Age-dependent Leaf Senescence Research Articles

WRKY47 transcription factor modulates leaf senescence through regulating PCD-associated genes in Arabidopsis

Transcription factors play crucial roles in almost all physiological processes including leaf senescence. Cell death is a typical symptom appearing in senescing leaves, which is also classified as developmental programmed cell death (PCD). However, the link between PCD and leaf senescence still remains unclear. Here, we found a WRKY transcription factor WRKY47 positively modulates age-dependent leaf senescence in Arabidopsis (Arabidopsis thaliana). WRKY47 was expressed preferentially in senescing leaves. A subcellular localization assay indicated that WRKY47 was exclusively localized in nuclei. Overexpression of WRKY47 showed precocious leaf senescence, with less chlorophyll content and higher electrolyte leakage, but loss-of-function mutants of WRKY47 delayed this biological process. Through qRT-PCR and dual luciferase reporter assays, we found that WRKY47 could activate the expression of senescence-associated genes (SAGs) and PCD-associated genes to regulate leaf senescence. Furthermore, through electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-qPCR, WRKY47 was found to bind to W-box fragments in promoter regions of BFN1 (Bifunctional Nuclease 1) and MC6 (Metacaspase 6) directly. In general, our research revealed that WRKY47 regulates age-dependent leaf senescence by activating the transcription of two PCD-associated genes.

Phosphorylation of Thr-225 and Ser-262 on ERD7 Promotes Age-Dependent and Stress-Induced Leaf Senescence through the Regulation of Hydrogen Peroxide Accumulation in Arabidopsis thaliana.

As the final stage of leaf development, leaf senescence is affected by a variety of internal and external signals including age and environmental stresses. Although significant progress has been made in elucidating the mechanisms of age-dependent leaf senescence, it is not clear how stress conditions induce a similar process. Here, we report the roles of a stress-responsive and senescence-induced gene, ERD7 (EARLY RESPONSIVE TO DEHYDRATION 7), in regulating both age-dependent and stress-induced leaf senescence in Arabidopsis. The results showed that the leaves of erd7 mutant exhibited a significant delay in both age-dependent and stress-induced senescence, while transgenic plants overexpressing the gene exhibited an obvious accelerated leaf senescence. Furthermore, based on the results of LC-MS/MS and PRM quantitative analyses, we selected two phosphorylation sites, Thr-225 and Ser-262, which have a higher abundance during senescence, and demonstrated that they play a key role in the function of ERD7 in regulating senescence. Transgenic plants overexpressing the phospho-mimetic mutant of the activation segment residues ERD7T225D and ERD7T262D exhibited a significantly early senescence, while the inactivation segment ERD7T225A and ERD7T262A displayed a delayed senescence. Moreover, we found that ERD7 regulates ROS accumulation by enhancing the expression of AtrbohD and AtrbohF, which is dependent on the critical residues, i.e., Thr-225 and Ser-262. Our findings suggest that ERD7 is a positive regulator of senescence, which might function as a crosstalk hub between age-dependent and stress-induced leaf senescence.

Senescence-related receptor kinase 1 functions downstream of WRKY53 in regulating leaf senescence in Arabidopsis.

Receptor-like kinases (RLKs) are the most important class of cell surface receptors, and play crucial roles in plant development and stress responses. However, few studies have been reported about the biofunctions of RLKs in leaf senescence. Here, we characterized a novel Arabidopsis RLK-encoding gene, SENESCENCE-RELATED RECEPTOR KINASE 1 (SENRK1), which was significantly down-regulated during leaf senescence. Notably, the loss-of-function senrk1 mutants displayed an early leaf senescence phenotype, while overexpression of SENRK1 significantly delayed leaf senescence, indicating that SENRK1 negatively regulates age-dependent leaf senescence in Arabidopsis. Furthermore, the senescence-promoting transcription factor WRKY53 repressed the expression of SENRK1. While the wrky53 mutant showed a delayed senescence phenotype as previously reported, the wrky53 senrk1-1 double mutant exhibited precocious leaf senescence, suggesting that SENRK1 functions downstream of WRKY53 in regulating age-dependent leaf senescence in Arabidopsis.

ANAC087 transcription factor positively regulates age-dependent leaf senescence through modulating the expression of multiple target genes in Arabidopsis.

Leaf senescence is the final stage of leaf development and appropriate onset and progression of leaf senescence are critical for reproductive success and fitness. Although great progress has been made in identifying key genes regulating leaf senescence and elucidating the underlining mechanisms in the model plant Arabidopsis, there is still a gap to understanding the complex regulatory network. In this study, we discovered that Arabidopsis ANAC087 transcription factor (TF) positively modulated leaf senescence. Expression of ANAC087 was induced in senescing leaves and the encoded protein acted as a transcriptional activator. Both constitutive and inducible overexpression lines of ANAC087 showed earlier senescence than control plants, whereas T-DNA insertion mutation and dominant repression of the ANAC087 delayed senescence rate. A quantitative reverse transcription-polymerase chain reaction (qRT-PCR) profiling showed that the expression of an array of senescence-associated genes was upregulated in inducible ANAC087 overexpression plants including BFN1, NYE1, CEP1, RbohD, SAG13, SAG15, and VPEs, which are involved in programmed cell death (PCD), chlorophyll degradation and reactive oxygen species (ROS) accumulation. In addition, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) assays demonstrated that ANAC087 directly bound to the canonical NAC recognition sequence (NACRS) motif in promoters of its target genes. Moreover, mutation of two representative target genes, BFN1 or NYE1 alleviated the senescence rate of ANAC087-overexpression plants, suggesting their genetic regulatory relationship. Taken together, this study indicates that ANAC087 serves as an important regulator linking PCD, ROS, and chlorophyll degradation to leaf senescence.

Transcriptional Programs and Regulators Underlying Age-Dependent and Dark-Induced Senescence in Medicago truncatula.

In forage crops, age-dependent and stress-induced senescence reduces forage yield and quality. Therefore, delaying leaf senescence may be a way to improve forage yield and quality as well as plant resilience to stresses. Here, we used RNA-sequencing to determine the molecular bases of age-dependent and dark-induced leaf senescence in Medicago truncatula. We identified 6845 differentially expressed genes (DEGs) in M3 leaves associated with age-dependent leaf senescence. An even larger number (14219) of DEGs were associated with dark-induced senescence. Upregulated genes identified during age-dependent and dark-induced senescence were over-represented in oxidation–reduction processes and amino acid, carboxylic acid and chlorophyll catabolic processes. Dark-specific upregulated genes also over-represented autophagy, senescence and cell death. Mitochondrial functions were strongly inhibited by dark-treatment while these remained active during age-dependent senescence. Additionally, 391 DE transcription factors (TFs) belonging to various TF families were identified, including a core set of 74 TFs during age-dependent senescence while 759 DE TFs including a core set of 338 TFs were identified during dark-induced senescence. The heterologous expression of several senescence-induced TFs belonging to NAC, WKRY, bZIP, MYB and HD-zip TF families promoted senescence in tobacco leaves. This study revealed the dynamics of transcriptomic responses to age- and dark-induced senescence in M. truncatula and identified senescence-associated TFs that are attractive targets for future work to control senescence in forage legumes.

TaWRKY13-A Serves as a Mediator of Jasmonic Acid-Related Leaf Senescence by Modulating Jasmonic Acid Biosynthesis.

Leaf senescence is crucial for crop yield and quality. Transcriptional regulation is a key step for integrating various senescence-related signals into the nucleus. However, few regulators of senescence implicating transcriptional events have been functionally characterized in wheat. Based on our RNA-seq data, we identified a WRKY transcription factor, TaWRKY13-A, that predominately expresses at senescent stages. By using the virus-induced gene silencing (VIGS) method, we manifested impaired transcription of TaWRKY13-A leading to a delayed leaf senescence phenotype in wheat. Moreover, the overexpression (OE) of TaWRKY13-A accelerated the onset of leaf senescence under both natural growth condition and darkness in Brachypodium distachyon and Arabidopsis thaliana. Furthermore, by physiological and molecular investigations, we verified that TaWRKY13-A participates in the regulation of leaf senescence via jasmonic acid (JA) pathway. The expression of JA biosynthetic genes, including AtLOX6, was altered in TaWRKY13-A-overexpressing Arabidopsis. We also demonstrated that TaWRKY13-A can interact with the promoter of AtLOX6 and TaLOX6 by using the electrophoretic mobility shift assay (EMSA) and luciferase reporter system. Consistently, we detected a higher JA level in TaWRKY13-A-overexpressing lines than that in Col-0. Moreover, our data suggested that TaWRKY13-A is partially functional conserved with AtWRKY53 in age-dependent leaf senescence. Collectively, this study manifests TaWRKY13-A as a positive regulator of JA-related leaf senescence, which could be a new clue for molecular breeding in wheat.

Gene expression changes occurring at bolting time are associated with leaf senescence in Arabidopsis.

In plants, the vegetative to reproductive phase transition (termed bolting in Arabidopsis) generally precedes age‐dependent leaf senescence (LS). Many studies describe a temporal link between bolting time and LS, as plants that bolt early, senesce early, and plants that bolt late, senesce late. The molecular mechanisms underlying this relationship are unknown and are potentially agriculturally important, as they may allow for the development of crops that can overcome early LS caused by stress‐related early‐phase transition. We hypothesized that leaf gene expression changes occurring in synchrony with bolting were regulating LS. ARABIDOPSIS TRITHORAX (ATX) enzymes are general methyltransferases that regulate the adult vegetative to reproductive phase transition. We generated an atx1, atx3, and atx4 (atx1,3,4) triple T‐DNA insertion mutant that displays both early bolting and early LS. This mutant was used in an RNA‐seq time‐series experiment to identify gene expression changes in rosette leaves that are likely associated with bolting. By comparing the early bolting mutant to vegetative WT plants of the same age, we were able to generate a list of differentially expressed genes (DEGs) that change expression with bolting as the plants age. We trimmed the list by intersection with publicly available WT datasets, which removed genes from our DEG list that were atx1,3,4 specific. The resulting 398 bolting‐associated genes (BAGs) are differentially expressed in a mature rosette leaf at bolting. The BAG list contains many well‐characterized LS regulators (ORE1, WRKY45, NAP, WRKY28), and GO analysis revealed enrichment for LS and LS‐related processes. These bolting‐associated LS regulators may contribute to the temporal coupling of bolting time to LS.

A Jasmonate-Activated MYC2-Dof2.1-MYC2 Transcriptional Loop Promotes Leaf Senescence in Arabidopsis.

DNA binding-with-one-finger (Dof) proteins are plant-specific transcription factors closely associated with a variety of physiological processes. Here, we show that the Dof protein family in Arabidopsis (Arabidopsis thaliana) functions in leaf senescence. Disruption of Dof2 1, a jasmonate (JA)-inducible gene, led to a marked reduction in promotion of leaf senescence and inhibition of root development as well as dark-induced and age-dependent leaf senescence, while overexpression of Dof2 1 promoted these processes. Additionally, the dof2 1 knockout mutant showed almost no change in the transcriptome in the absence of JA; in the presence of JA, expression of many senescence-associated genes, including MYC2, which encodes a central regulator of JA responses, was induced to a lesser extent in the dof2 1 mutant than in the wild type. Furthermore, direct activation of the MYC2 promoter by Dof2.1, along with the results of epistasis analysis, indicated that Dof2.1 enhances leaf senescence mainly by promoting MYC2 expression. Interestingly, MYC2 was also identified as a transcriptional activator responsible for JA-inducible expression of Dof2 1 Based on these results, we propose that Dof2.1 acts as an enhancer of JA-induced leaf senescence through the MYC2-Dof2.1-MYC2 feedforward transcriptional loop.

Disruption of a Upf1-like helicase-encoding gene OsPLS2 triggers light-dependent premature leaf senescence in rice.

The OsPLS2 locus was isolated and cloned by map-based cloning that encodes a Upf1-like helicase. Disruption of OsPLS2 accelerated light-dependent leaf senescence in the rice mutant of ospls2. Leaf senescence is a very complex physiological process controlled by both genetic and environmental factors, however its underlying molecular mechanisms remain elusive. In this study, we report a novel Oryza sativa premature leaf senescence mutant (ospls2). Through map-based cloning, a G-to-A substitution was determined at the 1st nucleotide of the 13th intron in the OsPLS2 gene that encodes a Upf1-like helicase. This mutation prompts aberrant splicing of OsPLS2 messenger and consequent disruption of its full-length protein translation, suggesting a negative role of OsPLS2 in regulating leaf senescence. Wild-type rice accordingly displayed a progressive drop of OsPSL2 protein levels with age-dependent leaf senescence. Shading and light filtration studies showed that the ospls2 phenotype, which was characteristic of photo-oxidative stress and reactive oxygen species (ROS) accumulation, was an effect of irritation by light. When continuously exposed to far-red light, exogenous H2O2 and/or abscisic acid (ABA), the ospls2 mutant sustained hypersensitive leaf senescence. In consistence, light and ROS signal pathways in ospls2 were activated by down-regulation of phytochrome genes, and up-regulation of PHYTOCHROME-INTERACTING FACTORS (PIFs) and WRKY genes, all promoting leaf senescence. Together, these data indicated that OsPLS2 played an essential role in leaf senescence and its disruption triggered light-dependent leaf senescence in rice.

A 22-bp deletion in OsPLS3 gene encoding a DUF266-containing protein is implicated in rice leaf senescence.

Key message The OsPLS3 locus was isolated by map-based cloning that encodes a DUF266-containing protein. OsPLS3 regulates the onset of leaf senescence in rice. Glycosyltransferases (GTs) are one of the most important enzyme groups required for the modification of plant secondary metabolites and play a crucial role in plant growth and development, however the biological functions of most GTs remain elusive. We reported here the identification and characterization of a novel Oryza sativa premature leaf senescence mutant (ospls3). Through map-based cloning strategy, we determined that 22-bp deletion in the OsPLS3 gene encoding a domain of unknown function 266 (DUF266)-containing protein, a member of GT14-like, underlies the premature leaf senescence phenotype in the ospls3 mutant. The OsPLS3 mRNA levels progressively declined with the age-dependent leaf senescence in wild-type rice, implying a negative role of OsPLS3 in regulating leaf senescence. Physiological analysis, and histochemical staining and transmission electron microscopy assays indicated that the ospls3 mutant accumulated higher levels of ethylene and reactive oxygen species than its wild type. Furthermore, the ospls3 mutant showed hypersensitivity to exogenous 1-aminocyclopropane-1-carboxylic acid, H2O2 and high level of cytokinins. Our results indicated that the DUF266-containing gene OsPLS3 plays an important role in the onset of leaf senescence, in part through cytokinin and ethylene signaling in rice.

Ectopic expression of VpSTS29, a stilbene synthase gene from Vitis pseudoreticulata, indicates STS presence in cytosolic oil bodies.

Stilbene synthase (STS) and its metabolic products are accumulated in senescing grapevine leaves. Ectopic expression of VpSTS29 in Arabidopsis shows the presence of VpSTS29 in oil bodies and increases trans-piceid in developing leaves. Stilbenes are the natural antimicrobial phytoalexins that are synthesised via the phenylpropanoid pathway. STS is the key enzyme catalysing the production of stilbenes. We have previously reported that the VpSTS29 gene plays an important role in powdery mildew resistance in Vitis pseudoreticulata. However, the synthesis and accumulation of these stilbene products in plant cells remain unclear. Here, we demonstrate that VpSTS29 is present in cytosolic oil bodies and can be transported into the vacuole at particular plant-developmental stages. Western blot and high-performance liquid chromatography showed that STS and trans-piceid accumulated in senescent grape leaves and in pVpSTS29::VpSTS29-expressing Arabidopsis during age-dependent leaf senescence. Subcellular localisation analyses indicated VpSTS29-GFP was present in the cytoplasm and in STS-containing bodies in Arabidopsis. Nile red staining, co-localisation and immunohistochemistry analyses of leaves confirmed that the STS-containing bodies were oil bodies and that these moved randomly in the cytoplasm and vacuole. Detection of protein profiles revealed that no free GFP was detected in the pVpSTS29::VpSTS29-GFP-expressing protoplasts or in Arabidopsis during the dark-light cycle, demonstrating that GFP fluorescence distributed in the STS-containing bodies and vacuole was the VpSTS29-GFP fusion protein. Intriguingly, in comparison to the controls, over-expression of VpSTS29 in Arabidopsis resulted in relatively high levels of trans-piceid, chlorophyll content and of photochemical efficiency accompanied by delayed leaf senescence. These results provide exciting new insights into the subcellular localisation of STS in plant cells and information about stilbene synthesis and storage.

A Tripartite Amplification Loop Involving the Transcription Factor WRKY75, Salicylic Acid, and Reactive Oxygen Species Accelerates Leaf Senescence.

Leaf senescence is a highly coordinated, complicated process involving the integration of numerous internal and environmental signals. Salicylic acid (SA) and reactive oxygen species (ROS) are two well-defined inducers of leaf senescence whose contents progressively and interdependently increase during leaf senescence via an unknown mechanism. Here, we characterized the transcription factor WRKY75 as a positive regulator of leaf senescence in Arabidopsis thaliana. Knockdown or knockout of WRKY75 delayed age-dependent leaf senescence, while overexpression of WRKY75 accelerated this process. WRKY75 transcription is induced by age, SA, H2O2, and multiple plant hormones. Meanwhile, WRKY75 promotes SA production by inducing the transcription of SA INDUCTION-DEFICIENT2 (SID2) and suppresses H2O2 scavenging, partly by repressing the transcription of CATALASE2 (CAT2). Genetic analysis revealed that the mutation of SID2 or an increase in catalase activity rescued the precocious leaf senescence phenotype evoked by WRKY75 overexpression. Based on these results, we propose a tripartite amplification loop model in which WRKY75, SA, and ROS undergo a gradual but self-sustained rise driven by three interlinking positive feedback loops. This tripartite amplification loop provides a molecular framework connecting upstream signals, such as age and plant hormones, to the downstream regulatory network executed by SA- and H2O2-responsive transcription factors during leaf senescence.

A single cytosine deletion in the OsPLS1 gene encoding vacuolar-type H+-ATPase subunit A1 leads to premature leaf senescence and seed dormancy in rice.

Leaf senescence is a programmed developmental process orchestrated by many factors, but its molecular regulation is not yet fully understood. In this study, a novel Oryza sativa premature leaf senescence mutant (ospls1) was examined. Despite normal development in early seedlings, the ospls1 mutant leaves displayed lesion-mimics and early senescence, and a high transpiration rate after tillering. The mutant also showed seed dormancy attributable to physical (defect of micropyle structure) and physiological (abscisic acid sensitivity) factors. Using a map-based cloning approach, we determined that a cytosine deletion in the OsPLS1 gene encoding vacuolar H(+)-ATPase subunit A1 (VHA-A1) underlies the phenotypic abnormalities in the ospls1 mutant. The OsPSL1/VHA-A1 transcript levels progressively declined with the age-dependent leaf senescence in both the ospls1 mutant and its wild type. The significant decrease in both OsPSL1/VHA-A1 gene expression and VHA enzyme activity in the ospls1 mutant strongly suggests a negative regulatory role for the normal OsPLS1/VHA-A1 gene in the onset of rice leaf senescence. The ospls1 mutant featured higher salicylic acid (SA) levels and reactive oxygen species (ROS) accumulation, and activation of signal transduction by up-regulation of WRKY genes in leaves. Consistent with this, the ospls1 mutant exhibited hypersensitivity to exogenous SA and/or H2O2 Collectively, these results indicated that the OsPSL1/VAH-A1 mutation played a causal role in premature leaf senescence through a combination of ROS and SA signals. To conclude, OsPLS1 is implicated in leaf senescence and seed dormancy in rice.

Precocious leaf senescence by functional loss of PROTEIN S-ACYL TRANSFERASE14 involves the NPR1-dependent salicylic acid signaling.

We report here that Arabidopsis PROTEIN S-ACYL TRANSFERASE14 (PAT14), through its palmitate transferase activity, acts at the vacuolar trafficking route to repress salicylic acid (SA) signaling, thus mediating age-dependent but not carbon starvation-induced leaf senescence. Functional loss of PAT14 resulted in precocious leaf senescence and its transcriptomic analysis revealed that senescence was dependent on salicylic acid. Overexpressing PAT14 suppressed the expression of SA responsive genes. Introducing the SA deficient mutants, npr1-5 and NahG, but not other hormonal mutants, completely suppressed the precocious leaf senescence of PAT14 loss-of-function, further supporting the epistatic relation between PAT14 and the SA pathway. By confocal fluorescence microscopy, we showed that PAT14 is localized at the Golgi, the trans-Golg network/early endosome, and prevacuolar compartments, indicating its roles through vacuolar trafficking. By reporter analysis and real time PCRs, we showed that the expression PAT14, unlike most of the senescence associated genes, is not developmentally regulated, suggesting post-transcriptional regulatory mechanisms on its functionality. We further showed that the maize and wheat homologs of PAT14 fully rescued the precocious leaf senescence of pat14-2, demonstrating that the role of PAT14 in suppressing SA signaling during age-dependent leaf senescence is evolutionarily conserved between dicots and monocots.

Gene regulatory cascade of senescence-associated NAC transcription factors activated by ETHYLENE-INSENSITIVE2-mediated leaf senescence signalling in Arabidopsis

Leaf senescence is a finely tuned and genetically programmed degeneration process, which is critical to maximize plant fitness by remobilizing nutrients from senescing leaves to newly developing organs. Leaf senescence is a complex process that is driven by extensive reprogramming of global gene expression in a highly coordinated manner. Understanding how gene regulatory networks involved in controlling leaf senescence are organized and operated is essential to decipher the mechanisms of leaf senescence. It was previously reported that the trifurcate feed-forward pathway involving EIN2, ORE1, and miR164 in Arabidopsis regulates age-dependent leaf senescence and cell death. Here, new components of this pathway have been identified, which enhances knowledge of the gene regulatory networks governing leaf senescence. Comparative gene expression analysis revealed six senescence-associated NAC transcription factors (TFs) (ANAC019, AtNAP, ANAC047, ANAC055, ORS1, and ORE1) as candidate downstream components of ETHYLENE-INSENSITIVE2 (EIN2). EIN3, a downstream signalling molecule of EIN2, directly bound the ORE1 and AtNAP promoters and induced their transcription. This suggests that EIN3 positively regulates leaf senescence by activating ORE1 and AtNAP, previously reported as key regulators of leaf senescence. Genetic and gene expression analyses in the ore1 atnap double mutant revealed that ORE1 and AtNAP act in distinct and overlapping signalling pathways. Transient transactivation assays further demonstrated that ORE1 and AtNAP could activate common as well as differential NAC TF targets. Collectively, the data provide insight into an EIN2-mediated senescence signalling pathway that coordinates global gene expression during leaf senescence via a gene regulatory network involving EIN3 and senescence-associated NAC TFs.

Knockdown of OsHox33, a member of the class III homeodomain-leucine zipper gene family, accelerates leaf senescence in rice

The class III homeodomain-leucine zipper (HD-Zip III) gene family plays important roles in plant growth and development, including regulation of apical embryo patterning, embryonic shoot meristem formation, leaf polarity, vascular development, and meristem function, with a particularly crucial function in leaf development. Although HD-Zip III members are highly conserved in land plants, previous studies, such as genetic analyses based on multiple mutants in Arabidopsis and other plants, suggest that various HD-Zip III family genes have evolved with distinct functions and pleiotropic effects on plant growth and development. In this study, we analyzed a HD-Zip III member, OsHox33, and demonstrated that it plays an important role in age-dependent leaf senescence in rice. We constructed two specific RNAi vectors derived from the 5'-end region and 3'-UTR of OsHox33 to knockdown its expression. Transgenic plants harboring either RNAi construct displayed similar phenotypes of precocious leaf senescence symptoms, suggesting that knockdown of OsHox33 accelerates leaf senescence in rice. pOsHox33::GUS fusion expression and RT-PCR revealed that OsHox33 is highly expressed in young organs, especially in young meristems such as shoot apical meristems, intercalary meristems, and young callus. In addition, real-time PCR indicated that OsHox33 was more highly expressed in young leaves than in old leaves. To further investigate OsHox33 function, we analyzed chloroplast ultrastructure in different-aged leaves of RNAi plants, and found that OsHox33 knockdown accelerated chloroplast degradation, which is consistent with RNAi phenotypes. Finally, real-time PCR studies showed that OsHox33 can regulate the expression of GS1 and GS2, two senescence-associated genes. Taken together, the work presented here provides new insights into the function of HD-Zip III members in plants.

Role of ARABIDOPSIS A-FIFTEEN in regulating leaf senescence involves response to reactive oxygen species and is dependent on ETHYLENE INSENSITIVE2.

Leaf senescence is a highly regulated developmental process that is coordinated by several factors. Many senescence-associated genes (SAGs) have been identified, but their roles during senescence remain unclear. A sweet potato (Ipomoea batatas) SAG, named SPA15, whose function was unknown, was identified previously. To understand the role of SPA15 in leaf senescence further, the orthologue of SPA15 in Arabidopsis thaliana was identified and characterized, and it was named ARABIDOPSIS A-FIFTEEN (AAF). AAF was expressed in early senescent leaves and in tissues with highly proliferative activities. AAF was localized to the chloroplasts by transient expression in Arabidopsis mesophyll protoplasts. Overexpression of AAF (AAF-OX) in Arabidopsis promoted, but the T-DNA insertion mutant (aaf-KO), delayed age-dependent leaf senescence. Furthermore, stress-induced leaf senescence caused by continuous darkness was enhanced in AAF-OX but suppressed in aaf-KO. Transcriptome analysis of expression profiles revealed up-regulated genes related to pathogen defence, senescence, and oxidative stress in 3-week-old AAF-OX plants. Indeed, elevated levels of reactive oxygen species (ROS) and enhanced sensitivity to oxidative and dark stress were apparent in AAF-OX but reduced in aaf-KO. ETHYLENE INSENSITIVE2 (EIN2) was required for the dark- and ROS-induced senescence phenotypes in AAF-OX and the induction of AAF expression by treatment with the immediate precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid. The results indicate the functional role of AAF is an involvement in redox homeostasis to regulate leaf senescence mediated by age and stress factors during Arabidopsis development.

Knock-out of ferritin AtFer1 causes earlier onset of age-dependent leaf senescence in Arabidopsis

Ferritins are iron-storage proteins involved in the regulation of free iron levels in the cells. Arabidopsis thaliana AtFer1 ferritin, one of the best characterized plant ferritin isoforms to date, strongly accumulates upon treatment with excess iron, via a nitric oxide-mediated pathway. However other environmental factors, such as exposure to oxidative stress or to pathogen attack, as well as developmental factors regulate AtFer1 transcript levels. In particular, recent findings have highlighted an accumulation of the ferritin transcript during senescence. To investigate the physiological relevance of AtFer1 ferritin during senescence we isolated an Arabidopsis mutant knock-out in the AtFer1 gene, which we named atfer1-2. We analyzed it together with a second, independent AtFer1 KO mutant, the atfer1-1 mutant. Interestingly, both atfer1-1 and atfer1-2 mutants show symptoms of accelerated natural senescence; the precocious leaf yellowing is accompanied by accelerated decrease of maximal photochemical efficiency and chlorophyll degradation. However, no accelerated senescence upon dark treatment was observed in the atfer1 mutants with respect to their wt. These results suggest that AtFer1 ferritin isoform is functionally involved in events leading to the onset of age-dependent senescence in Arabidopsis and that its iron-detoxification function during senescence is required when reactive oxygen species accumulate.

A Novel Nuclear-Localized CCCH-Type Zinc Finger Protein, OsDOS, Is Involved in Delaying Leaf Senescence in Rice

Leaf senescence is a developmentally programmed degeneration process, which is fine tuned by a complex regulatory network for plant fitness. However, molecular regulation of leaf senescence is poorly understood, especially in rice (Oryza sativa), an important staple crop for more than half of the world population. Here, we report a novel nuclear-localized CCCH-type zinc finger protein, Oryza sativa delay of the onset of senescence (OsDOS), involved in delaying leaf senescence in rice. The expression of OsDOS was down-regulated during natural leaf senescence, panicle development, and pollination, although its transcripts were accumulated in various organs. RNAi knockdown of OsDOS caused an accelerated age-dependent leaf senescence, whereas its overexpression produced a marked delay of leaf senescence, suggesting that it acts as a negative regulator for leaf senescence. A genome-wide expression analysis further confirmed its negative regulation for leaf senescence and revealed that, in particular, the jasmonate (JA) pathway was found to be hyperactive in the OsDOS RNAi transgenic lines but impaired in the OsDOS overexpressing transgenic lines, indicating that this pathway is likely involved in the OsDOS-mediated delaying of leaf senescence. Furthermore, methyl JA treatments of both seeds and detached leaves from the RNAi and the overexpressing transgenic lines showed hyper- and hyporesponses, respectively, consistent with the negative regulation of the JA pathway by OsDOS. Together, these results indicate that OsDOS is a novel nuclear protein that delays leaf senescence likely, at least in part, by integrating developmental cues to the JA pathway.

Extended leaf longevity in the ore4-1 mutant of Arabidopsis with a reduced expression of a plastid ribosomal protein gene.

The longevity of plant leaf organs is genetically determined. However, the molecular mechanisms underlying the control of longevity are still largely unknown. Here, we describe a T-DNA-insertional mutation of Arabidopsis thaliana that confers extended leaf longevity. The mutation, termed ore4-1, delays a broad spectrum of age-dependent leaf senescence, but has little effect on leaf senescence artificially induced by darkness, abscisic acid (ABA), methyl jasmonate (MeJA), or ethylene. The T-DNA was inserted within the promoter region of the plastid ribosomal small subunit protein 17 (PRPS17) gene, and this insertion dramatically reduced PRPS17 mRNA expression. In the ore4-1 mutant, the leaf growth rate is decreased, while the maturation timing is similar to that of wild-type. In addition, the activity of the photosystem I (PSI) is significantly reduced in the ore4-1 mutant, as compared to wild-type. Thus, the ore4-1 mutation results in a deficiency in various chloroplast functions, including photosynthesis, which may decrease leaf growth. Our results suggest a possible link between reduced metabolism and extended longevity of the leaf organs in the ore4-1 mutation.