Mutation of Oryza sativa CORONATINE INSENSITIVE 1b (OsCOI1b) delays leaf senescence

Jasmonic acid (JA) functions in plant development, including senescence and immunity. Arabidopsis thaliana CORONATINE INSENSITIVE 1 encodes a JA receptor and functions in the JA‐responsive signaling pathway. The Arabidopsis genome harbors a single COI gene, but the rice (Oryza sativa) genome harbors three COI homologs, OsCOI1a, OsCOI1b, and OsCOI2. Thus, it remains unclear whether each OsCOI has distinct, additive, synergistic, or redundant functions in development. Here, we use the oscoi1b‐1 knockout mutants to show that OsCOI1b mainly affects leaf senescence under senescence‐promoting conditions. oscoi1b‐1 mutants stayed green during dark‐induced and natural senescence, with substantial retention of chlorophylls and photosynthetic capacity. Furthermore, several senescence‐associated genes were downregulated in oscoi1b‐1 mutants, including homologs of Arabidopsis thaliana ETHYLENE INSENSITIVE 3 and ORESARA 1, important regulators of leaf senescence. These results suggest that crosstalk between JA signaling and ethylene signaling affects leaf senescence. The Arabidopsis coi1‐1 plants containing 35S:OsCOI1a or 35S:OsCOI1b rescued the delayed leaf senescence during dark incubation, suggesting that both OsCOI1a and OsCOI1b are required for promoting leaf senescence in rice. oscoi1b‐1 mutants showed significant decreases in spikelet fertility and grain weight, leading to severe reduction of grain yield, indicating that OsCOI1‐mediated JA signaling affects spikelet fertility and grain filling.     

NYC4, the rice ortholog of Arabidopsis THF1, is involved in the degradation of chlorophyll – protein complexes during leaf senescence

Yellowing/chlorophyll breakdown is a prominent phenomenon in leaf senescence, and is associated with the degradation of chlorophyll – protein complexes. From a rice mutant population generated by ionizing radiation, we isolated nyc4‐1, a stay‐green mutant with a defect in chlorophyll breakdown during leaf senescence. Using gene mapping, nyc4‐1 was found to be linked to two chromosomal regions. We extracted Os07g0558500 as a candidate for NYC4 via gene expression microarray analysis, and concluded from further evidence that disruption of the gene by a translocation‐related event causes the nyc4 phenotype. Os07g0558500 is thought to be the ortholog of THF1 in Arabidopsis thaliana. The thf1 mutant leaves show variegation in a light intensity‐dependent manner. Surprisingly, the F_v/F_m value remained high in nyc4‐1 during the dark incubation, suggesting that photosystem II retained its function. Western blot analysis revealed that, in nyc4‐1, the PSII core subunits D1 and D2 were significantly retained during leaf senescence in comparison with wild‐type and other non‐functional stay‐green mutants, including sgr‐2, a mutant of the key regulator of chlorophyll degradation SGR. The role of NYC4 in degradation of chlorophyll and chlorophyll – protein complexes during leaf senescence is discussed.     

Effects of cytokinin and senescence-inducing factors on expression of P ARR5 -GUS gene construct during leaf senescence in transgenic Arabidopsis thaliana plants

ARR5-gene expression was studied in the course of natural leaf senescence and detached leaf senescence in the dark using Arabidopsis thaliana plants transformed with the P _ARR5 -GUS gene construct. GUS-activity was measured as a marker of ARR5-gene expression. Chlorophyll and total protein amounts were also estimated to evaluate leaf senescence. Natural leaf senescence was accompanied by the progressive decline in the GUS-activity in leaves of the 2nd and 3rd nodes studied, and this shift of GUS-activity was more pronounced than the loss of chlorophyll content. The ability of the ARR5-gene promoter to respond to cytokinin was not eliminated during natural leaf senescence, as was demonstrated by a cytokinin-induced increase in GUS activity in leaves after their detachment and incubation on benzyladenine (BA, 5 × 10⁻⁶ M) in the dark. Leaf senescence in the dark was associated with the further decrease in the GUS-activity. The ARR5-gene promoter response to cytokinin was enhanced with the increase of the age of plants, taken as a source of leaves for cytokinin treatments. Hence, although the expression of the ARR5 gene reduces during natural and dark/detached leaf senescence, the ARR5-gene sensitivity to cytokinin was maintained in both cases and even increased with the leaf age. This data suggest that the ARR5 gene, which belongs to the type-A negative regulators of plant response to cytokinin, could be a feedback regulator able to prevent retardation by cytokinin of leaf senescence when it is important for plant life. Growth regulators either reduced ARR5 gene response to cytokinin during senescence of mature detached leaves in the dark (SA, meJA, ABA, SP) or increased it (IAA), thus modifying the resulting rate of its expression.     

A senescence-associated gene of Arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence

We have characterized the structure and expression of a senescence-associated gene (sen1) of Arabidopsis thaliana. The protein-coding region of the gene consists of 5 exons encoding 182 amino acids. The encoded peptide shows noticeable similarity to the bacterial sulfide dehydrogenase and 81% identity to the peptide encoded by the radish din1 gene. The 5-upstream region contains sequence motifs resembling the heat-shock- and ABA-responsive elements and the TCA motif conserved among stress-inducible genes. Examination of the expression patterns of the sen1 gene under various senescing conditions along with measurements of photochemical efficiency and of chlorophyll content revealed that the sen1 gene expression is associated with Arabidopsis leaf senescence. During the normal growth phase, the gene is strongly induced in leaves at 25 days after germination when inflorescence stems are 2–3 cm high, and then the mRNA level is maintained at a comparable level in naturally senescing leaves. In addition, dark-induced senescence of detached leaves or of leaves in planta resulted in a high-level induction of the gene. Expression of the sen1 gene was also strongly induced in leaves subjected to senescence by 0.1 mM abscisic acid or 1 mM ethephon treatment. The induced expression of the gene by dark treatment was not significantly repressed by treatment with 0.1 mM cytokinin or 50 mM CaCl₂ which delayed loss of chlorophyll but not that of photochemical efficiency.     

A combined 15N tracing/proteomics study in Brassica napus reveals the chronology of proteomics events associated with N remobilisation during leaf senescence induced by nitrate limitation or starvation

Our goal was to identify the leaf proteomic changes which appeared during N remobilisation that were associated or not associated with senescence of oilseed rape in response to contrasting nitrate availability. Remobilisation of N and leaf senescence status were followed using ¹⁵N tracing, patterns of chlorophyll level, total protein content and a molecular indicator based on expression of senescence‐associated gene 12/Cab genes. Three phases associated with N remobilisation were distinguished. Proteomics revealed that 55 proteins involved in metabolism, energy, detoxification, stress response, proteolysis and protein folding, were significantly induced during N remobilisation. Four proteases were specifically identified. FtsH, a chloroplastic protease, was induced transiently during the early stages of N remobilisation. Considering the dynamics of N remobilisation, chlorophyll and protein content, the pattern of FtsH expression indicated that this protease could be involved in the degradation of chloroplastic proteins. Aspartic protease increased at the beginning of senescence and was maintained at a high level, implicating this protease in proteolysis during the course of leaf senescence. Two proteases, proteasome beta subunit A1 and senescence‐associated gene 12, were induced and continued to increase during the later phase of senescence, suggesting that these proteases are more specifically involved in the proteolysis processes occurring at the final stages of leaf senescence.     

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.

     

大豆microRNA基因GmMIR160A负调控植物叶片衰老进程

:叶片衰老是受内外多种因子影响的遗传发育进程.生长素、细胞分裂素和乙烯等多种植物激素是调 控叶片衰老的重要内部因子,它们通过长或短距离运输形成叶片组织内特定的区域分布和浓度梯度,从而直 接或间接参与植物叶片衰老过程.分子遗传学表明,细胞分裂素和乙烯分别是叶片衰老的抑制子和正调节 子,而生长素如何参与叶片衰老的分子机制目前还不清晰.植物体内成熟小分子RNA 由小RNA 基因转录 并通过特定酶加工形成的２１~２３bp的双链RNA分子.这些小分子通过不完全配对方式抑制其靶基因转录 和/或表达,参与植物生长发育多个过程,然而这类小RNA 分子如何调控植物叶片衰老发育过程目前则还鲜 有报告.大豆是重要的油料作物,具有典型的单次结实性衰老特征.研究大豆叶片衰老具有重要的科学意义 和深远的应用价值.该文采用实时荧光定量PCR(qPCR)技术分析大豆(Glycinemax)microRNA基因Gm- MIR１６０A 的表达模式,发现大豆第一复叶中GmMIR１６０A 表达受外源生长素和黑暗处理的诱导,暗示该基 因是生长素快速响应的叶片衰老相关基因.为进一步探究GmMIR１６０A 在大豆叶片发育中的功能,构建了 肾上腺皮质激素(Glucocorticoid,GR)类似物地塞米松(Dexamethasone,DEX)诱导表达GmMIR１６０A 双元表 达载体并通过农杆菌介导的子叶节方法转化野生型大豆.通过抗性筛选和基因组PCR 鉴定并结合表型分 析,共获得了４株诱导表达的稳定遗传转基因植株(株系OXＧ３、OXＧ５、OXＧ７和OXＧ８).GmMIR１６０A 过表达 植株根、茎、叶、花和果实在形态学上与野生型相比无显著差异,但叶片的叶绿素含量增加、最大光量子效率 (Fv/Fm)增强.进一步分子分析发现,转基因大豆叶片中GmARFs 和衰老标记基因(GmCYSP１)表达明显下 降,表明大豆Gma-miR１６０通过抑制靶基因GmARFs 的表达来负调控植物叶片的衰老进程.该文揭示了生 长素通过小分子RNA调控叶片发育一条新途径,为研究植物激素调控植物叶片衰老提供了新的思路.     

Correlation of leaf senescence and gene expression/activities of chlorophyll degradation enzymes in harvested Chinese flowering cabbage (Brassica rapa var. parachinensis)

Chinese flowering cabbage is one of the main leafy vegetables produced in China. They have a rapid leaf yellowing due to chlorophyll degradation after harvest that limits their marketing. In the present study, leaf senescence of the cabbages was manipulated by ethylene and 6-benzyl aminopurine (6-BA) treatment to investigate the correlation of leaf senescence and chlorophyll degradation related to gene expression/activities in the darkness. The patterns of several senescence associated markers, including a typical marker, the expression of senescence-associated gene SAG₁₂, demonstrated that ethylene accelerated leaf senescence of the cabbages, while 6-BA retarded this progress. Similar to the trends of BrSAG₁₂ gene expression, strong activation in the expression of three chlorophyll degradation related genes, pheophytinase (BrPPH), pheophorbide a oxygenase (BrPAO) and red chlorophyll catabolite reductase (BrRCCR), was detected in ethylene treated and control leaves during the incubation, while no evident increase was recorded in 6-BA treated leaves. The overall dynamics of Mg-dechelatase activities in all treatments displayed increasing trends during the senescence process, and a delayed increase in the activities was observed for 6-BA treated leaves. However, chlorophyllase activity as well as the expression of BrChlase1 and BrChlase2 decreased with the incubation in all treatments. Taken together, the expression of BrPPH, BrPAO and BrRCCR, and the activity of Mg-dechelatase was closely associated with the chlorophyll degradation during the leaf senescence process in harvested Chinese flowering cabbages under dark conditions.     

Changes in activities of antioxidant enzymes and their gene expression during leaf development of sweetpotato

To understand the functions of antioxidant enzymes during leaf development in sweetpotato, we investigated the activities of several antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POX), ascorbate peroxidase (APX) and catalase (CAT). Significant increases were observed in the activities of SOD, POX and APX during the late stage of leaf development, whereas CAT activity increased during the early developmental stage. By RT-PCR analysis, various POX and APX genes showed differential expression patterns during leaf development. Four POX genes swpa3, swpa4, swpa6, swpb4 and one APX gene swAPX1 exhibited high levels of gene expression during the senescence stage of leaf development, but two POX genes, swpa1 and swpa7 were preferentially expressed at both the mature green and the late senescence stages of leaf development. These results indicate that hydrogen peroxide (H₂O₂)-related antioxidant enzymes are differentially regulated in the process of leaf development of sweetpotato.     

Identification and functional characterization of a leucine-rich repeat receptor-like kinase gene that is involved in regulation of soybean leaf senescence

We report here the cloning and characterization of a soybean receptor-like kinase (RLK) gene, designated GmSARK (Glycine max senescence-associated receptor-like kinase), which is involved in regulating leaf senescence. The conceptual protein product of GmSARK contains typical domains of LRR receptor-like kinases: a cytoplasmic domain with all the 11 kinase subdomains, a transmembrane domain and an extracelullar domain containing 9 Leucine-Rich Repeat (LRR) units that may act as a receptor. The expression of GmSARK in soybean leaves was up-regulated in all the three tested senescence systems: senescing cotyledons, dark-induced primary leaf senescence and the natural leaf senescence process after florescence. Furthermore, the RNA interference (RNAi)-mediated knocking-down of GmSARK dramatically retarded soybean leaf senescence. A more complex thylakoid membrane system, higher foliar level of chlorophyll content and a very remarkable delay of senescence-induced disintegration of chloroplast structure were observed in GmSARK-RNAi transgenic leaves. A homolog of maize lethal leaf-spot 1 gene, which has been suggested to encode a key enzyme catalyzing chlorophyll breakdown, was isolated and nominated Gmlls1. The expression level of Gmgtr1 gene, which encodes a key enzyme of chlorophyll synthesis, was also analyzed. It was found that Gmlls1 was up-regulated and Gmgtr1 was down-regulated during senescence in wild-type soybean leaves. However, both of the up-regulation of Gmlls1 and down-regulation of Gmgtr1 were retarded during senescence of GmSARK-RNAi transgenic leaves. In addition, over-expression of the GmSARK gene greatly accelerated the senescence progression of CaMV 35S:GmSARK transgenic plants. Taken together, these results strongly suggested the involvement of this LRR-RLK in regulation of soybean leaf senescence, maybe via regulating chloroplast development and chlorophyll accumulation. Multiple functions of GmSARK besides its regulation of leaf senescence were also discussed. Electronic Supplementary Material Supplementary material is available for this article at Rui Gan, Peng-Li Li and Yuan-Yuan Ma contributed equally to this work.