Signal transduction in leaf senescence

Leaf senescence is a complex developmental phase that involves both degenerative and nutrient recycling processes. It is characterized by loss of chlorophyll and the degradation of proteins, nucleic acids, lipids, and nutrient remobilization. The onset and progression of leaf senescence are controlled by an array of environmental cues (such as drought, darkness, extreme temperatures, and pathogen attack) and endogenous factors (including age, ethylene, jasmonic acid, salicylic acid, abscisic acid, and cytokinin). This review discusses the major breakthroughs in signal transduction during the onset of leaf senescence, in dark- and drought-mediated leaf senescence, and in various hormones regulating leaf senescence achieved in the past several years. Various signals show different mechanisms of controlling leaf senescence, and cross-talks between different signaling pathways make it more complex. Key senescence regulatory networks still need to be elucidated, including cross-talks and the interaction mechanisms of various environmental signals and internal factors.     

Molecular aspects of leaf senescence

Senescence is the last stage of leaf development and one type of programmed cell death that occurs in plants. The relationships among senescence programs that are induced by a variety of factors have been addressed at a molecular level in recent studies. Furthermore, an overlap between the pathogen-response and senescence programs is beginning to be characterized. The complexity of the senescence program is also evident in studies of senescence-specific gene regulation and the role of photosynthesis and plant hormones in senescence regulation. New molecular-genetic approaches are expected to be useful in unraveling the molecular mechanisms of the leaf senescence program.     

Molecular regulation of leaf senescence

Leaf senescence is a process of programmed cell death, which is induced in an age-dependent manner and by various environmental cues. The mechanisms that regulate the induction and progression of leaf senescence remain unclear because of their complexity. However, recent genetic and reverse-genetic approaches have identified key components of the regulation of leaf senescence and have revealed glimpses of the underlying molecular mechanisms.     

Cytokinin inhibition of leaf senescence

The senescence delaying effect of cytokinin is well known, however, the details behind how this process occurs remain unclear. Efforts to improve understanding of this phenomenon have led to the identification in Arabidopsis of specific cytokinin signaling components through which senescence signal responses are regulated. These include the cytokinin receptor (AHK3), the type-B response regulator (ARR2) and the recently identified cytokinin response factor (CRF6). At the mechanistic end of this process, it was found that increased cell-wall invertase activity which occurs in response to cytokinin is both necessary and sufficient for the inhibition of senescence. Yet, a direct link between the signaling and mechanistic steps of a cytokinin regulated senescence process has yet to be demonstrated. This may be in part because the relationship between senescence and primary metabolism implied by the key role of cell-wall invertase is the subject of two apparently opposing bodies of evidence. Here we briefly summarize and propose a model in which cytokinin mediated changes in sink/source relationships leads to delayed senescence which is consistent with existing evidence both for and against sugars as a trigger for developmental senescence.     

Influence of senescence and of carbohydrate levels on the pattern of leaf proteases in purple nutsedge (Cyperus rotundus)

Cyperus rotundus L. is a monocotyledonous perennial weed, which forms large numbers of tubers during its vegetative growth. Since these tubers represent major sinks, source/sink interactions are more complex, and leaf senescence and proteolytic processes in this species may be different from the situation in the well‐investigated annual crop plants characterized by monocarpic senescence. Judged by native PAGE and by inhibitor studies, three different aminopeptidases, one iminopeptidase, two or more carboxypeptidases and two or more different endopeptidases were present in mature green leaves. Exo‐ and endoproteolytic activities increased during the senescence of excised leaf segments. A marked change was observed in the endopeptidase pattern, since a cysteine proteinase activity was strongly induced during senescence of the segments. This endopeptidase was also found in naturally senescing leaves and may, therefore, participate in nitrogen salvage from these organs. An increase of different protease activities was demonstrated in leaf segments of C. rotundus in the presence of high carbohydrate levels. The mechanisms involved, and the importance of this phenomenon for the interaction between source/sink relations and senescence, remain to be demonstrated.     

Hormonal regulation of leaf senescence in Lilium

In addition to floral senescence and longevity, the control of leaf senescence is a major factor determining the quality of several cut flowers, including Lilium, in the commercial market. To better understand the physiological process underlying leaf senescence in this species, we evaluated: (i) endogenous variation in the levels of phytohormones during leaf senescence, (ii) the effects of leaf darkening in senescence and associated changes in phytohormones, and (iii) the effects of spray applications of abscisic acid (ABA) and pyrabactin on leaf senescence. Results showed that while gibberellin 4 (GA(4)) and salicylic acid (SA) contents decreased, that of ABA increased during the progression of leaf senescence. However, dark-induced senescence increased ABA levels, but did not affect GA(4) and SA levels, which appeared to correlate more with changes in air temperature and/or photoperiod than with the induction of leaf senescence. Furthermore, spray applications of pyrabactin delayed the progression of leaf senescence in cut flowers. Thus, we conclude that (i) ABA plays a major role in the regulation of leaf senescence in Lilium, (ii) darkness promotes leaf senescence and increases ABA levels, and (iii) exogenous applications of pyrabactin inhibit leaf senescence in Lilium, therefore suggesting that it acts as an antagonist of ABA in senescing leaves of cut lily flowers.     

Apple leaf senescence: leaf disc compared to attached leaf

Attached apple leaves (Pyrus malus L., Golden Delicious) began to lose protein in early August as the first sign of senescence. Apple leaf discs prepared from samples before early August gained protein for up to 7 days after detachment. After early August, the loss of protein from leaf discs was no greater than the loss from attached leaves in 7 days. The loss of chlorophyll from leaf discs began over 2 months before attached leaves began to lose chlorophyll naturally and before leaf discs lost protein. Leaf discs from presenescent leaves did not senesce significantly faster when maintained in darkness instead of 12 hours of light. In general, the loss of protein and chlorophyll from apple leaf discs after 7 days was much less than for most other leaf types studied.     

Molecular genetics of leaf senescence in Arabidopsis

Leaf senescence is a developmentally programmed degeneration process that constitutes the final step of leaf development and is controlled by multiple developmental and environmental signals. In addition to the information obtained from other plants, Arabidopsis has, as a model system, contributed to our understanding of this complex phenomenon in molecular genetic terms. Recent discoveries have identified several genetic mutants and potential regulatory components in Arabidopsis. Identifying further mutants that exploit novel biological resources, screening Scheme and a global functional analysis of senescence-associated genes in Arabidopsis should increase our understanding of the complex regulatory networks.     

Role of other plant organs in gibberellic acid-induced delay of leaf senescence in alstroemeria cut flowers

Chlorophyll loss in leaves of cut flowers of alstroemeria (Alstroemeria pelegrina L. cv. Westland) was rapid in darkness and counteracted by irradiation and treatment of the flowers with gibberellic acid (GA₃). The mechanism of the effect of GA₃ under dark conditions was investigated. The content of various carbohydrates in the leaves under dark conditions rapidly decreased; this was not influenced by treatment with GA_3. indicating that the loss of carbohydrates in the leaves did not induce the loss of chlorophyll. Placing the cut flowers in various solutions of organic and inorganic nutrients exhibited no significant effect on the retention of chlorophyll in leaves of dark-senescing flowers. The total nitrogen content in leaves of dark-senescing cut flowers decreased with time. Leaves of GA₃-treated flowers retained more nitrogen. In contrast, the buds of GA₃-treated flowers retained less nitrogen during senescence in the dark than control buds. To investigate whether GA₃ affects export of assimilates from the leaf to various parts of control and GA₃-treated flowers, we labelled one leaf with radioactive carbon dioxide. ¹⁴C-assimilates accumulated preferentially in the flowers, in which the relative specific activity of the youngest floral buds was highest. No significant differences were observed in the distribution of ¹⁴C-labelled compounds between the buds of control and GA₃-treated flowers. To establish the importance of source-sink relations for the loss of leaf chlorophyll we removed the flower buds (i. e. the strongest sink) from the cut flowers. This removal only slightly delayed chlorophyll loss as compared to the large delay caused by GA₃-treatment. In addition, detached leaf tips exhibited chlorophyll loss in the dark, which was delayed by GA₃-treatment in a fashion comparable with that in flowers. Together these data demonstrate that interactions of the leaves with other plant organs are not essential for chlorophyll loss during senescence in the dark. Additionally, we have found no evidence that GA₃ delays the loss of chlorophyll by affecting the transport of nutrients within the cut flowers.     

Vacuolar cysteine proteases of wheat (Triticum aestivum L.) are common to leaf senescence induced by different factors

Cellular proteins are extensively degraded during leaf senescence, and this correlates with an up-regulation of protease gene expression, particularly cysteine proteases. The objectives of this work were (i) to detect cysteine proteases associated with senescence of wheat leaves under different conditions and (ii) to find out their subcellular location. Activity labelling of cysteine proteases with the biotinylated inhibitor DCG-04 detected five bands at 27, 36, 39, 42, and 46 kDa in leaves of wheat senescing under continuous darkness. In-gel activity assays showed that these proteases are only active in an acid milieu (pH 4), and their activity increased several-fold in senescing leaves. Fractionation experiments showed that the senescence-associated cysteine proteases of 36, 39, 42, and 46 kDa localize to a vacuolar-enriched fraction. The vacuolar cysteine proteases of 36, 39, and 42 kDa increased in activity in attached flag leaves senescing naturally during post-anthesis, and in attached leaves of plants subjected to a period of water deficit. Thus, the activity of these vacuolar cysteine proteases is associated with developmental (post-anthesis) senescence and with senescence induced by stress factors (i.e. protracted darkness or drought). This suggests that vacuoles are involved in senescence-associated cellular degradation, and that different senescence-inducing factors may converge on a single degradation pathway.     

Enhanced expression of serine proteases during floral senescence in Gladiolus

Programmed cell death during senescence in plants is associated with proteolysis that helps in remobilization of nitrogen to other growing tissues. In this paper, we provide one of the few reports for the expression of specific serine proteases during senescence associated proteolysis in Gladiolus grandiflorus flowers. Senescence in tepals, stamens and carpels results in an increase in total protease activity and a decrease in total protein content. Of the total protease activity, serine proteases account for about 67-70% while cysteine proteases account for only 23-25%. In-gel assays using gelatin as a substrate and specific protease inhibitors reveal the enhanced activity of two trypsin-type serine proteases of sizes 75 kDa and 125 kDa during the course of senescence. The activity of the 125 kDa protease increases not only during tepal senescence but also during stamen and carpel senescence indicating that it is responsive to general senescence signals.     

Chloroplasts regulate leaf senescence: delayed senescence in transgenic ndhF-defective tobacco

Mitochondrial involvement has not been identified in the programmed cell death (PCD) of leaf senescence which suggests that processes such as those involving reactive oxygen species (ROS) are controlled by chloroplasts. We report that transgenic tobacco (DeltandhF), with the plastid ndhF gene knocked-out, shows low levels of the plastid Ndh complex, homologous to mitochondrial complex I, and more than a 30-day-delay in leaf senescence with respect to wt. The comparison of activities and protein levels and analyses of genetic and phenotypic traits of wtxDeltandhF crosses indicate that regulatory roles of mitochondria in animal PCD are assumed by chloroplasts in leaf senescence. The Ndh complex would increase the reduction level of electron transporters and the generation of ROS. Chloroplastic control of leaf senescence provides a nonclassical model of PCD and reveals an unexpected role of the plastid ndh genes that are present in most higher plants.     

The molecular biology of leaf senescence

Senescence is a complex, highly regulated, developmental phasein the life of a leaf that results in the co-ordinated degradationof macromolecules and the subsequent mobilization of componentsto other parts of the plant. The application of molecular biologytechniques to the study of leaf senescence has, in the lastfew years, enabled the isolation and characterization of a largerange of cDNA clones representing genes that show increasedexpression in senescing leaves. The analysis of these genesand identification of the function of the encoded proteins willallow a picture of the complex processes that take place duringsenescence to be assembled. To date, genes encoding degradativeenzymes such as proteases and nucleases, enzymes involved inlipid and carbohydrate metabolism and enzymes involved in nitrogenmobilization have all been identified as senescence-enhancedgenes. A variety of other genes of no obvious senescence-relatedfunction have also been identified; their role in senescencemay be less predictable and, possibly, more interesting. The combined action of several internal and external signalsmay be involved in the induction of senescence. Analysis ofthe regulatory mechanisms controlling the expression of senescence-inducedgenes will allow the signalling pathways that are involved inthe regulation of senescence to be elucidated. Experiments withtransgenic plants and mutants are already shedding light onthe role played by cytokinins and ethylene in regulating senescencein leaves. Key words: Senescence, cDNA clones, gene expression, signals     

Deferral of leaf senescence with calcium

In view of the possibility that senescence may be a consequence of the deterioration of membrane compartments in the cells of leaves, calcium was studied as a possible agent which might defer senescence. The senescence of corn leaf discs was deferred by added calcium, and the effect was additive to the cytokinin deferral of senescence. Likewise, the senescence of Rumex leaf discs was deferred by added calcium, and the effect was additive to the gibberellin deferral of senescence. Detailed experiments with corn leaf discs established that the increase in apparent free space associated with senescence was completely prevented by calcium. An increase in hydraulic permeability during senescence was likewise demonstrated, and this increase was deferred by calcium; calcium plus benzyladenine was even more effective. Each of the measured functions of leaf senescence (chlorophyll content, protein decrease, apparent free space increase, and hydraulic permeability increase) was suppressed by calcium, and the interpretation is offered that the effects are a consequence of the calcium function in maintaining cellular membranes.     

Role of ethylene action in ethylene production and poststorage leaf senescence and survival of pelargonium cuttings

This study investigated the role of ethylene action in ethylene production and in poststorage performance of pelargonium cuttings. Cuttings of zonal pelargonium (Pelargonium x hortorum L.H. Bailey) of the cultivars Isabell and Mitzou were treated with ethylene and with the ethylene action inhibitors 1-methylcyclopropene (MCP), silver-thiosulfate (STS) and silver nitrate (SN) and were stored in the dark at different temperatures (5, 12, and 20 °C) for 48 h. Ethylene concentrations in the storage boxes were monitored and poststorage leaf senescence, survival and root formation of cuttings were determined. Applications of MCP resulted in a significant increase of ethylene evolution by cuttings of both cultivars which was more pronounced with increasing storage temperature. After 48 h of storage at 20 °C, ethylene concentrations were more than 20-fold higher for the MCP-treated cuttings as compared to those of the untreated controls. Also preharvest applications of STS and SN increased ethylene evolution by cuttings, even though these effects were less pronounced. However, application of these inhibitors caused severe poststorage leaf injury. Application of ethylene during storage had no effect on subsequent leaf damage. Leaf senescence during rooting and decay of cuttings, which raised with increasing storage temperature, could only partially been reduced by MCP. The results strongly support the conclusion, that in zonal pelargonium cuttings, ethylene production is controlled by autoinhibition, and clearly indicate, that temperature dependent processes other than ethylene action are substantially involved in storage-induced leaf senescence and decay.     

Role of rhomboid proteases in bacteria

The first member of the rhomboid family of intramembrane serine proteases in bacteria was discovered almost 20 years ago. It is now known that rhomboid proteins are widely distributed in bacteria, with some bacteria containing multiple rhomboids. At the present time, only a single rhomboid-dependent function in bacteria has been identified, which is the cleavage of TatA in Providencia stuartii. Mutational analysis has shown that loss of the GlpG rhomboid in Escherichia coli alters cefotaxime resistance, loss of the YqgP (GluP) rhomboid in Bacillus subtilis alters cell division and glucose uptake, and loss of the MSMEG_5036 and MSMEG_4904 genes in Mycobacterium smegmatis results in altered colony morphology, biofilm formation and antibiotic susceptibilities. However, the cellular substrates for these proteins have not been identified. In addition, analysis of the rhombosortases, together with their possible Gly-Gly CTERM substrates, may shed new light on the role of these proteases in bacteria. This article is part of a Special Issue entitled: Intramembrane Proteases.