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Keywords:

  • Cereals;
  • leaf senescence;
  • nitrogen remobilisation;
  • protein degradation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cereals as model plants for studies on leaf senescence
  5. Dismantling of the photosynthetic apparatus and chloroplast protein degradation
  6. Plastidial and extraplastidial proteases involved in plastid protein degradation
  7. Nitrogen metabolism and re-translocation
  8. Senescence-associated gene expression
  9. Genetic modulation of senescence phenotypes
  10. An ideal senescence phenotype of cereals
  11. Acknowledgements
  12. Conflicts of Interest
  13. References

Extensive studies have been undertaken on senescence processes in barley and wheat and their importance for the nitrogen use efficiency of these crop plants. During the senescence processes, proteins are degraded and nutrients are re-mobilised from senescing leaves to other organs, especially the developing grain. Most of the proteins degraded reside in the chloroplasts, with Rubisco constituting the most dominant protein fraction. Despite intensive studies, the proteases responsible for Rubisco degradation have not yet been identified. Evidence for degradation of stromal proteins outside of chloroplasts is summarised. Rubisco is thought to be released from chloroplasts into vesicles containing stroma material (RCB = Rubisco-containing bodies). These vesicles may then take different routes for their degradation. Transcriptome analyses on barley and wheat senescence have identified genes involved in degradative, metabolic and regulatory processes that could be used in future strategies aimed at modifying the senescence process. The breeding of crops for characters related to senescence processes, e.g. higher yields and better nutrient use efficiency, is complex. Such breeding has to cope with the dilemma that delayed senescence, which could lead to higher yields, is correlated with a decrease in nutrient use efficiency. Pinpointing regulatory genes involved in senescence might lead to tools that could effectively overcome this dilemma.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cereals as model plants for studies on leaf senescence
  5. Dismantling of the photosynthetic apparatus and chloroplast protein degradation
  6. Plastidial and extraplastidial proteases involved in plastid protein degradation
  7. Nitrogen metabolism and re-translocation
  8. Senescence-associated gene expression
  9. Genetic modulation of senescence phenotypes
  10. An ideal senescence phenotype of cereals
  11. Acknowledgements
  12. Conflicts of Interest
  13. References

High nutrient mobilisation efficiency is an intrinsic feature of plant senescence. This complex process, leading eventually to the death of the photosynthetic and other vegetative organs, involves a well-orchestrated activation of genes encoding catabolic enzymes that gradually dismantle cellular components that mainly reside in the chloroplast. At the same time, basic metabolic activities are kept intact until cell death to ensure the processing of high molecular weight components and the subsequent export of the degradation products and minerals to the phloem. Accordingly, senescence is an intriguing basic biological process essential for the life cycle of plants. Most of the mechanisms are still elusive but it is apparent that plants share a common programme for senescence. This programme can be modulated by a range of abiotic and biotic factors such as light, UV, ozone, hormones, drought and various pests (Lim et al. 2006). It is, therefore, a major scientific challenge to obtain further insight into the signal transduction events and regulatory mechanisms underlying senescence.

From the applied perspective, the importance of well-orchestrated and robust senescence in crop plants can hardly be overestimated as it is a central component in the nutrient use efficiency, i.e. the ability of the plant to mobilise and translocate nutrients to sinks such as grains, tubers and roots. This parameter overlays and is tightly integrated with the plant nutrient uptake efficiency. The relative importance of the two processes appears to differ between different plant species. Hence, small-grained cereals like barley, wheat and rice may mobilise up to 90% of the nitrogen from the vegetative plant parts to the grain, while in maize 35–55% of the grain nitrogen is derived from soil uptake after anthesis. In recent years, substantial efforts have been invested in understanding the complex genetics underlying, in particular, nitrogen use efficiency in cereals (Mickelsen et al. 2003; Habash et al. 2004; Hirel et al. 2007).

Barley and wheat have been subjected to breeding over the last several thousand years to produce higher yields, initially by selecting for many and large grains and later for plant architecture and responsiveness to fertilisers. In consequence, modern cultivars are characterised by a high degree of nutrient use efficiency, where nutrients are mobilised from old to new leaves and eventually into the developing grain. Due to their economic importance, barley and wheat have been studied intensely with respect to senescence, and these two species can in many ways be regarded as model plants for studying this particular process. These studies have currently been intensified due to a growing demand for improved barley and wheat productivity, quality, environmental robustness and nutrient use efficiency.

In the present review we will present available evidence on the underlying mechanisms, the course and the characteristics of leaf senescence in barley and wheat, and will discuss how these processes are initiated and regulated. We will focus primarily on nitrogen metabolism and current breeding efforts aimed at improving nitrogen use efficiency and yield, as well as the quality of barley and wheat grains.

Cereals as model plants for studies on leaf senescence

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cereals as model plants for studies on leaf senescence
  5. Dismantling of the photosynthetic apparatus and chloroplast protein degradation
  6. Plastidial and extraplastidial proteases involved in plastid protein degradation
  7. Nitrogen metabolism and re-translocation
  8. Senescence-associated gene expression
  9. Genetic modulation of senescence phenotypes
  10. An ideal senescence phenotype of cereals
  11. Acknowledgements
  12. Conflicts of Interest
  13. References

In cereals, senescence appears to be regulated at the level of the individual leaf. Nutrients are thus mobilised from the older leaves to the younger leaves and eventually to the flag leaf, which contributes the majority of the nutrients and photoassimilates used for charging of the grain (Stamp & Herzog 1976; Wiedemuth et al. 2005). As opposed to dicotyledonous species, leaves of cereals have a basal meristem, and the leaf tip consists of the oldest cells while the youngest cells are found at the leaf base (Robertson & Laetsch 1974; Boffey et al. 1980; Mullet 1988). Due to this organisation, cereal leaves are ideal for studies on the progression of senescence. Excised cross sections of leaves provide samples of cells at the same developmental stage that can be studied in vitro and subjected to various treatments under controlled conditions. Different aspects of senescence can thus be studied in such in vitro systems, eliminating potential effects from the other parts of the plant (Fischer & Feller 1994).

Decline of chloroplast populations

Up to 75% of the reduced nitrogen in photosynthetically active cells of cereal leaves is located in chloroplasts (Peoples & Dalling 1988), mainly as Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) (Hörtensteiner & Feller 2002). Accordingly, the dismantling of the photosynthetic apparatus is the major event of senescence, providing most of the nitrogen to be translocated to the grain. The process commences by a decline in photosynthesis. So far, it is unknown to what extent the reduced photosynthetic capacity results from a reduction in the number of chloroplasts or whether the performance of the entire chloroplast population is affected (Krupinska 2006). Evidence for both scenarios has been presented. Several studies have shown that during wheat leaf senescence there is a decrease in the number of chloroplasts per cell (Wittenbach et al. 1980, 1982; Wardley et al. 1984; Ono et al. 1995). In field-grown wheat the number of chloroplasts per mesophyll cell was found to decrease gradually until the last stage of leaf senescence, when a rapid degradation of chloroplasts takes place (Ono et al. 1995). In contrast, in barley leaves the number of chloroplasts appeared to be largely constant until late senescence, while the chlorophyll and protein concentration was reduced earlier (Martinoia et al. 1983; Matile 1992). However, as discussed by Krupinska (2006), the reported discrepancies between chloroplast dynamics during leaf senescence in barley and wheat may be attributed to different methodologies. Senescence appears to proceed at different rates among neighbouring cells (K. Krupinska and M. Mulisch, unpublished observations) and the more intact cells may be those most amenable to analysis; in particular when the analysis is based on protoplasts, as was the case in the study by Martinoia et al. (1983).

Ultrastructural characterisation of changes occurring in chloroplasts persisting during senescence

As described above, cereal leaves provide a unique source for leaf cells at a specific developmental stage. The gradient in cell age is accompanied by a corresponding gradient in chloroplast development, with proplastids in the cells of the basal meristem and mature chloroplasts in cells at the leaf tip (Mullet 1988; Krupinska 1992) leading to detailed ultrastructural studies of chloroplast development during senescence in leaves of wheat (Mittelhäuser & Van Stevenick 1971; Hurkman 1979), rice (Hashimoto et al. 1989) and barley (Kolodziejek et al. 2003). The senescing chloroplasts have been reported to undergo differentiation into so-called gerontoplasts (Sitte 1977). The general changes observed at the ultrastructural level comprise a reduction of the thylakoid membrane system, a loosening of grana stacks, swelling of the intrathylakoidal space and an increase in the number and size of plastoglobuli (Fig. 1) (Thomas et al. 2003). In a recent study, plastoglobuli were shown to be attached to thylakoid membranes and to contain degradation products of the thylakoids (Austin et al. 2006).

image

Figure 1.  A: TEM images showing mesophyll cells in flag leaves from field-grown barley plants before senescence, after loss of 40% of the chlorophyll content and after loss of 90% of the chlorophyll content. All images are enlarged to the same extent. Bar = 5 μm. B: Representative chloroplasts from the same stages of development. Bar = 1 μm in each figure.

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In flag leaves of wheat a progressive disruption of the thylakoids and an increase in osmiophilic globules were observed to precede the loss of integrity of the plastid envelope. At later stages of senescence the outer membrane adjacent to the tonoplast appeared to break while the inner membrane remained intact (Peoples et al. 1980). Two biochemical studies have revealed distinct changes in the protein composition of envelope membranes during senescence (Höinghaus & Feierabend 1985; Guéra et al. 1989). Since, even at a late stage of senescence, protein turnover in the envelope membranes is high, it has been proposed that these changes may facilitate the degradation of chloroplasts by permitting the leakage of chloroplast components and probably the uptake of degradative molecules that are synthesised in the cytoplasm (Guéra et al. 1989).

Heat was shown to accelerate the typical ultrastructural changes in chloroplasts of wheat flag leaves (Xu et al. 1995). When ultrastructural changes in chloroplasts were compared between attached and detached ageing primary foliage leaves of wheat, distinct differences in the sequence of events were observed (Hurkman 1979). While in attached leaves the envelope ruptures after degradation of thylakoids, in detached leaves the envelope was observed to rupture before thylakoids were degraded. These differences may be attributed to differences in the concentrations of effectors such as phytohormones and metabolites in the detached and the attached leaves (Fischer & Feller 1994).

Dismantling of the photosynthetic apparatus and chloroplast protein degradation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cereals as model plants for studies on leaf senescence
  5. Dismantling of the photosynthetic apparatus and chloroplast protein degradation
  6. Plastidial and extraplastidial proteases involved in plastid protein degradation
  7. Nitrogen metabolism and re-translocation
  8. Senescence-associated gene expression
  9. Genetic modulation of senescence phenotypes
  10. An ideal senescence phenotype of cereals
  11. Acknowledgements
  12. Conflicts of Interest
  13. References

A general review of chloroplast protein degradation during senescence in plants is given by Martinez et al. (2008). Here, only the general aspects will be presented, together with highlights of the quite comprehensive literature on protein degradation during senescence in barley and wheat. As described above, the photosynthetic apparatus is the major source for nitrogen and carbon that is recycled during senescence, and stromal enzymes, mainly ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), represent the major fraction of chloroplast nitrogen (Hörtensteiner & Feller 2002). Pigment protein complexes in thylakoid membranes, which include the reaction centres of photosynthesis and the antenna system, account for 30% of the total chloroplast protein (Matile 1992). Studies with wheat and barley leaves have shown that photosynthesis declines faster than Rubisco activity (Wittenbach et al. 1982; Humbeck et al. 1996). This suggests that Rubisco is not the primary factor limiting photosynthesis during senescence, and that the disassembly of the photosynthetic apparatus is not a simple process, but rather a complex set of different processes that may be regulated independently (Noodén 1988; Humbeck et al. 1996). During heat-promoted senescence of wheat leaves it has been observed that the decline in PSII activity precedes the decline in PSI activity. However, the persisting PSII centres in a senescing leaf are working at high efficiency (Humbeck et al. 1996), and thus stringent control of the breakdown processes is required to avoid production of excess reactive oxygen species, which would be detrimental to the cell integrity.

Extreme shading of leaves or darkness and also daylength are important signals for initiation of the senescence process (Krupinska et al. 2003). A change in light intensity may influence the development at different levels and can change the timing of senescence or even reverse the senescence process (Humbeck & Krupinska 2003). High temperatures also promote a decline in photosynthesis during senescence, causing heat-specific injuries to the photosynthetic apparatus (Alkhatib & Paulsen 1990; Xu et al. 1995). The effect of environmental factors on photosynthesis during senescence may, however, also indirectly be due to changes in the source–sink relationship (Feller & Fischer 1994; Fischer & Feller 1994). Treatments that limit transport of carbohydrate and nitrogen out of wheat flag leaves were found to promote senescence. This was shown with flag leaves removed from the plant while the vascular connections to the stem and ear were preserved (Lazan et al. 1983). Senescence was observed to be most rapid when the flag leaf was detached in isolation (Lazan et al. 1983). Senescence was shown to occur prematurely in leaves with enhanced carbohydrate levels (Feller & Fischer 1994; Parrott et al. 2005) due to the experimental treatment known as ‘steam girdling’, which interrupts the sieve tubes at the base of the leaf lamina (Parrott et al. 2005).

The loss of the apoproteins of the pigment protein complexes is well coordinated with the degradation of chlorophyll, which is actually a prerequisite for degradation of these proteins (Hörtensteiner & Matile 2004). Initial studies on chlorophyll catabolism were performed on barley primary leaves (Matile et al.1988; Scheumann et al. 1999). Chlorophyll b was shown to be reduced to chlorophyll a before degradation (Scheumann et al. 1999). There is evidence for an export of fluorescing chlorophyll catabolites into the cytoplasm (Hörtensteiner & Matile 2004). After modification, they finally accumulate in the vacuole (Matile et al. 1999; Hörtensteiner & Matile 2004). Chlorophyll decline was observed to proceed at a slower rate than the loss of Rubisco and has therefore been considered to be a secondary alteration in metabolism during senescence (Friedrich & Huffaker 1980). While degradation of the pigment-binding membrane proteins is affected by light, this is not the case for degradation of Rubisco (Mae et al. 1993). Also, different kinetics of Rubisco degradation and degradation of thylakoid membrane proteins point towards different mechanisms of protein degradation (Krupinska 2006).

The protease activities involved in the degradation of Rubisco have long been a major objective of senescence studies (Friedrich & Huffaker 1980; Lamattina et al. 1985). In flag leaves of wheat, a biphasic degradation of Rubisco has been observed during grain filling (Peoples et al. 1980). The peptide-hydrolase enzymes that can degrade Rubisco are characterised by a pH optimum of about 5 (Peoples & Dalling 1978; Wittenbach 1979). The major portion of the activity is found in the cytoplasm, and it is increasing at a time when the tonoplast is still intact. A vacuolar localisation of proteases involved in Rubisco degradation has been suggested (Wittenbach et al. 1982), but the question still remains as to how substrate and enzyme are brought together during senescence. Rubisco appears to be degraded at the same low rate in non-senescent and senescent leaves (Peterson et al. 1973). Attempts to identify major proteases involved in Rubisco degradation in chloroplasts have failed so far (Mae et al. 1989; Miyadai et al. 1990; Ishida et al. 1997). Primary Rubisco degradation products have defined molecular masses. Desimone et al. (1998) identified fragments of 44, 41, 36 and 20 kDa, and Parrott et al. (2007) found fragments of 46, 40, 34.7, 31.5 and 21.6 kDa; the last two fragments were deduced to result from a single cleavage event. The well-defined fragmentation of Rubisco in intact chloroplasts and stroma fractions indicates that the primary cleavage is highly specific and is initiated by an unknown protease inside the chloroplast. The process appears to require ATP (Desimone et al. 1998).

The selective loss of Rubisco that occurs at later stages of senescence (Peoples et al. 1980) could at least in part be due to the formation of Rubisco-containing bodies (RBCs), as recently characterised in naturally senescing wheat leaves as well as in leaves induced to senesce by darkness (Feller et al. 2007). These bodies appear to form at the onset of senescence in the cytoplasm and to include Rubisco and/or Rubisco degradation products and also glutamine synthetase, another stromal protein. Some RCBs have been shown to be surrounded by a double membrane and further membranous structures in the cytoplasm. This suggests that RCBs are autophagosome-like structures (Chiba et al. 2003). Autophagy is usually considered to be a non-selective method of bulk protein degradation (Bassham 2007). In mammalian cells, however, a selective chaperone-mediated type of autophagy has been shown to be involved in degradation of oxidised proteins (Massey et al. 2004). Already in 1982, Wittenbach et al. suggested that Rubisco may be altered during senescence because its activity declines at a faster rate than its protein level. It is also possible that, in plants, a method for selective elimination of oxidised proteins from chloroplasts may exist (Xiong et al. 2007). Since mutants impaired in autophagy still degrade plastid proteins, and the abundance of mRNAs for ATG (autophagy, also called APG) proteins was shown to increase rather late in senescence (Doelling et al. 2002), it has been suggested that only a part of Rubisco degradation may occur by autophagy of RCB (Chiba et al. 2003).

When senescence of barley leaves was characterised under field conditions, a preferential early degradation of the large subunit of Rubisco was observed (Humbeck et al. 1996). Therefore, it is possible that the two subunits belong to different stroma protein populations with respect to their degradation timing and mechanism. This would suggest a sorting mechanism. It is possible that some stromal proteins are more sensitive to oxidative modification and are excluded from the stroma thereafter. Oxidative modification has been shown to occur in the case of the large subunit of Rubisco (Ishida et al. 1997; Desimone et al. 1998) and for glutamine synthetase (Ishida et al. 2002).

Plastidial and extraplastidial proteases involved in plastid protein degradation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cereals as model plants for studies on leaf senescence
  5. Dismantling of the photosynthetic apparatus and chloroplast protein degradation
  6. Plastidial and extraplastidial proteases involved in plastid protein degradation
  7. Nitrogen metabolism and re-translocation
  8. Senescence-associated gene expression
  9. Genetic modulation of senescence phenotypes
  10. An ideal senescence phenotype of cereals
  11. Acknowledgements
  12. Conflicts of Interest
  13. References

Plastid localised proteases

Chloroplasts contain a number of proteases, such as the ClpC/P system, FtsH, and the Deg and Lon proteases (Adam & Clarke 2002). Due to constitutive expression of the plastid-encoded clpP gene, it has been suggested that in barley the Clp protease does not play a role during leaf senescence, but rather has a function in non-senescing chloroplasts (Humbeck & Krupinska 1996). However, the erd1 gene encoding a plastid targeted homologue of the ClpA subunit is up-regulated during senescence of Arabidopsis (Nakashima et al. 1997). There is evidence that metallo-endoproteinases may be involved in the first steps of plastidial protein degradation (Bushnell et al. 1993), and a large fraction of plant cell aminopeptidases is located in plastids of senescing barley leaves (Thayer et al. 1988). Recently, the Zn+-dependent metalloprotease FtsH6 was shown to be involved in degradation of the major light harvesting complex (LHCII) during senescence and high light stress conditions in Arabidopsis (Zelisko et al. 2005). Partial degradation of LHCII apoprotein from a 25 to a 24 kDa protein may be due to proteolytic cleavage at its stroma-exposed part and to the loss of stacking observed during senescence (Xu et al. 1995).

A number of protease genes have been shown to have enhanced expression during steam girdling-induced senescence in barley leaves. Among these is the plastidial aspartic protease with homology to CND41 that, in tobacco, has been implicated to be involved in Rubisco degradation (Kato et al. 2004, 2005). This observation supports the notion that photosynthesis-associated proteins are at least partially degraded in the intact chloroplasts (Parrott et al. 2007). On the other hand, transcript levels of several other plastidial proteases (Clp, Fts, Spp, etc.) suggested to play a role in senescence (Gepstein et al. 2003) were found to be down-regulated (Parrott et al. 2007) or to be expressed constitutively (Zelisko et al. 2005). It seems that the proteolytic activity in chloroplasts is not regulated at the level of gene expression, but possibly by conformational changes due to substrate availability.

Extraplastidial proteases

Using QTL analysis, it was recently shown that neither the major endopeptidases nor aminopeptidases are instrumental in leaf nitrogen remobilisation and control of grain protein accumulation in barley (Yang et al. 2004). Identification of endoproteinases (EP1, EP2) by isoelectric focusing in extracts from barley leaves, either attached or detached (Miller & Huffaker 1985), showed that the amount of these proteases did not change during senescence of attached leaves, that they are evenly distributed in one leaf, and that detached leaves had additional proteases present adjacent to the cut site.

Early studies indicated that the main proteases present in the leaves and responsible for the senescence-associated increase in proteolytic activity were thiol proteases with a high affinity for Rubisco and a pH optimum of 4.8–5.0 (Wittenbach 1979). Such an increase in proteolytic activity was shown during dark-induced senescence of wheat leaves (Wittenbach 1978) as well as during senescence of flag leaves from field-grown wheat plants (Wittenbach 1979). Subsequent work has shown that the vacuoles of wheat (Lin & Wittenbach 1981; Waters et al. 1982) and barley (Heck et al. 1981; Thayer & Huffaker 1984) leaves contain the major proteolytic activity. The low pH requirement for Rubisco degradation suggests that the vacuole may be the site of Rubisco degradation (Matile 1997; Marty 1999; Otegui et al. 2005). A recent QTL analysis has strongly indicated that one or several carboxypeptidase isoenzymes are involved in nitrogen retranslocation from senescing barley leaves (Yang et al. 2004). This result is intriguing, considering that these proteases have been localised to the vacuole. Considering, however, that most of the proteolytic activity has been detected in association with the cytoplasm, other mechanisms also have to be envisaged. In this respect, the senescence-associated vacuoles (SAV) detected in the cytoplasm are of great interest (see review by Martinez et al., this issue). It is possible that the SAVs contribute to formation of autophagosome-like membrane whorls surrounding RCBs (Fig. 2).

image

Figure 2.  Scheme for the release of Rubisco-containing bodies (RCB) from chloroplasts and different methods for their degradation involving SAVs. a: RCBs may be engulfed by the central vacuole; b: RCBs may fuse with SAVs and fusion products may fuse with the tonoplast and c: RCBs may form autophagosomes with membrane material derived from SAVs.

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So far, the significance of the high activity of cysteine endoproteinases and carboxypeptidases for senescence-associated plastid protein degradation has not been elucidated. However, gene expression analyses with both wheat and barley indeed identified several cysteine protease genes with enhanced transcript levels during leaf senescence (Gregersen & Holm 2007; Parrott et al. 2007). It has been suggested that vacuolar proteases may degrade part of the plastid proteins after vacuolar autophagy of chloroplasts or chloroplast parts (Hörtensteiner & Feller 2002). As early as 1982, Wittenbach et al.. presented evidence for an engulfment of chloroplasts by the vacuole and hypothesised that the final disintegration of chloroplasts may occur within the vacuoles. In that case, the engulfing of the chloroplasts by vacuoles may represent the rate-limiting step in loss of chloroplasts, rather than the concentration of vacuolar proteases.

Discussion on the compartmentalisation of plastid protein proteolysis during senescence is still ongoing. The first steps of protein degradation are likely to occur in the plastid, involving proteases that are constitutively present and are activated by conformational changes of the substrate, e.g. by oxidation. Partially degraded proteins are then removed from the plastids by release of vesicles. So far, little is known about the mechanisms of vesicle formation by plastids. Shedding of tip regions from stromules could be a possible mechanism (Gunning 2005). In Fig. 2 several methods for degradation of stromal proteins outside of chloroplasts are presented. Either RBCs are engulfed by the central vacuole, as proposed previously (Krupinska 2006), and they fuse with SAVs before their content is delivered to the central vacuole. Alternatively, SAVs may provide the material for the formation of autophagosomes (Fig. 2).

Nitrogen metabolism and re-translocation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cereals as model plants for studies on leaf senescence
  5. Dismantling of the photosynthetic apparatus and chloroplast protein degradation
  6. Plastidial and extraplastidial proteases involved in plastid protein degradation
  7. Nitrogen metabolism and re-translocation
  8. Senescence-associated gene expression
  9. Genetic modulation of senescence phenotypes
  10. An ideal senescence phenotype of cereals
  11. Acknowledgements
  12. Conflicts of Interest
  13. References

The many different proteolytic activities associated with senescence at the whole leaf level ensure that during senescence proteinaceous components of leaf cells are degraded into amino acids, amides and ammonium. The major part of the ammonium is re-assimilated into amino acids for export from the senescing leaf, whereas a minor part is evaporated as ammonia from the leaf canopy (Schjoerring et al. 1993). Free amino acids are exported via the phloem to the developing grain, either directly or indirectly via the root or the glumes (Simpson et al. 1983). The major phloem-exported amino acid in barley and wheat is glutamate, followed by varying levels of aspartate, glutamine, threonine and serine (Winter et al. 1992; Caputo et al. 2001; Kichey et al. 2006). Thus, glutamate, originating in 2-oxoglutarate, plays a central role in the remobilisation of nitrogen (Forde & Lea 2007). Glutamine also seems to be important since its relative abundance in the phloem increases during late senescence in wheat (Simpson & Dalling 1981). Accordingly, glutamine synthetase (GS) activity appears to be of major importance for the re-assimilation of ammonium into exported amino acids during senescence, in particular the cytosolic GS forms (Habash et al. 2001; Miflin & Habash 2002).

During the period of highly active photosynthesis, the GS activity is due to the dominant GS2 form located in the chloroplast, primarily assimilating the ammonium that is derived from nitrate reductase activity and photorespiration. The activity of GS2 declines during senescence, when chloroplasts are degraded, and the cytosolic GS1 forms take over and contributes a relatively higher part of the GS activity. These changes in the relative proportions of the two forms are also reflected at the transcriptome level (Gregersen & Holm 2007). Furthermore, GS activity in wheat flag leaves has been shown to be a good marker for high remobilisation of nitrogen to the grain (Kichey et al. 2007). This is most probably due to GS2, since the activity is highly correlated to leaf longevity, i.e. negatively correlated to senescence. A recent QTL study has confirmed the importance of the total GS activity of wheat flag leaves for nitrogen remobilisation, since the GS1 locus was associated with QTLs for nitrogen amount in grains and in grain weight (Habash et al. 2007). The roles of the different isoforms of the cytosolic GS1, located in different cell types, are still unresolved (Kichey et al. 2006; Habash et al. 2007). However, the specific manipulation of GS activity in leaves seems to be a useful instrument for achieving improved nitrogen remobilisation in cereals (Hirel et al. 2007). Indeed, preliminary results from wheat indicated that over-expression of a GS1 can increase grain yield (Habash et al. 2001). In addition, recent results with maize over-expressing different isoforms of GS1 strongly support this approach to obtain higher yield (Martin et al. 2006). Kernel number and kernel size increased following over-expression of different isoforms of GS.

The metabolism of carboxylic and amino acids, which is part of the senescence-associated recycling of nitrogen, is complex and comprises a range of enzyme activities and genes in addition to GS. Limited information is available on these aspects for barley and wheat, but transcriptome analysis of senescence in wheat flag leaves (Gregersen & Holm 2007) clearly showed an up-regulation of genes involved in different parts of this metabolism. Real-time PCR validation of these results is presented in Fig. 3. Thus, e.g. phosphoenolpyruvate carboxylase, an essential enzyme for organic acid production (Foyer et al. 2003), might be of importance for the formation of carbon skeletons participating in the formation of exported amino acids. The transcriptomics results also indicate that cytosolic/peroxisomal isoforms of citrate synthase, aconitase and isocitrate dehydrogenase could be important in this context (Gregersen & Holm 2007). These enzymes are probably involved in the breakdown of fatty acids from cellular membranes and link this to carbohydrate metabolism via the formation of 2-oxoglutarate, a key player in the carboxylic acid metabolism.

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Figure 3.  Real-time PCR validation of gene expression results from cDNA microarrays for selected genes involved in carboxylic and amino acid metabolism during flag leaf senescence in wheat. The expression fold difference is the log2 ratio between the expression levels in fully senescing flag leaves (approximately 25 days after pollination) and non-senescing leaves (at pollination). Genes encoding Aco, aconitate hydratase; CS(per), peroxisomal citrate synthase; CS(mit), mitochondrial citrate synthase; ICDH(nadp), NADP-dependent isocitrate dehydrogenase; PEPC, phosphoenolpyruvate carboxylase; GS1, glutamine synthetase 1; tubulin, beta-tubulin; HP68, 68 kDa protein HP68; GOGAT, glutamate synthetase; GS2, glutamine synthetase 2; Cab, Cab-binding protein; Rubisco, ribulose-1,5-bisphophate carboxylase (small subunit). Tubulin and HP68 served as reference genes for the normalisation of the PCR data. Grey scale colours indicate relative gene expression levels, with dark indicating high expression.

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Senescence-associated gene expression

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cereals as model plants for studies on leaf senescence
  5. Dismantling of the photosynthetic apparatus and chloroplast protein degradation
  6. Plastidial and extraplastidial proteases involved in plastid protein degradation
  7. Nitrogen metabolism and re-translocation
  8. Senescence-associated gene expression
  9. Genetic modulation of senescence phenotypes
  10. An ideal senescence phenotype of cereals
  11. Acknowledgements
  12. Conflicts of Interest
  13. References

It has been evident for many years that the extensive physiological changes taking place during senescence in cereal leaves also involve changes at the gene transcription level (Becker & Apel 1993). Over the last decade, several specific senescence-associated genes have been isolated and characterised, especially from barley, using differential screening techniques. A number of cDNA clones from barley leaves induced to senesce by dark treatment represent genes that are also expressed during natural senescence (Kleber-Janke & Krupinska 1997). Two of these were identified as genes encoding a proteinase inhibitor and the 4-hydroxyphenylpyruvate dioxygenase. One of the genes suggested as a senescence marker gene for barley encodes the small nucleus targeted protein HvS40, which is expressed in necrotising leaf areas during senescence and pathogen attack (Krupinska et al. 2002). Three additional barley senescence-associated genes were described by Scharrenberg et al. (2003), as encoding a glycosyltransferase, a cysteine protease and an NAC domain transcription factor (GRAB2 homologue). Several barley genes induced by heavy metal stress are also induced during natural leaf senescence. These comprise genes that encode an HMA domain-containing protein (Barth et al. 2004), a calcium-dependent C2 domain protein (Quelhadj et al. 2006), a receptor-like protein kinase (Quelhadj et al. 2007) and a metallothionein (Heise et al. 2007).

In a global analysis of the transcriptome of wheat flag leaves undergoing senescence, up-regulation of the genes described above was confirmed (Gregersen & Holm 2007). In total, a few hundred genes were shown to be up-regulated during senescence. Among these genes, 140 could be categorised into functional groups mainly involved in degradative processes, regulatory processes, stress responses, transport and secondary metabolism. Overall, this work supports the notion that a generic senescence programme exists across monocot and dicot plant species. Thus, there is a substantial overlap with results from studies on the transcriptome of senescing leaves of Arabidopsis thaliana (Buchanan-Wollaston et al. 2005). In a recent study on the transcriptome of barley leaves induced to senesce by accumulation of carbohydrates as a result of ‘steam girdling’ (Parrott et al. 2007), the genes specifically up-regulated during senescence comprise a range of protease genes that have also been shown to be up-regulated during senescence of wheat flag leaves (Gregersen & Holm 2007). In addition, several thousands of other genes were determined to be differentially up-regulated in the barley study, comprising most of the few hundred genes designated as up-regulated by Gregersen & Holm (2007). Differences in the number of regulated genes between these two studies presumably relate to the different microarray platforms used, the 22 k Affymetrix array of barley and a 9 k cDNA array of wheat, respectively. It is possible that the differences may also be related to the different biological materials used, i.e. senescence induced by steam girdling at the base of leaves as opposed to natural senescence.

So far, the transcriptomic studies primarily confirm previous more scattered results for the senescence process in barley and wheat. However, the comprehensive data available from these studies illustrate the dynamic interactions between a range of different metabolic pathways and will serve as a platform for future detailed studies on senescence-associated proteases, transcription factors, secondary metabolites and other aspects of senescence. Recently, additional results in this direction were obtained in a study where gene expression profiling of near-isogenic barley lines differing in the allelic state of a major grain protein content locus identified genes with possible roles in leaf senescence and nitrogen reallocation, albeit the exact gene functions remained undetermined (Jukanti et al. 2008).

Genetic modulation of senescence phenotypes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cereals as model plants for studies on leaf senescence
  5. Dismantling of the photosynthetic apparatus and chloroplast protein degradation
  6. Plastidial and extraplastidial proteases involved in plastid protein degradation
  7. Nitrogen metabolism and re-translocation
  8. Senescence-associated gene expression
  9. Genetic modulation of senescence phenotypes
  10. An ideal senescence phenotype of cereals
  11. Acknowledgements
  12. Conflicts of Interest
  13. References

From a plethora of studies, it is evident that there is a genetic basis for variation in timing and rate of leaf senescence in wheat and barley. Modulation of the senescence phenotype is therefore a breeding objective (Joshi et al. 2007) that has indirectly been included in breeding for the time of seed maturation. Cultivars are rather invariable within a geographic area, indicating an optimal maturity date for a given area that is unintentionally selected for by breeders. However, if targeted breeding efforts or genetic manipulations could further prolong the time until maturity is achieved, productivity could presumably be raised (Thomas & Howarth 2000). Several efforts have been made to achieve delayed senescence in cereals, but also, accelerated senescence has been achieved, as a consequence of selection for other traits.

Delayed senescence (‘stay green’)

Several previous reports (Simpson 1968; Rawson et al. 1983) clearly demonstrated a genetically determined correlation between yield and flag leaf area duration. The leaf area duration, i.e. delayed senescence, is closely correlated with leaf size. A delayed leaf senescence, or ‘stay green’ phenotype, is thus in general believed to have the potential for higher plant productivity due to a longer period of active photosynthesis (Thomas & Howarth 2000). This has been confirmed in several recent reports. Thus, a positive correlation between chlorophyll content (or visual senescence) and grain yield, and also total nitrogen content of the grain, has been found (Kichey et al. 2007). In a hybrid ‘stay green’ winter wheat cultivar (Gong et al. 2005), high grain yield was observed to correlate with delayed senescence. Another high-yielding wheat cultivar was shown to have a delayed decline in the components of the photosynthetic apparatus (Zhang et al. 2006).

In a targeted approach, several mutant lines with a considerable delay in senescence were obtained having a delay of up to 10 days in the onset of chlorophyll loss (Spano et al. 2003). Total grain yield was increased in these lines; however, the remobilisation of nitrogen was not increased to the same extent, probably due to a higher retention of chloroplastic proteins in the ‘stay green’ leaves. These results strongly indicate that it is feasible to interfere with general regulatory components of the senescence process. Such components could comprise hormonal regulation or important gene transcription factors. Indeed, as described below, a delay of senescence was achieved in wheat using RNA interference to silence selected putative transcription factors of the NAC domain family (Uauy et al. 2006b) .

Accelerated senescence (high grain protein content genes)

The protein content of barley and wheat grains is important in relation to different end product use of the harvested grains, e.g. low protein content is desirable in malting processes whereas high protein content is desirable for bread making from wheat grains. Thus, targeted breeding efforts have been undertaken for both barley and wheat to change the protein content of grains and to characterise genes affecting this character. Unintentionally, this has also turned out to involve senescence characters.

In tetraploid durum wheat a trait for high grain protein content has been introduced from a wild tetraploid wheat species and the QTL-mapped to the Gpc-B1 locus (Olmos et al. 2003). In addition to a higher content of protein, wheat lines with this locus also have a higher grain content of micronutrients (Distelfeld et al. 2007). The Gpc-B1 locus was demonstrated to confer an accelerated flag leaf senescence correlating with a shorter grain filling period (Uauy et al. 2006a). In barley, a similar locus associated with grain protein content was characterised from a cross of two lines with high and low grain protein contents (See et al. 2002). Likewise, in barley this trait also affected leaf senescence properties (Mickelsen et al. 2003), although not as distinct as shown for the wheat Gpc-B1 locus. The barley and wheat loci are situated in the same region of the homoeologous group 6, but whether they represent homologues of the same gene still remains unclear (See et al. 2002).

The gene conferring the high grain protein content trait in tetraploid wheat was cloned by positional cloning procedures (Uauy et al. 2006b). It was found to encode a member of the NAC transcription factor family, belonging to the NAM subgroup. It was accordingly designated NAM-B1. Interestingly, the corresponding allele to NAM-B1 in tetraploid and hexaploid wheat has a 1-bp frameshift mutation in the 5′ end, generating a non-functional polypeptide. It is thus conceivable that early domestication of wheat involved a selection for delayed senescence originating from a non-functional Gpc-B1 gene, possibly due to the higher yield. The NAC transcription factors comprise a large, plant-specific family with several members that have been ascribed functions in stress responses of plants (Olsen et al. 2005). Since gene expression analyses have shown transcriptional up-regulation of a number of NAC transcription factors during senescence in both barley and wheat (Scharrenberg et al. 2003; Gregersen & Holm 2007), the novel characterisation of the Gpc-B1 gene as a NAC transcription factor has opened very interesting possibilities for a deeper understanding of the regulation of senescence. For example, it has recently been shown that NAC transcription factors might be associated with ABA signalling in plants (Jensen et al. 2007), and they could thus be involved in the ABA-dependent regulation of senescence in wheat (Yang et al. 2001, 2003).

An ideal senescence phenotype of cereals

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cereals as model plants for studies on leaf senescence
  5. Dismantling of the photosynthetic apparatus and chloroplast protein degradation
  6. Plastidial and extraplastidial proteases involved in plastid protein degradation
  7. Nitrogen metabolism and re-translocation
  8. Senescence-associated gene expression
  9. Genetic modulation of senescence phenotypes
  10. An ideal senescence phenotype of cereals
  11. Acknowledgements
  12. Conflicts of Interest
  13. References

Although the literature has repeatedly stated that manipulation of the senescence process could result in higher yields, the method to obtain these is not straightforward. The overall dilemma that must be solved is that, although delayed senescence may lead to a high grain yield, it meanwhile leads to inefficient nitrogen remobilisation and a lower harvest index (Gong et al. 2005). The inefficient nitrogen remobilisation means more nitrogen in the residual crop and a higher need for nitrogen input by fertilisation, potentially causing environmental problems. In addition, if slow grain filling is associated with delayed senescence, the cultivar may be vulnerable to damage by heat stress and drought during the late stages of crop development (Mi et al. 2002; Yang & Zhang 2006). Delayed senescence, which may be caused by environmental factors, in particular excess nitrogen input and water supply, can lead to heavy lodging problems (Yang & Zhang 2006). On the other hand, acceleration of senescence confers efficient nitrogen remobilisation and a high protein content, but also a lower total grain yield, presumably due to a shorter period of active photosynthesis (See et al. 2002). This reflects the overall strong negative correlation observed between total grain yield and grain protein concentration, which seems very difficult to break in the breeding of cereals (Simmonds 1995). An ideal senescence phenotype should cope with this dilemma, and should also be robust towards the high influence of environmental factors on senescence, which can lead to unfavourably delayed senescence. How this can be achieved in practice still remains to be solved.

One aspect to address could be the speed of senescence development (Hafsi et al. 2000), as compared to just the attenuation of senescence. This means that large and highly productive green stem and leaf areas should be kept as long as possible, but should also be able to respond as quickly as possible to environmental stress such as heat or desiccation and mobilise the nutrients with high efficiency. This could be described as a shift in senescence, i.e. a type A as opposed to a type B stay-green phenotype as designated by Thomas & Howarth (2000). The difficulties with this approach are to ensure an efficient senescence and nutrient remobilisation under low-stress conditions in order to avoid unfavourably delayed senescence causing lodging and harvest problems. Whether this is at all plausible in a plant-breeding context for barley and wheat presumably has to await further elucidation of the genetically determined regulation of senescence. Thus, the elucidation of the role for senescence-associated transcription factors, e.g. NAC transcription factors, could open up novel ways to more specifically manipulate the senescence phenotypes. In addition, manipulation of key factors in the nitrogen remobilisation, e.g. proteases, cell death mechanisms or glutamine synthetase, seems a promising strategy to modulate senescence parameters.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cereals as model plants for studies on leaf senescence
  5. Dismantling of the photosynthetic apparatus and chloroplast protein degradation
  6. Plastidial and extraplastidial proteases involved in plastid protein degradation
  7. Nitrogen metabolism and re-translocation
  8. Senescence-associated gene expression
  9. Genetic modulation of senescence phenotypes
  10. An ideal senescence phenotype of cereals
  11. Acknowledgements
  12. Conflicts of Interest
  13. References
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