Genetic improvement of a number of crops including soybean (Glycine max L. [Merr]) has been associated with stay-green. Research on stay green genes has focused primarily on genes involved with photosynthesis and chlorophyll degradation. The current study explores the impact of a group of developmental genes, known as the E gene series, on the rate of soybean leaf senescence. The objective of this experiment was to determine the role of E-genes in the control of leaf senescence in soybean. The experiment was conducted in a split-plot design with three replications. The main plots were two photoperiods imposed following R1; i) natural day length (Amb) and ii) incandescent day length extension of 3 hours (Amb+3). The split plots were five E-gene near-isogenic lines (NILs), planted on different dates to obtain synchronous flowering. Phenology, photosynthesis, normalized difference vegetative index (NDVI) and fluorescence measurements were taken including, dark adapted photosynthetic efficiency (Fv/Fm), electron transport rate (ETR), and leaf chlorophyll concentration (SPAD). Leaf tissues were also analyzed for gene expression patterns among Harosoy isolines. Yield parameters like dry matter accumulation, harvest index and grain yields were recorded. The leaf net photosynthesis was more closely related to ETR than to SPAD values, suggesting that visual observation of stay-green may not be as effective in evaluating functional senescence as measurement of ETR. Cultivars with the dominant E1 allele maintained functional photosynthesis for longer, such that full senescence was delayed by 10-15 days in these cultivars. This phenomenon was observed under both photoperiod treatments and irrespective of the genetic background (Clark and Harosoy) in which the alleles appeared. Maintenance of functional photosynthesis by the E1 dominant allele can be attributed to maintenance of high ETR, and Fv/Fm, as well as delayed decline in leaf chlorophyll concentrations. Expression of senescence related genes were delayed in the isoline which had delayed leaf senescence phenotype. Consistent with the effect on leaf senescence, the dominant alleles also reduced the rate of phenological development, such that R5 occurred later in genotypes with dominant alleles and under the Amb+3 treatment. Cultivars with the dominant E1 allele under extended photoperiod treatment accumulated more biomass and had decreased apparent harvest index which caused no change in grain yields. The dominant E allele may delay leaf senescence directly or indirectly, through its delay of reproductive development.

Ethylene is a plant growth regulator and plays a key role in leaf senescence. Its signaling, however, remains mostly unclear in sweet potato. Ethephon, an ethylene releasing compound, induced sweet potato detached leaf senescence and associated gene expression, and the effects were repressed by mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059. These data suggest that MAPK cascade is likely involved in ethylene signaling leading to leaf senescence and associated gene expression. With gene-specific primers and RT-PCR methods, a full-length cDNA, SPMAPK, was isolated from ethephon-treated sweet potato leaves. SPMAPK contained 1098 nucleotides (365 amino acids) in the open reading frame. Sweet potato SPMAPK also exhibited high amino acid sequence identities (ca. 79.8% to 83.4%) with plant MAPKs, and was most close to Arabidopsis MPK3 and MPK6 in phylogenetic tree analysis. RT-PCR analysis showed that SPMAPK gene expression was detected in roots, stems, and leaves. The mature and partial yellowing leaves expressed higher amount. SPMAPK gene expression was also inducible and significantly enhanced by ethephon. Results from studies with inhibitors or effectors showed that ethephon treatment resulted in acceleration of leaf senescence in detached sweet potato leaves, promotion of leaf chlorophyll content reduction and decrease of photochemical Fv/Fm, and induction of associated gene expression. These ethephon-mediated effects were all delayed or repressed by pretreatment with ethylene receptor inhibitor 1-methylcyclopropene (1-MCP), MAPK kinase inhibitor PD98059, NADPH oxidase inhibitor diphenyleneiodonium (DPI), antioxidant reduced glutathione, calcium ion chelator EGTA, and de novo protein synthesis inhibitor cycloheximide, respectively. Based on these results we conclude that an ethylene-inducible mitogen-activated protein kinase SPMAPK was isolated from sweet potato leaves, and expressed higher amount in mature and partial yellowing leaves. Ethephon-induced sweet potato SPMAPK expression was significantly repressed by 1-MCP, PD98059, DPI, reduced glutathione, EGTA and cycloheximide. These data also suggest that the possible signal components in ethephon-mediated leaf senescence and associated gene expression in sweet potato leaves likely include ethylene receptor, MAPK cascade, elevated H2O2 , external calcium influx, and de novo synthesized proteins. A possible ethylene signaling model leading to sweet potato leaf senescence and associated gene expression was also proposed.

Ethylene is a gaseous growth regulator, and plays an important role in response to plant developmental and environmental stimuli. Ethylene also plays a key role in leaf senescence. Calcium is a second message, and participates in the signal transduction pathways of many plant physiological responses. In this research, ethephon, an ethylene-releasing compound, was used to induce sweet potato leaf yellowing, chlorophyll content reduction, photochemical Fv/Fm decrease, H2O2 elevation and senescence-associated gene expression. These ethephon-mediated effects were all delayed or repressed by pretreatment of a calcium ion chelator, EGTA. Treatment with a calcium ionophore A23187 also induced senescence-associated gene expression in sweet potato detached leaves, and the induction was repressed by EGTA pretreatment. Calcium signaling in general is transmitted by calcium sensor proteins, including calmodulin to translate into appropriate responses to developmental and environmental stimuli. Therefore, pretreatment with calmodulin inhibitor chlorpromazine (CPZ) delayed or repressed ethephon-mediated leaf senescence, H2O2 elevation and senescence-associated gene expression. These CPZ-mediated effects were reversed by the exogenous application of an ethephon-inducible calmodulin SPCAM fusion protein. These results suggest that external Ca2+ influx and calmodulin SPCAM play a role in ethephon signaling leading to leaf senescence, H2O2 elevation and senescence-associated gene expression. In addition, NaCl salt stress also caused sweet potato leaf senescence, H2O2 elevation and senescence-associated gene expression. Pretreatment with CPZ delayed or repressed NaCl salt stress-mediated leaf senescence, H2O2 elevation and senescence-associated gene expression. These CPZ-mediated effects were also reversed by the exogenous application of calmodulin SPCAM fusion protein. These results suggest that calmodulin SPCAM may play a role in NaCl salt stress signaling leading to leaf senescence, H2O2 elevation and senescence-associated gene expression. Based on these results, external Ca2+ influx is required for ethephon induced leaf senescence. Ethephon-inducible calmodulin SPCAM likely participates in ethylene and NaCl salt stress signaling leading to leaf senescence, H2O2 elevation and senescence-associated gene expression in sweet potato in order to cope with different developmental cues or environmental stimuli.

Ethylene has long been considered as the main plant growth regulator that plays a key role in the regulation of leaf senescence. In sweet potato, ethephon, an ethylene releasing compound, promoted leaf senescence and H2O2 elevation. These ethephon-mediated effects were alleviated or attenuated by exogenous reduced glutathione and ascorbic acid. Ethephon treatment gradually increased endogenous total and reduced glutathione and ascorbic acid levels in sweet potato detached leaves 3 days after treatment. The H2O2 amount, however, was also increased at 72 h after treatment. Sweet potato detached leaves pretreated with reduced glutathione did significantly increased endogenous total and reduced glutathione levels at 24 h and remarkably decreased H2O2 amount at 72 h after ethephon application compared to that of ethephon alone control. Ethephon caused quick elevation of a small H2O2 peak at about 4 h after application, and the enhancement was eliminated by reduced glutathione pretreatment in treated sweet potato leaves. Pretreatment of diphenylene iodonium (DPI), an NADPH oxidase inhibitor, also repressed leaf senescence and H2O2 elevation at day 3 after ethephon treatment in sweet potato detached leaves, and the attenuation was effective within the first 4 h after ethephon treatment. For senescence-associated gene expression, ethephon and L-buthionine sulfoximine (BSO), an endogenous glutathione synthase inhibitor, did induced asparaginyl endopeptidase (SPAE) and cysteine proteases (SPCP1, SPCP2 and SPCP3) gene expression and the activation was repressed by reduced glutathione pretreatment. Based on these data we conclude that ethephon treatment may cause quick elevation of a small H2O2 peak likely via the NADPH oxidase, which may function as a signal component leading to leaf senescence, H2O2 elevation and senescence-associated gene expression in sweet potato detached leaves. The rate of endogenous antioxidant such as reduced glutathione elevation is also important and affects leaf senescence, H2O2 elevation and senescence-associated gene expression in sweet potato leaves.

Proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. Similarities and differences between the proteases expressed in different species may give valuable insights into their physiological roles and evolution. Systematic comparative analysis of the available sequenced genomes of two model organisms led to the identification of an increasing number of protease genes, giving insights about protein sequences that are conserved in the different species, and thus are likely to have common functions in them and the acquisition of new genes, elucidate issues concerning non-functionalization, neofunctionalization and subfunctionalization. The involvement of proteases in senescence and PCD was investigated. While PCD in woody tissues shows the importance of vacuole proteases in the process, the senescence in leaves demonstrate to be a slower and more ordered mechanism starting in the chloroplast where the proteases there localized become important. The light-harvesting complex of Photosystem II is very susceptible to protease attack during leaf senescence. We were able to show that a metallo-protease belonging to the FtsH family is involved on the process in vitro. Arabidopsis knockout mutants confirmed the function of FtsH6 in vivo.

<p>Recent changes in the timing of annual events are a sign that climate change is already impacting ecosystems. Carbon sequestration by forests increases with longer growing seasons. Biodiversity can be affected by mis-timing of events through shading interactions and frost damage. Projecting forests' ability to provide these ecosystem services in the future requires an understanding of trees' phenological responses to a new climate. I begin by proposing a first order definition of an `optimal' phenological response to warming: that the mean temperature following budburst should remain essentially constant. Analogously, the temperature preceding senescence can serve the same role. </p><p>To understand which environmental cues will drive future changes in phenology, I assimilate clues from observational and experimental literature. For budburst in woody plants, spring warmth, over-winter chilling and light drive nearly all behavior, but species' responses vary widely. Species using chilling or light as safety mechanisms against budburst during mid-winter thaws are thought to be less able to phenologically track a warming climate. However, I show that even species cued solely by spring warmth are likely to under-track temperature changes. Fall cues are more idiosyncratic, and a plant's driver of senescence is likely to vary from year to year. </p><p>Models are a tempting method to untangle species budburst cues and forecast phenology under warmer climate scenarios. I tested two models' ability to recover parameters used to simulate budburst data. The simpler model was cued only by spring warmth while the complex one modulated warmth requirements with chilling exposure. For the simple model, parameters could be recovered consistently from some, but not all, regions of parameter space. The complex model's parameters were largely unrecoverable. To understand the consequences of parameter uncertainty, I applied both models to an 18 year phenological record of 13 deciduous tree species. While a few species fell into identifiable regions of the simple model's parameter space, most did not, and projected budburst dates had wide parameter-derived uncertainty intervals. These bands were wider still under a 5°C warming scenario. Even greater uncertainty resulted from the complex model.</p><p>To better understand plants' potential for growing season extension I subjected seedlings to warmer climates in a series of open-topped chambers in sites at each end of the eastern deciduous biome. Soil and air were heated to 3 or 5°C above ambient, or left unheated. For nearly all species, warming hastened budburst and germination and delayed senescence. However, these events failed to track temperature changes, happening at warmer temperatures in hotter chambers. Individual species showed a remarkable variability of all events' dates within treatments, and even within chambers. Because phenological traits are heritable, this offers a potential for evolutionary response to climate change.</p><p>This research has shown that while individual trees extend their growing seasons under warmer temperatures, they typically under-respond to the magnitude of warming, suggesting forests' capacity for increased carbon sequestration may reach a limit. However, within populations, trees vary substantially in their phenological responses, forming a possibility for evolutionarily adaptation to changing cues.</p> / Dissertation

Leaf senescence is an active process required for plant survival, and it is flexibly controlled, allowing plant adaptation to environmental conditions. Although senescence is largely an age-dependent process, it can be triggered by environmental signals and stresses. Leaf senescence coordinates the breakdown and turnover of many cellular components, allowing a massive remobilization and recycling of nutrients from senescing tissues to other organs (e.g., young leaves, roots, and seeds), thus enhancing the fitness of the plant. Such metabolic coordination requires a tight regulation of gene expression. One important mechanism for the regulation of gene expression is at the transcriptional level via transcription factors (TFs). The NAC TF family (NAM, ATAF, CUC) includes various members that show elevated expression during senescence, including ORE1 (ANAC092/AtNAC2) among others. ORE1 was first reported in a screen for mutants with delayed senescence (oresara1, 2, 3, and 11). It was named after the Korean word “oresara,” meaning “long-living,” and abbreviated to ORE1, 2, 3, and 11, respectively. Although the pivotal role of ORE1 in controlling leaf senescence has recently been demonstrated, the underlying molecular mechanisms and the pathways it regulates are still poorly understood. To unravel the signaling cascade through which ORE1 exerts its function, we analyzed particular features of regulatory pathways up-stream and down-stream of ORE1. We identified characteristic spatial and temporal expression patterns of ORE1 that are conserved in Arabidopsis thaliana and Nicotiana tabacum and that link ORE1 expression to senescence as well as to salt stress. We proved that ORE1 positively regulates natural and dark-induced senescence. Molecular characterization of the ORE1 promoter in silico and experimentally suggested a role of the 5’UTR in mediating ORE1 expression. ORE1 is a putative substrate of a calcium-dependent protein kinase named CKOR (unpublished data). Promising data revealed a positive regulation of putative ORE1 targets by CKOR, suggesting the phosphorylation of ORE1 as a requirement for its regulation. Additionally, as part of the ORE1 up-stream regulatory pathway, we identified the NAC TF ATAF1 which was able to transactivate the ORE1 promoter in vivo. Expression studies using chemically inducible ORE1 overexpression lines and transactivation assays employing leaf mesophyll cell protoplasts provided information on target genes whose expression was rapidly induced upon ORE1 induction. First, a set of target genes was established and referred to as early responding in the ORE1 regulatory network. The consensus binding site (BS) of ORE1 was characterized. Analysis of some putative targets revealed the presence of ORE1 BSs in their promoters and the in vitro and in vivo binding of ORE1 to their promoters. Among these putative target genes, BIFUNCTIONAL NUCLEASE I (BFN1) and VND-Interacting2 (VNI2) were further characterized. The expression of BFN1 was found to be dependent on the presence of ORE1. Our results provide convincing data which support a role for BFN1 as a direct target of ORE1. Characterization of VNI2 in age-dependent and stress-induced senescence revealed ORE1 as a key up-stream regulator since it can bind and activate VNI2 expression in vivo and in vitro. Furthermore, VNI2 was able to promote or delay senescence depending on the presence of an activation domain located in its C-terminal region. The plasticity of this gene might include alternative splicing (AS) to regulate its function in different organs and at different developmental stages, particularly during senescence. A model is proposed on the molecular mechanism governing the dual role of VNI2 during senescence. / Der Alterungsprozess lebender Organismen wird seit vielen Jahren wissenschaftlich untersucht. In Pflanzen wird der Alterungsprozess Seneszenz genannt. Er ist für das Überleben der Pflanze von großer Bedeutung. Dennoch ist unser Wissen über die molekularen Mechanismen der Blattseneszenz, dessen komplexe Steuerung und die Wechselwirkungen mit Umweltsignale noch sehr limitiert. Ein wichtiges Steuerungselement besteht in der Aktivierung bestimmter Transkriptionsfaktoren (TFs) die während der Seneszenz unterschiedlich exprimiert werden. Aus der Literatur ist bekannt, dass Mitglieder der NAC TF Familie (NAM/ATAF/CUC) an der Regulation der Seneszenz bei Pflanzen beteiligt sind. ORE1 (ANAC092/AtNAC2), ein NAC TF mit erhöhter Genexpression während der Seneszenz, wurde erstmals in Mutanten mit verzögerte Seneszenz beschrieben, die molekularen Mechanismen, wie ORE1 die Seneszenz kontrolliert und die Stoffwechselwege reguliert, sind aber noch weitgehend unbekannt. Die Arbeiten im Rahmen dieser Dissertation wurden durchgeführt, um einen tieferen Einblick in die Regulationsmechanismen von ORE1 auf natürliche, dunkel induzierte sowie Salzstress-induzierte Seneszenz zu erhalten. Ergebnisse von Untersuchungen an zwei unterschiedlichen Pflanzenspezies (Arabidopsis thalinana und Nicotiana tabacum) deuten auf ein ähnliches Expressionsmuster von ORE1 während der natürlichen als auch der Salz-induzierten Seneszenz hin. In der Promotorregion von ORE1 wurde ein für natürliche Seneszenz charakteristisches Muster identifiziert. In vivo Analysen ergaben darüber hinaus. Hinweise auf zwei weitere ORE1 Regulatoren. Debei handelt es sich umeinen weiteren NAC TF (ATAF1) und (ii) CKOR, einer Calcium-abhängige Protein-Kinase (CDPK).In weiteren Studien wurden sechs Gene identifiziert, die durch ORE1 reguliert werden. In den Promotoren dieser Gene wurden entsprechende Bindestellen für ORE1 lokalisiert. Die ORE1-Bindung an die Promotoren wurde daraufhin sowohl in vitro als auch in vivo verifiziert. Zwei dieser Gene, die BIFUNCTIONAL Nuclease I (BFNI) und VND-Interacting2 (VNI2), wurden zudem auf molekularer und physiologischer Ebene untersucht.

Ethephon treatment caused SPCP3 gene expression (Chen et al., 2006), reduction of chlorophyll content, decrease of Fv/Fm value, increase of H2O2 amount, and more cell death, and accelerated leaf senescence in detached sweet potato leave. Exogenous application of modulators such as reduced glutathione, EGTA or cycloheximide delay leaf senescence and cell death caused by ethephon. These data suggest that oxidative stress, calcium influx and de novo synthesized protein may influence ethephon-mediated leaf senescence and cell death. When ethephon induced leaf senescence and cell death, granulin-containing cysteine protease SPCP3 gene was induced. Transgenic Arabidopsis system was used to explore the possible physiological role and function of SPCP3. The results showed that ectopic expression of SPCP3 in transgenic Arabidopsis plants caused earlier flowering, less rosette leaves when flowering, higher yellowing silique percentage during harvest, and lower germination percentage than that in control. During drought treatment, transgenic plants also exhibited reduction of Fv/Fm value and relative water content, but an increase in H2O2 content and cell death. These data suggest that ecopic expression of SPCP3 caused altered developmental characteristics and drought stress sensitivity. Previous report suggests that granulin-like domain may play a role in regulating enzymatic activity of granulin-containing cysteine protease (Yamada et al., 2001). In this report we demonstrate that pre-removal of granulin-like domain of SPCP3 does not affect significantly drought stress sensitivity compared to full-length SPCP3 in transgenic Arabidopsis plants. Based on these data we conclude that oxidative stress, calcium influx, and de novo synthesized proteins may be involved in ethylene signaling leading to leaf senescence and SPCP3 gene expression in detached sweet potato leaves, and ectopic SPCP3 expression in transgenic Arabidopsis plants caused altered developmental characteristics and enhanced drought sensitivity. Granulin-like domain may have no significant influence on SPCP3-mediated effect on drought stress sensitivity.

A major sweet potato leaf-type catalase was detected and identified from fullyexpandedmature leaves using in-gel activity staining assay with native- andSDS-PAGEs. The putative catalase activity band was inhibited by a catalaseinhibitor 3-amino-1,2,4-triazole. The major leaf-type catalase activity wasoptimal over 8, and was significantly repressed by £]-mercaptoethanol. However,its activity was much less affected by temperature within the range of 5 to 450C.Temporal and spatial expression showed that it was specifically detected inleaves, but not in roots and stems. Its activity increased from the immature L2leaves, and reached the maximal at the fully-expanded mature L3 leaves, thenslightly decreased in partial yellowing senescent L4 leaves, and was almost notdetected in completely yellowing L5 leaves similar to folding unopenedimmature L1 leaves. The catalase level showed approximately inversecorrelation with the H2O2 amounts in leaves of different developmental stages.Dark and ethephon, an ethylene-releasing compound, also temporarily enhancedthe catalase activities from 6 h to 24 h, however, the enhanced activitydecreased from 24 h to 48 h in detached leaves after treatment. The catalaselevel also showed approximately negative correlation with the H2O2 amounts intreated leaves. The major leaf-type catalase activity was repressed by EGTA,and the repression can be reversed by exogenous CaCl2. The major leaf-typecatalase activity was also repressed by calmodulin inhibitor chlorpromazine,and the repression can be reversed by exogenous purified SPCAM calmodulinfusion protein. Chlorpromazine-treated leaves also elevated H2O2 amount.Based on these data we conclude that a major leaf-type catalase with maximalactivity in L3 leaf was identified in sweet potato. Its activity was temporarilyenhanced by dark and ethephon, and was modulated by external calcium ion(Ca2+) and calmodulin. A possible physiological role and function in associationwith cellular H2O2 homeostasis in cope with developmental and environmentalcues in sweet potato leaves is suggested.

According to our previous results, ethephon-induced sweet potato leaf senescence and senescence-associated gene SPCP1 expression was affected by reduced glutathione, EGTA, and cycloheximide (Chen et al., 2009). These data suggest that calcium influx, reactive oxygen species (ROS) and de novo synthesized proteins can affect ethephon-mediated effects. Therefore, PCR-selective substractive hybridization and RACE-PCR methods were used to clone 5 full-length cDNAs encoded putative calmodulin (SPCAM), catalase (SPCATA), anionic peroxidase (SPPA), ACC oxidase (SPACO), and DSS1-like protein (SPDSS1) from mixed samples of ethephon-treated leaves for 6 and 24 hours. The ORF of SPCAM contains 450 nucleotides and encodes 149 amino acids. There are 4 putative EF-motifs in the deduced protein structure. SPCAM exhibited amino acid sequence identity with isolated Arabidopsis calmodulins from 48% to 100%, and was completely the same as CaM7 calmodulin. The ORF of SPCATA contains 1479 nucleotides and encodes 492 amino acids. SPCAM exhibited high amino acid sequence identity with other plant catalases from 71.2% to 80.9%, and had the highest identity with mangrove catalase. The ORF of SPPA contains 1068 nucleotides and encodes 355 amino acids. SPPA exhibited amino acid sequence identity with other published sweet potato peroxidase isoforms from 28.7% to 97.5%, and had the highest identity with anionic peroxidase SWPA4. The ORF of SPACO contains 930 nucleotides and encodes 309 amino acids. SPACO exhibited high amino acid sequence identity with other plant ACC oxidases from 62.3% to 81.5%, and had the highest identity with tobacco ACC oxidase. The ORF of SPDSS1 contains 228 nucleotides and encodes 75 amino acids. SPDSS1 exhibited amino acid sequence identity with other DSS1 from 25.2% to 62.3%, and had the highest identity with maize DSS1. The chlorophyll contents and Fv/Fm values were significantly reduced, however, the isolated gene expression was remarkably enhanced in natural senescent leaves. DAB staining showed that H2O2 amount was remarkably elevated at S3 senescent leaves compared to leaves of the other developmental stages. Evan blue staining also demonstrated that S3 senescent leaf had more cell death compared to S0 young leaves. In addition ethephon-induced leaf senescence exhibited similar results. The chlorophyll contents and Fv/Fm values were significantly reduced, however, the isolated gene expression was remarkably enhanced in ethephon-treated leaves compared to dark control. DAB staining showed that H2O2 amount was remarkably elevated at 72 hours in ethephon-treated leaves compared to dark control. Evan blue staining also demonstrated that ethephon-treated leaf for 72 hours had more cell death compared to dark control. Based on these data we conclude that SPCAM, SPCATA, SPPA, SPACO and SPDSS1 gene expression were significantly increased in natural and ethephon-induced senescent leaves. The possible functions of these isolated genes in association with events in ethephon-induced leaf senescence, including calcium influx, ROS elevation or scavenge, and following signaling will be discussed.