photoautotrophic salt tolerate 1 (pst1) mutant has increased superoxide dismutase (SOD) and ascorbate peroxidase (APX) activity resulting in increased salt and drought tolerance (Tsugane et al., 1999). Thus, the rapid detoxification of ROS during salt or drought stress is an important factor in determining tolerance. Detoxification of ROS can also be achieved by increasing the level of non-enzymatic antioxidant compounds. Synthesis of the sulfur-containing anti-oxidant glutathione (GSH) is upregulated during oxidative stress (Noctor and Foyer, 1998). Ozone is a potent inducer of oxidative stress which results in accumulation of glutathione and up-regulation of sulfur assimilation (Bick et al. 2001). Therefore the increased sulfate content in old13 might be the direct consequence of oxidative stress. However, the 80 observed changes in anion content can be explained as a general change in iontransport (Frachisse et al., 1999) and thus may not be related to salt stress. Excess nitrate and chloride are stored by the same mechanism in the vacuole and therefore the OLD13 gene product might function directly in ion transport (Harada et al., 2004). Although the mechanism might relate to storage or transport of anions, in old13 the increased anion content might cause the drought sensitive phenotype. Next to increased cellular ROS and anion concentration the old13 mutant also displays lesion formation. The physiology of old13 resembles that of the edr1 mutation which results in early ethylene-induced senescence, spontaneous lesion formation during drought stress and enhanced resistance to pathogens. Although, pathogen response of old13 has not been tested we did find that the pathogenesis-related transcripts of PR2 and PAD3 accumulate in the mutant. EDR1 encodes for a MAPKKK suggesting that different stress factors might initiate the same signaling cascade (Tang and Innes, 2005). By double mutant analysis it was shown that the drought-induced phenotype of edr1 is depending on SA while the senescence phenotype requires ethylene signaling. Our double mutant analysis shows that lesion formation in old13 is also dependent on SA-signaling pathway while ein2 blocks the senescence phenotype. In contrast to the role of PAD4 during developmental senescence in which it modulates the switch to PCD during the final phase (Morris et al., 2000) it is necessary for the onset of senescence during pathogen attack (Pegadaraju et al., 2005). This suggests that only a part of the old13 senescence response is related to defense since the lesion and senescence phenotype are regulated by different pathways. Interestingly crossing the late senescence mutant ore9 with edr1 results in suppression of ethylene and drought induced senescence but not plant defense responses (Tang et al., 2005), confirming that the different phenotypes can be separated in the edr1 mutant as well and are the result of different signaling pathways. Previously, the early leaf senescence mutant old1 has been implicated as an integration node of different stimuli including pathogen response, ethyleneinduced senescence and sugar signaling (Jing et al., 2007). Taken together, our analysis suggests that OLD13 might represent another OLD protein that regulates plant development and senescence by monitoring the environment and endogenous status of plants and adjusts development accordingly. The characterization of the old13 mutation reveals a novel locus involved in modulating the developmental program of Arabidopsis during stress conditions. One 81 important concept is that during stress a plant has to decide if a certain organ can be sacrificed or not. Although most of the current research focuses on delayed senescence to improve productivity in several crops it is important to note that a rapid and localized senescence response is essential for stress tolerance and survival. Therefore, we would like to encourage future research that aims at senescence modulators that control plant fitness. Methods Plant materials and growth conditions Arabidopsis thaliana ecotype Landsberg erecta (Ler-0) was used in this study. The old9 mutant was obtained from an EMS mutagenized collection (Jing et al., 2005). Plants were grown on either soil or half-strength Murashige and Skoog medium at 23°C and 65% relative humidity with a day length of 16 h. The light intensity was set at 120 µmol·m-2·s-1. An organic-rich γ-ray radiated soil was used (Hortimea Groep, Elst, The Netherlands). Plants for ethylene exposure were treated in a flow-through chamber at 20 °C and a humidity of 40% under continuous illumination. The ethylene dosage was set at 10 µl l–1 as suggested by Chen and Bleecker (1995). Cotyledons or rosette leaves with over 5% yellow area of the leaf blade were judged as yellow as suggested by Lohman et al. (1994). The mutant alleles and transgenic plants used for the double mutants were ein2-1 (Guzman and Ecker, 1990), pad4-1 (Jirage et al., 1999), and abi4-1 (Finkelstein et al., 1998). The old13 mutation was crossed into the mutant lines that have a Col-0 background, thus the F2 double mutant population contains a mixed Ler-0 and Col-0 background. NBT staining The first leaf pair was harvested and placed in an aqueous solution of 0.05% nitroblue tetrazolium; blue color indicated superoxide (O2−) generation (Flohe and Otting, 1984). After 2 hours leaves were fixed and decolorized in 96% ethanol overnight. Decolorized and cleared leaves were mounted in saturated chloral hydrate and analyzed under a light microscope. Subsequently, photographs were taken of representative leaves. 82 Tryphan blue staining Appearance of cell death was studied in whole leaf mounts stained with lactophenoltryphan blue (10 mL of lacticacid, 10 mL of glycerol, 10 g of phenol, 10 mg of tryphan blue, dissolved in 10 mL of distilled water) (based on Keogh et al.,1980). Whole leaves were boiled for 1 min in the staining solution and then overnight decolorized in chloral hydrate. Subsequently the leaves were mounted in chloral hydrate and viewed under a light microscope. Pigment determination and measurement of photochemical efficiency For extraction of chlorophyll and carotenoids samples were incubated overnight with N,N-dimethylformamide at 4°C in darkness. The chlorophyll content was quantified spectrophotometrically according to Wellburn (1994) at 647 and 664nm. Chlorophyll fluorescence emission was measured from the upper surface of the first leaf, at room temperature (23 °C) with a pulse-amplitude modulation portable fluorometer (PAM-2000; H. Walz, Effeltrich, Germany) according to Maxwell and Johnson, 2000. Plants were dark-adapted for 1 to 2 hr before measurements to ensure complete relaxation of the thylakoid pH gradient. An attached, fully expanded rosette leaf was placed in the leaf clip, allowing air to circulate freely on both sides of the leaf. At the start of each experiment, the leaf was exposed to 2 min of far-red illumination (2 to 4 µmol photons m-2 s-1) for determination of Fo (minimum fluorescence in the dark-adapted state). Saturating pulses of white light (8000 µmol photons m-2 s-1) were applied to determine Fm or Fm' values. PSII efficiency was calculated as (Fm - Fo)/Fm. RNA-isolation and RT-PCR Total RNA was isolated using TRIZOL reagent (Sigma) according to the manufacturer's protocol. Five hundred nanograms of RNA were used as template for first-strand cDNA synthesis using 200U of RevertAid H-minus MMuLV reverse transcriptase (Fermentas, USA) and an oligo(dT21) primer. Primer pairs for real-time PCR were designed with open-source PCR primer design program PerlPrimer v1.1.10 (Marshall, 2004). The primer sequences are available upon request. Briefly, real-time PCR amplification was performed with 50 µL of reaction solution, containing 2 µL of 10-fold–diluted cDNA, 0.5 µl of a 10 mM stock of each primer, 1 µl of 25mM stock MgCl2 (Fermentas), 5 µl PCR buffer +Mg (Roche), 1 µl of a 1000x diluted 83 SYBR-green stock (Sigma), 0.5 µl 100xBSA (New England Biolabs), and 1u of Roche Taq Polymerase. The PCR program was 2’ at 94, 40x (94-10”/60-10”/72-25”). Obtained data was analyzed with BioRad software. CNS measurements The first leaf pair of wild type and mutant was collected after treatment and oven dried at 80°C. Subsequently the tissue was weighted and grinded to a fine powder. For determination of the C, N, and S content we made use of a LECO CHNS 932 (USA) analyzer by combustion at 1200°C. Detachment-induced senescence Leaves were incubated in light on two layers of Whatman filter papers saturated with MES solution (pH 5.7) and collected after 0, 3, 6, 9 and 12 days for chlorophyll content measurement. Three replicates of 6 pairs of leaves were analyzed for each data point. Intracellular anion content For chloride, nitrate and sulfate analysis, fresh first leaf pairs were harvested and homogenized in demineralized water, with an Ultra Turrax (T25 Basic Ika Labortechnik, Staufen, Germany). The homogenate was incubated at 100 °C for 10 min, filtered and centrifuged at 30 000 g for 15 min. 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Dijkwel1 1 Molecular Biology of Plants, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands 2 Warwick HRI, University of Warwick, Wellesbourne CV35 9EF, UK. 89 90 Abstract The expression of several Arabidopsis senescence-associated genes (SAGs) was found to be upregulated during leaf development of the onset of leaf death 9 mutant under standard growth conditions. Treatment of the mutant with ethylene resulted in a severe senescence response both phenotypically as well as on transcript level. A RNAi-based strategy to downregulate the expression of a SAG encoding for a hydroxyproline-rich glycoprotein, attenuated the ethylene induced senescence response of old9. Comparison of the old9 transcriptome revealed that 67% of the differentially expressed genes with wild type are cytokinin responsive. Cytokinins are well known for their effect to retard leaf senescence. However, by applying cytokinin early in development the response to ethylene-induced senescence could be enhanced. We argue that the cytokinin effect we observed is the result of increasing the sink strength of young tissue while opposite to that ethylene promotes source strength of old leaves. Therefore we propose that the balance between sink and source tissue can modulate the progression rate and timing of senescence. Introduction The development of a leaf starts with the formation of a leaf primordium from the shoot apical meristem (Van Lijsenbettens and Clarke, 1998). A subsequent differentiation and expansion of the different cells gives rise to a mature leaf consisting of an epidermis, a mesophyll layer and vascular tissue (Pyke et al., 1991). The determinate program of leaf development ends with the age dependent onset of senescence resulting in the death of the tissue. However, this final stage of leaf development is critical for plant fitness as nutrient relocation to young developing parts is achieved through this process. Leaf senescence is thus an evolutionary selected developmental process and represents an important phase in the plant life 91 cycle (Bleecker, 1998; Buchanon-Wollaston et al., 2003). The study of leaf senescence will not only increase our understanding of a fundamental biological process, but also contributes to means to control leaf senescence to improve traits of crops and ornamental plants including postharvest storage. The development of a leaf is as a mitotic event which occurs prior to unfolding and cell expansion is initiated. The mitotically active cells that form the leaf primordium arise from the shoot apical meristem (SAM), which contains a pluripotent stem cell population (Bowman and Eshed, 2000). The initiation of a leaf from this population of stem cells involves the action of several phytohormones. First the maintenance of the stem cells requires biosynthesis of cytokinin (Bowman and Eshed, 2000). However, the initiation of a leaf primordium requires gibberellin (GA), which accumulates due to the repression of cytokinin biosynthesis (Yanai et al., 2005; Jasinski et al., 2005). Plants with reduced cytokinin production have less leaves and contain fewer leaf cells (Werner et al., 2001) which was interestingly also shown in the triple mutant for a cytokinin receptor arabidopsis histidine kinase2 (ahk2), ahk3, ahk4 (Nishimura et al., 2004). On the other hand transgenic Arabidopsis plants which overproduce cytokinin have more leaf cells (Rupp et al., 1999). Thus cytokinin has a regulatory function in leaf cell formation. The cell division in the leaf primordium of Arabidopsis sets an age-gradient from “old” cells at the leaf tip and margins to younger cells at the base of the leaf (Ferreira et al., 1994). The post mitotic phase of leaf development involves cell expansion until the final size is reached at maturity. The expansion is controlled in part by the ANGUSTIFOLIA gene in Arabidopsis which acts through arrangement of cortical microtubules and the expression of cell-wall loosening genes (Kim et al., 2002). During the expansion of mesophyll cells, partially differentiated chloroplasts start to divide actively (Pyke and 92 Leech, 1994) to attain photosynthetic competence of a leaf. Arabidopsis grown in medium containing cytokinin shows an accelerated photomorphogenesis phenotype coinciding with induced expression of photosynthetic genes (Schmülling et al., 1997; Brenner et al., 2005). Several effects of cytokinin can also be induced by light, including leaf expansion, chlorophyll accumulation and inhibition of hypocotyl elongation. Although both cytokinin and light can induce similar processes the action of light involves a phytochrome-dependent pathway while cytokinin in part requires the ethylene response pathway (Cary et al., 1995; Su and Howell, 1995). Interestingly, cytokinin induces the expression of nitrate reductase in plants (Samuelson et al., 1995; Taniguchi et al., 1998) and was suggested to have a regulatory role in nitrate assimilation (Tischner, 2000). Cytokinin affects source-sink relationships and causes nutrient accumulation in leaves treated with the phytohormone (Roitsch and Ehness, 2000). The transport of carbon and nitrogen compounds from source leaves to sink leaves requires glucose, sucrose, and amino acid transporters which have been identified in the Arabidopsis genome as large gene families (Lalonde et al., 2004). One of these sucrose transporters AtSUC2 was shown to be developmentally regulated and is induced during the sink-to-source transition of leaves (Truernit and Sauer, 1995). The application of cytokinin to seedlings results in a downregulation of AtSUC1 (Brenner et al, 2005) while overexpression of ARR22 (Kiba et al., 2004) results in down regulation of both AtSUC1 and AtSUC2 suggesting that cytokinin can modulate the transition of sourceto-sink. The final stage of leaf development results in a total commitment to source function and results in massive nutrient remobilisation by the onset of leaf senescence (Balibrea Lara et al., 2004). In Arabidopsis the onset of leaf senescence occurs in a leaf-age dependent manner (Gan and Amasino, 1997; Quirino et al., 93 2000). Ageing of the leaves starts with the initiation of a leaf primordium and occurs throughout the development resulting in senescence and death (Lim et al., 2003). The developmental changes during the lifespan of a leaf can be viewed as agerelated changes (ARCs) that set the developmental age and thus determine if a leaf can undergo senescence (Jing et al., 2005; Schippers et al., 2007). The onset of senescence can be delayed by the application of cytokinin on leaf senescence (Richmond and Lang, 1957) which is thus acting as an anti-ageing factor. In addition, expression of cytokinin biosynthesis genes under the promoter of a senescenceassociated gene (SAG) delays the initiation of leaf senescence in several plant species (Gan and Amasino, 1995; McCabe et al., 2001; Khodakovskaya et al., 2005). The SAM gene knotted1 (kn1) regulates cytokinin content and delays leaf senescence when expressed under the control of a SAG promoter in tobacco (Ori et al., 1999). The mechanism of cytokinin-mediated delay of senescence is dependent on an extracellular invertase. Activation of the invertase increases the sink strength of the tissue and the rate of sugar utilization which results in delayed onset of senescence (Balibrea Lara et al., 2004). These studies suggest that sugar metabolism mediated by cytokinin is an important factor in determining the onset of leaf senescence and that the transition of sink-to-source is an important ARC during the development of a leaf. In our attempt to elucidate factors that control the onset of leaf senescence we make use of the senescence inducing phytohormone ethylene. In the present study, we selected the Arabidopsis onset of leaf death 9 (old9) mutant. The mutant was shown in chapter 2 to senescence early upon ethylene treatment. Since the effect of ethylene is age-dependent the mutant presents a good starting point for determining regulation of age-related changes (ARCs). Here we present evidence for a role of 94 cytokinin on the timing of ethylene-induced senescence in Arabidopsis. The mutant old9 shows altered expression of cytokinin responsive genes. Furthermore we demonstrate that exogenous application of cytokinin can prime a leaf for ethyleneinduced senescence and argue it to be caused by changes in the sink/source strength of the leaves. Results old9 presents a co-dominant locus involved in ethylene-inducible senescence The old9 mutant of Arabidopsis was isolated initially as an early leaf senescence mutant upon ethylene treatment (Jing et al., 2005). old9 is classified as a class II mutant (Jing et al., 2002) and was found to develop as wild type under standard growth conditions. Since the induction of leaf senescence by ethylene is dependent on the age of the leaf (Grbić and Bleecker, 1995, Jing et al., 2002) we anticipate that old9 might be involved in the integration of ARCs and environmental stimuli to regulate the onset of senescence. Figure 1. Senescence response of old9 and wild type leaves to ethylene treatment. Wild type and old9 plants were grown for 21 days in air and were subsequently treated for 2 or 3 days with ethylene. Representative leaves from the first true leaf pair were photographed (A) or stained with tryphan blue (B) as described in the Methods section. (C). Transcript accumulation of the senescence-associated genes SAG12 and SAG13 after ethylene treatment in old9 (gray bars) and wild type (black bars). The values shown are the means of three repeats ± SD (indicated by error bars). 95 For the characterization of the mutant we first examined the senescence symptoms during ethylene-induced senescence. The first leaf pair of old9 has reduced leaf longevity upon ethylene treatment at 21 days as observed visually (Figure 1A). After 2 days the leaves become pale green and after 3 days the entire leaf turned yellow which indicated the complete execution of senescence (Grbić and Bleecker, 1995). In contrast, leaves of the wild type show visual senescence symptoms after 4 days of ethylene treatment (data not shown). After 3 days of treatment cell death is observed in the mutant but not in the wild type (Figure 1B). The appearance of cell death occurs in an ordered way starting from the leaf tip. The onset of leaf senescence is marked by increased expression of senescence associated genes (SAGs) (Nam, 1997). Expression of the SAG12 and SAG13 genes increased dramatically after 3 days of ethylene treatment in old9 mutants (Figure 1C). Taken together, the complete yellowing of the leaf, appearance of cell death and SAG gene expression upon ethylene treatment are consistent with the suggested occurrence of early ARCs in the mutant (Jing et al., 2005). A backcross of the old9 mutant revealed that it segregates as a co-dominant trait both in the original mutant accession Landsberg (Ler) and the mapping background Columbia (Col). The position of the old9 locus was narrowed down to a region on chromosome II of 146kb on BAC F6E13 and F4I1. The identified region contains 54 annotated genes which are summarized in Table 1. In an attempt to identify the mutation seven genes have been sequenced but were shown to be identical to the wild type gene. Table 1. List of genes in mapped old9 area. Shown is the data from the TAIR annotation and a Pubmed record when available. Asterisks indicate genes that were sequenced and found to be identical to the wild type. TAIR- ID at2g43960 at2g43970 at2g43980 at2g43990 at2g44000 at2g44010 at2g44020 at2g44030 at2g44040 at2g44050 at2g44060 96 Name ITPK4 COS1 Description PubMed similar to SWAP La domain-containing protein inositol 1,3,4,5-tetrakisphosphate isomerase Unknown protein Unknown protein Unknown protein mitochondrial transcription termination factor-related Kelch-repeat containing F box dihydrodipicolinate reductase; lysine biosynthesis 6,7-dimethyl-8-ribityllumazine (DMRL) synthase late embryogenesis abundant family protein NF NF 17698066 NF NF NF NF NF 15652176 10419541 NF at2g44065 at2g44070 at2g44080* at2g44090 at2g44100* at2g44110* at2g44120 at2g44130* at2g44140* at2g44150* at2g44160 at2g44170 at2g44175 at2g44180 at2g44190 at2g44195 at2g44200 at2g44210 at2g44220 at2g44230 at2g44240 at2g44250 at2g44260 at2g44270 at2g44280 at2g44290 at2g44300 at2g44310 at2g44320* at2g44330 at2g44340 at2g44350 at2g44360 at2g44370 at2g44380 at2g44390 at2g44400 at2g44410 at2g44420 at2g44430 at2g44440 at2g44450 at2g44460 at2g44470 at2g44480 ARL ATGDI1 MLO15 RPL7C ATG4a ASHH3 MTHFR2 NMT2 MAP2a ATCS Ribosomal protein L2 eukaryotic translation initiation factor 2B family protein AGROS-like gene involved in cell expansion; BR induced Unknown protein Guanoside diphosphate dissociation inhibitor 7 transmembrane (7TM) G-protein-coupled receptor 60S ribosomal protein L7 Kelch-repeat containing F box Autophagy 4a Histone-Lysine N methyl transferase methylenetetrahydrofolate reductase N-myristoyltransferase Unknown protein Methionine Aminopeptidase Unknown protein Unknown protein Unknown protein Unknown protein Unknown protein Unknown protein Unknown protein Unknown protein Unknown protein Unknown protein similar to lactose permease-related protease inhibitor/seed storage/lipid transfer protein lipid transfer protein-related calcium-binding EF hand family protein TRNA serine zinc finger (C3HC4-type RING finger) VQ motif-containing protein Citrate synthase 4 Unknown protein DC1 domain-containing protein DC1 domain-containing protein DC1 domain-containing protein DC1 domain-containing protein protein binding / zinc ion binding protein N-terminal asparagine amidohydrolase family protein DNA-binding bromodomain-containing protein emsy N terminus domain-containing protein glycosyl hydrolase family 1 protein putative thioglucosidase gene glycosyl hydrolase family 1 protein glycosyl hydrolase family 1 protein NF NF 16824178 NF 8953772 16525893 15821981 NF 15178341 17295027 NF 12912986 NF 11060042 NF NF NF NF NF NF NF NF NF NF NF NF 12805588 NF NF 15644464 NF 8979399 NF NF NF NF NF NF NF NF NF 16287169 17063378 NF NF Transcriptional analysis of air-grown old9 As mentioned before the induction of senescence by ethylene is age-dependent (Hensel et al., 1993; Jing et al., 2002) in Arabidopsis. Therefore old9 might represent a locus that monitors the age of a leaf to set the developmental program accordingly. Since the mutant shows normal sensitivity to ACC (see chapter 2), we suggest that 97 an early age-related change (ARC) occurs in the mutant priming the leaves for senescence. Micro-array analysis was used to identify differentially expressed transcripts which could function as a marker for possible ARCs in the mutant. Transcript levels were assessed in the first leaf pair of old9 and wild type after 21 days of growth under standard conditions thus when no visual difference between the two genotypes is observed. 89 genes were found to be differentially regulated in the combined biological and technical replicates of air-grown plants (Table 2). Table 2. Genes differentially expressed in old9 after 21 days of growth under standard conditions. The 89 genes listed show statistical significance versus the wild type control (P < 0.05, Student’s t test). Shown are TAIR ID, gene name, description, Pubmed ID and ratio. BA indicates ratio found during micro-array study on genes responsive to cytokinin (Brenner et al., 2005). (S). Senescence associated (-, no; +, yes) TAIR- ID At1g01090 At1g04820 At1g08200 At1g11840 At1g12840 At1g16850 At1g20510 At1g21312 At1g21520 At1g23040 At1g28380 At1g32470 At1g35710 At1g37130 At1g67105 At1g67740 At1g67865 At1g68190 At1g69935 At1g71030 At1g76990 At1g78710 At1g78995 At1g55490 At2g06850 At2g18700 At2g19620 At2g20120 At2g21130 At2g24850 At2g25490 At2g25950 At2g26560 At2g29630 At2g30230 98 Name PDH-E1 TUA4 AXS2 AtGLX1 DET3 OPCL1 NSL1 NR2 PSBY Description PubMed Ratio BA S pyruvate dehydrogenase alpha; Tubulin alphaL-4 chain; cytoskeleton UDP-D-apiose/UPD-D-xylose synthetase lactoylglutathione lyase activity V-ATPase; cell expansion and photomorphogenesis Role in NaCl and cold stress; anthocyanin synthesis Jasmonic acid biosynthesis; wound-induced RNA recognition motif (RRM)-containing protein Unknown protein hydroxyproline-rich glycoprotein Necrotic spotted lesions; plant defense glycine cleavage system leucine-rich repeat transmembrane protein kinase Nitrate reductase Senescence-associated Gene PsbY precursor Part of Photosystem II Unknown protein zinc finger (B-box type) family protein Unknown protein 9393637 1498608 12969423 1.61 0.57 0.29 0.30 0.41 0.47 1.66 3.58 0.46 3.01 1.42 0.61 2.61 3.52 19.56 1.72 1.65 1.57 0.61 1.36 1.53 0.31 0.65 0.59 0.60 1.62 0.45 1.54 0.54 6.94 1.93 0.32 1.93 1.66 0.31 1.51 0.71 ND 0.50 1.15 0.22 0.38 1.21 1.50 0.5 0.61 1.23 1.03 1.94 ND 1.48 1.19 1.74 1.13 5.27 1.77 0.56 0.09 0.59 1.08 8.17 0.68 1.58 0.97 1.73 1.73 0.69 0.48 1.27 ND + + + + + + + + + + + MYB transcription factor repressed by BZR1 ACR3 amino acid-binding protein Stress related; unknown protein CPN60B EXGT-1A TPS11 COV1 CYP1 TAT3 EBF1 PLP2 Unknown protein beta subunit of the chloroplast chaperonin 60 Endo-xyloglucan transferase; br responsive Trehalose biosynthesis Ndr family protein; cell differentiation Vascular patterning Cyclophyllin Tyr amino transferase F-box binds EIN3; OE ethylene insensitive Unknown protein Phospholipase induced by pathogen attack thiamine biosynthesis family protein Unknown protein NF 11604454 17888165 16963437 NF NF 12805588 16900325 14671022 NF 3393528 NF 9829828 NF NF NF 15681342 12481063 NF NF 12668771 11673616 11520870 17565583 12668628 17473866 9342878 14675533 NF 16297072 NF NF At2g31890 At2g32670 At2g40000 At2g40080 At2g40360 At2g43100 At2g46450 At2g47960 At2g29080 At3g01490 At3g06710 At3g10020 At3g10260 At3g11410 At3g16520 At3g18490 At3g20790 At3g22320 At3g23700 At3g23820 At3g48360 At3g55240 At3g57260 At4g12830 At4g14890 At4g17390 At4g18200 At4g19170 At4g19860 At4g28220 At4g32020 At4g34740 At4g35090 At4g36020 At4g36040 At5g02500 At5g03850 At5g08050 At5g08520 At5g10760 At5g15650 At5g16150 At5g17460 At5g18630 At5g19140 At5g22580 At5g24530 At5g35180 At5g37475 At5g40450 At5g43060 At5g47560 At5g55960 At5g57760 Unknown protein; endomembrane system VAMP275 Vesicle associated membrane protein Putative nematode resistance gene ELF4 Photoperiodism; red light response; PhyB signaling putative WD-40 repeat protein; BOP1 domain aconitase C-terminal domain-containing protein CNGC12 Cyclic nucleotide gated channel; fungus response Transglutaminase, putative FTSH3 Ftsh protease; repair PSII Putative MAPKKK, MRK1-like; sucrose phospho similar to protein binding / zinc ion binding similar to Os12g0147200 reticulon family protein AtPP2CA Protein phosphatase 2C, represses ABA UDP-glucoronosyl/UDP-glucosyl transferase protein aspartyl protease family protein oxidoreductase family protein RPABC24 RNA polymerase I, II and III 24.3 kDa subunit S1 RNA-binding domain-containing protein GAE6 UDP-D-glucuronate 4-epimerase; pectin synthesis BT2 Involved in TAC1-mediated telomerase activation Unknown protein; OE Pseudo Etiolation in Light BGL2/PR2 beta 1,3-glucanase hydrolase, alpha/beta fold family protein ferredoxin family protein; light protection 60S ribosomal protein L15 AtPUP7 Putative cytokinin and purine transporter NCED4 Nine-cis-epoxycarotenoid dioxygenase 4 lecithin:cholesterol acyltransferase NDB1 NAD(P)H DEHYDROGENASE B1 Unknown XRN4 target ATASE2 de novo purine biosynthesis CAT2 peroxisomal catalase; photosynthetic active tissue CSDP1 Encodes a cold shock domain protein DNAJ heat shock like protein HSC70 Heat shock cognate 70 kDa protein 40S ribosomal protein S28 Thylakoid protein MYB family transcription factor aspartyl protease family protein RGP2 alpha-1,4-glucan-protein synthase GLT1 plastidic glucose exporter, trehalose responsive Unknown mitochondrial protein triacylglycerol lipase auxin-responsive genes Putative monooxygenase flavanone 3-hydroxylase like protein EDR2-like (enhanced disease resistance 2) translation initiation factor-related Unknown protein RD21-like cysteine proteinase, sugar responsive ATTDT malate transmembrane transporter Unknown protein DNA binding protein, MPK4 dependent NF 14500793 15448178 14605220 NF NF 16461580 NF 14630971 NF NF NF NF 16361522 NF NF NF NF NF 15225656 17220202 17227551 1824335 NF NF NF 10662864 15862093 NF 17673460 NF 15266056 17080932 17169986 NF 12805626 NF NF NF NF 9536051 7031512 NF NF NF 15213437 NF NF NF NF NF 12947042 NF 16813576 1.86 3.24 1.57 0.59 0.19 2.18 3.61 1.61 0.65 0.64 2.08 1.51 0.08 1.66 1.99 1.85 0.14 0.62 1.35 1.59 0.23 1.53 5.48 0.13 0.65 0.66 1.61 2.02 1.44 1.87 1.70 0.60 1.46 0.63 1.92 4.61 0.62 0.63 1.37 2.07 0.49 1.51 0.53 1.60 1.84 0.43 2.43 1.71 0.30 4.60 1.36 2.12 0.66 0.24 0.82 1.67 4.31 0.13 0.61 0.08 0.78 1.74 0.89 ND 1.24 4.18 0.65 0.58 1.21 0.78 0.54 1.25 1.05 0.82 9.87 ND 0.31 0.60 1.46 1.20 nd 1.88 1.33 1.23 0.45 0.88 1.47 1.44 4.73 1.82 1.37 1.30 1.03 0.91 ND 1.51 0.60 1.32 1.20 1.13 1.27 0.63 ND 0.62 1.20 3.46 0.81 3.68 99 + + + + + + + + + + + + + - To confirm the microarray data we performed real-time PCR analysis of several identified genes as shown in Figure 2. NIA2, TAT3 and EBF1 were confirmed to be up regulated in air-grown old9 compared to the wild type. Moreover the cell wall Dapiose synthetase (AXS2) is downregulated according to the RT-PCR analysis confirming the microarray data. Thus the RT-PCR analysis validated the genes identified by the microarray experiment. Figure 2. Real-Time PCR validation of transcripts identified by microarray analysis of old9. Transcript levels are shown as ratio between wild type and mutant. The values shown are the means of three repeats ± SD (indicated by error bars). Experiments were repeated once and similar results were obtained. Asterisks indicate statistical significance versus the wild type control in each case (* P < 0.05, Student’s t test). Microarray analysis results in large datasets of differentially expressed genes, however, these datasets are insignificant without a biological-orientated analysis. Therefore the identified genes that are differentially expressed in the mutant were studied by their annotation and classification in the TAIR database and by literature search. The developmental program of young leaves is regulated and controlled by many genes, interestingly several early leaf developmental genes are differentially expressed in old9 plants as compared to the wild type. DET3 (At1g12840), encodes for a subunit of a vacuolar ATPase involved in cell elongation during leaf development (Schumacher et al., 1999). The expression of DET3 decreases during the development of the leaf, and the mutant has a more than 2-fold decreased expression compared to the wild type. Formation of vascular tissue is repressed by COV1 (At2g20120) which when mutated results in increased vascular tissue in Arabidopsis (Parker et al., 1999). In old9 an increased expression of this repressor is detected at 21 days. Telomerase activity is shut down after mitotic division of cells by decreasing the expression of its activator BT2 (At3g48360) (Ren et al., 2007), BT2 is 4 fold downregulated in old9 compared to wild type. The altered expression of these 100 transcripts in old9 is consistent with the suggestion that certain ARCs occur earlier in the mutant. Next to that transcripts were identified that are involved in genetic pathways implicated in the biosynthesis or response to phytohormones. Ethylene promotes the onset of leaf senescence in many plant species and in old9 an increased expression of the negative regulator of ethylene responses EBF1 (At2g25490) (Potuschak et al., 2003) was observed. Besides that OPCL1 (At1g20510), involved in the biosynthesis of Jasmonic acid (Koo et al., 2006) shows increased expression in old9. Furthermore a negative regulator of ABA signaling, PP2CA (At3g11410), is more abundantly expressed in the mutant (Kuhn et al., 2006). Thus, the old9 mutation may cause a partial repression of ABA and ethylene signaling after 21 days of growth in air. Noteworthy is the observation that several genes encoding for cell-wall modifying enzymes are differentially expressed in old9. Two genes involved in cross-linking of the cell wall are differentially expressed; a D-apiose gene is downregulated (At1g08200) while a hydroxyproline-rich glycoprotein (HRGP) is up regulated (At1g23040). Next to that a xyloglucan transferase (At2g06850) is down regulated while a gene involved in pectin synthesis (At3g23820) is up regulated. Furthermore, an alpha-1,4-glucan synthase (At5g15650) is down regulated while beta-1,3glucanase is more than 5-fold increased in expression. This suggests that the cell wall of old9 might have a different composition than the wild type. Interesting to note is the increased expression of a putative cytokinin transporter (At4g18200) and the up regulation of nitrate reductase (At1g37130). To gain further insight in the air-grown transcriptome of old9 a comparison with other microarray experiments was carried out by making use of the GENEVESTIGATOR tool (Zimmerman et al., 2004). The analysis revealed that 67% of the differentially expressed genes in old9 also respond to a 2 hour cytokinin treatment in 8 day old seedlings (Brenner et al., 2005) (Table 2). Since cytokinin prevents senescence this observation might explain the absence of early senescence under normal growth conditions. However, it can not be ruled out that altered expression of cytokinin responsive genes might cause the early onset of senescence by ethylene. Of the 89 transcripts, 24 are senescence associated as shown by Guo et al., (2004). Moreover, At1g67105 is 19.5 fold increased in old9 and was shown to be upregulated during leaf senescence (data not show). Also TAT3 and BGL2 are induced during leaf senescence and are strongly up regulated in old9. These results are consistent with 101 the notion that old9 mutants may experience early ARCs and suggests that old9 leaves may be primed for senescence or that the senescence program may be partially induced during normal growth conditions. To determine a common regulation between the differentially expressed genes in old9, we used coexpression analysis within the ATTED-II database (Obayashi et al., 2007). The coexpression analysis reveals gene-to-gene relationships and is therefore a useful tool to identify gene networks. 29 of the 89 identified genes have an association with one or more other genes. Their association is presented in Figure 3 and shows 3 gene networks containing 3 or more genes and 6 interactions between 2 genes. The largest network contains 8 genes of which 2 are involved in lipid metabolism (AT4g19860 and At5g18630), 3 chloroplastic genes (NCED4, At4g36040 and At5g19140), a gene involved in trehalose metabolism (TPS11) and a MYB transcription factor (At1g71030). The second network is built around the BGL2 (At3g57260) which encodes a 1,3-beta-glucosidase involved in hydrolyzing fungal cell walls (Mauch et al., 1988) and TAT3 gene that encodes for an enzyme concerned in the synthesis of radical scavengers tocopherols from tyrosine (Hématy et al., 2007). These 2 networks consist for 50 to 66% out of SAG genes which are late leaf developmental genes and thus suggest an advanced leaf developmental program in old9. The third network is built around a sucrose-regulated putative MAPKKK (Niittylä et al., 2007). Figure 3. Gene-gene relation between the identified old9 microarray transcripts was determined by coexpression analysis. Genes are shown as green spheres and a relation between two genes is represented by lines. Lines are only drawn between genes with Pearson correlations >0.6. An S indicates that the gene is senescence associated. 102 The other 2 genes encode a putative CONSTANS-like transcription factor (At1g68190) and an unknown DNA-binding domain-containing protein (At5g57760). Taken together this analysis suggests that on the transcriptional level the leaf developmental program of old9 is more progressed. Furthermore it suggests that cytokinin might be an important factor in controlling the old9 phenotype. Onset of senescence in old9 after ethylene treatment The accelerated execution of senescence in old9 by ethylene was investigated by making use of microarray analysis. We performed the microarray after 8 hours of ethylene treatment when both wild type and mutant show no sign of visible yellowing. We prefer this timepoint because ethylene can induce thousands of transcripts within 24 hours and next to that the progression of senescence involves a massive transcriptional change. We anticipated that by limiting the length of the ethylene exposure only genes involved in the start of leaf senescence and ethylene signaling will show up, facilitating the analysis of the dataset. However, after ethylene treatment hundreds of transcripts were identified that showed altered expression between the mutant and the wild type. To focus our analysis we only investigated genes that are more than 3-fold changed in expression (Table 3). Table 3. Genes differentially expressed in old9 after 8 hours of ethylene treatment. 38 transcripts are more than 3-fold induced (P < 0.05, Student’s t test). Shown are TAIR ID, gene name, description, Pubmed ID and ratio. E indicates ethylene-inducible ; S indicates senescence associated; D indicates defense related. Genes less than 1.5-fold change are considered as not changed represented by 0. +, positive correlation, - negative correlation. TAIR- ID Name Description At1g03850 At1g06000 At1g16260 At1g19380 At1g33960 At1g56660 At1g73650 At1g74590 At1g75830 At2g29350 At2g29720 At2g30140 At2g32240 At2g32670 At2g43570 At2g43680 Glutaredoxin family protein, response cytokinin flavonol-7-O-rhamnosyltransferase WAKL8 Wall-associated kinase like 8 Unknown protein AIG1 Avirulence induced gene Similar to wound-inducible KED of tobacco 3-oxo-5-alpha-steroid 4-dehydrogenase like ATGSTU10 Glutathione S-transferase PDF1.1 Plant defensin protein SAG13 Senescence associated; alcohol dehydrogenase CTF2B flavoprotein monooxygenase UDP-glucoronosyl/UDP-glucosyl transferase similar to kinesin VAMP275 Vesicle associated membrane protein Putative endochitinase IQD14 calmodulin binding, calcium signaling PubMed Ratio E S D 16212609 17314094 12068092 3.2 3.1 3.9 3.2 9.4 4.3 3.3 4.3 8.2 3,5 4,6 3,3 3,3 3,6 3,2 3,2 0 0 + 0 0 0 0 + + 0 + + 0 0 + + + + + + + + + - + 0 + + 0 0 + + + 0 + + 0 + 0 NF 8742710 10945337 NF 12090627 15955924 10444084 NF NF NF 14500793 15915637 16368012 103 At3g13950 At3g18250 At3g22060 At3g23550 At3g25882 At3g44700 At3g54920 At3g62770 At4g14450 At4g17500 At4g25110 At4g33050 At4g34390 At4g35180 At4g37290 At5g13080 At5g26690 At5g48540 At5g54610 At5g58300 At5g58350 At5g64120 Ndr1 dependent Unknown protein NIMIN-2 PMR6 ATG18 ATERF1 ATMC2 EDA39 XLG2 LHT7 WRKY75 ANK WNK4 Unknown protein Multi antimicrobial extrusion protein a kinase that physically interacts with NPR1/NIM1 Unknown protein Powdery mildew resistant 6; pectate lyase-like autophagosome formation C terminal similar to hydroxyporline rich glycoprotein Ethylene responsive element binding factor 1 Metacaspase 2 calmodulin-binding protein Extra-large GTP binding protein; LYS/HIS transporter 7 Unknown protein WRKY Transcription Factor Heavy metal transport/detoxification protein 33 kDa secretory protein-related Ankyrin; SA induced leucine-rich repeat transmembrane protein kinase WNK kinase cell wall bound peroxidase; oxidative burst 17181774 NF 17761682 NF 9,1 4,8 3,2 3,3 0 + + + + - + + + + 15749762 3,7 N - N NF 12215508 15860012 132.0 160.0 5,0 0 0 0 - 0 NF 3,1 N - N 10715325 15326173 15634699 10394945 16607029 NF 17322336 NF 16115070 16307367 3,3 4,6 3,4 3,2 5,9 3,2 7,5 7.0 4,1 4,4 + + + 0 + + + N 0 0 + + + + - + + + 0 + + + N + + 16280546 3,2 - - - 12506983 16551688 3.0 5,7 0 + + + + + With these parameters we identified 40 genes whose transcripts are increased in the mutant compared to the wild type. We evaluated our data to other microarray experiments and the senescence transcripts identified by Guo et al. (2004). In Figure 4 a VENN diagram illustrates the overlap between the old9 genes and the different experiments. Figure 4. A Venn-diagram is used to visualize the overlap between the old9 microarray dataset and that of other experiments. E: Microarray dataset of plants treated for 3 hours with ethylene; P: microarray dataset of plants exposed to Phytophthora; S: EST dataset of senescence associated transcripts. The NASCARRAYS-32 was performed with seedlings treated for 3 hours of ethylene by F.F. Millenaar. The identified transcripts correlate for 36.8% with the old9 transcriptome data. Next to that, 60% of the genes induced by ethylene in old9 were previously identified during a plant defense experiment NASCARRAYS-123 by D. Scheel. Interestingly, the experiments of Millenaar and Scheel share an overlap of 104 65%, indicating that ethylene induces many plant defense associated transcripts. Importantly, 44.7% of the induced genes in old9 have been implicated before in leaf senescence (Guo et al., 2004). Thus, as expected we find many ethylene-inducible genes but the notion that almost 50% of the genes are senescence-associated suggests early onset of leaf senescence in the mutant. Ten transcripts are specifically up regulated in the old9 mutant as compared to the other experiments. One of these encodes a flavonol 7-O-rhamnosyltransferase which is involved in the biosynthesis of flavonols that function as either cell protectant, signaling molecules or regulator of auxin transport (Yonekura-Sakakibara et al., 2007). Next to that the ABA synthesis gene CTF2b is up regulated in old9 (Bilodeau et al., 1999). ABA was shown to antagonize ethylene induced cell death in maize and barley (Young and Gallie, 2000; Wang et al., 1999). The microarray analysis of ethylene treated plants further supports the notion that in old9 cell wall associated transcripts are differentially expressed as compared to the wild type. In old9 we observed up regulation of ATVAMP275 (At2g32670) a SNARE-encoding gene involved in vesicle transport of membrane proteins as well as cell wall materials from the plasma membrane (Uemera et al., 2004). PMR6 encodes a pectate-lyase gene which is 160-fold induced after ethylene treatment of the mutant and is involved in cell-wall modification (Vogel et al., 2002). The degradation products of the cell wall were shown to trigger a signaling cascade including a putative leucine-rich repeat transmembrane protein kinase (At5g58300) which is specifically induced in old9 (Moscatiello et al., 2006). Another interesting gene that is up regulated in old9 encodes a G-protein (At4g34390) which serves as a physical coupler between cell surface, 7 transmembrane (7TM) G-protein-coupled receptors (GPCRs) and downstream targets (Temple and Jones, 2007). Noteworthy is the up regulation of IQD14 (At2g43680) which is involved in calcium-signaling. During nutrient starvation and senescence, autophagy is used to breakdown cellular components and regulate nutrient relocalization. The autophagy gene ATG18 is 5 fold induced after ethylene treatment of old9 and was shown before to function in nutrient remobilization during leaf senescence of Arabidopsis (Xiong et al., 2005). To determine the relation between old9 transcripts identified during air growth and after ethylene treatment we performed a coexpression analysis (Figure 5). The analysis revealed that 15 of the genes identified after ethylene treatment correlate to the genetic networks identified in air-grown old9 mutant plants. The BGL2 network covers 15 genes of which 9 after 105 ethylene treatment, and contains in total 10 identified SAGs (Guo et al., 2004). The second group that is built around TPS11 covers 16 genes of which 12 have been identified as SAG (Guo et al., 2004). Figure 5. Correlation between transcripts of the microarray on air-grown old9 and ethylene treated. Genes of the air-grown microarray dataset are represented by green spheres and those of the ethylene microarray dataset are shown in red. The relation between two is represented by lines. Lines are only drawn between genes with Pearson correlations >0.6. An S indicates that the gene is senescence associated. Taken together this data shows that within 8 hours of ethylene treatment a senescence program is initiated in old9. Cytokinin primes leaves for ethylene-induced senescence Microarray analysis of air-grown old9 mutants revealed that more than two third of the identified transcripts is responsive to cytokinin (Table 2). However, more than 2000 genes are responsive to cytokinin (Brenner et al., 2005) and only a small fraction was identified in the microarray. This suggest that only a specific set of cytokinin-responsive genes are important for the old9 phenotype. Cytokinins are wellknown for their ability to delay leaf senescence when applied exogenously (Lim et al., 2003) or when overproduced in transgenic plants (Gan and Amasino, 1995). However, the application or production of cytokinin in these experiments is always restricted to the last phase of leaf development just before or during the onset of leaf 106 senescence. The observation that long-term ethylene exposures of old9, starting at 8 days result in an early senescence phenotype (Chapter 2) suggests that the role of cytokinin in the old9 phenotype is related to changes at early leaf development phase. Therefore we designed an experiment in which we tested the application of cytokinin at early and late developmental stages of the wild type and old9. If cytokinin promotes the onset of senescence by ethylene than application to the wild type should result in a phenocopy of the mutant. Plants were given a cytokinin treatment at day 10, day 17 or at both days and grown in air till 21 days and subsequently treated for 3 days with ethylene. The visual senescence was assessed (Lohman et al., 1994) and presented in Figure 6 for wild type and old9. Figure 6. Cytokinin promotes the onset of ethylene-induced leaf senescence. Wild type and old9 plants were treated with cytokinin at day 17, day 10 or at both days as described in methods. After 21 days plants were treated with ethylene to induce senescence and 3 days later the number of yellow leaves per plant was scored. Experiments were repeated 3 times and similar results were obtained. Asterisks indicate statistical significance versus the mock treatment in each case (P < 0.05, Student’s t test). Application of cytokinin at 17 days resulted in a delay in the onset of senescence in both wild type and old9 when compared to mock treatment, consistent with Lim et al. (2003) and Gan and Amasino (1995). Interestingly this effect disappeared when plants were treated at both 10 and 17 days for both wild type and old9. This suggests that early application of cytokinin results in either insensitivity to the application at day 17 or the application at early stage promotes the onset of senescence by ethylene which is again counteracted by application at day 17. Application at day 10 resulted in an increased senescence response of the wild type accession while this had no 107 effect on the senescence of the mutant. Taken together these results demonstrate that cytokinin can result in a partial phenocopy of the mutant phenotype in wild type when applied early in leaf development. More importantly, these results suggest that cytokinin can result in an ARC required for ethylene-inducible senescence when applied early in development. Role of a hydroxyproline-rich glycoprotein in old9 response The old9 mutant plant has induced expression of several SAG genes when grown in air. The expression of these SAG genes might either prime old9 for the induction of leaf senescence by ethylene or be a marker for this possible priming. Therefore we applied RNA interference (RNAi) to down-regulate one of these SAG genes and test whether this influences the induction of leaf senescence. RNAi constructs obtained from the Agrikola (Arabidopsis Genomic RNAi Knock-out Line Analysis) resource (Hilson et al., 2004) were screened and transformed to the mutant. This resulted in a stable RNAi line for the senescence associated hydroxyproline-rich glycoprotein (Hrgp) (At1g23040). Homozygous T2 plants of 3 independent lines were grown till 21 days and treated for 3 days with ethylene. The independent lines were obtained by transforming different copies of the same construct to different old9 plants. Interestingly, all 3 lines showed a reduced senescence response compared to old9 (Figure 7). Figure 7. Visible yellowing of old9 plants carrying a RNAi against HRGP. The number of yellow leaves of three independent RNAi lines is indicated and compared to old9 control plants. The visible yellowing was scored and expressed as means ±SD of at least three replicates of 30 plants each. Plants that were grown for 24 d in air did not show any sign of senescence (not shown). Asterisks indicate statistical significance versus the old9 mutant in each case (* P < 0.05, Student’s t test). Black bars, RNAi line; Grey bars, old9 mutant. However, the senescence response is enhanced when compared to the wild type. These results demonstrate that Hrgp is an important determinant for the senescence 108 response of old9 and supports the notion that at least one of the identified SAGs can prime a leaf for ethylene-inducible senescence. Discussion Plants are continuously interacting with the environment and adapt their growth and developmental strategy accordingly. During non-stress conditions the leaves of Arabidopsis undergo age-dependent senescence (Gan and Amasino, 1997; Quirino et al., 2000; Jing et al., 2002). The progression of senescence occurs in a coordinated manner starting from the tip of a leaf toward the base. The controlled breakdown of the leaf during senescence is in part to ensure effective remobilization of nutrients. Therefore it is not surprising that senescence is a highly regulated process involving many genetic programs (Buchanon-Wollaston et al., 2005; Keskitalo et al., 2005; van der Graaff et al., 2006). The two major plant hormones cytokinin and ethylene have well established opposite effects on leaf senescence. Here we report on the identification of an early ethylene-inducible senescence mutant that has increased expression of several SAG genes during air-grown conditions but no visual differences with the wild type. Interestingly, we demonstrate that cytokinin application at an early stage of plant development can promote the onset of leaf senescence by ethylene. Endogenous levels of cytokinin drop concomitant with the progression of leaf development (Gan and Amasino, 1996) suggesting that the hormone plays a role during the development of the young leaf. The retardation of leaf senescence by cytokinin has been demonstrated by applying the hormone just before or during the onset of leaf senescence (Gan and Amasino, 1995; Lim et al., 2003). However, in our study we demonstrate that cytokinin can also have the opposite effect on the onset of leaf senescence. First the delay of senescence by applying cytokinin at 17 days is make ineffective when plants are also treated at 10 days with cytokinin. This might imply that application of cytokinin early in development has an opposite effect to application late in development. This is evident from the application at 10 days which is sufficient to induce early senescence after ethylene treatment in the wild type. Cytokinin is known to delay senescence by increasing the sink strength of the tissue (Balibrea Lara et al., 2004) and thus reverses the leaf senescence program which mobilizes nutrients from the senescing leaf to other parts of the plant. Thus by influencing the sink-to-source transition of a leaf, cytokinin might modulate the onset 109 of senescence. In support to this it was demonstrated in tobacco that application of cytokinin to leaf blades of decapitated plants is much less effective than application to intact plants to delay senescence (Singh et al., 1992) which was explained by suggesting that an increase of the sink strength only is not sufficient to delay senescence but also remobilization of nutrients to the tissue is required. Interestingly, detached leaves of old9 senesce at the same time as wild type plants (Chapter 5), consistent with the idea that a sink tissue might be required for the accelerated senescence of old9 leaves. Since we sprayed whole plants with cytokinin we interfered with the sink/source strength of all leaves. Therefore we propose that cytokinin promotes the onset of leaf senescence both in old9 and treated wild type plants, by increasing the sink strength of young tissue. Endogenous cytokinin content and sensitivity to cytokinin regulates the developmental rate of Arabidopsis as mutants with a reduced cytokinin sensitivity had a slower rate of rosette leaf emergence and leaf expansion (Smalle et al., 2002), while plants with increased cytokinin sensitivity showed rapid chloroplast development and proliferation (Kubo and Kakimoto, 2000). Furthermore plants overproducing cytokinin develop more rosette leaves (Chaudhury et al., 1993). In old9 several genes involved in different leaf developmental programs including cell elongation, vascular tissue formation and telomerase activity are down regulated compared to the wild type at 21 days, suggesting that the leaf developmental program is in a different phase than the wild type. Overexpression of the cytokinin receptor AHK3 or a downstream target ARR2 delays leaf senescence in Arabidopsis (Kim et al., 2006). Both transformants have a constitutive overexpression of cytokinin responsive genes. It would be interesting to determine the effect of ethylene on leaf senescence in relation to cytokinin with these mutants. This might reveal whether or not the old9 phenotype is depending on a transient activation of cytokinin responsive genes or constitutive activation and if the subset of cytokinin responsive genes is important or all (Brenner et al., 2005). The orderly visual appearance of cell death is in accordance with the induction of a senescence program in old9. Interestingly, the senescence starts at the tip of the leaf and thus the oldest leaf cells in old9 (Ferreira et al., 1994) which is in agreement with the age-dependent progression of leaf senescence. The first leaf pair of 21 day old9 mutants has increased expression of several SAGs which is not sufficient to induce early senescence under normal conditions but might prime old9 for ethylene-induced 110 leaf senescence. Since SAG genes are associated with the last developmental phase they are a marker for an early ARC in the development of old9 leaves. That these SAGs play a role in the ability of ethylene to induce early senescence is at least confirmed for one transcript. Transformation of the mutant with RNAi constructs directed against HRGP transcripts result in a decreased senescence response. Since the induction of leaf senescence by ethylene is age-dependent this suggests that reducing the expression of HRGP might interfere with the ageing of the leaf and thus causes a reduced response. HRGPs constitute one of the most abundant structural proteins in the plant cell wall and can confer resistance to pathogens in Pearl millet (Deepak et al., 2007). However, in Arabidopsis they have been implicated in embryo development (Hall and Cannon, 2002) but also in response to ABA (van Hengel and Roberts, 2003) suggesting that they play a role in growth and development of Arabidopsis. The accumulation of HRGP might represent a marker for a developmental change which is related to the progression towards leaf senescence. The microarray analysis suggests important changes on the cell wall composition of old9. First of all a pectin synthesis gene is enhanced during air-growth, however during ethylene treatment a pectate lyase gene is induced 160 fold suggesting that the senescence of old9 also involves a rapid degradation of the cell wall. Interestingly pectate lyase activity has been related to fruit ripening in strawberry and banana (Medina-Escobar, 1997; Asif and Nath, 2005) and resistance to Powdery Mildew when knocked-out (Vogel et al., 2002). Recently it has been demonstrated that pectic fragments of plant cell walls are able to induce defense and developmental responses (Moscatiello et al., 2006). Therefore the increased expression of the pectate lyase gene after ethylene treatment of old9 might partially explain the abundance of plant defense genes after ethylene treatment. However, as shown, many plant defense genes are ethylene inducible and thus are probably not related to degradation of pectate. The effect of cell wall composition on the induction of leaf senescence has not been well described in literature and thus might be an aim for future research. One question that remains is why old9 does not show early leaf senescence during normal development in air. If the mutant is primed to senescence due to ARCs than it would be logical to conclude that this also affects the onset of developmental 111 senescence. A study by Jing et al. (2002) proposed the existence of a so-called senescence window. The senescence window is a developmental phase during which ethylene can induce premature leaf senescence. This model explains the absence of early developmental senescence in the mutant since OLD9 regulates at which developmental age a leaf can perceive ethylene to promote leaf senescence but not when to start to senesce. Since no early developmental senescence occurs this suggests that old9 is not involved in the direct execution of senescence. The conferred change by the old9 mutation might be useful for controlling the ripening of crops or fruits. For example the simultaneous start of fruit ripening will make it possible to harvest all products at the same time. Methods Plant materials and growth conditions Arabidopsis thaliana ecotype Landsberg erecta (Ler-0) was used in this study. The old9 mutant was obtained from an EMS mutagenized collection (Jing et al., 2005). Plants were grown on either soil or half-strength Murashige and Skoog medium at 23°C and 65% relative humidity with a day length of 16 h. The light intensity was set at 120 µmol·m-2·s-1. An organic-rich γ-ray radiated soil was used (Hortimea Groep, Elst, The Netherlands). Plants for ethylene exposure were treated in a flow-through chamber at 20 °C and a humidity of 40% under continuous illumination. The ethylene dosage was set at ~10 µl l–1 as suggested by Chen and Bleecker (1995). Cotyledons or rosette leaves with over 5% yellow area of the leaf blade were judged as yellow as suggested by Lohman et al. (1994). Tryphan blue staining Appearance of cell dead were studied in whole leaf mounts stained with lactophenoltryphan blue (10 ml of lacticacid, 10 ml of glycerol, 10 g of phenol, 10 mg of tryphan blue, dissolved in 10 ml of distilled water) (based on Keogh et al.,1980). Whole leaves were boiled for 1 min in the staining solution and then overnight decolorized in saturated solution of chloral hydrate. Subsequently the leaves were mounted in chloral hydrate and viewed under a light microscope. 112 RNA-isolation and RT-PCR Total RNA was isolated using TRIZOL reagent (Sigma) according to the manufacturer's protocol. Five hundred nanograms of RNA were used as template for first-strand cDNA synthesis using 200U of RevertAid H-minus MMuLV reverse transcriptase (Fermentas, USA) and an oligo(dT21) primer. Primer pairs for real-time PCR were designed with open-source PCR primer design program PerlPrimer v1.1.10 (Marshall, 2004). The primer sequences are available upon request. Briefly, real-time PCR amplification was performed with 50 µL of reaction solution, containing 2 µL of 10-fold–diluted cDNA, 0.5 µl of a 10 mM stock of each primer, 1 µl of 25mM stock MgCl2 (Fermentas), 5 µl PCR buffer +Mg (Roche), 1 µl of a 1000x diluted SYBR-green stock (Sigma), 0.5 µl 100xBSA (New England Biolabs), and 1u of Roche Taq Polymerase. The PCR program was 2’ at 94, 40x (94-10”/60-10”/72-25”), meltcurve. Obtained data was analyzed with BioRad software. Microarray analysis All the array experiments were carried out using the CATMA2 Arabidopsis GeneChip microarray containing 24576 gene-specific tags from Arabidopsis thaliana (http://www.catma.org). For each comparison, one technical replication with fluorochrome reversal was performed for each pool of RNA. cRNA were produced from 2 µg of total RNA from each sample using the Message Amp aRNA® kit (Ambion). Then 5 µg of cRNA was reverse transcribed in the presence of 300 units of SuperscriptTM II DNA polymerase (Invitrogen/Life technologies), cy3-dUTP and cy5dUTP (PerkinElmer Life Sciences) for each slide. Slides were prehybridized for 1 h and hybridized overnight at 42°C in 25% formamide. Slides were washed in 2× SSC and 0.1% SDS for 4 min, 1× SSC for 4 min, 0.2× SSC for 4 min, and 0.05× SSC for 1 min and dried by centrifugation. Raw data from each experiment were normalized in GeneSpring (Silicon Genetics, Redwood City, CA, USA) using the default procedure. GeneSpring was used to filter out genes detected in only one of the slides and to identify up- and down regulated genes for further analysis. The identified genes were compared to 2 experiments available from the Nottingham Arabidopsis Stock Centre (NASC) microarray database (http://affy.arabidopsis.info). Coexpression analysis 113 Coexpression analyses were performed by using a Coexpression Gene Search algorithm on the RIKEN PRIMe web site. The Coexpression Gene Search program is a web-based application designed to identify correlated genes from gene expression data produced using Affymetrix Gene-Chip technology by the AtGenExpress consortium (RIKEN Plant Science Center and the Max-Planck Institute for Molecular Plant Physiology) deposited in TAIR. The database can be searched by using the AGI annotation. The ATTED-II release directly gives the correlation coefficients (Obayashi et al., 2007). Cytokinin treatment Plants were grown on soil for 21 days and subsequently exposed to ethylene for 3 days. During the growth of the plants a foliar spray technique was used to apply cytokinin. The synthetic cytokinin, benzyladenine (BA) was applied exogenously in a concentration of 50 µM dissolved in 0.5% Tween-20. As a control plants were treated by spraying with a solution of 0.5% Tween-20. Plants were sprayed in the morning and approximately 10 ml of solution was applied to a tray containing 104 plants. RNAi lines A 175-bp HRGP-specific fragment (clone CATMA1a22110) (from +308 to +482 of HRGP cDNA) was cloned into a binary vector by the AGRICOLA consortium to create a hairpin construct (Hilson et al., 2004). The pAGRIKOLA plasmid was verified according to the validation protocols by Hilson et al. 2004. 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Zimmermann, P., Hirsch-Hoffmann, M., Hennig, L., and Gruissem, W. (2004). GENEVESTIGATOR: Arabidopsis microarray database and analysis toolbox. Plant Physiol. 136: 2621-2632. 119 120 Chapter 5 The Arabidopsis old5 mutation of quinolinate synthase affects NAD biosynthesis and causes early ageing Jos H.M. Schippers1, Adriano Nunes-Nesi2, Roxana Apetrei1, Jacques Hille1, Alisdair R. Fernie2 and Paul P. Dijkwel1. 1 Molecular Biology of Plants, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands 2 Max Planck Institute for Molecular Plant Physiology, Potsdam-Golm 14766, Germany. 121 122 Abstract Leaf senescence in Arabidopsis is regulated by a strict, genetically controlled nutrient recovery program which typically progresses in an age-dependent manner. To date, several genes altering the onset of senescence have been identified, suggesting that unigenic factors influence leaf lifespan. That said, the understanding of factors that control leaf ageing and the onset of senescence remain incomplete. Leaves of the previously identified Arabidopsis onset-of-leaf-death 5 (old5) mutant exhibit early developmental senescence. Here, we identify that OLD5 encodes quinolinate synthase, a key enzyme in the de novo synthesis of NAD. Despite harboring a lesion in this enzyme, the mutant contains increased NAD steady-state levels likely due to increased expression of enzymes in the NAD salvage pathway. NAD plays a key role in cellular redox reactions including those of the TCA cycle. Broad range metabolite profiling of the old5 mutant revealed that it contains higher levels of TCA cycle intermediates and nitrogen-containing amino acids. The increased TCA cycle intermediates coincide with a higher respiration rate for the mutant and increased expression of oxidative stress markers. Taken together the results of this study suggest that early ageing of the mutant is a consequence of mitochondrial oxidative stress. These observations are consistent with the “free-radical” theory of ageing that has been widely accepted as the mechanism behind animal ageing. Introduction In Arabidopsis thaliana the onset of leaf senescence proceeds within a predictable time-window (Jing et al., 2002). Only when a leaf is under attack or grows in unfavorable conditions the senescence program will be prematurely initiated (Schippers et al., 2007). Senescence is a critical component of the plant life cycle where it contributes to relocation of valuable resources from old, dying tissue to young developing parts of the plant. The transfer of nutrients supports the filling of grains in order to afford the plant reproductive success. Recent comprehensive genomics studies have demonstrated that senescence is under strict genetic control (Buchanan-Wollaston et al., 2003, 2005; Guo et al., 2004), representing a carefully maintained developmental program which ultimately results in cell death. Aside from the fundamental importance to understand senescence, the commercial implications of increasing crop yield or the longevity of ornamental plants are vast. In this light, a recently identified transcription factor in wheat, that was shown to affect crop yield by 123 30% (Uauy et al., 2006), underlines the impact of senescence research on future agriculture. Despite recent advances in the understanding of senescence itself, little is known regarding the factors that determine its onset. In Arabidopsis senescence occurs in a leaf-age dependent manner even when the plant is challenged by (a)-biotic stress (Gan and Amasino, 1997; Quirino et al., 2000). Whereas in animal and yeast research the terms senescence and ageing are interchangeable, in plants senescence refers to the process that leads to the death of the leaf, while ageing itself occurs throughout the development from initiation of a leaf primordium throughout senescence and death (Lim et al., 2003). Given that most animals reproduce early in life and die before they age no specific ageing program is required (Kirkwood, 2002). Although many genes have been identified that alter longevity in organisms ranging from yeast, to worms, fruit flies and mammals (Jing et al., 2003), no combination of genes has been found to abolish ageing. This can be explained in two ways, either no ageing program exists or many overlapping programs are present rendering the process highly robust (Kirkwood, 2005). Although ageing of Arabidopsis is influenced by its reproductive strategy there is a weak correlative control between the appearance of reproductive structures and leaf senescence. While male and female sterile mutant plants live 20 days longer (Nooden and Penney, 2001) the longevity of individual rosette leaves of these mutants is unaltered. Nutrient salvage during leaf senescence is a vital part of development and it is under evolutional selection (Bleecker, 1998). Thus the agedependent onset of senescence allows to study ageing in plants from an evolutionary point of view by looking at individual leaves. Processes that lead to ageing, termed, age-related changes occur as a result of the differential regulation of development and growth (Jing et al., 2005). In general, ageing is controlled by programs involved in life maintenance, stress responses and development (Schippers et al., 2007). From the moment leaves reach their full size they are subjected to post-mitotic ageing (Gan, 2003). If leaves are considered as independent systems we can compare their ageing with that of animal systems (Jing et al., 2003). In the mid-1950s it was postulated that Reactive Oxygen Species (ROS) are the main cause of animal ageing beginning with cumulative damage that results in loss of viability (Harman, 1956). In keeping with this theory the delayed leaf senescence mutants ore1, ore3 124 and ore9 (Woo et al., 2004) and the late flowering and longevity mutant gigantea of Arabidopsis (Kurepa et al., 1998) all show higher tolerance to oxidative stress. These combined data support the notion that ROS influence both the ageing process and the lifespan of Arabidopsis leaves. Whilst in animals ROS are produced by mitochondria, the main ROS source in a senescent leaf is the chloroplast (Quirino et al., 2000). That said plant mitochondria produce considerable ROS during the hypersensitive response invoked by plant-pathogen interactions (Finkel and Holbrook, 2000), and under a variety of other cellular circumstances (Sweetlove et al., 2006). Increased tissue contents of ROS (Navabpour et al., 2003) and exogenously applied causal agents of oxidative stress such as UV-B and ozone (John et al., 2001; Miller et al., 1999) lead to expression of senescence-associated genes (SAGs). SAGs were previously identified on the basis of their characteristic up-regulation during leaf senescence. Furthermore, reducing the expression of SAG101 or phopholipase Dα has been demonstrated to delay the onset of senescence in Arabidopsis (Fan et al., 1997; He and Gan, 2002), providing further evidence of a link between ROS induced SAG activation, ageing and the onset of leaf senescence. An important source of ROS production in animals and plants are the membraneassociated nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidases that generate ROS via lipid oxidation (Finkel and Holbrook, 2000; Mittler, 2002). NAD(P)H are well-studied redox molecules which mediate hundreds of reactions and are at the basis of almost every metabolic pathway in the cell (Noctor et al., 2006). ROS production in the chloroplast has been clearly demonstrated to be under the influence of NADP/NADPH ratios in this compartment (Asada, 2006) whereas production of mitochondrial ROS by the electron transport chain is largely dependent on NAD(P) status (Dutilleul et al., 2005; Shen et al., 2006, Sweetlove et al., 2006). The turn-over of anti-oxidants pools such as those of glutathione and ascorbate are also maintained by NAD(P)H (Noctor et al., 2006). Recently an NAD–dependent histone deacetylase, SIR2 was found in yeast to control longevity (Lin et al., 2000) and the NAD-consuming enzyme poly(ADP-ribose)polymerase (PARP) was described to protect cells from oxidative stress induced DNA damage (De Block et al., 2005). Pyridine nucleotides are the main redox regulators since they react slowly with oxygen species in an enzyme dependent way. On the contrary, the small antioxidant 125 molecules glutathione and ascorbate react spontaneously with ROS. The regeneration of these small molecules is operated by a high-capacity system that depends on the in vivo activities of enzymes that require the reducing power of NADH or NADPH in order to catalyze the reactions that they mediate (Noctor, 2006). Thus glutathione and ascorbate are part of the NAD(P)-dependent buffering system that regulates the cellular targets with which ROS are allowed to interact. This function, alongside that NAD homeostasis plays in the regulation of central metabolism (Dutilleul et al., 2005), makes NAD a key player in regulating metabolism and development. The availability of senescence mutants has given us the opportunity to discover which processes play a role in plant ageing and determine the onset of leaf senescence (Jing et al., 2002; Jing et al., 2005). Here we report a mutation in de novo pyridine nucleotide synthesis which results in an early onset of developmental senescence. Despite the fact that the genetic legion of this mutant is in the pathway of NAD synthesis the mutant exhibits increased content of pyridine nucleotides. The most likely explanation for this was provided by RT-PCR-based transcript analysis, which revealed an up regulation of the NAD salvage pathway. Further characterization of the mutant revealed many changes in the levels of transcripts that are suggestive of oxidative stress whilst the metabolic profile obtained for the mutant shows increased levels of metabolites of central metabolism. The results are discussed in the context of current theories of ageing and senescence that have been previously documented in the plant and mammalian literature. Results Characterization of the old5 mutant during developmental senescence The old5 mutant was identified as a monogenic recessive trait during a screen of an EMS treated population of Arabidopsis plants (Jing et al., 2002) and was shown to display an early onset of senescence and an enhanced senescence response after ethylene treatment (Jing et al., 2005). The mutant shows yellowing of the first leaf pair after 30 days (Figure 1A), whereas the wild type shows senescence symptoms after 40 days (data not shown). The visual yellowing runs parallel with a decline in chlorophyll content and photochemical efficiency (Figure 1B). Thus the old5 mutant 126 exhibits the physiological hallmarks of leaf senescence. The early senescence of the mutant results in a decrease in lifespan of approximately 2 weeks (Figure 1C). Figure 1. The onset of leaf senescence in old5 mutant Arabidopsis plants. (A) Visible leaf yellowing in the old5 mutant line. The first rosette leaves are shown from 21 till 33 day old plants of wild type (WT) and old5. (B) Photochemical efficiency (PSII) and chlorophyll levels of the old5 mutant line decrease at the onset of senescence (old5 open circles, wild type closed circles). The photochemical efficiency was determined with a PAM-2000. The values shown are the means of three repeats ± SD (indicated by error bars). Asterisks indicate statistical significance versus the wild type control in each case (* P < 0.05, Student’s t test). (C) Six-week-old adult plants. The old5 mutant plants display strong senescence phenotype compared to wild type. The expression of two established senescence marker genes was followed by realtime PCR. First, we examined SAG13 whose expression is associated with oxidative stress and senescence (Weaver et al., 1998). SAG13 expression was detected from day 24 in both the mutant and the wild type. However, the expression in the mutant was 10 (day 24) and progresses to a 10000 fold (day 33) higher than in the wild type (Figure 2). In addition we examined the hallmark of age-induced senescence, SAG12. SAG12 was detected after 30 days only in old5 but not the wild type. The SAG12 expression correlates with the first symptoms of yellowing at 30 days implying together with the expression of the SAG13 marker, that senescence occurs in an age-dependent way. 127 Figure 2. Transcript accumulation of the senescence-associated genes, SAG12 and SAG13, in old5 mutant plants. The relative levels of SAG12 and SAG13 were examined by semiquantitative RT-PCR analysis, using ACT2 as internal control, of first rosette leaves of 21 to 33 day old plants (old5 gray bars, wild type solid black bars). For SAG13 the relative transcript levels detected in the wild type plants were arbitrarily assigned a value of 1 after normalization to the ACT2 samples. For SAG12 the relative transcript level detected at day 30 in old5 was set at 1 and used for comparison with day 33. The values shown are the means of three repeats ± SD (indicated by error bars). Experiments were repeated and showed the same results. Asterisks indicate statistical significance versus the wild type control in each case (* P < 0.05, Student’s t test). OLD5 encodes a quinolinate synthase The old5 mutant was identified in the Landsberg (Ler) accession and was crossed to Columbia (Col) to facilitate mapping. The mapping population segregated in a 1:3 ratio, demonstrating that the mutant phenotype is encoded by a single locus. In a population of 2500 F2 plants, the location of old5 could be narrowed down to a 60kb region, containing 19 genes, in the overlap on BACs K6A12 and MXI22 on chromosome V between markers K6A12-23K and MXI22-12K. From these 19 genes, eleven candidate genes were selected for sequence comparison between the mutant and the Ler wild type. In the sequence of At5g50210 a C to T change in the first exon of the old5 mutant was detected. This gene has previously been annotated as quinolinate synthase (QS) (Katoh et al., 2006), which is a component of the pathway of the de novo synthesis of NAD from aspartate. A homozygous T-DNA knock-out of this enzyme is embryo lethal (Katoh et al., 2006), suggesting that the old5 mutation does not result in a complete loss of functionality. The identity of At5g50210 as the OLD5 gene was confirmed by a complementation test. A 5.5 kb Ler genomic fragment containing the coding sequence, a 2.1-kb 5’ 128 promoter sequence according to the AGRIS database (Davuluri et al., 2003) and a 0.5 kb 3’ sequence was transformed into old5 mutant plants. Figure 3. OLD5 encodes for quinolinate synthase. (A) Schematic representation of the protein domain organization of OLD5. The first domain is a transit peptide that targets OLD5 to the chloroplast. The other 2 domains are a quinolinate synthase domain and a SufE/YgdK domain. (B) The OLD5 SufE domain shows similarity to SufE proteins from E. coli (SufE and YgdK). This domain is also present in 2 other Arabidopsis proteins, At4g26500 encoding for AtSufE, and At1g67810, a putative pollen specific SufE protein. The amino-acid change in old5 mutant protein is in a proline residue that is conserved between all three Arabidopsis proteins (indicated by arrow). (C) Modeling by 3D-Jigsaw server predicts an effect of the mutated proline on the structure of the SufE/YgdK domain (indicated by arrow). Yellow: E.coli YgdK structure (Liu et al., 2005). Magenta: wild type Arabidopsis OLD5. Cyan: old5 mutant protein. 129 10 T1 lines were selected on kanamycin, these all showed absence of the old5 phenotype, as expected since the mutation is recessive. By PCR we showed that the lines contained both the KANr gene and the homozygous parental old5 point mutation (see methods). The T2 segregated 3:1 (wildtype:mutant) confirming the identity of the OLD5 mutant gene. old5 mutants have an amino acid change in the SufE/YgdK domain The OLD5 protein consists of 3 domains: a chloroplast targeting signal, a SufE/YgdK domain and a quinolinate synthase domain (Figure 3A). The old5 mutation results in a proline to serine amino-acid substitution in the SufE/YgdK domain. This domain is highly homologous to the previously identified AtSufE/CpSufE (Xu and Møller, 2006; Ye et al., 2006), the Ygdk and SufE proteins of E.coli (Loiseau et al., 2005) and a putative gene, At1g67810, which was found by a BLAST search of the OLD5 SufE/YgdK domain (residues 84 to 214) against the Arabidopsis genome (Figure 3B). AtSufE/CpSufE, SufE, and YgdK share functional homology, as acceptors of sulfur atoms and stimulators of cysteine desulfurase activity. SufE of E.coli (Loiseau et al., 2003) and CpSufE of Arabidopsis (Ye et al., 2006) stimulate SufS cysteine desulfurase activity. The Fe-S cluster which is inserted by SufS into QS is absolutely required for enzyme activity in E.coli (Ollagnier-de Choudens et al., 2005). In the mutated Arabidopsis old5 protein proline-101 is replaced by a serine. The proline-101 residue is present in all Arabidopsis SufE/YgdK containing proteins, suggesting a conserved role. Utilizing the Jigsaw 3D protein modeling server (Bates et al., 2001) the OLD5 protein could be modeled with high confidence on the resolved structure of YgdK (Liu et al., 2005). The resultant model suggests that the mutation of the proline residue into serine affects the loop between helix I and II of the protein (Figure 3C). OLD5 interacts with a SufS-like protein The YgdK/SufE proteins of E.coli interact with their SufS counterpart (Louiseau et al., 2003; Loiseau et al., 2005). By analogy we therefore expected the SufE/YgdK domain of OLD5 to interact with a SufS-like protein. Recent studies show that the Arabidopsis SufS-like protein, CpNifS interacts with AtSufE (Xu and Møller, 2006; Ye et al., 2006). Furthermore, it has been suggested that quinolinate synthase might work in an enzyme complex with aspartate oxidase (Sakuraba et al., 2005) since the product of aspartate oxidase is unstable (Yang et al., 2003). 130 By a database search we found that the Arabidopsis genome contains 2 other putative plastidic SufS-like proteins, CpNifS2 and CpNifS3, respectively. In order to test whether OLD5 can interact with a cysteine desulfurase, a yeast-two hybrid assay was performed. As a positive control we tested the interaction between CpNifS and AtSufE. The results of our screen are summarized in Table 1. On selection medium lacking either histidine or adenine we found that the OLD5 protein interacts with CpNifS and CpNifS3. These interactions were found for the mutated old5 protein only on His- selection medium but not on the more stringent Ade- plates. Interestingly, an interaction with aspartate oxidase was also detected, suggesting that OLD5 works as part of an enzyme complex. Table 1. Result of yeast-two-hybrid analysis Interaction OLD5 - OLD5 OLD5 - CpNIFS OLD5 - CpNIFS2 OLD5 - CpNIFS3 OLD5 - NADB old5 – old5 old5 - CpNIFS old5 - CpNIFS2 old5 - CpNIFS3 old5 - NADB AtSufE - CpNIFS Empty - Empty His- +2mM 3 AT ++ ++ ++ ++ ++ ++ ++ - Ade++ ++ ++ ++ - Taken together these results suggest that OLD5 has a functional SufE/YgdK domain. Recently, Murthy et al., (2007) demonstrated that the SufE/YgdK domain can indeed increase the cysteine desulfurase activity of CpNifS by 70-fold. The old5 mutation affects steady state pyridine nucleotide levels Since the OLD5 SufE/Ygdk domain is mutated we expect the enzyme to be less active. Moreover, it has previously been demonstrated that the QS domain is dependent on the biogenesis of an Fe-S cluster for optimal activity (Gardner and Fridovich, 1991). Moreover, replacing the cysteine residue of the SufE/YgdK domain with a serine disables the full length protein from complementing an E. coli ∆NadA strain (Murthy et al., 2007) indicating the necessity of this domain for QS activity. Given that a reduced QS activity may be anticipated to result in lower NAD steady 131 state levels we determined the pyridine nucleotide content in the first leaf pair of 21 and 27 day old plants. At these time points the old mutant leaves show no phenotypical difference in comparison to the wild type (Figure 1A). Measurement of pyridine nucleotide content revealed, however, that the mutants displayed a significantly increased NAD, NADH, NADP and NADPH content after 21 and 27 days (Figure 4A). The values we report for NAD, NADP and NADPH in wild type are similar to those of previous studies (Wang and Pichersky, 2007; Chai et al., 2005). Interestingly, the NAD/NADH and NADP/NADPH ratios in mutant do not differ from the wild type suggesting a similar redox balance for the pyridine nucleotides. The maintenance of the NAD pool results from the balance of de novo synthesis, active pyridine salvage and degradation (Wang and Pichersky, 2007). The de novo synthesis and the pyridine salvage pathway result in the production of Nicotinate mononucleotide (NaMN) which by 2 enzymatic steps is converted to NAD (Figure 4B). The increased NAD pool suggests an increased expression of enzymes in the salvage pathway to compensate for insufficient biosynthesis via the de novo pathway. Such a mechanism was previously observed for the enzymes of the salvage pathway of pyrimidine nucleotides following the antisense inhibition of an enzyme of the de novo pathway of pyrimidine biosynthesis (Geigenberger et al., 2005). The salvage of nicotinamide (NIM), released from NAD, starts with deamidation by nicotinamidases to nicotinate (Wang and Pichersky, 2007) and subsequent conversion by nicotinate phosphoribosyltransferase (NaPRT). In Arabidopsis, AtNIC1 is the best characterized nicotinamidase which, when knockedout, almost completely abolishes the salvage of NIM. Overexpression of AtNIC1 result in an increase in NAD levels, thus the expression level of AtNIC1 correlates with NAD content (Wang and Pichersky, 2007). The expression of AtNIC1 is constitutively higher in the old5 mutant (Figure 4B). The product of AtNIC1, nicotinate is converted by NaPRTases, the Arabidopsis genome encodes 2 homologes, in old5 At2g23420 (NaPRT2) is up regulated when compared to the wild type, while the other putative NaPRTase (At4g36940) is expressed at similar levels as the wild type (data not shown). The increase in expression of both AtNIC1 and At2g23420, together with the increased pyridine levels, suggests an increased activity of the salvage pathway in the old5 mutant. 132 Figure 4. Pyridine nucleotide biosynthesis is increased during leaf development and ageing of old5. (A) Pyridine nucleotide content in old5 and wild type at day 21 and day 27 (old5 gray bars, wild type solid black bars). The values shown are the means of six repeats ± SD (indicated by error bars). Asterisks indicate statistical significance versus the wild type control (* P < 0.05, Student’s t test). The ratio of pyridine nucleotides is not affected by the old5 mutation. (B) Schematic representation of NAD biosynthetic pathways. Transcripts of AO, AtNIC1 and NaPRT2 accumulate in old5 mutants (old5 open circles, wild type closed circles). The values shown are the means of three repeats ± SD (indicated by error bars). Asterisks indicate statistical significance versus the wild type control (* P < 0.05, Student’s t test). Aspartate oxidase (AO), Quinolinate Synthase (QS), Quinolinate Phosporibosyltransferase 133 (QPRT), Nicotinamidase (AtNIC1), Nicotinate Phosporibosyltransferase (NaPRT2), NAD synthase (NAD-S). In addition to our analysis of the salvage pathway we determined the expression of genes encoding key enzymes in the pathway of the de novo synthesis (Figure 4B). The first step in this pathway is the conversion of L-aspartate into iminoaspartate mediated by L-aspartate oxidase (AO). The transcript level of AO was 3-fold higher in old5 mutant than in the wild type at all time points measured. The next step, of the de novo biosynthesis, the synthesis of the pyridine ring is controlled by OLD5/QS. The expression of QS decreases with age in both the wild type and the mutant. However, during senescence the expression of QS is upregulated in the mutant, reflecting a possible increased demand for NAD during the process. Finally, NaMN are produced by quinolinate phosphoribosyltransferase (QPRT), which for both the wild type and the mutant gradually increase in expression with age but do not differ between the two. Taken together we observe an increased AO expression in de novo synthesis and induction of expression of key salvage enzymes. This is consistent with a block in the de novo synthesis in old5 mutants compensated for by the salvage pathway. old5 is characterized by increased TCA intermediates Since previous studies have shown that alterations in NAD/NADH levels can have dramatic consequences on cellular metabolism (Shen et al., 2006; Dutilleul et al., 2005) we performed a comprehensive metabolite profiling using an established GCMS protocol capable of quantifying the relative levels of over 80 metabolites (Lisec et al., 2006). For this purpose leaves were harvested, in the middle of the light period, from both the old5 mutant and the wild type at days 21 and 27. Interestingly although sugar levels in the old5 mutant were similar to the wild type at 21 days, maltose and sucrose levels were significantly higher at 27 days (Figure 5A). Increased sugar levels have been observed previously before the onset of senescence (Diaz et al., 2005; Wingler et al., 2006), suggesting that the old5 lesion invokes an early change in metabolism reminiscent of senescence. Sugars are subsequently processed through glycolysis to form the pyruvate needed to support mitochondrial respiration through TCA cycle (Fernie et al., 2004). After 21 days we found an increase in the TCA cycle intermediate succinate in the mutant in comparison to the wild type, 134 whereas other intermediates of this cycle were invariant (Figure 5B). However, at this time point amino acids derived from TCA cycle intermediate α-ketoglutarate accumulate in old5 to higher levels than in the wild type. These amino acids include Figure 5. Metabolite profiling of old5 leaves along development. Relative metabolite content of leaves at day 21 and day 27 is given together with change in time as indicated by ratio of 27/21 (old5 gray bars, wild type solid black bars). Metabolite contents were identified and quantified by GC-MS, and their relative amounts were calculated as described by RoesnerTunali et al., (2003) relative to wild type control. Histograms show the relative amount of (A) soluble sugars, (B) TCA-cycle intermediates and (C) amino acids. glutamate, glutamine, proline and the polyamine spermidine (Figure 5C). Importantly, aspartate, which is the precursor for the de novo NAD synthesis was also significantly up regulated in old5 after 21 days. From aspartate several other compounds are synthesized of which asparagine was observed to accumulate after 21 days in old5. At 27 days all the measured α-ketoglutarate derived compounds including GABA (4-amino-butyric acid), ornithine, putrescine and β-alanine were more abundant in old5. Furthermore the branched-chain amino acids isoleucine and valine, that derive from pyruvate (Binder et al., 2007), are significantly higher after 27 days of growth in the old5 mutant. Isoleucine and valine have previously been documented to accumulate in mutants of the electron-transfer flavoprotein complex which display accelerated dark induced senescence (Ishizaki et al., 2005; 2006). Moreover, aspartate precursor oxaloacetate, as well as succinate and malate are 135 more abundant at 27 days in the mutant. These changes might be indicative for an increased mitochondrial metabolic rate in old5 mutants (Noctor et al., 2007). Previously it was shown that redox manipulation by DTT treatment of Arabidopsis leaves increases the level of succinate, malate and oxaloacetate (Kolbe et al., 2006), suggesting that the changes in NAD/NADH level maybe the direct cause of the increase found in TCA cycle intermediates for the old5 mutant. Comparison of differences in the metabolite levels observed between 21 and 27 days may reveal differential metabolic regulation in the mutant with respect to the wild type. Although the levels of the TCA cycle intermediates are higher in old5 than in the wild type at both time points, the pattern of change (i.e. decrease) between the time points is conserved across the genotypes (Figure 5B). However, that is not true for pryridine nucleotide levels which in the wild type decrease with time (Figure 4A) while in old5 they increase (Figure 4A). Increased respiration rate and antioxidant accumulation in old5 Both increased amounts of organic acids and higher levels of pyridine nucleotide can be attributed to an increased rate of respiration (Priault et al., 2007).The metabolite profiling results suggested that the changed redox state in old5 leads to an altered respiration rate. To analyze that possibility we determined the oxygen consumption of Figure 6. Increased respiration of old5 coincides with increased glutathione levels. (A) The oxygen consumption in the dark was measured and quantified with a clark-type electrode (old5 gray bars, wild type solid black bars). (B) Elevated levels of glutathione (GSH) are observed in old5 when compared to the wild type. GSH was extracted from leaf tissues of 21 and 27 days old plants and the analysis was performed using reverse phase (RP)-HPLC. Asterisks indicate statistical significance versus the wild type control (* P < 0.05, Student’s t test). 136 whole leaves in the dark. Figure 6A shows oxygen consumption at 21 and 27 days. At both time points the old5 mutant consumes approximately 2-fold more oxygen than the wild type demonstrating an altered respiration rate. An over reduction of the mitochondrial electron transport chain (mETC) gives rise to increased ROS production which can be alleviated by activation of the alternative oxidase 1 (AOX1) (Noctor et al., 2007). The transcript abundance of AOX1 is also activated directly by TCA cycle intermediates (Vanlerberghe et al., 1997; Gray et al., 2004). Figure 7 shows that AOX1 is constitutively higher expressed in old5 further supporting the notion that the mutant has a higher respiration rate than the wild type. Higher respiratory rates are generally coupled to an increased production of ROS by complex I and III of the mETC (Apel and Heribert, 2004; Noctor et al., 2007). The first Figure 7. Relative expression of marker genes indicative of oxidative stress. The relative expression of several genes was determined by real time PCR and calculated relative to the wild type. The alternative oxidase is constitutively higher expressed (old5 gray bars, wild type solid black bars). Two oxidative stress marker genes were tested and found to be upregulated in the mutant (At2g23420 and At3g13610). Next to that GDH is more abundant than in the wild type. The values shown are the means of three repeats ± SD (indicated by error bars). Experiments were repeated and showed the same results. Asterisks indicate statistical significance versus the wild type control in each case (* P < 0.05, Student’s t test). 137 indication that old5 suffers from ROS stress is SAG13 expression (Figure 2) which is associated with oxidative stress (Miller et al., 1999) and low antioxidants levels (Conklin and Barth, 2004) as well as with senescence. Moreover the mutant showed a change in expression of two other well-established ROS marker genes: At2g43510 and At3g13610. The defensin-like gene At2g43510 which has previously been shown to be ubiquitously induced by various ROS (Gadjev et al., 2006) was constitutively expressed at higher levels in old5 leaves and was strongly upregulated after 30 days (Figure 7). The second marker, 2-oxoglutarate-dependent dioxygenase At3g13610 which is specifically expressed during H2O2-related stress (Gadjev et al., 2006) showed a distinct expression after 24 days in old5 which increased rapidly with age. ROS are continuously generated as a by-product of aerobic metabolism and need to be rapidly detoxified by either enzymatic or non-enzymatic pathways since they are highly toxic (Apel and Heribert, 2004). Protection against oxidative stress is partially provided for by low molecular weight antioxidants such as glutathione and ascorbate. The thiol glutathione, which is synthesized from its constituent amino acids, L-Glu, LCys, and Gly was more abundant in the mutant. Moreover glutathione levels increased from 21 to 27 days in old5, but decreased in the wild type (Figure 6B). Finally ROS stress in Arabidopsis has previously been reported to affect nitrate assimilation catalyzed by glutamate dehydrogenase (Skopelitis et al., 2006). The altered metabolite profile and increased expression of ROS marker genes coincide with an increased expression of GDH from day 27 in old5 mutant (Figure 7). Taken together, these results show that the old5 mutation results in both a different metabolite profile as well as oxidative stress. Discussion old5 reveals a role for NAD in plant ageing Plants differ fundamentally from animals in the sense that they do not have a rigid body plan and their organs can easily be added or removed making them highly adaptive to the environment. As a result of developmental and adaptive strategies that resist, avoid and anticipate ageing, it is suggested that plants do not age at all in any sense recognizable in animals (Thomas, 2002). However, the lifespan of individual leaves clearly resembles animal ageing (Gan, 2003; Jing et al., 2003; Lim et al., 2003; Schippers et al., 2007). That is especially visualized in Arabidopsis 138 where the onset of leaf senescence occurs in an age-dependent way (Gan and Amasino, 1997; Quirino et al., 2000; Jing et al., 2002). Moreover, the recent identification of genes that affect the onset of leaf senescence (Woo et al., 2001; Jing et al., 2005; Jing et al, 2007) reveals common mechanisms in the regulation of ageing between plants and animals. In this study we observed early leaf senescence in the mutant old5 induced by age, as indicated by the expression of SAG12. The cloning of old5 identified a mutation in the gene encoding the quinolinate synthase (QS). QS is an essential enzyme in the de novo synthesis of the pyridine nucleotide NAD (Katoh et al., 2006). Pyridine nucleotides have well-characterized roles as redox carriers in processes such as oxidative phosphorylation, the TCA cycle, and as electron acceptors in photosynthesis (Hunt et al., 2004). Next to their role in metabolism they play a role during several stress conditions including oxidative stress (Moller, 2001; Hayashi et al., 2005), wound-response (Sinclair et al., 2000), ABA-signaling and salt stress (Shen et al., 2006; Wang and Pichersky, 2007). Recently it was found that the NADdependent histone deacylase SIR2 influences the lifespan for several model organisms including yeast, C. elegans, mice and human cells (Lin et al., 2000; Tissenbaum and Guarente, 2001; Lim et al., 2006). Remarkably, SIR2 is involved in both mitotic and post-mitotic ageing as demonstrated by yeast and human cells affecting replicative lifespan and C.elegans, respectively. The current view is that NAD levels serve as a metabolic sensor which is integrated in the developmental program by the effect of SIR2 on chromatin remodeling (Grewal and Moazed, 2003). Here we show that a mutation that causes alteration in the level of NAD influences the lifespan of Arabidopsis suggesting a possible conservation for the role of NAD on development and ageing in plants. old5 mutation results in a metabolite profile characteristic for early ageing Redox reactions are the fundamental metabolic processes through which cells convert and distribute the energy that is necessary for growth and maintenance (Noctor, 2006). Changes in the availability of pyridine nucleotide levels were shown before to have a dramatic effect on the metabolite profile of plants (Dutilleul et al., 2005; Shen et al., 2006). Next to that, increased levels of NADH affect other reactions that produce NADH, and disturb the cellular oxide/redox balance to cause oxidative stress (Shen et al., 2006; Moller, 2001). The increased NAD levels in the 139 old mutant coincide with increases in the level of TCA cycle intermediates and the precursor for NAD biosynthesis aspartate. In addition nitrogen metabolism is strongly affected since the amino-acids deriving from ketoglutarate are more abundant. A previous study on the Tobacco nad7 mutant revealed a similar result, in which the mutant has NAD/NADH levels higher than the wild type and accumulation of nitrogen containing amino acids (Dutilleul et al., 2005). Given that the onset of leaf senescence in old5 is age-induced it could be anticipated that certain metabolites are characteristic for an age-related change (ARC) of the leaf (Diaz et al., 2005; Jing et al., 2005). The difference in metabolite profile between the mutant and the wild type is more pronounced with increasing age. Several ARCs of metabolism specific for the onset of leaf senescence are observed in old5. First the accumulation of the branched chain amino acids valine and isoleucine in old5 mutants was previously shown to occur before the onset of senescence (Masclaux et al., 2000; Diaz et al., 2005; Ishizaki et al., 2005). One of the most intriguing metabolites that shows an age-related accumulation is GABA (4-amino-butyric acid), which accumulates in old5, but not in the wild type at day 27. Studies on Osl2 a mitochondrial GABA transaminase in rice (Ansari et al., 2005) suggest a switch in metabolism at the onset of senescence. Moreover, it seems likely that in plants, as in animals, the GABA shunt is up regulated in times of high respiratory demand (Sweetlove et al., 2007). In addition to this it has been found that GABA induces senescence in sunflower by enhancing the synthesis of ethylene (Kathiresan et al., 1997). This is in line with a previous study in which we suggested that the ethyleneinduced leaf senescence of old5 is depending on ARCs (Jing et al., 2005). The identified metabolites might serve as useful biomarkers to determine the viability of leaves and progression of senescence. In addition to previously documented markers of ageing we observed the accumulation over development of the polyamines in the old5 mutant. Spermidine has the same precursor as aminocyclopropane (ACC), an important source of ethylene which can induce premature senescence (Imai et al., 2004). However, the accumulation of the polyamines might not be an ARC perse but may also reflect a protection against ROS generation and programmed cell death (Papadakis et al., 2005). Moreover polyamines as cytokinins are well-known for their senescence retarding effect. 140 Interaction between de novo synthesis and the salvage pathway of NAD and their role in stress signaling The analysis of old5 quinolinate synthase mutant protein showed that it weakly interacts with components of Fe-S biogenesis complex, CpNifS and CpNifS3 in a yeast two-hybrid experiment. As the QS is dependent on a functional Fe-S biogenesis complex (Loiseau et al., 2005), the observed weak interaction likely leads to a lower in vivo activity of the whole protein. Thus we anticipated that the recessive mutant allele old5 results in a decreased activity of the enzyme and we expected lower levels of NAD/NADH. However, the analysis revealed an increased NAD/NADH level for the mutant. The maintenance of the NAD pool is both due to the de novo synthesis and pyridine salvage pathway. Whereas the de novo synthesis is absolutely required for plant survival (Katoh et al., 2006) the recycling pathway is mainly important during stress to recycle the degraded NAD in an energy efficient manner (Wang and Pichersky, 2007). In apparent response to the retardation of QS, old5 plants exhibit high expression of aspartate oxidase the first enzyme in de novo synthesis of NAD (Figure 4). We furthermore observed an increased expression of the two key enzymes involved in the salvage pathway, AtNiC1 and NaPRT2. The results point out that old5 compensates for the decreased de novo synthesis of NAD by up regulation of the salvage pathway of pyridine nucleotides. That is in agreement with results of Geigenberger et al., (2005) who previously reported that inhibition of de novo pyrimidine biosynthesis results in a compensatory stimulation of the salvage pathway. Moreover enhanced expression of the salvage pathway enzyme AtNIC1 has previously been shown to increase the NAD levels in Arabidopsis (Wang and Pichersky, 2007; Hunt et al., 2007). The increased expression of the NAD salvage pathway is reminiscent to the need to replenish the NAD pool during stress conditions (Berglund et al., 1996; Wang and Pichersky, 2007; Hunt et al., 2007). Therefore the adjustment in NAD metabolism of old5 might result in stress signaling. One example of a NAD-consuming enzyme induced by oxidative stress is PARP (De Block et al., 2005) linking increased salvage metabolism to stress. Next to that the salvage pathway was shown to be activated and necessary for response to osmotic stress in Arabidopsis (Wang and Pichersky, 2007). 141 Role for mitochondria in early ageing of old5 Longevity is clearly genetically controlled in many species including yeast, C. elegans, mice and man (Jing et al., 2003). Almost 100 years ago it was postulated that there is an inverse relationship between metabolic rate and longevity (Rubner, 1908; Pearl, 1928). Nowadays it is clear that caloric restriction (CR) can greatly extend the maximum lifespan of many species (Sohal and Weindruch, 1996). The discovery of the first gene (PHA-4) absolutely required for lifespan extension by CR in C. elegans supports a role for ROS generated by mitochondrial oxidative energy metabolism in lifespan determination. This is especially evident by the fact that reduced PHA-4 expression does not suppress the long lifespan of animals with defective electron transport chains (Panowski et al., 2007). These results fit within the free-radical theory of ageing, which proposes that ROS produced by respiration contribute to the ageing of all organisms (Harman, 1956). In our study we show that the mutant old5 has an increased respiration rate together with increased oxidative stress. The reducing equivalents that are generated from TCA-cycle activity are used by the mitochondrial electron transport chain to power the synthesis of ATP (Fernie et al., 2004). The increased NADH levels in the mutant might directly influence the rate of mitochondrial electron transport (Hunt et al., 2004). This results in expression of the alternative oxidase which by-passes the phosporylation and does not contribute to ATP production but alleviates over reduction of the chain (Moller, 2001). Transgenic tobacco cells that fail to induce alternative oxidase go rapidly into programmed cell death (PCD) during oxidative stress (Vanlerberghe et al., 2002). The increased glutathione levels and increased expression of H202-induced stress markers in our mutant fit well with previous studies that during oxidative stress mitochondria produce increased amounts of H202 (Sweetlove et al., 2002; Tiwari et al., 2002; Gadjev et al., 2006). ROS have a role in the onset of developmental senescence. The natural senescence of pea leaves coincides with increases in the levels of superoxide and H202 (Pastori and del Rio, 1997). Treatment of Arabidopsis leaves with the herbicide 3-AT inhibits catalase activity and causes H202 stress resulting in the expression of SAG genes (Navapbour et al., 2003). Next to that the senescence-specific transcription factor WRKY53 is induced by H202 (Miao et al., 2004; Miao and Zentgraf, 2007). Taken together this demonstrates that oxidative stress can function as a signal for the onset of leaf senescence. Thus the possible accumulation of damage to the mitochondria by 142 increased ROS in old5 might be at the basis of early ageing of the mutant as denoted by the “free-radical“ theory of ageing (Harman, 1956). In general it is believed that oxidative stress results in damage to the cell which loses its viability. To determine if oxidative stress in our mutant results in damage or serves as a signal for the onset of senescence additional experiments are required. Making use of Arabidopsis leaves as a model system for plants we have exploited the opportunity to characterize the mechanisms and genetics of ageing. Our study supports the idea that an increased mitochondrial respiration rate affects lifespan as denoted by the “free-radical” theory of ageing. It suggested that factors important for mammalian ageing appear to be conserved across kingdoms. Since ageing is manifested in many ways, cloning and physiological characterization of other old mutants will allow a fuller elucidation of the subtle differences between the various mechanisms of ageing and senescence. Methods Plant materials and growth conditions Arabidopsis thaliana ecotype Landsberg erecta (Ler-0) was used throughout this study. The old5 mutant was obtained from an EMS mutaganized collection (Jing et al., 2005). T-DNA insertion mutant QS-1 (SALK_079205) was obtained from the Nottingham Arabidopsis Stock Centre (NASC). Complementation of the old5 mutation was completed by making use of the binary vector pGreen029 (Hellens et al., 2000). Plants were grown on either soil or half-strength Murashige and Skoog medium at 23°C and 65% relative humidity with a day length of 16 h. The light intensity was set at 120 µmol·m-2·s-1. An organic-rich γ-ray radiated soil was used (Hortimea Groep, Elst, The Netherlands). Pigment determination and measurement of photochemical efficiency For extraction of chlorophyll and carotenoids samples were incubated overnight with N,N-dimethylformamide at 4°C in darkness. The chlorophyll content was quantified spectrophotometrically according to Wellburn (1994) at 647 and 664nm. Chlorophyll fluorescence emission was measured from the upper surface of the first leaf, at room temperature (23 °C) with a pulse-amplitude modulation portable 143 fluorometer (PAM-2000; H. Walz, Effeltrich, Germany) according to Maxwell and Johnson, 2000. Plants were dark-adapted for 1 to 2 hr before measurements to ensure complete relaxation of the thylakoid pH gradient. An attached, fully expanded rosette leaf was placed in the leaf clip, allowing air to circulate freely on both sides of the leaf. At the start of each experiment, the leaf was exposed to 2 min, of far-red illumination (2 to 4 µmol photons m-2 s-1) for determination of Fo (minimum fluorescence in the dark-adapted state). Saturating pulses of white light (8000 µmol photons m-2 s-1) were applied to determine Fm or Fm' values. PSII efficiency was calculated as (Fm - Fo)/Fm. RNA-isolation and RT-PCR Total RNA was isolated using TRIZOL reagent (Sigma) according to the manufacturer's protocol. Five hundred nanograms of RNA were used as template for first-strand cDNA synthesis using 200U of RevertAid H-minus MMuLV reverse transcriptase (Fermentas, USA) and an oligo(dT21) primer. Primer pairs for real-time PCR were designed with open-source PCR primer design program PerlPrimer v1.1.10 (Marshall, 2004). The primer sequences are available upon request. Briefly, real-time PCR amplification was performed with 50 µL of reaction solution, containing 2 µL of 10-fold–diluted cDNA, 0.5 µl of a 10 mM stock of each primer, 1 µl of 25mM stock MgCl2 (Fermentas), 5 µl PCR buffer +Mg (Roche), 1 µl of a 1000x diluted SYBR-green stock (Sigma), 0.5 µl 100xBSA (New England Biolabs), and 1u of Roche Taq Polymerase. The PCR program was 2’ at 94, 40x (94-10”/60-10”/72-25”), meltcurve. Obtained data was analyzed with BioRad software. Determination of metabolite levels Leaf samples were taken at the time points indicated, immediately frozen in liquid nitrogen and stored at –80°C until further analysis. Extraction was performed by rapid grinding of tissue in liquid nitrogen and immediate addition of the appropriate extraction buffer. The relative levels of metabolites were determined using an established gas chromatography coupled to a time-of-flight mass analyzer (GC-TOFMS) protocol as described by Lisec et al., 2006. Data are presented normalized as detailed by Roessner et al., 2001. The procedure of extraction and assay of nicotinamide adenine dinucleotides was performed according to the method 144 described by Gibon and Larher, 1997. The determination of glutathione contents was performed as described Kreft et al., 2003. Yeast two-hybrid experiments The coding sequence of the selected genes were amplified from RNA and cloned in frame with the GAL4 binding domain of the pGBKT7 and GAL4 activation domain of the pGAD424 vector (Clonetech). For protein-protein interaction screening, PJ69-4A (James et al., 1996) was transformed according to the Clonetech yeast protocols handbook (PR13103) and selected on synthetic dropout (SD) medium lacking Leu and Trp for transformants. Subsequent colonies were dissolved in SD and spotted on selection medium for interaction by testing growth on SD lacking either Ade or medium lacking His with the addition of 2mM 3-amino-1,2,4-triazole. Plates were incubated at 28 degrees for 2-5 nights. Respiratory measurements Assays of oxygen consumption by whole leaves were performed using a Clark-type oxygen electrode. Leaves were placed on a filter containing CO2 buffer and subsequently the measuring room was filled after calibration with a 2% oxygen containing gas before measurement. Measurements were performed in 6 fold and fresh weight and chlorophyll content of the leaves was determined. Accesion numbers The Arabidopsis Genome Initiative locus numbers for the major genes discussed in this article are At1g08490 for CpNifS1, At1g18490 for CpNifS2, At1g55090 for NADS, At2g01350 for QPRT, At2g22570 for AtNIC1, At2g23420 for NaPRT2, At2g29350 for SAG13, At2g43510 for defensin-like, At3g13610 for oxidoreductase, At4g26500 for AtSufE, At4g36940 for NaPRT1, At5g07440 for GDH, At5g14760 for AO, At5g26600 for CpNifS3, At5g50210 for QS/OLD5 and At5g45890 for SAG12. Acknowledgements We would like to thank Bert Venema and Margriet Ferwerda for their excellent technical support. We also show gratitude to Anne de Jong at the Laboratory of Molecular Genetics of the University of Groningen for support with RT-PCR. 145 Furthermore we would like to thank Ying Miao and Marcel Proveniers for their support with the yeast two hybrid experiments. 146 References Ansari, M.I., Lee, R-H., and Chen S-C.G. (2005). 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Ye, H., Abdel-Ghany, S.E., Anderson, T.D., Pilon-Smits, E.A.H., and Pilon, M. (2006). CpSufE activates the cysteine desulfurase CpNifS for chloroplastic Fe-S cluster formation. J. Biol. Chem. 281: 8958-8969. 152 Summary Leaf senescence is a genetically regulated process which enables plants to relocate valuable resources from dying tissue to other parts of the plant. The notion that thousands of genes are associated with leaf senescence illustrates the complex biological mechanisms underlying this process. The onset of this program is agedependent in Arabidopsis. Recent studies have led to the isolation of several genes that alter the timing of leaf senescence. Therefore the leaf is an excellent model to study the genetic network that underlies the ageing process. The study presented in this thesis is focussed on isolating genes that regulate the timing of leaf senescence. In addition, hormonal responses and metabolic processes are characterized to identify age-related changes that are at the basis of the onset of leaf senescence. The combination of these results allow for defining important insights into the onset of leaf senescence. Hormones are universal regulators of developmental and metabolic processes in all living organisms. Plant hormones can have senescence retarding or senescence promoting. One of the best characterized senescence-promoting hormones is the gaseous molecule ethylene which is involved in several physiological processes throughout the plant's life cycle. Ethylene promotes the onset of leaf senescence after a certain developmental stage (Chapter 1). This relation between ethyleneinduced senescence and leaf age was comprehensively studied in Chapter 2. Interestingly, it was found that an increase in the length of the ethylene exposure results in a more pronounced senescence response. However, when the exposure to ethylene is more than 12 days, the senescence response is reduced. Thus the timing and length of the ethylene treatment influences the senescence response in plants with the same final age. It is argued that certain age-related changes are necessary to either promote or reduce the effect of ethylene on the induction of leaf senescence. The use of eight early senescing onset of leaf death (old) mutants 153 indicates that individual genes can modulate the outcome of ethylene-induced senescence. An important notion here is that the loci influence age-related changes which constitute molecular checkpoints for the onset of leaf senescence. Thus, the induction of senescence by ethylene is the result of a complex interaction between leaf age and ethylene which can be modulated by multiple genetic loci. Three of the eight mutants presented in chapter 2 were chosen for a detailed characterization to identify novel components involved in the onset of leaf senescence. Two of the three selected mutants showed only early leaf senescence upon ethylene treatment. Since the action of ethylene is age-dependent it was anticipated that specific mechanisms involved in the interaction between leaf age and ethylene could be identified. Most senescence mutants described in literature have additional defects like the delayed senescence mutant ore9, which has a highly branched shoot. Another example is old1, which is smaller than wild type and is hypersensitive to sugar and ethylene. Therefore it is unlikely that genes have evolved that solely have a function in the regulation of leaf senescence. However, the three mutants were mainly selected for their similar development as the wild type and might therefore represent loci specific for the onset of leaf senescence Results obtained with early senescence mutant old13, indicate that the gene product is involved in several plant programs that can result in senescence. In chapter 3 it is presented that the mutant displays a hypersensitive response-like phenotype after treatment with ethylene. Moreover, expression data indicates that the mutant is involved in plant defense. Next to that the mutant displays early senescence upon lack of watering and has an increased intracellular anion concentration. Common signaling molecules involved in biotic and abiotic stress are the reactive oxygen species (ROS) which are enhanced in the mutant. Taken together, the OLD13 gene product is suggested to function as a modulator of leaf senescence upon stress in an age-dependent way. The distinct role of the old13 gene product in senescence processes is an exciting finding that might indicate that specific regulatory genes exist. Chapter 4 reports on the characterization of the early leaf senescence mutant old9. The mutant displays specifically early ethylene-inducible senescence. The analysis of the old9 mutant reveals two novel insights in the regulation of the onset of leaf senescence. Transcriptome analysis of the mutant points at a role for the plant 154 hormone cytokinin in the early senescence in the mutant. Cytokinins are known for their effect to retard the onset of leaf senescence (Chapter 1). However, in this study it is shown that exogenous application of cytokinin to wild type, early in development results in an enhanced response to ethylene-induced senescence. Thus, cytokinin can both induce and delay the timing of leaf senescence depending on the age of the plant. The cytokinin mediated senescence response is argued to be related to sinkto-source change of the leaf. Another interesting finding is that a cell wall associated protein can modulate the senescence response of the old9 mutant. A gene encoding for a hydroxyproline rich glycoprotein is upregulated in air-grown old9 mutants. Downregulation of the expression level of the transcript by RNA-interference results in a decreased senescence response of the mutant. The role for cell wall proteins in the onset of leaf senescence has not been described in literature and represents a novel pathway involved in the process. In chapter 5 the characterization and identification of old5 is presented. OLD5 encodes quinolinate synthase, a key enzyme in the de novo synthesis of NAD. Recently it was found that the NAD-dependent histone deacylase SIR2 influences the lifespan of several model organisms including yeast, C. elegans, mice, and human. It is proposed that NAD levels serve as a metabolic sensor which is integrated in the developmental program by the effect of SIR2. This implies that ageing in plants can be compared with animal ageing when individual leaves are used. It has been argued before that plant and animal ageing share similar strategies; however, this is the first study that reports on a common molecular pathway. Moreover, it is argued that the early ageing of the mutant is the result of oxidative stress in the mitochondria, thus, implying that the “free-radical” theory of ageing can be applicable to plants. The results illustrate that the leaf is an excellent model system for ageing research in plants that will increase the understanding of senescence mechanisms in animals as well. The identification of old5 shows that central metabolism is involved in ageing. The recent developments in the field of metabolomics add a new tool for the identification of age-related processes. In chapter 5 it is presented that several metabolites have a distinct age-related accumulation in leaves of Arabidopsis. These metabolites include 155 valine, isoleucine, and GABA. Thus next to transcripts, metabolites appear to mark age-related changes during development. A combination of metabolite profiling together with transcriptome data will reveal novel mechanisms and pathways that underlie ageing processes in plants. Ten years ago senescence was described as almost too complex and difficult biological phenomenon to fully comprehend. However, the development of novel molecular biological techniques has revolutionized the field and the understanding of the process. Next to that, a continuous increasing number of genes that regulate leaf senescence are being described. The dedication of more and more research to leaf senescence will advance the understanding of the process. Therefore the coming years will emerge as exciting and inspiring when the mechanisms underlying senescence are being unravelled. 156 Samenvatting De kleurverandering van bladeren is een van de opvallendste kenmerken van de herfst voordat ze afsterven en afvallen. Tijdens de herfst worden de bouwstoffen die aanwezig zijn in het blad gerecycled en opgeslagen om na afloop van de winter gebruikt te worden om nieuwe bladeren te laten uitlopen. Ons woord herfst is afgeleid van het oud Germaanse woord ‘harbista’, dat via het Hoogduitse ‘herbist’ en het Middelhoogduitse ‘herbest’ uiteindelijk het Middelnederlandse ‘hervest’ werd. Het Engelse woord harvest wat oogsten betekent heeft echter niet meer de betekenis van jaargetijde behouden zoals in Nederland. De herfst is het jaargetijde van het oogsten en dat doen zowel mensen als planten. Het afsterven van bladeren is niet passief. Voordat ze afsterven, halen veel planten de voedingsstoffen terug uit hun bladeren. Ze worden opgeslagen in de wortels en de stengel voor het volgende voorjaar of worden gebruikt voor de aanmaak van zaden en vruchten. Tijdens mijn onderzoek heb ik gewerkt met de modelplant Arabidopsis thaliana, oftewel een onkruid dat vrijwel overal in Nederland is te vinden en ook wel zandraket wordt genoemd. De zandraket wordt gekenmerkt door een korte levenscyclus wat het mogelijk maakt om tijdens een promotieonderzoek een bepaalde eigenschap te volgen in opeenvolgende generaties. Sinds de ontdekking van de structuur van het DNA zijn wetenschappers in staat om eigenschappen van een organisme te herleiden tot een gen. Tegenwoordig is de volledige erfelijke informatie die is opgeslagen in het DNA van de zandraket bekend en weten we dat het voor ongeveer 32000 genen codeert. Een aantal studies heeft aangetoond dat ruim tweeduizend genen betrokken zijn bij het afsterven van bladeren. Dit toont aan dat bladafsterving een genetisch gecontroleerd proces is en dus een belangrijk onderdeel vormt van de levenscyclus van de plant. De bladeren van de zandraket sterven af zodra ze een bepaalde leeftijd hebben bereikt, daarnaast kan de plant er voor zorgen dat een blad afsterft zodra deze stress krijgt. Dus een deel van de plant kan worden opgeofferd voor het behoud van de rest. Een belangrijk plantenhormoon dat het afsterven van 157 bladeren bevordert is ethyleen. Daarnaast zorgt ethyleen voor het rijp worden van bananen en appels maar heeft ook als effect dat snijbloemen snel verwelken. Daarom mogen vruchten en bloemen nooit samen vervoerd worden. Naast ethyleen is er een ander plantenhormoon, cytokinine wat juist een anti-verouderings effect op bladeren heeft en ervoor zorgt dat ze langer groen blijven. Mijn onderzoek is voornamelijk gericht op veranderingen in bladeren die optreden tijdens het verouderen. Deze wijzigingen kunnen een signaal zijn voor de plant om een blad te laten afsterven. Omdat het afsterven van de bladeren van de zandraket wordt gereguleerd door de leeftijd van het blad vormt het een uitstekend testsysteem. De studie die hier beschreven is heeft als doel genen te identificeren die betrokken zijn bij het verouderingsproces van de bladeren. Daarnaast is het effect van het plantenhormoon ethyleen onderzocht op de ontwikkeling van de zandraket. Naast het genetisch onderzoek is er ook bepaald hoe het metabolisme van de bladeren verandert tijdens het ouder worden. De combinatie van de onderzoeksresultaten heeft nieuwe inzichten opgeleverd over de regulatie van de bladveroudering. Resultaten van het onderzoek kunnen worden toegepast om uiteindelijk de opbrengst van gewassen te verbeteren. Hormonen zijn universeel betrokken bij de ontwikkeling, groei en metabolische processen van alle organismen. Plantenhormonen kunnen zowel bladafsterving stimuleren als voorkomen. Het plantenhormoon ethyleen is betrokken bij vele aspecten van de ontwikkeling van de plant waarvan het aanzetten van bladafsterving een aspect is. Voor het identificeren van genen die van invloed zijn op de bladveroudering is er gebruik gemaakt van een mutant analyse. Door zaden van de zandraket chemisch te behandelen kunnen er mutaties optreden in het DNA. Het veranderen van een genetische eigenschap op deze manier kan gevolgen hebben voor de bladveroudering. Met behulp van een ethyleen behandeling is het mogelijk om mutanten te selecteren die verschillen in hun bladveroudering ten opzichte van de controle planten. Op deze manier zijn er ruim 100 onafhankelijke mutanten geïsoleerd. De grote vraag die overblijft is dan, welke van de 32000 genen zijn er veranderd in deze mutanten? Naast het toepassen van ethyleen voor het identificeren van genen die mogelijk betrokken zijn bij de bladveroudering is het effect van ethyleen ook onderzocht tijdens de ontwikkeling van controle planten. 158 In hoofdstuk 1 en 2 van dit proefschrift wordt de relatie tussen de leeftijd van een blad en het effect van ethyleen op de inductie van bladafsterving uitgebreid beschreven. Ethyleen kan alleen bladafsterving induceren wanneer het blad een bepaalde leeftijd heeft bereikt, dus jonge bladeren zijn ongevoelig voor het hormoon. Het onderzoek toonde aan dat het effect van ethyleen op bladveroudering afhankelijk is van de tijdsduur van blootstelling aan het hormoon. Een langere ethyleen behandeling resulteert in sterkere verouderingssymptomen, echter een extra lange behandeling vermindert de effecten van ethyleen op veroudering. Dus het effect van ethyleen op de inductie van bladveroudering hangt af van de leeftijd van een blad en de lengte van de behandeling. Naast dit effect van ethyleen op de controle planten zijn er ook acht mutanten getest. De mutanten worden old (onset of leaf death) genoemd. De behandeling van de mutanten met ethyleen toont aan dat een enkele genetische eigenschap een rol kan spelen bij de regulatie van de bladveroudering. Drie van de acht mutanten die beschreven zijn in hoofdstuk 2 zijn gekozen voor een gedetailleerde studie om nieuwe genetische componenten te identificeren die betrokken zijn bij bladveroudering. Twee mutanten, old13 en old9 vertonen alleen vervroegde bladveroudering tijdens een behandeling met ethyleen. Omdat het effect van ethyleen leeftijdsafhankelijk is verwachten wij door het bestuderen van deze mutanten het mechanisme van de interactie tussen leeftijd en ethyleen beter te begrijpen. Tot nog toe zijn er geen specifieke bladverouderingsgenen gevonden en hebben de bekende mutanten vaak een veelvoud aan extra defecten. Vanuit het perspectief van evolutie is het ook minder aannemelijk dat er specifiek genen zijn die alleen tijdens de laatste ontwikkelingsfase van een blad een rol spelen. De old13 mutant wordt beschreven in hoofdstuk 3 van dit proefschrift en is betrokken bij verscheidene processen in de plant. Wanneer een blad wordt aangevallen door een pathogeen dan kan er een lokale celdood optreden waardoor de invasie kan worden verhinderd. De old13 mutant vertoont deze eigenschap tijdens de ethyleen behandeling. Ondanks dat de mutant een normale ontwikkeling vertoont is hij overgevoelig voor droogte stress. Dit heeft waarschijnlijk te maken met het feit dat de interne concentratie van anionen (chloride, nitraat en sulfaat) sterk verhoogd is. Belangrijke signaalmoleculen tijdens zowel pathogeen detectie, droogte stress en bladafsterving zijn zuurstof radicalen. Tijdens de groei van de mutant is er een 159 constante verhoogde aanwezigheid van deze signaalmoleculen. Samenvattend kan het old13 gen product worden gekarakteriseerd als een belangrijke modulator tussen omgevingsstress en het aanschakelen van het blad afstervingsproces. In hoofdstuk 4 wordt de old9 mutant beschreven. De resultaten van het onderzoek naar de mutant resulteerden in 2 nieuwe inzichten. Ten eerste is er bepaald welke genen actief zijn in de mutant ten opzichte van de controle planten. Dit heeft aangetoond dat een grote groep genen die kunnen reageren op het hormoon cytokinine zich anders gedragen dan in de controle planten. Dit is opmerkelijk omdat cytokinine is gerelateerd aan het voorkomen van veroudering. Echter, een experiment waarin controle planten worden behandeld met cytokinine tijdens verschillende fasen van ontwikkeling toont aan dat het anti-verouderingshormoon ook veroudering kan veroorzaken. Wanneer planten op jonge leeftijd een behandeling krijgen met cytokinine dan resulteert dit in een verhoogde bladafsterving tijdens de ethyleen behandeling. Een ander interessant resultaat is dat een eiwit dat een onderdeel vormt van de celwand betrokken is bij de vervroegde bladafsterving in de old9 mutant. In de mutant is het gen dat voor de aanmaak van dit eiwit codeert sterk actief, echter wanneer dit gen wordt geblokkeerd vermindert de reactie van de mutant op de ethyleen behandeling. De rol van celwand eiwitten in de regulatie van de bladveroudering is een nieuw inzicht dat om verder onderzoek vraagt. In het laatste hoofdstuk van dit proefschrift wordt de old5 mutant gepresenteerd. De mutant toont vervroegde veroudering tijdens normale groei omstandigheden en heeft een enkele weken kortere levenscyclus. De mutant wordt veroorzaakt door een mutatie in een gen dat betrokken is bij de productie van het energiemolecuul NAD waarvan vitamine B3 een onderdeel is. NAD is belangrijk voor vrijwel alle metabole processen in de cel. Recent onderzoek in muizen, gist en mensencellen toont aan dat NAD direct is gekoppeld aan veroudering. Het onderzoek geeft aan dat de ontwikkeling en groei van cellen wordt aangepast aan de hand van de aanwezige hoeveelheid NAD. De old5 mutant vertoont vervroegde veroudering die waarschijnlijk wordt veroorzaakt door vergelijkbare mechanismen die betrekking hebben op veroudering in dieren. Het was eerder geopperd dat planten en dieren een zelfde verouderingsstrategie zouden kunnen hebben, echter dit is de eerste studie die dat inderdaad ook aantoont. De verandering in NAD niveaus in de mutant gaan gepaard 160 met een verhoogd cel metabolisme. Metabolisme afhankelijk van zuurstof gaat altijd hand in hand met oxidatieve stress. Oxidatieve stress ontstaat voornamelijk in de mitochondriën waar de verbranding plaatsvindt en wordt gezien als de belangrijkste oorzaak van veroudering in dieren. Deze studie toont aan dat de mitochondriën in planten wel degelijk een rol spelen bij veroudering in tegenstelling tot de algemeen aanvaarde opvatting dat de bladgroenkorrels de belangrijkste bron van oxidatieve stress zijn. Gezien de parallel tussen dieren- en plantenveroudering toont dit onderzoek aan dat het blad als een uitstekend model kan dienen voor het onderzoek naar veroudering. Door het toepassen van een nieuwe techniek waarbij de concentratie van honderden verschillende verbindingen in de cel tegelijk kan worden bepaald hebben we nieuwe factoren geïdentificeerd die wellicht de leeftijd van een blad bepalen. Verscheidene aminozuren vertonen een sterke ophoping vlak voor de start van de bladafsterving. Deze componenten kunnen dienen als kenmerken voor oude bladeren en dus nuttig zijn voor het bepalen van de ontwikkelingsfase van een blad. Daarnaast kan de combinatie van informatie van het metabolisme met die van de genexpressie worden gebruikt om nieuwe mechanismen te ontdekken die betrokken zijn bij de bladveroudering. Ongeveer 10 jaar geleden werd bladveroudering beschreven als een te complex biologisch proces om volledig te kunnen begrijpen. Echter de ontwikkeling van nieuwe moleculair biologische technieken heeft een ware revolutie te weeg gebracht in het onderzoek. Dit heeft geleid tot een continue toename van publicaties over genen die betrokken zijn bij bladveroudering. De groei in het aantal studies aan bladveroudering zal zeker leiden tot nieuwe inzichten in het proces. Daarom zullen de komende jaren opwindend en inspirerend zijn wanneer de processen die ten grondslag liggen aan bladveroudering worden onthuld. 161 162 Acknowledgements I don’t really like to thank people from this position but I will do my best: Firstly, I would like to thank my Supervisor, Dr. Paul Dijkwel. I could not have imagined having a better advisor for my PhD, and without his common-sense, knowledge, perceptiveness, rock-n-roll motivation and cracking-of-the-whip I would never have finished. I would like to say a big 'thank-you' to Prof. Dr. Jacques Hille for his support and help during the whole period of the study, especially for staying calm everytime I exceed a deadline. Of course I would like to acknowledge the colleagues from MBP and Plant Physiology for creating a pleasurable atmosphere during my Ph.D. research. Than I can not forget to mention that due to the enthusiasm of Hai-Chun Jing and his fantastic work I was able to start a wonderfull fascinating project. Finally, I have to say 'thank-you' to: all my friends and family, wherever they are, particularly my Mum and Dad; and, most importantly of all, to my Girlfriend, for everything. 163 164