Delayed leaf senescence in ethylene-deficient ACC-oxidase antisense tomato plants: molecular and physiological analysis

To determine the role of ethylene during tomato (Lycopersicon esculentum Mill. cv. Alisa Craig) leaf senescence, transgenic ACC oxidase antisense plants were analysed. Northern analysis of wild-type plants indicated that ACC oxidase mRNA accumulation normally begins in pre-senescent green leaves but was severely reduced in the antisense plants. Although the levels of ethylene evolved by wild-type and transgenic leaves increased during the progression of senescence, levels were extremely low in transgenic leaves. Leaf senescence, as assessed by colour change from green to yellow, was clearly delayed by 10–14 days in the antisense plants when compared with wild-type plants. Northern analysis of the photosynthesis-associated genes, cab and rbcS, indicated that levels of the corresponding mRNAs were higher in transgenic leaves which were not yet senescing compared with senescing wild-type leaves of exactly the same age. Northern analysis using probes for tomato fruit ripening-related genes expressed during leaf senescence indicated that once senescence was initiated the expression pattern of these mRNAs was similar in transgenic and wild-type leaves. In the antisense plants chlorophyll levels, photosynthetic capacity and chlorophyll fluorescence were higher when compared with senescing wild-type plants of the same age. Photosynthetic capacity and the quantum efficiency of photosystem II were maintained for longer in the transformed plants at values close to those observed in wild-type leaves prior to the visible onset of senescence. These results indicate that inhibiting ACC oxidase expression and ethylene synthesis results in delayed leaf senescence, rather than inducing a stay-green phenotype. Once senescence begins, it progresses normally. Onset of senescence is not, therefore, related to a critical level of ethylene. The correlation between higher levels prior to senescence and early onset, however, suggests that ethylene experienced by the plant may be a significant contributing factor in the timing of senescence.     

Ectopic expression of wheat expansin gene TaEXPA2 improved the salt tolerance of transgenic tobacco by regulating Na+/K+ and antioxidant competence

High salinity is one of the most serious environmental stresses that limit crop growth. Expansins are cell wall proteins that regulate plant development and abiotic stress tolerance by mediating cell wall expansion. We studied the function of a wheat expansin gene, TaEXPA2, in salt stress tolerance by overexpressing it in tobacco. Overexpression of TaEXPA2 enhanced the salt stress tolerance of transgenic tobacco plants as indicated by the presence of higher germination rates, longer root length, more lateral roots, higher survival rates and more green leaves under salt stress than in the wild type (WT). Further, when leaf disks of WT plants were incubated in cell wall protein extracts from the transgenic tobacco plants, their chlorophyll content was higher under salt stress, and this improvement from TaEXPA2 overexpression in transgenic tobacco was inhibited by TaEXPA2 protein antibody. The water status of transgenic tobacco plants was improved, perhaps by the accumulation of osmolytes such as proline and soluble sugar. TaEXPA2‐overexpressing tobacco lines exhibited lower Na⁺ but higher K⁺ accumulation than WT plants. Antioxidant competence increased in the transgenic plants because of the increased activity of antioxidant enzymes. TaEXPA2 protein abundance in wheat was induced by NaCl, and ABA signaling was involved. Gene expression regulation was involved in the enhanced salt stress tolerance of the TaEXPA2 transgenic plants. Our results suggest that TaEXPA2 overexpression confers salt stress tolerance on the transgenic plants, and this is associated with improved water status, Na⁺/K⁺ homeostasis, and antioxidant competence. ABA signaling participates in TaEXPA2‐regulated salt stress tolerance.     

Increased cytokinin levels in transgenic PSAG12–IPT tobacco plants have large direct and indirect effects on leaf senescence, photosynthesis and N partitioning

We studied the impact of delayed leaf senescence on the functioning of plants growing under conditions of nitrogen remobilization. Interactions between cytokinin metabolism, Rubisco and protein levels, photosynthesis and plant nitrogen partitioning were studied in transgenic tobacco (Nicotiana tabacum L.) plants showing delayed leaf senescence through a novel type of enhanced cytokinin syn‐thesis, i.e. targeted to senescing leaves and negatively auto‐regulated (P_SAG12–IPT), thus preventing developmental abnormalities. Plants were grown with growth‐limiting nitrogen supply. Compared to the wild‐type, endogenous levels of free zeatin (Z)‐ and Z riboside (ZR)‐type cytokinins were increased up to 15‐fold (total ZR up to 100‐fold) in senescing leaves, and twofold in younger leaves of P_SAG12–IPT. In these plants, the senescence‐associated declines in N, protein and Rubisco levels and photosynthesis rates were delayed. Senescing leaves accumulated more (¹⁵N‐labelled) N than younger leaves, associated with reduced shoot N accumulation (–60%) and a partially inverted canopy N profile in P_SAG12–IPT plants. While root N accumulation was not affected, N translocation to non‐senescing leaves was progressively reduced. We discuss potential consequences of these modified sink–source relations, associated with delayed leaf senescence, for plant productivity and the efficiency of utilization of light and minerals.     

TFT6 and TFT7, two different members of tomato 14-3-3 gene family, play distinct roles in plant adaption to low phosphorus stress

14‐3‐3 proteins are a large family of proteins but exact roles of their members in plant response to abiotic stresses are not clear, especially under nutrient deficiency. We investigated the expressions of all the tomato 14‐3‐3 gene family members (TFT1–TFT12) under low phosphorus stress (LP) and found that TFT6 belongs to the later responsive gene while TFT7 belongs to the early responsive gene. When the two genes were separately introduced into Arabidopsis and overexpressed, their plant growth under LP was much enhanced compared with wild‐type plant. TFT6 overexpressing plants showed reduced starch synthase activity, reduced starch content but enhanced sucrose loading into phloem in the shoot under LP. TFT7 overexpressing plants had much enhanced H⁺ flux along their root tip and activity of plasma membrane H⁺‐ATPase in the roots under LP. Our results suggest that TFT6 and TFT7 play different roles in plant adaption to LP. TFT6 acts mainly in leaves and is involved in the systemic response to LP by regulating leaf carbon allocation and increasing phloem sucrose transport to promote root growth, while TFT7 directly functions in root by activating root plasma membrane H⁺‐ATPase to release more protons under LP.     

Ethylene regulates the timing of leaf senescence in Arabidopsis

The plant hormone ethylene influences many aspects of plant growth and development, including some specialized forms of programmed senescence such as fruit ripening and flower petal senescence. To study the relationship between ethylene and leaf senescence, etr1-1, an ethylene-insensitive mutant in Arabidopsis, was used. Comparative analysis of rosette leaf senescence between etr1-1 and wild-type plants revealed that etr1-1 leaves live approximately 30% longer than the wild-type leaves. Delayed leaf senescence in etr1-1 coincided with delayed induction of senescence-associated genes (SAGs) and higher expression levels of photosynthesis-associated genes (PAGs). In wild-type plants, exogenous ethylene was able to further accelerate induction of SAGs and decrease expression of PAGs. The extended period of leaf longevity in etr1-1 was associated with low levels of photosynthetic activity. Therefore, the leaves in etr1-1 functionally senesced even though the apparent life span of the leaf was prolonged.     

Influence of cytokinins and novel cytokinin antagonists on the senescence of detached leaves of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Cytokinins N⁶-benzyladenine (BA) and 1-(2-chloropyridin-4-yl)-3-phenylurea (4PU-30) delayed the senescence of detached leaves (3^rd to 7^th leaf node) of wild and ethylene insensitive eti5 mutant of Arabidopsis thaliana. The novel anticytokinins, structural analogues of purine and phenylurea cytokinins also affected the senescence of detached rosette leaves of A. thaliana. They diminished to a significant extent the cytokinin-induced delay of chlorophyll destruction, but without a considerable difference in their action against both types of cytokinins. These results correlated with changes observed in ribonuclease (RNase) activity.     

Mobilization of metabolites from leaves to grains as the cause of monocarpic senescence in rice

The pattern of senescence was studied by following the changes in chlorophyll and protein in the leaves and by measuring ³²P retention and export from source to sink during development of the rice plant (Oryza sativa L. cv. Jaya) subjected to different manipulative treatments. With the advance of reproductive development, the chronological sequence of leaf senescence was changed, so that the flag and the third leaf senesced earlier than did the second leaf. In presence of the daughter shoot of defruited plants, senescence was delayed in all three leaves of the mother plant, as compared to the same leaves of intact plants. Senescence of all three leaves was further delayed when both panicle and daughter shoots were removed from the plant. The above manipulative treatments caused the initial sequential pattern of senescence of leaves to persist. Removal of both panicle and daughter shoots caused little export of ³²P between leaves. In the presence of daughter shoots of defruited plants, export of ³²P was maximum from leaves of the mother plant to the nearest daughter shoots. This led to earlier senescence of such mother plant leaves than that of plants from which both panicle and daughter shoots were removed. The pattern of senescence and export of ³²P in the flag and the second leaf of the daughter shoot was essentially the same as that of the intact plant. Based on these findings, it was concluded that mobilization of metabolites from source to sink is the primary cause of monocarpic senescence in rice.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis: implications of the stay-green mutation for photosynthesis, growth and nitrogen nutrition

Mutation of the nuclear gene sid disables chlorophyll degradation during leaf senescence in the pasture grass Festuca pratensis. This study investigated the effect of the mutation on photosynthesis and on leaf and whole plant growth under a range of nitrogen regimes. When plants were cultivated in a static hydroponic system, the chlorophyll content of fourth leaves of the stay-green mutant Bf993 remained virtually unchanged from full expansion to complete senescence, while tissue of the wild-type (cv. Rossa) became completely yellow. The retention of chlorophyll in Bf993 was not associated with maintenance of photosynthetic activity as shown by rates of light-saturated CO₂ fixation and apparent quantum efficiency. Higher levels of total N in senescing leaves of Bf993 than in Rossa indicated reduced nitrogen remobilization in the mutant. When using a range of [NH₄NO₃], dry matter production and tillering Mere lower for Bf993 at all but the highest [NH₄NO³, which was supra-optimal for the wild type. In contrast to the static system, where fluctuations in N supply occurred, growth and [NO₃^?] uptake were similar in mutant and wild type when [NO₃^?] was continuously maintained by a flowing solution culture system. The results are discussed in relation to the role of N supply and the effect of the stay-green mutation on N recycling.     

Defence responses regulated by jasmonate and delayed senescence caused by ethylene receptor mutation contribute to the tolerance of petunia to Botrytis cinerea

Ethylene and jasmonate (JA) have powerful effects when plants are challenged by pathogens. The inducible promoter‐regulated expression of the Arabidopsis ethylene receptor mutant ethylene‐insensitive1‐1 (etr1‐1) causes ethylene insensitivity in petunia. To investigate the molecular mechanisms involved in transgenic petunia responses to Botrytis cinerea related to the ethylene and JA pathways, etr1‐1‐expressing petunia plants were inoculated with Botrytis cinerea. The induced expression of etr1‐1 by a chemical inducer dexamethasone resulted in retarded senescence and reduced disease symptoms on detached leaves and flowers or intact plants. The extent of decreased disease symptoms correlated positively with etr1‐1 expression. The JA pathway, independent of the ethylene pathway, activated petunia ethylene response factor (PhERF) expression and consequent defence‐related gene expression. These results demonstrate that ethylene induced by biotic stress influences senescence, and that JA in combination with delayed senescence by etr1‐1 expression alters tolerance to pathogens.     

Effect of Ethylene and its Antagonist 1-MCP on the Senescence of Detached Leaves of Arabidopsis thaliana

1-Methylcyclopropene (1-MCP) applied alone did not influence significantly the chlorophyll and carotenoid content of the older leaves of Arabidopsis thaliana (L.) Heynh., but retarded the senescence of the younger ones (6^th and 7^th leaf nodes). However, 1-MCP effectively blocks the ethylene induced senescence of excised rosette leaves. The preliminary application of 1-MCP (3 h in advance to the treatment by Ethrel) almost totally eliminated the ethylene action. Similar trend was also observed after simultaneous application of Ethrel and 1-MCP, and the effects of both treatments on the chlorophyll and carotenoid destruction are comparable.     

The dependence of leaf senescence on the balance between 1‐aminocyclopropane‐1‐carboxylate acid synthase 1 (ACS1)‐catalysed ACC generation and nitric oxide‐associated 1 (NOS1)‐dependent NO accumulation in Arabidopsis

• Ethylene and nitric oxide (NO) act as endogenous regulators during leaf senescence. Levels of ethylene or its precursor 1‐aminocyclopropane‐1‐carboxylate acid (ACC) depend on the activity of ACC synthases (ACS), and NO production is controlled by NO‐associated 1 (NOA1). However, the integration mechanisms of ACS and NOA1 activity still need to be explored during leaf senescence.
• Here, using experimental techniques, such as physiological and molecular detection, liquid chromatography‐tandem mass spectrometry and fluorescence measurement, we investigated the relevant mechanisms.
• Our observations showed that the loss‐of‐function acs1‐1 mutant ameliorated age‐ or dark‐induced leaf senescence syndrome, such as yellowing and loss of chlorophyll, that acs1‐1 reduced ACC accumulation mainly in mature leaves and that acs1‐1‐promoted NOA1 expression and NO accumulation mainly in juvenile leaves, when compared with the wild type (WT). But the leaf senescence promoted by the NO‐deficient noa1 mutant was not involved in ACS1 expression. There was a similar sharp reduction of ACS1 and NOA1 expression with the increase in WT leaf age, and this inflection point appeared in mature leaves and coincided with the onset of leaf senescence.
• These findings suggest that NOA1‐dependent NO accumulation blocked the ACS1‐induced onset of leaf senescence, and that ACS1 activity corresponds to the onset of leaf senescence in Arabidopsis.

     

Interrelationship between ethylene and growth regulators in the senescence of lettuce leaf discs

The interrelationship between ethylene and growth regulators in the senescence of romaine lettuce (Lactuca sativa L.) leaves was studied. Gibberellic acid (GA₃), kinetin, and 3-indoleacetic acid (IAA) retarded chlorophyll loss from leaf discs which were floated on hormone solutions. Abscisic acid (ABA) and ethephon enhanced chlorophyll loss and antagonized the senescence-retarding effect of GA₃ and kinetin. A high concentration of IAA (10^–4 M) caused accelerated chlorophyll loss, whereas a similar concentration of kinetin neither retarded nor promoted chlorophyll loss. The ineffectiveness of IAA and kinetin at their supraoptimal concentrations in retarding leaf senescence was related to increased production of ethylene induced in the treated leaf discs. GA₃ was the most effective in retarding chlorophyll loss and did not stimulate ethylene production at all. The senescence-enhancing effect of ABA was not mediated by ethylene. However, the moderately increased production of ethylene, induced by relatively high concentrations of ABA, could act synergistically with the latter to accelerate chlorophyll loss. It is proposed that the effectiveness of exogenously applied hormones, both in enhancing and retarding senescence, is greatly affected by the endogenous ethylene concentration of the treated plant tissue.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 2571-E, 1988 series.     

Overexpression of OsRab7B3, a Small GTP-Binding Protein Gene,Enhances Leaf Senescence in Transgenic Rice

Rab family proteins are small GTP-binding proteins involved in intracellular trafficking. They play critical roles in several plant development processes. Different expression patterns of 46 Rabs in the rice genome were examined in various rice tissues and in leaves treated with plant growth regulators and under senescence conditions. One of the OsRab genes, OsRab7B3, closely associated with senescence in expression pattern, was chosen for functional analysis. Expression of sGFP under the control of the OsRab7B3 promoter increased in leaves when ABA and NaCl were applied or when kept in dark. In transgenic rice overexpressing OsRab7B3, the senescence-related genes were upregulated and leaf senescence was significantly enhanced under dark conditions. Moreover, leaf yellowing occurred earlier in the transgenic plants than in the wild type at the ripening stage. Hence it is suggested that OsRab7B3 act as a stress–inducible gene that plays an important role in the leaf senescence process.     

Premature leaf senescence 3, encoding a methyltransferase,is required for melatonin biosynthesis in rice

Premature leaf senescence in rice is one of the most common factors affecting the plant's development and yield. Although methyltransferases are involved in diverse biological functions, their roles in rice leaf senescence have not been previously reported. In this study, we identified the premature leaf senescence 3 (pls3) mutant in rice, which led to early leaf senescence and early heading date. Further investigations revealed that premature leaf senescence was triggered by the accumulation of reactive oxygen species. Using physiological analysis, we found that chlorophyll content was reduced in the pls3 mutant leaves, while hydrogen peroxide (H₂O₂) and malondialdehyde levels were elevated. Consistent with these findings, the pls3 mutant exhibited hypersensitivity to exogenous hydrogen peroxide. The expression of other senescence‐associated genes such as Osh36 and RCCR1 was increased in the pls3 mutant. Positional cloning indicated the pls3 phenotype was the result of a mutation in OsMTS1, which encodes an O‐methyltransferase in the melatonin biosynthetic pathway. Functional complementation of OsMTS1 in pls3 completely restored the wild‐type phenotype. We found leaf melatonin content to be dramatically reduced in pls3, and that exogenous application of melatonin recovered the pls3 mutant's leaf senescence phenotype to levels comparable to that of wild‐type rice. Moreover, overexpression of OsMTS1 in the wild‐type plant increased the grain yield by 15.9%. Our results demonstrate that disruption of OsMTS1, which codes for a methyltransferase, can trigger leaf senescence as a result of decreased melatonin production.     

Over-expression of gsh1 in the cytosol affects the photosynthetic apparatus and improves the performance of transgenic poplars on heavy metal-contaminated soil

Recent studies of transgenic poplars over‐expressing the genes gsh1 and gsh2 encoding γ‐glutamylcysteine synthetase (γ‐ECS) and glutathione synthetase, respectively, provided detailed information on regulation of GSH synthesis, enzymes activities and mRNA expression. In this experiment, we studied quantitative parameters of leaves, assimilating tissues, cells and chloroplasts, mesophyll resistance for CO₂ diffusion, chlorophyll and carbohydrate content in wild‐type poplar and transgenic plants over‐expressing gsh1 in the cytosol after 3 years of growth in relatively clean (control) or heavy metal‐contaminated soil in the field. Over‐expression of gsh1 in the cytosol led to a twofold increase of intrafoliar GSH concentration and influenced the photosynthetic apparatus at different levels of organisation, i.e., leaves, photosynthetic cells and chloroplasts. At the control site, transgenic poplars had a twofold smaller total leaf area per plant and a 1.6‐fold leaf area per leaf compared to wild‐type controls. Annual aboveground biomass gain was reduced by 50% in the transgenic plants. The reduction of leaf area of the transformants was accompanied by a significant decline in total cell number per leaf, indicating suppression of cell division. Over‐expression of γ‐ECS in the cytosol also caused changes in mesophyll structure, i.e., a 20% decrease in cell and chloroplast number per leaf area, but also an enhanced volume share of chloroplasts and intercellular airspaces in the leaves. Transgenic and wild poplars did not exhibit differences in chlorophyll and carotenoid content of leaves, but transformants had 1.3‐fold fewer soluble carbohydrates. Cultivation on contaminated soil caused a reduction of palisade cell volume and chloroplast number, both per cell and leaf area, in wild‐type plants but not in transformants. Biomass accumulation of wild‐type poplars decreased in contaminated soil by more than 30‐fold, whereas transformants showed a twofold decrease compared to the control site. Thus, poplars over‐expressing γ‐ECS in the cytosol were more tolerant to heavy metal stress under field conditions than wild‐type plants according to the parameters analysed. Correlation analysis revealed strong dependence of cell number per leaf area unit, chloroplast parameters and mesophyll resistance with the GSH level in poplar leaves.     

Effects of 1-MCP on the vase life and ethylene response of cut flowers

Pretreatment for 6 h with low concentrations of 1-MCP (1-Methylcyclopropene, formerly designated as SIS-X), a cyclic ethylene analog, inhibits the normal wilting response of cut carnations exposed continuously to 0.4 μl·l^?1 ethylene. The response to 1-MCP was a function of treatment concentration and time. Treatment with 1-MCP was as effective in inhibiting ethylene effects as treatment with the anionic silver thiosulfate complex (STS), the standard commercial treatment. Other ethylene-sensitive cut flowers responded similarly to carnations. In the presence of 1 μl·l^?1 ethylene, the vase life of 1-MCP-treated flowers was up to 4 times that of the controls.     

Effect of inhibitors of ethylene biosynthesis and action, as well as calcium and magnesium on rose shoot rooting, shoot-tip necrosis and leaf senescence in vitro

Microcuttings of easy-to-root dwarf rose cv. Starina, showing early symptoms of leaf senescence and shoot-tip necrosis in rooting stage, were chosen for the study. The effects of inhibitors of ethylene biosynthesis (AOA, AIB) and action (AgNO₃), and Ca²⁺ and Mg²⁺ were studied in relation to rooting, leaf senescence and shoot-tip necrosis. The effects of these substances were examined with respect to IAA presence in a medium, which stimulated leaf yellowing and shoot-tip necrosis. AOA strongly inhibited rooting of microcuttings, but did not affect ethylene biosynthesis. AIB at 250 mg·l⁻¹ and AgNO₃ 2.5 mg·l⁻¹ in the presence of IAA did not affect rooting but effectively prevented leaf senescence. Ca²⁺ alone or combined with Mg²⁺ at raised concentration, or an ethylene action inhibitor Ag⁺, reduced shoot-tip necrosis in microcuttings treated with IAA. Addition of Ag⁺ to IAA medium drastically increased ethylene production by the shoots. Interaction between endogenous levels of auxin, ethylene and calcium in relation to rooting, shoot-tip necrosis and leaf senescence was discussed. Ethylene could enhance tissue sensitivity to auxin. Moreover, the tissue of rose shoots is very sensitive in the in vitro condition on standard medium because of the calcium deficiency. Thus, the raised Ca/Mg level counteracted shoot-tip necrosis through enhancing cell membrane and wall resistance to ethylene and IAA.