Hormonal control of senescence
Senescence in leaves is induced by many different factors and it is obvious that there are many different pathways involved in controlling the process (Gan and Amasino, 1997; Nam, 1997). The best evidence for hormonal involvement in the control of senescence is for the hormones cytokinin and ethylene. The signal that initiates the onset of developmental senescence appears to involve cytokinin. It has been known for many years that treatment with cytokinin can delay leaf senescence, and several reports in the last few years have illustrated the importance of cytokinin in the control of senescence. Tobacco plants that express a cytokinin biosynthesis gene (the Agrobacterium ipt gene) from a senescence-enhanced promoter (SAG12) were shown to remain green and non-senescent for an extended period of time (Gan and Amasino, 1995). All aspects of senescence are delayed in the leaves of these transgenic plants, including chlorophyll degradation, protein degradation and loss of photosynthetic status (Wingler et al., 1998). This indicates that the senescence process is delayed at an early stage. Transgenic tobacco plants in which the maize homeobox gene knotted1 was expressed from the same senescence specific promoter showed a similar phenotype and increased levels of cytokinin in the older leaves (Ori et al., 1999). However, it is not clear whether this homeobox gene, which is normally expressed in developing meristems and not in leaves, has a role in controlling senescence in normal plants. In an activation tagging experiment in petunia, delayed senescence has been shown in a tagged line that over-expresses Sho, a gene that has similarity to IPT genes from Arabidopsis and is therefore likely to encode a cytokinin biosynthesis gene (Zubko et al., 2002). Premature senescence was detected in Arabidopsis lines which over-expressed the mevalonic pathway gene, farnesyl diphosphate synthase (FPS) (Masferrer et al., 2002). Overproduction of FPS led to a reduction in the substrates required for cytokinin biosynthesis and a consequent reduction in cytokinin levels presumably led to the premature senescence-like symptoms and early expression of SAG12 that was reported.
Ethylene is essential for the ripening of many fruit, but it has also been shown to have a role in leaf senescence in some plants. Plants that are exposed to ethylene show premature senescence and the older leaves on the plant are induced to yellowing. The leaves of an ethylene insensitive mutant of Arabidopsis (Etr1) were delayed in their onset of senescence (Grbic and Bleecker, 1995); similarly, an antisense tomato plant that synthesized very low levels of ethylene showed delayed leaf senescence (Picton et al., 1993). In addition, certain Arabidopsis mutant lines that have been identified as showing delayed senescence turn out to have defects in genes in the ethylene signalling pathway (Oh et al., 1997). However, in all these cases senescence occurs normally once the process has begun. Hence, it has been concluded that ethylene is a modulator of leaf senescence; its presence will speed up the senescence process but it is not essential for senescence to occur. Leaves have to be a certain age to be ready for the ethylene signal, young leaves treated with ethylene do not senesce.
The role of stress response pathways in senescence
Premature senescence in a plant can be induced by a number of different environmental stresses such as pathogen infection, nutrient or water stress or oxidative stresses induced by ozone or UV-B. Recent analysis of the signalling pathways involved with different stress responses has indicated that these have considerable cross-talk with senescence related gene expression. One question to be addressed is whether stress is causing the onset of senescence, or whether the senescence itself is inducing stress responses. Premature senescence induced by drought stress in the pea was shown to follow a pattern similar to that seen in developmental senescence (Pic et al., 2002). In this case, the stress imposed by the environment has initiated the onset of premature senescence. However, in most cases, stress response pathways appear to be involved after the onset of senescence, indicating that they occur downstream of the senescence induction signal. Links between gene expression and pathogen responses were initially indicated by the discovery that pathogenesis-related (PR) genes (genes that are expressed in response to pathogens) are expressed during the senescence of healthy leaves (Hanfrey et al., 1996; Quirino et al., 1999). Conversely, genes identified as being senescence-enhanced have been shown to be expressed in leaves exposed to many different stresses such as pathogen infection (Butt et al., 1998; Pontier et al., 1999), ozone treatment (Miller et al., 1999), UV-B exposure (John et al., 2001, and see below) and others. A number of different senescence enhanced genes were induced in response to different stresses, but they did not all show the same patterns of expression (Park et al., 1998; Weaver et al., 1998). The GeneChip experiments of Chen et al. (2002), described above have shown that many common regulatory factors are expressed in senescence and in stress responses. Overall, it can be concluded that many signalling pathways control gene expression in response to different stresses and some of these are involved in leaf senescence in Arabidopsis.
The signalling molecules SA, JA and ethylene have been implicated in complex interconnecting pathways that control gene expression in plant pathogen responses as well as in plant responses to stress (reviewed in Turner et al., 2002; Wang et al., 2002). These pathways may also be involved in regulating gene expression during senescence. Morris et al. (2000) showed that the expression of certain genes during leaf senescence depended on the presence of an active SA pathway, and this is also illustrated in Figure 4. SA levels were shown to increase in senescing leaves and this increase could account for the senescence-enhanced expression of some of the genes examined. Genes encoding the PR proteins PR1a and a chitinase are also induced in SA treated green leaves. In contrast, expression of SAG12, a senescence-specific cysteine protease (Lohman et al., 1994), requires the SA pathway for senescence-enhanced expression but is not expressed in SA treated green leaves. Expression of this gene during senescence therefore requires the presence of an ‘age’ related factor in addition to SA (Morris et al., 2000). Senescence appears to occur normally in SA deficient plants, indicating that the genes controlled by SA are not essential for senescence. However, there may be some role for this pathway in the final death phase of senescence (see above).
Jasmonic acid (JA) and related compounds play an important role in regulating a number of plant responses such as wounding and pathogen infection (reviewed in Turner et al., 2002). JA has also been implicated in senescence. It was shown that exogenous treatment of barley leaves with JA or MeJA (methyl jasmonate) led to a loss of chlorophyll and reduced levels of RBCS, indicating that senescence was induced (Parthier, 1990). Recently, the role of the JA pathway in senescence has been investigated in Arabidopsis (He et al., 2002). These authors showed that treatment of Arabidopsis with JA resulted in typical premature senescence symptoms, and these did not occur on the JA insensitive mutant Coi1 (Xie et al., 1998). In addition, JA levels were shown to increase during senescence and several enzymes involved in JA biosynthesis showed senescence-enhanced expression. Studies on genes that may be involved in the chlorophyll degradation pathway have shown that the expression of a potential chlorophyllase gene (AtCHL1), is enhanced by MeJA treatment (Tsuchiya et al., 1999). However, senescence in the JA insensitive mutant Coi1, or in plants which produce low levels of JA due to a knock-out mutation in OPR3, does not appear to be impaired (He et al., 2002; Stintzi and Browse, 2000). Therefore, as is the case for the SA pathway and ethylene resistant mutants described above, although the JA pathway appears to have a role in senescence, it is not essential for the process to take place. Neither is it essential for the timing of the process.
Increased levels of ROS (reactive oxygen species) are a common factor between different stress responses as well as in senescence. Macromolecule degradation is likely to increase the levels of ROS in senescing tissues (del Rio et al., 1998; Thompson et al., 1998) and lipid peroxidation products increase in senescing Arabidopsis leaves (John et al., 2001; Ye et al., 2000). We have shown that the expression of many different senescence enhanced genes is induced in green leaves treated with silver nitrate; co-treatment with the ROS quencher, ascorbate, attenuated that expression, indicating that ROS were involved (Navabpour et al., manuscript in preparation). A similar result was obtained following treatment with 3-amino triazole, a catalase inhibitor. Therefore, the enhanced expression of certain genes during senescence may be mediated by increased ROS levels and this could also account for the expression of these genes in response to stress treatments such as ozone or pathogen infection. The tissues treated with silver nitrate undergo a cell death-like process. They do not enter senescence, since many senescence-enhanced genes are not expressed.
How similar are the events that occur in stress-induced senescence to those that take place in developmental senescence? To address this question, we have used an Affymetrix GeneChip experiment to compare gene expression in response to UVB irradiation with that seen during developmental senescence. Arabidopsis plants treated with UV-B show senescence-like symptoms (Figure 5A), chlorophyll levels drop and photosynthesis rates decrease. UV-B irradiation results in increased ROS (Surplus et al., 1998) and there is also an indication that senescence is induced by this treatment (John et al., 2001). We have recently shown that many of the treated leaves recover from the stress, that gene expression reverts close to that seen before treatment and that photosynthesis is restored (Earl et al., manuscript in preparation). This result also indicates that senescence is being induced rather than cell death. The Affymetrix data indicates that, although there are many genes that show a similar expression pattern in both senescence and in UV-B responses, there are also groups that show different expression patterns (Figure 5B). Therefore, many signalling pathways are common between stress responses and senescence but there are some pathways that may be senescence specific. In addition, different stress treatments may well result in the induction of other signalling pathways not involved with the UV-B stress response. Dissection of the components of the signalling pathways that are senescence specific will identify genes that regulate the metabolic events that are unique to senescence.
Figure 5. The effects of UV-B irradiation on Arabidopsis. (A) Arabidopsis plants (4 weeks old) have been exposed to UV-B radiation for 3 days. Senescence-like symptoms are visible on the treated leaves. (B) GeneTree clustering as described for Figure 3. Expression patterns in green, senescent and UV-B treated leaves are shown. At least six different profiles are visible which differentiate the expression profile in UV-B treated leaves with those in senescent leaves. 1: Expression repressed by UVB and in senescence; 2: Expression induced in senescence and not by UVB; 3: Expression enhanced by both senescence and UVB but less expression in UVB treated leaves; 4: Low expression in UVB; 5: Expression enhanced in both senescence and UVB; 6: Expression enhanced in UVB only.
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Stress response pathways obviously have a role in the senescence process, but this may be of relatively low importance compared to the signalling pathways that initiate the onset of senescence and control the degradative processes that occur. Sugar sensing of signalling pathways have been implicated in the control of aspects of plant metabolism and development including plant senescence, and a central role for hexokinase as a glucose sensor modulating multiple signalling pathways has been proposed (reviewed in Rolland et al., 2002). Transgenic plants over-expressing hexokinase exhibit premature senescence, indicating that this protein may have a role in controlling senescence (Dai et al., 1999; Xiao et al., 2000). Reduced photosynthetic activity is an early event in senescence and is accompanied by a loss of protein, followed by chlorophyll and lipid degradation. Senescence is also induced in dark treated tissues, where sugar levels rapidly become diminished. It has been postulated that the reduction in efficiency of photosynthesis, resulting in sugar starvation in the leaf, may be an early signal for the induction of senescence (Hensel et al., 1993). This is supported by the observation that the dark induced expression of many senescence-enhanced genes is repressed in the presence of sucrose (Chung et al., 1997; Fujiki et al., 2001). Interestingly, sugar treatment of Arabidopsis leaves that were in a mid-senescent stage reduced the expression of the senescence specific gene, SAG12 but not another senescence enhanced gene SAG13 (Noh and Amasino, 1999). However, in green leaves, low sugar levels enhance photosynthesis, and the accumulation of glucose and sucrose represses the transcription of photosynthetic genes (Rolland et al., 2002). Moreover, senescing leaves have been found to accumulate glucose and fructose, rather than exhibiting sugar starvation (Stessman et al., 2002; Wingler et al., 1998). The hys1 mutant (described above) has an increased sensitivity to exogeneously applied sugars as well as showing accelerated senescence, and the authors suggest that this gene might have a role in a sugar sensing pathway controlling the onset of senescence (Yoshida et al., 2002). It is obvious that the relationship between sugar levels and senescence is not at all clear. Sugar may be involved in the regulation of some genes during senescence, but a combination of other developmental signals must also be involved (Ono et al., 2001).
Regulatory genes and promoters
The identification of regulatory genes that could be manipulated to regulate the onset or progression of senescence is of key interest and importance. Initially, the approach to this has been the isolation of senescence-enhanced genes, although early signal receptors or transcriptional repressors are unlikely to fall into this class. In the differential and other screens that have been applied to identify senescence enhanced genes (described above), a number of groups have identified potential regulatory genes. Genes that encode receptor kinase proteins that may serve as receivers or transducers of external or external signals, or genes that may encode transcription factors have been isolated (Table 1). A senescence-associated receptor kinase (SARK) gene, identified in bean, was shown to be expressed early in leaf senescence before the first signs of chlorophyll degradation or loss of chlorophyll a/b binding protein (Hajouj et al., 2000). This early expression implicates this protein as having a role in the early steps in senescence initiation rather than in downstream stress-related pathways.
Several classes of senescence-enhanced transcription factors have been isolated. A member of the Arabidopsis WRKY family of transcription factors, WRKY6, showed strong expression during leaf senescence (Robatzek and Somssich, 2001). Senescence and pathogen induced expression of the PR1 gene depended on the presence of the WRKY6 protein, which also appears to induce expression of NPR1, the transcriptional regulator required for PR1a expression (Zhou et al., 2000). A senescence specific receptor kinase gene, SIRK, that was dependent on WRKY6 for expression was also identified (Robatzek and Somssich, 2002). Although WRKY6 itself has a role in pathogen defence as well as senescence, the SIRK gene appeared to be expressed solely during senescence. The WRKY6 knock-out mutant resulted in an altered expression of several genes but no obvious senescence-related phenotype was observed. Another WRKY gene, WRKY53, was identified after a suppressive subtractive hybridization experiment designed to isolate genes expressed early in senescence (Hinderhofer and Zentgraf, 2001). This gene showed an interesting pattern of expression. It was expressed only in the oldest leaves of a 6-week-old plant but by the 7th week, expression was detected in all the rosette leaves, independent of their age. This implies that the gene is initially controlled by an age-related signal which is masked by a different signal when the plant reaches a certain developmental stage.
Two members of the leucine zipper (b-ZIP) family of transcription factors that show senescence-enhanced expression in tobacco have been identified (Yang et al., 2001). One of the genes, TBZF, was expressed in senescing leaves and flowers while the other, TBZ17, only accumulated in ageing leaves. The proteins were shown to accumulate in the guard cells and vascular tissues of senescing leaves. Both these cell types need to remain active until the very last stages of senescence, the guard cells to allow responses to environmental stimuli and the vascular tissues to maximize mobilization from the senescing leaf. The authors suggest that these genes may retard senescence by activating the genes that are required to retain cellular activity in these specific cell types.
The array experiments of Chen et al. (2002), described above identified many transcription factors that showed senescence-enhanced expression. In addition, we have identified around 40 different senescence-enhanced genes encoding proteins such as MYB, zinc finger, MADS box and leucine zipper as well as a number of different kinases. The functional analysis of these genes, to find the signalling pathway they are a component of and the downstream gene(s) that they regulate, will help to elucidate the complex pathways that control senescence. This is a key challenge for the next few years.
Little progress has been made in the identification of a ‘Senescence Box’, i.e. an upstream sequence that is required for senescence activated transcription. The involvement of multiple signalling pathways complicates the search for this sequence. Increased knowledge of individual pathways and the identification of smaller groups of genes that require the activity of a specific pathway for expression will increase the chances of identifying common senescence promoter sequences. A sequence identified in the upstream region of the ArabidopsisSAG12 gene was shown to bind different proteins in extracts from young or old leaves (Noh and Amasino, 1999). This result suggests that a repressor or inactive transcriptional activator may inhibit SAG12 expression in young leaves, to be replaced by a newly transcribed or modified activator during senescence.
A relatively simple model designed to illustrate the complexities of the signals and pathways that have a role in the control of gene expression during senescence is presented in Figure 6. There are obviously many other signalling factors and hormones that are not considered here, and multiple combinations of factors are possible. In addition, the relative importance of the particular pathways may well vary in different plant species.
Figure 6. A simple model to illustrate the network of pathways that are required for gene expression during senescence. Leaf senescence can be initiated by developmental signals that are related to the growth stage of the plant and the position of the leaf on the plant. Photosynthetic status, sugar levels and/or changes in cytokinin levels may be key initial signals in controlling the onset of developmental senescence. Many stresses induce premature senescence, including oxidative stress caused by environmental factors such as UVB irradiation or ozone, or nutrient stress due to lack of water, nitrogen, etc. Initiation of senescence results in the induction of multiple signalling pathways including stress response pathways involving ethylene, SA and JA, resulting in new gene expression. Levels of ROS, SA, JA and ethylene increase during senescence and the expression of some senescence-enhanced genes is solely dependent on one or a mixture of these factors. For example, PR1a is expressed in green leaves treated with SA; other senescence-enhanced genes are expressed in green leaves treated with JA, etc. However, many senescence-enhanced genes are dependent on other factors for expression, which may be senescence specific (age specific factors). Some of these genes may also require the presence of factors such as SA. For example, for expression of SAG12, the SA signalling pathway must be active but also expression only occurs in yellowing leaves, showing that an age specific factor is also required. Therefore, senescence enhanced genes can be divided into groups depending on signals that control them. However, only some of the classes of gene may be essential for the senescence process to occur. For example, SA treatment induces the expression of senescence enhanced genes but does not induce senescence. Ethylene treatment only induces visible senescence in leaves that have reached a certain developmental stage, indicating that age related factors are also necessary. Senescence appears to occur normally in plants that are defective in each of the three stress-related signalling pathways.
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