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PLANT SENESCENCE PROCESS

md AND P RODUCTIVITY iter

VIJAY PAUL, AJAY ARORA AND G.C. SRIVASTAVA

yin >eed INTRODUCflONb.9 ­

Senescence, like growth and reproduction, is a norm al phase of plant development -orld except that, in this case, death is the culminating event. Nearly all parts of a plan t senesce,

although which and when are dicta ted by autonomous and environmental factors. Petals and sepals senesce afte r pollina tion or fertilization. In annuals, leaves senesce during seed lasm or grain development. In perennials, leaves may show yearly cycles of senescence and p for abscission or, as in evergreens, senesce and abscise after 2-3 years although not all at the I curu, same time. Some plants produce flowers once in their life and then die. These plants, known as monocarpic plants, include annuals (e.g, cereals, soybean, Arabidopsis); all biennials, (e.g. carrot, celery) and some perennials (e.g. Agavespp, bamboos). In comparison to senescence, ageing encompasses a wide array of passive or non-regulated, degenerative processes driven primarily by exogenous factors (Leopold 1975). As the biochemical nature of senescence and ageing is not known precisely, even today, so in view of some researchers it wo uld therefore be premature to attempt to define these two processes more exactly or to draw a fine line between them. .

Plants exhibit two basic types of senescence: (1) Mitotic senescence (proli ferative or replicative senescence) and (2) Post-mitotic senescence (Gan & Amasino 1999). A shoot

216 VJJAY PAUL, A JAY A RO RA AND G.C. SRIVASTA VA

apical meristem cell can undergo a certain number of mitotic divisions to produce organs such as leaves and flowers. Cessation of the cell division in the meristem is called mitotic senescence (Hensel et al 1994). Telomere shortening has been implicated in controlling replicative senescence in mammal (Bodnar et aI1998). In contrast, post-mitotic senescence occurs in organs such as leaves and petals. Once formed, cells in these organs rarely undergo cell division (Colon-Carmona et a11999) but these cells undergo cell growth and ultimately cell degeneration or senescence. Telomere length in these cells remains stable during leaf growth and senescence (Riha et al 1998, Zentgraf et aI2000).

Our interest is basically in understanding mechanisms and control of leaf senescence (a type of post-mitotic senescence). Studying senescence is interesting from much theoretical perspectives however, it is also of practical value as a factor in plant productivity and also in post-harvest longevity. Better understanding of senescence, a process that limits yield, nutritional values and marketability of many crops, will lead to ways of manipulating senescence for agricultural application (Iones 2004). Leaf senescence is undesirable in vegetable crops in which the leaf is the part consumed and for producers of fresh cut flowers, petal senescence reduces the commercial value of the crop. Moreover, if leaves are kept photosynthetically active for a longer period of time, this may have a positive influence on seed yield . At this point, the critical issue is whether retention of green area during post­anthesis (usually drought prone period) will also increase the gr ain yield. Positive associations have been observed in range.of cereals, including wheat (Evans et al 1975), maize (Wolfe et al1988), oat (Helsel & Frey 1978), sorghum (Henzell et al 1992) and other crops (Thomas & Smart 1993, Thomas & Howarth 2000). For example, the record yield of maize gram, almost 24,000 kg ha-l, was achieved in 1985 on a farm in Illinois, with a variety FS854, a stay green maize line (Tollenaar 1985). Further, it is not only delayed senescence trait but its incorporation with early leaf development -...ould enable us to get maximum benefits of manipulating leaf/plant senescence (Thomas & Howarth 2000).For crops of dual purpose like sorghum and millets in regions of semi arid tropics, extended period of leaf greenness is of vital importance in the struggle to feed populations living under harsh economic and environmental circumstances, besides they possess leaves with higher nutritional quality and attractiveness to grazing animals (Van Oosterom et aI1996).

In view of above advantages, it is important to understand the process of senescence first and then to man ipulate it for economic purposes.

SENESCENCE

Leaf sen escen ce

After a photosynthetically productive period, the leaves contr ib u tion of photosynthate to plant diminishes and the leaf then enters into its last stage of development i.e, senescence. Indeed, leaf senescence can be viewed as a recycling programme at the plant organizationa l level (Quirino et al 2000). During senescence, nutrient such as nitrogen, phosphorus and metals that were earlier invested in the leaf are reallocated to younger leaves and growing seeds or stored for the next growing season (Buchanan-Wollaston 1997).

Monocarpic senescence

Monocarpic senescence, which follows the reproductive phase of many plants, is one of the most dramatic and probably most complex forms of senescence (Nooden et al

216 VIJAY PAUL, A JAY A RO RA AND G.c. SRIVASTA VA

apical meristem cell can undergo a certain number of mitotic divisions to produce organs such as leaves and flowers. Cessation of the cell division in the meristem is called mitotic senescence (Hensel et al 1994). Telomere shortening has been implicated in controlling replicative senescence in mammal (Bodnar et aI 1998). In contrast, post-mitotic senescence occurs in organs such as leaves and petals. Once formed, cells in these organs rarely undergo cell division (Colen-Carmona et a11999) but these cells undergo cell growth and ultimately cell degeneration or senescence. Telomere length in these cells remains stable during leaf growth and senescence (Riha et al 1998, Zentgraf et al 2000).

Our interest is basically in understanding mechanisms and control of leaf senescence (a type of post-mitotic senescence). Studying senescence is interesting from much theoretical perspectives however, it is also of practical value as a factor in plant productivity and also in post-harvest longevity. Better understanding of senescence, a process that limits yield, nutritional values and marketability of many crops, will lead to ways of manipulating senescence for agricultural application (Iones 2004). Leaf senescence is undesirable in vegetable crops in which the leaf is the part consumed and for producers of fresh cut flowers, petal senescence reduces the commercial value of the crop. Moreover, if leaves are kept photosynthetically active for a longer period of time, this may have a positive influence on seed yield. At this point, the critical issue is whether retention of green area during post­anthesis (usually drought prone period) will also increase the grain yield. Positive associations have been observed in range,of cereals, including wheat (Evans et al 1975), maize (Wolfe et aI1988), oat (Helsel & Frey 1978), sorghum (Henzell et a11992) and other crops (Thomas & Smart 1993, Thomas & Howarth 2000). For example, the record yield of maize gram, almost 24,000 kg ha-l, was achieved in 1985 on a farm in Illinois, with a variety FS 854, a stay green maize line (Tollenaar 1985). Further, it is not only delayed senescence trait but its incorporation with early leaf development would enable us to get maximum benefits of manipulating leaf/plant senescence (Thomas & Howarth 2000).For crops of dual purpose like sorghum and millets in regions of semi arid tropics, extended period of leaf greenness is of vital importance in the struggle to feed populations living under harsh economic and environmental circumstances, besides they possess leaves with higher nutritional quality and attractiveness to grazing animals (Van Oosterom et aI1996).

In view of above advantages, it is important to understand the process of senescence first and then to manipulate it for economic purposes.

SENESCENCE

Leaf senescence

After a photosynthetically produ ctive period, the leaves con tribution of photosynthate to plant diminishes and the leaf then enters into its last stage of development i.e. senescence. Indeed, leaf senescence can be viewed as a recycling programme at the plant organizati onal level (Quirino et al 2000). During senescence, nutrient such as nitrogen, phosphorus and metals that were earlier invested in the leaf are reallocated to younger leaves and growing seeds or stored for the next growing season (Buchanan-Wollaston 1997).

Monocarpic senescence

Monocarpic senescence, which follows the reproductive phase of many plants, is one of the most dramatic and probably most complex forms of senescence (Nooden et al

P LANT S ENESCENCE P ROCESS AND P RODUCTIVITY 217

1997). Usually, Monocarpic plants stop replacing their old vegetative organs, for example, leaves, during reproductive development and the remaining organs are signaled to die (Nooden 1980).The reproductive structures often govern senescence of whole plant with especially striking effects on leaves. Soybean, for example, shows a decreased in growth rate as flowering gets initiated. So, usually correlative controls (i.e, control by specific plants organs) play an important regulatory role in monocarpic senescence (Nooden 1988a). Further, removal of pods before the seeds mature delays the senescence and death of the plant. In soybean, cessation of growth in the shoot and induction of senescence are clearly separate activities, and depodding does not reinstate muc h vegetative growth . This correlative control therefore plays major roles in coordinating development including senescence at the whole plant level. Due to this the factors those usually delay reproductive development in monocarpic plants can thereby also delay the death of the plant (Nooden 1988a). It should be noted however that Arabidopsis thaliana, which has become a model for studying developmental processes, does not show the control of monocarpic senescence by the reproductive structures that is so evident in soybean and many other monocarpic species (Hensel et a11993,Nooden et a11996). In Arabidopsis excision of the reproductive structures promotes both bolt and leaf production, whereas, the sterility mutations mainly increase bolt production. Thus, it appears that the reproductive structures do exert correlative controls of whole plant senescence in Arabidopsis; however, it is different from most other flowering plants in that they act on regenerative growth (rosette leaves and photosynthetic bolts) rather than on leaf senescence (Nooden & Penney 2001).

Factors affecting senescence Like other genetically programmed developmental processes, leaf senescence,

particularly its initiation, is subject to regulation by many environmental and autonomous (internal) factors (Nooden 1988b, Smart 1994, Gan & Amasino 1997, Bleecker & Patterson 1997, Nam 1997, Dai et a11999). The environmental cues include stresses such as extremes of temperature, drought, ozone, nutrient deficiency, pathogen infection, wounding, darkness, shading and salicylic acid whereas the au tonomous factors inclu de age, reprodu ctive development, levels of phytohormone and sugar, and decline in photosynthesis. It has been shown that age is the major factor that controls leaf senescence in many plant species such as Arabidopsis (Hensel et al 1993) and soybean Giang et al 1993). Although the effect of brassinosteroids on plant senescence has not been extensively examined bu t certain genetic evidences indicate their involvement in leaf senescence. The det2 (de-etiolaled2) mutation has a defect at an early step in brassinosteroids biosynthesis and this was reported to confer delayed leaf senescence symptoms i.e. delayed leaf yellowing (Chory et a11991).

Plant responds to adverse environmental/external factors by initiating changes that may result in leaf senescence and abscission, precocious seed development and a reduced plant life span. These changes can be reversed if grow th conditions once again become favou rable at suitable time during plant development. In contrast, natural senescence (induced by endogenous factors), which is integral part of developmental programme, occurs even under the most optimal environmental conditions and could not be reversed. Likewise, it is also imp ortant to distinguish senescence mutan t from life cycle or environm ental response mutation, which alter senescence only indirectly (Nooden 1988a). For example, a single gene mutation can convert biennial su gar beets to ann uals by elimin ating the vernalization requirement and male sterility inhibits monocarpic senescence in soybean by blocking pod development (Nooden & Guiamet 1989).

V IJAY P AUL, AJAY ARO RA AND c.c, SRIVASTAVA218

Ch anges occurri n g d u rin g senesce n ce

Interconnections operate between major metabolic pa thways op erate duri ng senescence (Buchanan-Wollaston 1997, Srivastava 2002). Sugars are broken down to provide energy. Acetyl-CcA derived from the ~-oxidation of fatty acids also enters mitochondria and participate in Krebs cycle. Acetyl-CoAmay also enter into glyoxylate cycle. Acetate that entered into the mitochondria can condense with succinate to give rise to oxaloacetate and eventually sucrose by the process known as gluconeogenesis. Sucrose synthesized in this manner is used for respiration or is exported. Amino acids obta ined from hydrolysis of proteins are transaminated and give rise to glutamine and asparagine, which are the major forms in which amino nitrogen is transported in the phloem tissue. To carry out above said metabolic activities, following enzymes are expressed during the senescence phase: 1. Fructose-I, 6-biphosphate aldolase, 2. Glyceraldehyde-3-phosphate dehydrogenase, 3. Pyruvate orthophosphate dikinase, 4. PEP carboxykinase, 5. Glutamine synthase, 6. Asparagine synthetase, 7'. Isocitrate lyase, 8. Malate synthase, 9. NAD-malate dehydrogenase, 10. Various proteases and lipid-degrading enzymes such as phospholipase D and /),,9 desaturase. The reported expression of a gene encoding a monosaccharide transporter may implicate the role of thisprotein in the mobilization of sugars from the cell (Quirino et al 2001). Besides this, many genes that encode PR proteins (e.g. chitinase) are upregulated in senescing leaves. Other upregulated genes encode proteins for scavenging free radicals and detoxification of H20 2• These proteins include superoxide dismutases, which convert the superoxide radical to H20 2. H20 2, in tum, is detoxified by catalases, peroxidases and enzymes of ascorbate glutathione cycle. Genes encoding cytochrome P 450­type monooxygenases and metallothioneins that scavenge metal ions are also expressed in senescingorgans.

Which of the changes associated with senescence are central (or primary) and which are peripheral (or secondary)? This is the topic of interest even today as precise understanding underlying the processes of senescence remain poorly understood (He et al 2001). But, collectively, the central and periphera l changes form a syndrome of changes associated with senescence. So, the term senescence is used loosely to refer to the senescence syndrome (Nooden 1988b). The senescence process takes place in a highly regulated manner and the cell constituents are dismantled in an ordered progression. Chlorophyll degradation is the first visible symptom of senescence but by the time yellowing of the leaf can be seen, the majority of the senescence process has occurred.

Chlorophyll degradati on: The pathway for chlorophyll degradation has been elucldated in the last few years (Matile et a11999) and a number of the genes in the pathway have been cloned. None of these however, show an enhanced expression during senescence (Takamiya et al 2000). The key enzyme in the pa thway appears to be pheophorbide a oxygenase that cleaves the tetrapyrrole ring to produce RCC (Hortensteiner et aI 1998). A stay green mutant of Festuca pratensis (sid) accumulates pheophorbide a and is assumed to have a defect in the gene encoding pheophorbide a oxygenase (Vicentini et aI1995). The activity of pheophorbide a oxygenase increases dramatically during senescence, implicating this enzyme as a control point in the process. Cloning and characterization of this gene would be a key step in the elucidation of the control mechanisms for chlorophyll degradation. The final products of chlorophyll catabolism, known as non-fluorescent chlorophyll catabolites (NCCs) are deposited in the vacuole with no recycling of any of the nitroge n contained within them (Hinder et al1996, Tommasini et al 1998). Therefore, the energy

P LA NT S ENESCENCE PROCESS AN D P RODUCTIVITY 219

expensive chlorophyll degradation steps are not carried out in order ~o mobilize the nutrients, but take place to detoxify this highly reactive compound as it is released from the pigment­protein complexes. This is essential to maintain the viability of the plant cell while senescence is taking place. The importance of chlorophyll degradation has been well illustrated by the identification of the accelerated cell death 2 (Acd2) gene product as an enzyme involved in chlorophyll degradation (RCC reductase) (Mach et aI 2001). In the absence of this enzyme activity, the accumulation of phytotoxic chlorophyll products such as RCC causes rapid cell death.

Protein degradation: A major question in plant senescence is the conundrum of how the leaf protein, up to 75 % of which is located within the chloroplast, is degraded and mobilized. In addition, what signals act to initiate this process? Many protease genes show induced expression during senescence, but these appear to encode enzymes localized in the vacuole and are therefore not in contact with chloroplast proteins until the membranes disrupt late in senescence. There have been a number of reports to indicate that degradation of stromal proteins such as Rubisco and glutamine synthetase can be initiated non­-enzymatically by reactive oxygen species (ROS) when chloroplasts are incubated in photo­oxidative stress conditions (Ishida et a11999,2002,Roulin & Feller 1998). However, it is not clear whether increased ROS could initiate the early degradation of Rubisco during senescence. Although ROS levels do increase during senescence, this is likely to be the result of macromolecule degradation processes and thus occur after protein and lipid degradation is initiated . There are reports of the activity of aminopeptidases and metalloendopeptidases in the chloroplast and also chloroplast localization of members of the caspase-like proteases family (Roulin & Feller 1998, Shanklin et aI1995). These enzymes may have a role in protein turnover during leaf development but there is no clear evidence to show that they control protein degradation during senescence (Majeran et al2000, Shikanai et al 2001). Degradation of thylakoid proteins such as LHCP II appears to follow a different route. This protein exists as a pigment-protein complex with chlorophyll and its degradation requires the parallel detoxification of the released chlorophyll, as described above. This is shown in the stay green Festuca mutant where the catabolism.of chlorophyll is blocked, the LHCP protein is stabilized and does not get degraded (Thomas & Donnison 2000). The first step in the degradation of proteins such as LHCP may be the removal of chlorophyll catabolite that destabilizes the protem complex, allowing degradation by chloroplast proteases. Vacuolar proteases may not have a role in protein degradation until the final lytic stages after the membranes have disrupted . There is evidence that some senescence-enhanced proteases accumulate in the vacuole as an inactive aggregate, which slowly matur es to produce a soluble active enzyme at later stages of senescence (Yamada et aI 2001).

The ubiquitin pathway for targeted protein degradation is important to control protein turnover during normal development. Increased expression of a polyubiquitin gene SEN3 has been detected in the senescing leaves of Arabidopsis (Park et a1 1998) indic ating that ubiquitin dependent proteolysis may be an important aspect of non-chloroplast protein degrad ation during senescence. The analysis of the Arabidopsis delayed senescence mutant Ore9showed that the ORE9 protein is an F-box protein which interacts with a component of the plant SCF complex which controls selective ubiquitination and subsequent proteolysis of target proteins (Woo et al 2001). This result ind icates that the ubiquitin-mediated degradation of specific proteins has an important role in the control of senescence. The authors postulate that the ORE9 protein may be involved in the degradation of a key

220 VIJAY P AUL,. A JAY A RO RA AN D G.C. SRIVASTAVA

regulatory repressor of senescence and the continuous presence of this may inhibit the onset of senescence. The identification of the proteins tha t are the targets for the ORE9 complex is therefore of great interest to improving our understanding of the control of senescence.

Lipi d degradation: Turnover of membranes occurs regularly in healthy cells. Free radicals accumulate in ageing tissues are known to have deleterious effects on membrane by initiating lipid peroxida tion and in tum induce membrane rigid ification (Mazliak 1983). So, during senescence there is a decline in the structural and functional integrity of cellular membranes that is the result of the accelerated metabolism of membrane lipids (Thompson et al 1998). Enz ymes such as phospholipase D, phosphatid ic acid phosphatase, lytic acyl hydrolase and lipoxygenase have been implicated, certain genes that ma y encode enzymes with these activities show senescence-enhanced expression (Thompson et al 1998, He & Gan 2002). Thylakoid membranes provide an abunda n t source of carbon that can be mobilized for use as an energy source during senescence. Enzymes that carry out peroxisomal fatty acid ~-oxidation are present in ma ture leaves (Graham & Eastmond 2002).

AGE only

identified in carna tion petals (Hong et a12000) showed delayed leaf senescence (Thompson et al 2000). A simila r study wi th an acyl hydrolase gene from Arabidopsis (SAG101) showed that the antisense suppression of this gene delayed leaf senescence by a few days (He & Gan 2002). In addition, over-expression of this gene accelerated the onset of senescence. It is not clear from these reports whether it is the same gene that is being studied; both enz;ymes

Multiple classes of sene scence enhanced genes

I ~ ~

ISymplons of senesence

Fig. 1 : A simple mode l to ill ustrate the ne twork of pathways that are require d for gene expression during senescence (Buchanan-Wollas ton et aI2003).

There have been two reports indicating that altered levels of acyl hydrolase genes that could be involved in lipid de grad ation may affect senescence. Firstly, transgenic Arabidopsis plants that had reduced levels of senescence enhanced lipase, originally

PLANT S ENESCENCE PROCESS AN D P ROD UCTIVITY 221

had fairly weak acyl hydrolase activity when tested with an artificial substrate. The authors suggested th at this enzyme might act to initiate the senescence-rela ted degradation of mE:mbranes. Release of free fatty acids and the consequent pertu rbation of th e lipid bilayer could make it susceptible to further degradation by other lipolytic enzymes. An:alternative explanation for the altered senescence phenotype seen in the under- and over-exp ressing plants may be tha t ±-linolenic acid released from the membranes by the action of thisenzyme could be made available for the synthesis of JA. Altered levels of JA in the leaves could affect the initiation of senescence (Fig. 1) (Buchanan-Wollaston et al 2003).

O th er important events: Nucleic acids, especially RNA, form a valuable source of phosphorus in a mature leaf. Senescence-enhanced expression of genes enc oding several different nucleases has been reported and these presumably act to degrade nucleic acids during senescence. Total RNA levels fall rap idly wi th the p rogress of senescence but nuclear DNA is maintained to allow gene expression to continue, until late in the process. Other valuable leaf constituents include metal ions such as K, Mo, Cu and Fe, and it is likely that much of these, are also mobilized from leaves during senescence. Himmelblau & Amasino (2001) analysed nutrient mobilization from Arabidopsis leaves and showed that the levels of many compounds measured (Mo, Cr, S, Fe, Cu and Zn) were reduced by over 50 % in senescent leaves when compared to green leaves. Levels of the valuable nutr ien ts N, P and K were reduced by at least 80 %. Little is known about the genes that encode enzymes that carry out the mobilization processes. Levels of cytosolic GS increase during senescence and the role of thisenzyme is likely to be in the conversion of amino acids to glutamine to increase the efficiency of ni trogen transport. Increased levels of glutamine have been measured in Brassica napus leaves and phloem in the late stages of senescence (Finnemann & Schjoerring 2000). The cytosolic form of GS is predominantly located in the vascular bundles in senescing rice leaves (Sakurai et a11996) indicating its role in nitrogen transport. Trans crip t levels of GS1 increase during senescence, but post-translational control of GS activity has also been shown to occur by both phosphorylation, which protects the protein from degradation, and by interaction with 14-3-3 p rotein that increases enzyme activity (Finnemann & Schjoerring 2000).

Regulation of senescence Genes expressed during leaf senescence have been isolated from several species,

notably Arabidopsis (SAG), Brassica (LSC),maize (See) and tomato (SEN U) etc. Usually SAG (senescence-associated gene) is the term used to rep resent the genes linked to the process of senescence. Ripening-associated genes from fruitsuch as tomato, banana and melon include homology of SAG. About 50 cDNAs have been assigned possible functions in senescence on the basis of sequence homology. But even today, only in a few cases their functions have been conform ed biochemically or physiologically. Till today, no clear p attern emerges that might allow us to devise a simple model for regulation of gene expression during senescence. It has been postula ted that there may be multipl e p athw ays that respond to various autonom ous and environmental factors, and that these pathways are possibly interconnected to form a regulatory ne twork to control leaf senescence (Gan & Amasino 1997, He et a12001). A pu tative leaf senescence regulatory network in Arabidopsis has been p roposed by He et al (2001) where, effect of senescence p romotive factors, external and in ternal (abscisic acid, jasmonic acid, darkness, ethylene, epibrassinolide, dehydration, age and other), were examined on senescence associated up regu lation -of genes . Senescence is also reported to

222 V IJAY PAUL, AJAY ARORA AND G.C. SRIVASTAVA

be modulated by gibberellins (Kappers et al 1998) and cytokinins (Smart et al 1991): Transformants created in Arabidopsisand tobacco with tmr or ipt genes clearly showed that over production or senescence induced production of cytokinin delayed the process of senescence (Smart et a11991, Gan & Amasino 1995).

Recently, Cowan et al (2005) studied the effect of altered cytokinin metabolism caused by senescence-specificautoregulated expression of the Agrobacterium tumefaciens 1PT gene under control of the promoter of SAG12 (PSAG12-1PT) on seed germination and the response to a water-deficit stress in tobacco. Cytokinin levels, sugar content and composition of the leaf strata within the canopy of wild-type and PSAG12-1PT plants confirmed the reported altered source-sink relations. No measurable difference in sugar and pigment content of discs harvested from apical and basal leaves was evident 72 h after incubation with ABA or in darkness, indicating that expression of the transgene was not 'restricted to senescing leaves. No difference in quantum efficiency, photosynthetic activity, accumulation of ABA, and stomatal conductance was apparent in apical, middle and basal leaves of either wild-type or PSAG12-IPT plants after imposition of a mild water stress. However, compared to wild-type plants, PSAG12-1PT plants were slower to adjust biomass allocation. A stress­induced increase in root:shoot ratio and specific leaf area occurred more rapidly in wild­type than in PSAG12- 1PT plants reflecting delayed remobilization of leaf reserves to sink organs in the transformants. PSAG12-1PT seeds germinated more slowly even though ABA content was 50 % that of the wild-type seeds confirming cytokinin-induced alterations in reserve remobilization. Thus, senescence is integral to plant growth and development and an increased endogenous cytokinin content impacts source-sink relations to delay ontogenic transitions.

Stillwe do not know how the various cellular senescence programmes are integrated during the development and life history of organs or whole plants. Perhaps a 'd ie now' signal is constantly present and a particular cell, tissue or organ becomes competent to respond to the signal at a time dictated by its own individual developmental programme. Alternatively, certain cell-specific 'die now' stimuli may inv olve senescence or death programme in their targets (Wilson 1997). ­

As discussed above, senescence is closely associated with reproductive development and nutrients are often redistributed from the vegetative parts to the developing reproductive sinks, so various forms of nutrient starvation have been invoked as causal in senescence of vegetative parts . The nutrient starvation ideas provide an attractive and easy to understand explanation for a very complex process however, there are some direct evidence against it (Nooden 1988a, Mauk & Nooden 1992). The results obtained by Zhou et al (2000) indicated that intra-plant competition for reduced carbon play an important role in plant senescence. Technology like enhancer trap lines (He et al 2001) start to reveal the complexity of the network of senescence regu lated pathways and will allow for the identification of many additional SAG. The identification of senescence specific promoter elements (Noh &: Amasino 1999) and generation of mutants and transgenic plant will help us to better understand the regulation of senescence related genes during senescence. An increased understanding of the genes that control plant senescence is very important for future agronomic improvements in many crop species (Lim & Nam 2005). Recent approaches like, enhancer trap lines, identificati on of senescence specific promoter elements, genera tions of mutant and transgenics and DNA microarrays will lead to decipher temporal and spatial expression patterns for hundreds of genes involved in senescence. Increased understanding to the

PLANT S ENESCENCE P ROC ESS AND PRODUCTIV ITY 223

initiation and execution of senescence would allow us to increase vase life and horticultural performance of ornamen tals, increase yield in agronomic crops and decrease postharvest loses of fruits and vegetable (Jones 2004, Woo et a12004).

Emerging role of sugars and the rate of photosynthesis as such in regulating the senescence are discussed later in this ar ticle.

Senescence as a p rogrammed process Senescence and death serve very diverse functions, and often, death is the end result

of internally programmed degeneration termed senescence (Nooden et al 1997). Even before much was known about the biochemistry of senescence, it was considered to be internally programmed, because it is specific and orderly in terms of when, where and how it occurs (Nooden & Leopold 1978). Senescence is not a case of passive decay of structural and biochemical machinery of cells. Rather it is a highly regulated, ordered series of events in which organelles, membrane and macromolecules are broken down and nutrien ts (amino acids, sugars and minerals) are reclaimed for export out of senescing leaf to be reused in other parts of plants. It is important to mention here that certain cell organelles and tissues remain intact and functional until after mobilization is completed. As chloroplasts are rich reservoirs of pro teins and lipids, they are degraded first. Mitochondria and peroxisomes remain functional till late. Nucleus also remains functional and transcriptionally active. Guard cells in epidermis and phloem tissue remain functional for gas exchange and transport respectively, until the metabolic breakdown of chloroplast and export of me tabolites is mostly completed. Studies with enucleation, selective inhibitors, mutations and genetic engineering have supported the idea that senescence is an active process and is controlled by nucleus (Nooden 1988c, Buchanan-Wollaston 1997, Gan & Amasino 1997, Nam 1997).

Although, the expression of many genes is down regulated in senescing tissues (Brady 1988) but senescence does not seem to be caused by turning genes off (Nooden 198&). Nonetheless, down regulation may be necessary to prevent regeneration of cell components broken down in the senescence process. The available evidences the refore strongly supp ort the concept that senescence is a form of pe D, which in broad term refers to a process by which cells promote their own death throu gh the activation of self-destru ction systems. However, it is unclear whether leaf senescence share any biochemical or genetic pathways with other types of PCD in plants or an imal (Gan & Amasino 1997).

SENESCENCE AND PHOTOSYNTHESIS

Changes in chloroplast and photosynthetic apparatus Chloroplasts, which contain most of the proteins in a leaf cell, are one of the first

organelles to be targeted for breakdown. Disappearance of chlorophyll is one of the most prominent phenomena of senescence, and eventually the rate of chlorophyll degradation is usually considered to be a reliable criterion of leaf senescence and a measure of the age­related deteriorations of the photosynthetic capacity (Thomas & Stoddart 1980, Quirino et al 2000).

One of the cons picuous changes that occur during senescence is a rapid decline in photosynthesis (Jiang et al1993). Initially loss of integri ty of stroma thylakoids occurs and this is followed by gradual disintegration of granal thylakoids. Since, PS II is located mainly in the graria and PS I is distributed both in grana and stroma lamellae the early loss of

224 VIJAY PAUL, A JAY ARORA AN D G.c. SRIVASTAVA

stroma lamellae exp lains why PS I declines faster than PS II (Bricker & Newman 1982). Marked decline in the rates of non-cyclic electron transport with the progress of leaf senescence was observed (Holloway et aI 1983). Since, photosynthetic electron transport produces ATP and NADPH, both requi red for enzymatic reactions of the photosyn thetic carbon red uction cycle (Calvin cycle), a decline in this process would inevitably red uce the rate of CO2 fixation (Bricker & Newmann 1982, Holloway et aI1983) . Further loss of cyt b/ f complex limits the rate of electron transport, which in tum limits the rate of photosynthesis in the senescing leaf (Bricker & Newmann 1982, Holloway et aI1983).

It has been demonstrated that loss of photosynthetic activity is usually accompanie d by a concomitant decrease in the activity of Rubisco during senescence (Hall et al 1978, Camp et al 1984), however, in several species it was not found true (Secor et aI1983). Studies per taining to several regulatory enzymes of Calvin cycle during leaf development/ senescence (Batt & Woolhouse 1975, Sexton & Woolhouse 1984) allowe d them to conclude that, enzymes which are encoded in chloroplast and synthesized on 70S ribosomes within the organelle are lost earlier, whereas the second group of enzymes that are encoded in the nucleus and synthesized on 80S ribosome decline at later stages of senescence. Rubisco presents a particularly interesting problem because it is composed of two sub-units, the large subunit is encoded by the chloroplast on 70Sribosomes and synthesized within the organelle and the small subunit is synthesized on 80S cytoplasmic ribosomes.

Photosynthetic rate and senescence It has been proposed that ageing or stress that contributed to a loss of photosynthetic

output below a certain threshold level or loss in the integrity of chloroplast membrane might produce signal that initiate the senescence programme (Linda et aI1993, Smart 1994, Blacker & Patterson 1997,Quirino et al2000). Potential regulatory processes that drive the expression of PAG and SAG transcripts are proposed by Linda et al (1993) in Arabidopsis. The emphases of the proposed model were on the transcriptional control of PAG and SAG genes. Following sequence of events are proposed: (1) matur ation signals suppress PAG gene expression at full leaf exp ression, (2) photosynthetic function .declines as a result of inadequate maintenance, (3) metabolites of photosynthesis (chemical identity unknown) which act to repress SAG gene expression in young leaf, decline in concentration, resulting in derepression of SAG genes and (4) SAG gene products act to mobilize nutrients and as a consequence, promote the senescence of the leaf tissue.

Sugars as regulator of senescence

One of the earliest features of leaf senescence is the decline of photosyn thesis. So, it has been proposed that sugars (primary product of photosynthesis) if decrease in the leaves trigger the senescence programme (Noh & Amasino 1999, Quirino et a12000). The expression of several SAGs are induced by sugar starvation and suppressed by sugars (Noh & Amasino 1999, Fujiki et al 2001). However, recent physiological and genetic analysis favour an opposite hypotheses, that is, the increased sugars levels or sugar signals induce leaf senescence (Quirin o et al 2001, Stessman et al 2002). Since hexokinase is known to be a sugar sensor that med iates bot." the sugar signa ling and the catabolic activity of hexose phosphorylation, so its role has also been studied in senescence (Dai et al 1999, Noh & Amasino 1999, Xiao et al 2000). All of these results su ggest tha t an enhanced sugar signal Fan induce premature senescence, most likely via a hexokinase function . So, increased sugar levels and / or signals rep ress photosynthetic activity by negative feedback regulation.

P LANT SENESCENCE P ROCESS AND PROD UCTIV ITY 22 5

{Rolland et al 2002, Gibson 2005) and this system might be involved in inducing leaf senescence. However, studies in tobacco plants showed tha t the glucose and fructose levels increase as the leaves progress through senescence (Wingler et a11998). This increased sugar levels during tobacco-leaf senescence could not be explained in light of hexokinase-over expression and SAG12 expression studies (Dai et al 199C;, Noh & Amasino 1999). Thus, how sugars affect the induction of leaf senescence under natural conditions is still unclear . Source-sink balances ma y affect the pa rtitioning of sugar within a plant and hence induce leaf senescence. Youn g leaves develop their own photosynthetic machinery and so their demands for sugars begin to decrease after they mature. Such limited demands may lead to the accumulation of sugar and the induction of senescence in old leaves. This is supported by the fact that when young leaves are shaded, and therefore, cannot produce sugars and their senescence is retarded (Ono et aI 2oo1). Besides the source-sink balance, sugar mediated control of senescence is also influenced by other factors such as; nitrogen status, light conditions and developmental stage (Iordi et al 2000, Ono et al 2001, Pau l & Foyer 2001, Weaver & Amasino 2001). Thus, the integrated networking of signal in response to internal and external factors is important in induction and controlling the leaf senescence (Yoshida 2003).

Plasticity in senescence

Senescence is as responsive as any other physiological activity to sub-or supra­optimal environmental conditions . The patterns of SAG expression change in response to different treatments or conditions (Oh et a11996, He et a12001,). In addition, the expression level of individual SAG genes may also changes in response to diffe rent stimulus (Oh et al 1996). For example, shading a plant often greatly extends the green area duration of its mature leaves (Mae et a1 1993). In maize, detopping has been shown to down-regulate senescence­enhanced genes (Griffiths et al1997). A vast array of growth regulators, metabolic inhibitors and chemicals of unknown mode of action will also stop yellowing (Weaver et a11998).

Generally, the chloroplast env elope retains its integrity until very late when the inte rnal membranes are alread y completely broken , It has been suggested by Woolhouse (1984) that maintenance of envelop inte grity might assist in regreening as new thylakoids are initiated from the inner membranes of the chloroplast envelop even at advanced stages of senescence besides the major role of envelop in the p rocess of export of material (remobilization) via contro lled unloading. Thus, manipul ating pl an t /leaf senescence through breeding or genetic engineering might helps to improve crop yields by keeping leaves photosynthetically active for longer (Nelson 1988). This trait will increase the concentration of stem sugars and thereby improves quality of grain and fodder in dual-purpose crops like sorghum (Van Oos terom et aI 1996).

Leaf senescence at elevated CO 2 concentration

During past years many studies at elevated CO2 concentrations observed that follow ing an in it ial increase photosynthesis .often declines a t high CO2 levels . Th is acclimation at elevated CO2 is not a simple down-regulation of photosynthesis but rather results in a temporal shift of leaf 'development towards premature senescence (Millar et al 1997). The molecular reasons underlying prem~ture senescence at elevated CO2 are not clear. Most of the workers favour the idea that at elevated CO2 levels carbohydrate accumulation exerts a nega tive feedback inhibition on pho tosynthesis, which most likely occurs at the,

226 VIJA Y PAUL, AJAY ARORA AND G.e. SRIVASTAV A

le~el of RUbis ~ o (Miglietti et aI199?, Si?,er & Bunce 199? ,.On? & Watanabe, 1997). Study with transgenic tobacco plants havmg ipt gene for cytokinm biosynthesis under control of the senescence induced SAG 12 promoter showed that under high CO2 levels senescence and the down regulation of photosynthesis-rela ted genes are re tarded in these plants, while levels of soluble sugars were ind istingu ishable from wild type (Ludewig & Sonnewald 20(0). Th is result s trongly a rgu es against a ro le for soluble sugars in the regulation of photosyn thesis-related transcripts under elevated CO2, The author suggest th at high CO level accelerate senescence by increasing the flow of sugars through hexokinase. Indeed~ hexokinase over expression leads to accelerate senescence in tomato plants (Dai et aI1999). Comparative studies in senescence at different CO2 levels could give new insight into the in terrelationship of photosynthesis and senescence.

DELAYED SENESCENCE, STAY GREEN MUTANTS AND PLANT PRODUCTIVITY

Transgenic approach -Transgenic tobacco plants that show delayed leaf senescence because of the auto

regulated production of the senescence inhibiting horm one cytokinin also showed increased seed prod uction as well as inc reased biomass (Gan & Amasino 1995). Leaves of this transgenic plant (PSAG12-IPn exhibit a p rol onged photosynthetic life span, which was responsible for 50 % increase in dry wei ght and seed yield incomparison to wild-type. The use of a similar system to improve stress tolerance has been rep orted . Arabidopsis transformed wi th thesame SAG12: ipt fusion showed delayed senescence as well as an increased tolerance to flooding (Zhang et al 2000).

Besides making use of cytokinin, greenness can also be altered by down-regulating

I the production of a senescence promoting hormonal. Tomato plants in which ethylene biosynthesis is inhibited by antisense suppression of the gene for ACC-oxidase exh ibit

I delayed leaf senescence 0 000 et al 1995). A similar phenotype is apparent in ethylene­insensitive mutants of Arabidopsis (Grbic & Bleecker 1995).

Stay green mutants In contrast to transgen ic with delayed senescence, stay green is the term given to a

variant (natural mutant) in which senescence is de layed compared with a standard reference genotype. It has been shown by Thomas & Smart (1993) that stay green phenotype could arise in one of four fundamen tally dis tinct ways. Type A: Senescence is initiated late but then proceeds at normal rates. Type B: Senescence initiated on schedule but thereafter senesce comparatively slowly. Type C: Chlorophyll is retained more or less indefinitely but measures of physiological function such as photosynthetic capacity show that senescence is proceeding no rmally beneath the cosmetic su rface of retained green p igmentation and Type D: Colour of the leaf is retained due to rapid killing of leaf. In practice, stay green can be combinations of two or more different types as described above .

In a study, the net contribution over the lifetime of 4th leaf of Lolium temulentum was 36.8 mg of carbon and delaying the start of senescence jus t by 2 days increased the carbon contribution of the leaf by 4.1 mg or 11 % (Gay & Thomas 1995). The scale of response on productivity wi th such a rela tively small modification emphasizes the importance of delayed senescence trait in crop productivity . .

WA P LANT S ENESCENCE PROCESS AND PRODUCTIV ITY 227

idy Sorghum genotypes vary in the timing of senescence ini tiation and also in the lof subsequent rate of leaf senescence (Borrell et al 2000a). Some genotypes, however, not only nce remain green, but also contain significantly more carbohydrates in the stem at all matu rity tile stages (Me Bee et aI1983). Drought during grain filling hastens leaf senescence (Rosenow )0). & Clark 1981)however, stay green genotypes retain more green leaf area than do genotypes of not possessing this trait, and so they continue to fill grain normally under drought conditions

:°2 also (Rosenow et aI19S3). Positive associations between stay green and grain yield un der ed, water-limited environment has been shown by Borrell & Douglas (1996). Stay green also ~9) . reduces lodging, in view of more sugars in stem (Borrell et a12000b) and also resistance to the stem rots as well (Rosenow 1984) suggesting that stay green leaves remain photosynthetically

active.

In wheat duration of grain filling was the main factor limiting the yield (Gelang et aI2000). Physiological characterization of stay green mutants in duru m wheat (Spano et al 2003) showed that net photosynthesis of flag leaf was maintained longer in mutants than in parent line. The mutants had 10-12 % increase in seed weigh t when grown in greenhouse and it was presumably related to the extended duration of photosynthesis resulting in lIto increased translocation of photoassimilate to the grain. However, it is necessary to verify red

his the effects of the stay green mutation on yield of durum wheat under field conditions, particularly under drought stress which is common especially during the grain filling per iod.

,"he Further, as the significant proportion of protein N was retained in leaves of stay green mutant led so it may prove necessary for farmers to provide additional nitrogen fertilizer in order to nce produce grain with sufficiently high protein (Spano etaI2003).

The study with non-yellowing mutant of Festuca pratensis indicated that thylakoid ing membrane bound proteins are more stable in mutants (Hilditch et al 1989). Similar me observations were made on stay green lines of Phaseolus vulgaris (Bachmann et aI 1994). It bit has been speculated that the reduced chlorophyll breakdown in the mutant of Festuca ne- pratensis is due to altered lipid metabolism in the mutant was due to altered lipid metabolism

in the thylakoids of mutant which reduced the accessibility of chlorophyll and other membranes cons tituents proteins to the degradative systems (Thomas 1982, Harwood et al 1982).

~as

o a It is known that for foliar senescence genetic variations exist. Incidences of delayed ice

or inoperative senescence have been documented in maize, sorghum, oat, rice wheat, fescue, lid soybean, french bean, fruit crops, trees and other species (Thomas & Smart 1993). The search lUt for physiological and biochemical determinants of crop production has been domina ted by ice the belief that the rate of carbon assimilation is directly related to yield. But, it was also realized that most of the diversity in yield for most crops is a consequence of variation in the duration rather than the rate of photosynthetic activity. During past, concept of leaf area duration was correlated with production in a wide range of crops under a var iety of agronomic conditions . More effective concept of green area duration was put forward by Waggoner & Berger (1987), they introduced the concept of healthy area duration, ana logous ras

on to leaf area duration and green area duration and showed that this index is tightly coupled

on to yield in a number of crops. Since, de layed senescence or stay green trait represent most consistent and accessible physiological parameter that correla tes with productivity so this d extended greenness might be expected to be a well established objective in crop breeding and improvement strategies.

228 VIJAY P AUL, AJAY ARORA AND G.c. SRIVASTAVA

Stay green mutants are also valuable tool for the investigation of regulatory mechanisms underlying senescence specific changes in chloroplast. Mutation of the nuclear sid gene of Festuca disables chlorophyll degradation during leaf senescence. In this mutan t, in spite of unchanged chlorophyll content, CO2 fixation rate decreases similarly to that in the wild-type (Thomas 1987, Hauck et al1997) . While, some stay green mutants of soybean retrain substantial amounts of Rubisco and other exhibit a significant retardation in the loss of soluble proteins (Guiamet & Giannibelli, 1996). In addition, Oh et al (1997) have identified three genetic loci in stays-green mutants of Arabidopsis that show delay in breed range of senescence-associated events including chlorophyll breakdown, increases in RNase activity and decline in Rubisco.

It seems from survey of available literature on stay green and associated processes that different processes such as chlorophyll degradation, decrease in soluble proteins and the decrease in photosynthetic function are not strictly coupled during senescence and may be regulated independently. So, it will be important to identity the genes affected in various types of stay green mutants and to study their function with regard to the regulation of photosynthesis and chloroplast breakdown (Krupinska & Humbeck 2004). So far interest in stay green character has been largely empirical and incidental. But, real exploitation of the stay green phenomenon in future for enhancing plant productivity depends on our skill to attain knowledge of physiological, biochemical and genetic basis of this important tra it.

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