The senescence window
In an attempt to identify genetic loci that define the onset of leaf senescence, the interaction between ethylene and leaf age was studied in Arabidopsis. The effect of ethylene in inducing senescence varied largely, depending on the time when it was applied. Our results further confirmed the notion that ethylene can only induce senescence after a leaf reaches a certain developmental stage (Grbic and Bleecker, 1995; Weaver et al., 1998). In extreme situations such as in the ctr plants in which ethylene signalling is constantly switched on, or in wild-type plants that are grown in the continuous presence of exogenous ethylene, early leaf senescence is not induced (data not shown, Kieber et al., 1993). In opposite situations such as in the ein mutants in which the ethylene signalling is permanently blocked, or in the plants with antisense constructs that reduce ethylene biosynthesis, normal leaf senescence eventually occurs (Alonso et al., 1999; Bleecker et al., 1988; Chao et al., 1997; Grbic and Bleecker, 1995; John et al., 1995; Oh et al., 1997). These observations exhibit that ethylene has no effect in stimulating senescence before or after certain developmental stages. In conclusion, a leaf has a defined age window to perceive the effect of ethylene on senescence, which is termed here as the senescence window.
Ethylene is involved in almost every façade of plants development and responses to various stresses (reviewed by Johnson and Ecker, 1998). Ethylene response windows have also been described for other developmental events. Raz and Ecker (1999) showed that ethylene treatment does not affect the curvature of the apical hook in etiolated seedlings when given before or after certain growth stages. The effects of ethylene in releasing dormancy and promoting germination depend on the concentrations of exogenous ABA (Beaudoin et al., 2000). In tomato, ethylene treatment can induce epinasty in the young and middle-aged leaves, but not in older ones (Abeles et al., 1992). Tomato fruit ripening was induced by exogenously applied ethylene in mature green fruits, but not in immature fruits (Yang, 1987). These observations suggest that many such windows exist in plants to perceive ethylene action. When these windows open and how wide the windows are, may depend on the physiological context, developmental stage and the genetic make-up.
Many genes are involved in the biosynthesis, signalling and action of ethylene and are differentially regulated. In Arabidopsis, 5 genes encoding ACC synthase are differentially expressed in plant organs, during development and in response to various stress conditions (Liang et al., 1992). Throughout plant development, the expression of the ETR ethylene perception gene family is differentially regulated in tomato (Lashbrook et al., 1998). In both Arabidopsis and tomato a large gene family of EIN3 and EIL was identified (Chao et al., 1997; Tieman et al., 2001). Downstream are ethylene-response-factor1 and ethylene-responsive element binding factors whose differential regulation is also observed (Fujimoto et al., 2000; Solano et al., 1998). Such a sophisticated biosynthesis and signalling pathway is consistent with the existence of the diverse ethylene response windows.
The understanding of the relationship between the age-related factor and ethylene allowed us to formulate a model for the regulation of leaf senescence as shown in Figure 7. In this model, age-related factors are proposed to control the senescence window of a leaf and ethylene has to be recognised and integrated with the ageing signals. Perception of the signals leads to the activation of SAGs to accomplish senescence. This model provides clear implications: mutations causing perturbations in the control of age-related factors, in the integration of age and ethylene signals, or in the execution process of senescence, may give predicted phenotypes. By utilising this knowledge and defining the senescence window, a screening system was established and mutants with altered leaf senescence phenotypes were isolated.
Figure 7. A genetic pathway showing the proposed positions of the OLD genes in the regulation of leaf senescence.
The model is constructed by analysing the phenotypes, genetics and SAG expression of the old mutants. The emphasis is on the control of OLD genes and their interaction with ethylene. An arrow indicates a stimulatory effect, while a T-bar represents an inhibitory effect. See text for details.
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Functions of OLD genes
Bearing the model in mind, we tried to select those mutants in our collection with various senescence phenotypes that might locate at different positions in the regulating pathway. The phenotypes of the old1, old2 and old3 suggested that they potentially represented genes acting at the proposed positions (Figure 7). The physiological and molecular analyses were consistent with the suggested positions of the OLD genes, which were further confirmed by genetic interaction analysis.
The unique phenotype of old2 suggests that in old2 mutants the components of the regulatory pathway from ethylene signalling to the execution of senescence are not affected. Indeed, dark-grown old2 seedlings showed a normal triple response. The SAG expression profile of ethylene-treated old2 plants was not different from that of the wild-type, but advanced. Epistatic analysis also suggested that OLD2 might work upstream of OLD1 and OLD3. The simplest explanation could be that the old2 mutation abolishes suppression on age-related factors and shifts the ethylene response window to an earlier stage. Thus, we infer that OLD2 acts as a repressor of age-related factors upstream of ethylene action.
Air-grown old1-1 plants display a strongly advanced senescence syndrome, which was further accelerated by ethylene treatment, suggesting that old1 mutation generates alterations in two sets of pathways: age-regulated leaf senescence and ethylene signalling. This was more convincingly shown by the phenotypes of the old1-1 and old1-1etr1-1 double mutant. When ethylene perception was blocked in old1-1, the earlier onset of leaf senescence still occurred, but was not exaggerated by ethylene treatment. Thus, a plausible explanation for old1-1 phenotypes would be that OLD1 acts as repressor of the integration process.
It is important to notice that the old1 mutants were different from ctr and eto that show a constitutive ethylene response (Guzmàn and Ecker, 1990; Kieber et al., 1993). OLD1 is likely to define a novel link integrating ethylene actions into leaf senescence. Ethylene is shown to interact with many other signalling molecules such as with ABA in seed germination (Beaudoin et al., 2000), with jasmonic acid in conferring defence response (Penninckx et al., 1996), with glucose in regulating seedling growth (Zhou et al., 1998), or with light in controlling hypocotyl elongation (Smalle et al., 1997). Isolation and analysis of OLD1 may provide important insights on the interaction between senescence and ethylene signalling pathways.
In old3, leaf senescence occurred very rapidly, and ethylene treatment did not induce additional new leaves to senesce, suggesting that onset of leaf senescence in old3 is independent of ethylene. We also constructed the old3etr1-1 double mutants and observed that the lethal old3 phenotype was not affected when the etr1-1 mutation was introduced (data not shown). Therefore, OLD3 may act downstream of ethylene at a late step of the senescence-regulating network with pleiotrophic functions, which is consistent with the double mutant analysis that placed OLD3 downstream of OLD1. One complication of isolating early leaf senescence mutants is that mutations in homeostatic or housekeeping genes could also give an early senescence or lethal phenotype, which may not be distinguishable from the mutations in genes that specifically regulate leaf senescence. For instance, the premature senescence or lethal phenotype is generated from a mutation in a copper transport gene (Woeste and Kieber, 2000). It is hard to distinguish whether the lethal phenotype is the cause of the rapidly accelerated senescence, or the consequence. Nevertheless, the lethality of old3 can be prevented by culture in sucrose but the senescence is still accelerated in old3. This may argue against the first possibility. Together, we infer that OLD3 may be a pleiotrophic regulatory gene acting at a late step of the senescence-regulating networks.
In Arabidopsis, SAG12 expression is highly associated with age-regulated senescence, is not induced by several stress conditions and is believed to be a reliable marker for natural leaf senescence (Lohman et al., 1994; Noh and Amasino, 1999; Weaver et al., 1998). In old1-1 and old3, SAG12 consistently showed lower levels of mRNA upon ethylene treatment in comparison with the wild-type. These results raise two points. First, it is clear that high SAG12 expression is not essential for the progression of leaf senescence in old1-1 and old3. Second, these two OLD genes are likely to control a common pathway that is required but is not sufficient for the full expression of SAG12 in wild-type plants. Disruption of OLD2 alone did not induce SAG12 expression. However, old2 mutants showed higher levels of SAG12 expression than old1-1, old3 and the wild-type when treated with ethylene. This suggests that in old2 mutants all the pathways required for full expression of SAG12 are intact and active. In other words, OLD2 controls an additional pathway that is involved in regulation of SAG12 expression besides the OLD1-OLD3 pathway. Therefore, analysis of SAG12 expression in old mutants discloses at least two parallel regulatory pathways.
Previously, several factors required for the expression of SAG12 have been identified. Noh and Amasino (1999) proposed the existence of a transcription factor that can bind to the senescence-specific promoter region of SAG12 and initiate SAG12 transcription in senescing leaves. Morris et al. (2000) showed that SA is also required for the expression of SAG12. The inducible PR-1 expression demonstrated that the SA-mediated pathway is present in the old mutants. Thus OLD genes may be not involved in the SA-signalling pathway per se. Also, OLD gene products are unlikely to be the transcription factors only acting on SAG12, since a broad spectrum of SAG expression was altered. Nevertheless, it is clear that OLD genes are required for SAG12 expression. Thus, in this model, we propose at least three different pathways to regulate SAG12 expression.
In summary, we have presented a model in an attempt to explain the control of senescence by age-related factors, the integration of ethylene action, and the regulation of SAG expression during the execution process. In contrast to the wealth of knowledge on the ethylene signalling pathway, our current information on the molecular mechanisms of the regulation of leaf senescence and on the involvement of ethylene in senescence is scarce. Our model, although rudimentary, matches well the current knowledge available. The phenotypic, genetic and SAG expression analysis of the mutants provided information concerning at which steps the genes act and allowed for the construction of the model. Further refinements, through the isolation of mutants in additional steps, the analysis on double mutants from the OLD and the ethylene signalling pathways, and cloning and characterisation of the OLD genes, should yield more elaborate models for the regulation of leaf senescence.