Leaf Senescence In Arabidopsis Research Articles

HbWRKY82, a novel IIc WRKY transcription factor from Hevea brasiliensis associated with abiotic stress tolerance and leaf senescence in Arabidopsis.

WRKY group transcription factors of model plants and major crops are confirmed to play essential roles in stress responses, senescence, secondary metabolism processes and hormone signal transduction. Previous studies have identified 81 HbWRKY genes from Hevea brasiliensis (the Pará rubber tree), but the functions of HbWRKYs in response to abiotic stresses and leaf senescence are unclear. In this study, one novel group IIc WRKY transcription factor named HbWRKY82 was identified and characterized as a stress-associated WRKY in rubber tree. Transient expression and transcriptional activation analyses indicated that HbWRKY82 encoded a nuclear protein and functioned as a transcription activator. The transcription levels of HbWRKY82 were induced by exogenous Ethrel (ET) (ethylene releaser) and abscisic acid (ABA) stimulations, down-regulated in tapping panel dryness rubber trees, and also exhibits significant decrease during the progression of leaf senescence. Overexpression of HbWRKY82 in Arabidopsis improved the tolerance to dehydration and salinity, and decreased the sensitivity to exogenous ABA. Moreover, real-time quantitative PCR analysis demonstrated that HbWRKY82 regulated the transcriptional expression of several stress-responsive genes (DREB1A, ERD10, HKT1, P5CS, RD22, RD29B, SKOR), leaf senescence marker genes (EIN3, WRKY53, NAP), ROS-related genes (RbohD, CSD1, CSD2, FSD3) and hormone signaling genes (EIN3, ABF3, ABF4). Collectively, our findings suggested that HbWRKY82 might function as an important transcriptional regulator in ET- and ABA-mediated leaf senescence and abiotic stress responses, and also be involved in tapping panel dryness, latex flow and regeneration processes of rubber trees via participating in the ET and reactive oxygen species signaling pathways.

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.

Wheat Transcription Factor TaSNAC11-4B Positively Regulates Leaf Senescence through Promoting ROS Production in Transgenic Arabidopsis.

Senescence is the final stage of leaf development which is accompanied by highly coordinated and complicated reprogramming of gene expression. Genetic manipulation of leaf senescence in major crops including wheat has been shown to be able to increase stress tolerance and grain yield. NAC(No apical meristem (NAM), ATAF1/2, and cup-shaped cotyledon (CUC)) transcription factors (TFs) play important roles in regulating gene expression changes during leaf senescence and in response to abiotic stresses. Here, we report the characterization of TaSNAC11-4B (Uniprot: A0A1D5XI64), a wheat NAC family member that acts as a functional homolog of AtNAP, a key regulator of leaf senescence in Arabidopsis. The expression of TaSNAC11-4B was up-regulated with the progression of leaf senescence, in response to abscisic acid (ABA) and drought treatments in wheat. Ectopic expression of TaSNAC11-4B in Arabidopsis promoted ROS accumulation and significantly accelerated age-dependent as well as drought- and ABA-induced leaf senescence. Results from transcriptional activity assays indicated that the TaSNAC11-4B protein displayed transcriptional activation activities that are dependent on its C terminus. Furthermore, qRT-PCR and dual-Luciferase assay results suggested that TaSNAC11-4B could positively regulate the expression of AtrbohD and AtrbohF, which encode catalytic subunits of the ROS-producing NADPH oxidase. Further analysis of TaSNAC11-4B in wheat senescence and the potential application of this gene in manipulating leaf senescence with the purpose of yield increase and stress tolerance is discussed.

The transcription factor ZmNAC126 accelerates leaf senescence downstream of the ethylene signalling pathway in maize.

Leaf senescence is an integral part of plant development, during which, nutrients are remobilized from senescent leaves to fast-growing organs. The initiation and progression dynamics of leaf senescence is therefore vital not only to the maximal accumulation of assimilates but also to the efficient remobilization of nutrients. Senescence is a finely tuned process that involves the action of a large number of transcription factors (TFs). The NAC TFs play critical roles in regulating leaf senescence in Arabidopsis, wheat, rice and tomato. Here, we identified a NAC TF, ZmNAC126 that is responsive to leaf senescence in maize. Ectopic overexpression of ZmNAC126 in Arabidopsis and maize enhanced chlorophyll degradation and promoted leaf senescence. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that ZmNAC126 could directly bind to the promoters of major chlorophyll catabolic genes in maize. Dual-luciferase assay in maize protoplasts indicated that ZmNAC126 positively regulates these chlorophyll catabolic genes in maize. Moreover, ZmNAC126 could be induced by ethylene, and ZmEIN3, a major TF of ethylene signalling, could bind to its promoter to transactivate its expression. Taken together, ZmNAC126 may play a pivotal role in regulating natural and ethylene-triggered leaf senescence in maize.

Arabidopsis FAR-RED ELONGATED HYPOCOTYL3 Integrates Age and Light Signals to Negatively Regulate Leaf Senescence.

Leaf senescence is tightly regulated by numerous internal cues and external environmental signals. The process of leaf senescence is promoted by a low ratio of red to far-red (R:FR) light, FR light, or extended darkness and is repressed by a high ratio of R:FR light or R light. However, the precise regulatory mechanisms by which plants assess external light signals and their internal cues to initiate and control the process of leaf senescence remain largely unknown. In this study, we discovered that the light-signaling protein FAR-RED ELONGATED HYPOCOTYL3 (FHY3) negatively regulates age-induced and light-mediated leaf senescence in Arabidopsis (Arabidopsis thaliana). FHY3 directly binds to the promoter region of transcription factor gene WRKY28 to repress its expression, thus negatively regulating salicylic acid biosynthesis and senescence. Both the fhy3 loss-of-function mutant and WRKY28-overexpressing Arabidopsis plants exhibited early senescence under high R:FR light conditions, indicating that the FHY3-WRKY28 transcriptional module specifically prevents leaf senescence under high R:FR light conditions. This study reveals the physiological and molecular functions of FHY3 and WRKY28 in leaf senescence and provides insight into the regulatory mechanism by which plants integrate dynamic environmental light signals and internal cues to initiate and control leaf senescence.

Physiological and Proteomic Changes in the Apoplast Accompany Leaf Senescence in Arabidopsis.

The apoplast, i.e. the cellular compartment external to the plasma membrane, undergoes important changes during senescence. Apoplastic fluid volume increases quite significantly in senescing leaves, thereby diluting its contents. Its pH elevates by about 0.8 units, similar to the apoplast alkalization in response to abiotic stresses. The levels of 159 proteins decrease, whereas 24 proteins increase in relative abundance in the apoplast of senescing leaves. Around half of the apoplastic proteins of non-senescent leaves contain a N-terminal signal peptide for secretion, while all the identified senescence-associated apoplastic proteins contain the signal peptide. Several of the apoplastic proteins that accumulate during senescence also accumulate in stress responses, suggesting that the apoplast may constitute a compartment where developmental and stress-related programs overlap. Other senescence-related apoplastic proteins are involved in cell wall modifications, proteolysis, carbohydrate, ROS and amino acid metabolism, signaling, lipid transport, etc. The most abundant senescence-associated apoplastic proteins, PR2 and PR5 (e.g. pathogenesis related proteins PR2 and PR5) are related to leaf aging rather than to the chloroplast degradation program, as their levels increase only in leaves undergoing developmental senescence, but not in dark-induced senescent leaves. Changes in the apoplastic space may be relevant for signaling and molecular trafficking underlying senescence.

SAUR49 Can Positively Regulate Leaf Senescence by Suppressing SSPP in Arabidopsis.

The involvement of SMALL AUXIN-UP RNA (SAUR) proteins in leaf senescence has been more and more acknowledged, but the detailed mechanisms remain unclear. In the present study, we performed yeast two-hybrid assays and identified SAUR49 as an interactor of SENESCENCE SUPPRESSED PROTEIN PHOSPHATASE (SSPP), which is a PP2C protein phosphatase that negatively regulates Arabidopsis leaf senescence by suppressing the leucine-rich repeat receptor-like protein kinase SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE (SARK), as reported previously by our group. The interaction between SAUR49 and SSPP was further confirmed in planta. Functional characterization revealed that SAUR49 is a positive regulator of leaf senescence. The accumulation level of SAUR49 protein increased during natural leaf senescence in Arabidopsis. The transcript level of SAUR49 was upregulated during SARK-induced premature leaf senescence but downregulated during SSPP-mediated delayed leaf senescence. Overexpression of SAUR49 significantly accelerated both natural and dark-induced leaf senescence in Arabidopsis. More importantly, SAUR49 overexpression completely reversed SSPP-induced delayed leaf senescence. In addition, overexpression of SAUR49 reversed the decreased plasma membrane H+-ATPase activity mediated by SSPP. Taken together, the results showed that SAUR49 functions in accelerating the leaf senescence process via the activation of SARK-mediated leaf senescence signaling by suppressing SSPP. We further identified four other SSPP-interacting SAURs, SAUR30, SAUR39, SAUR41 and SAUR72, that may act redundantly with SAUR49 in regulating leaf senescence. All these observations indicated that certain members of the SAUR family may serve as an important hub that integrates various hormonal and environmental signals with senescence signals in Arabidopsis.

H2O2 as a Feedback Signal on Dual-Located WHIRLY1 Associates with Leaf Senescence in Arabidopsis.

Leaf senescence, either as a natural stage of development or as an induced process under stress conditions, incorporates multiple intricate signaling pathways. At the cellular level, retrograde signals have been considered as important players during the initiation and progression of senescence in both animals and plants. The plant-specific single-strand DNA-binding protein WHIRLY1 (WHY1), a repressor of leaf natural senescence, is dually located in both nucleus and plastids. Despite many years of studies, the myth about its dual location and the underlying functional implications remain elusive. Here, we provide further evidence in Arabidopsis showing that alteration in WHY1 allocation between the nucleus and chloroplast causes perturbation in H2O2 homeostasis, resulting in adverse plant senescence phenotypes. The knockout of WHY1 increased H2O2 content at 37 days post-germination, coincident with an early leaf senescence phenotype, which can be rescued by ectopic expression of the nuclear isoform (nWHY1), but not by the plastid isoform (pWHY1). Instead, accumulated pWHY1 greatly provoked H2O2 in cells. On the other hand, exogenous H2O2 treatment induced a substantial plastid accumulation of WHY1 proteins and at the same time reduced the nuclear isoforms. This H2O2-induced loss of nucleus WHY1 isoform was accompanied by enhanced enrichments of histone H3 lysine 9 acetylation (H3K9ac) and recruitment of RNA polymerase II (RNAP II) globally, and specifically at the promoter of the senescence-related transcription factor WRKY53, which in turn activated WRKY53 transcription and led to a senescence phenotype. Thus, the distribution of WHY1 organelle isoforms and the feedback of H2O2 intervene in a circularly integrated regulatory network during plant senescence in Arabidopsis.

PROTEIN PHOSPHATASE 2A-B'γ Controls Botrytis cinerea Resistance and Developmental Leaf Senescence.

Plants optimize their growth and survival through highly integrated regulatory networks that coordinate defensive measures and developmental transitions in response to environmental cues. Protein phosphatase 2A (PP2A) is a key signaling component that controls stress reactions and growth at different stages of plant development, and the PP2A regulatory subunit PP2A-B'γ is required for negative regulation of pathogenesis responses and for maintenance of cell homeostasis in short-day conditions. Here, we report molecular mechanisms by which PP2A-B'γ regulates Botrytis cinerea resistance and leaf senescence in Arabidopsis (Arabidopsis thaliana). We extend the molecular functionality of PP2A-B'γ to a protein kinase-phosphatase interaction with the defense-associated calcium-dependent protein kinase CPK1 and present indications this interaction may function to control CPK1 activity. In presenescent leaf tissues, PP2A-B'γ is also required to negatively control the expression of salicylic acid-related defense genes, which have recently proven vital in plant resistance to necrotrophic fungal pathogens. In addition, we find the premature leaf yellowing of pp2a-b'γ depends on salicylic acid biosynthesis via SALICYLIC ACID INDUCTION DEFICIENT2 and bears the hallmarks of developmental leaf senescence. We propose PP2A-B'γ age-dependently controls salicylic acid-related signaling in plant immunity and developmental leaf senescence.

CpWRKY71, a WRKY Transcription Factor Gene of Wintersweet (Chimonanthus praecox), Promotes Flowering and Leaf Senescence in Arabidopsis.

The WRKY transcription factors are one of the most important plant-specific transcription factors and play vital roles in various biological processes. However, the functions of WRKY genes in wintersweet (Chimonanthus praecox) are still unknown. In this report, a group IIc WRKY gene, CpWRKY71, was isolated from wintersweet. CpWRKY71 was localized to the nucleus and possessed transcriptional activation activity. qRT-PCR (quantitative real-time PCR) analysis showed that CpWRKY71 was expressed in all tissues tested, with higher expression in flowers and senescing leaves. During the flower development, the highest expression was detected in the early-withering stage, an obvious expression of CpWRKY71 was also observed in the flower primordia differentiation and the bloom stage. Meanwhile, the expression of CpWRKY71 was influenced by various abiotic stress and hormone treatments. The expression patterns of the CpWRKY71 gene were further confirmed in CpWRKY71pro:GUS (β-glucuronidase) plants. Heterologous overexpression of CpWRKY71 in Arabidopsis caused early flowering. Consistent with the early flowering phenotype, the expression of floral pathway integrators and floral meristem identity (FMI) genes were significantly up-regulated in transgenic plants. In addition, we also observed that the transgenic plants of CpWRKY71 exhibited precocious leaf senescence. In conclusion, our results suggested that CpWRKY71 may be involved in the regulation of flowering and leaf senescence in Arabidopsis. Our study provides a foundation for further characterization of CpWRKY genes function in wintersweet, and also enrich our knowledge of molecular mechanism about flowering and senescence in wintersweet.

NAP is involved in GA-mediated chlorophyll degradation and leaf senescence by interacting with DELLAs in Arabidopsis.

RGA/GAI and NAP interacted with each other, and NAP was involved in GA signaling as a role of regulating age-dependent and dark-induced leaf senescence in Arabidopsis. Leaf senescence is a significant biological process which is beneficial for plant growth, development, and generation alternation in Arabidopsis. Recent researches have shown gibberellins (GAs) could accelerate leaf senescence. Nevertheless, the GA signaling involved in leaf senescence process remains elusive. Here, we reported a new potential regulation mechanism of GA-mediated chlorophyll degradation and leaf senescence. In this study, we confirmed that NAP positively regulated age-dependent and dark-induced leaf senescence and NAP knockout mutant nap was hyposensitive to GA3 (an active form of GA) treatment. DELLA family proteins with highly conserved structural domain function as master growth repressors that integrated GA signaling and leaf senescence. We validated RGA and GAI could interact with NAP in vitro and in vivo, and subsequently impaired the transcriptional activities of NAP to induce SAG113 and AAO3 expression in nap protoplasts. Taken together, we suggest that NAP is a novel component of the regulatory network that modulates the progress of leaf senescence in GA signaling.

Transcriptome profiling of postharvest shoots identifies PheNAP2- and PheNAP3-promoted shoot senescence.

The juvenile shoots of Phyllostachys edulis have been used as a food source for thousands of years, and it is recognized as a potential source of nutraceuticals. However, its rapid senescence restricts bamboo production and consumption, and the underlying molecular mechanisms of rapid shoot senescence remain largely unclear. In the present study, transcriptome profiling was employed to investigate the molecular regulation of postharvest senescence in shoots, along with physiological assays and anatomical dissections. Results revealed a distinct shift in expression postharvest, specifically transitions from cellular division and differentiation to the relocation of nutrients and programmed cell death. A number of regulatory and signaling factors were induced during postharvest senescence. Moreover, transcription factors, including NAM, ATAF and CUC (NAC) transcription factors, basic helix-loop-helix transcription factors, basic region/leucine zipper transcription factors, MYB transcription factors and WRKY transcription factors, were critical for shoot postharvest senescence, of which NACs were the most abundant. PheNAP2 and PheNAP3 were induced in postharvest shoots and found to promote leaf senescence in Arabidopsis by inducing the expression of AtSAG12 and AtSAG113. PheNAP2 and PheNAP3 could both restore the stay-green Arabidopsis nap to the wild-type phenotype either under normal growth condition or under abscisic acid treatment. Collectively, these results suggest that PheNAPs may promote shoot senescence. These findings provide a systematic view of shoot senescence and will inform future studies on the underlying molecular mechanisms responsible for shoot degradation during storage.

AtWDS1 negatively regulates age-dependent and dark-induced leaf senescence in Arabidopsis

Although the involvement of ROS (reactive oxygen species) in leaf senescence is well known, the factors governing this accumulation of ROS are not fully characterized. In this study, analysis of transgenic overexpressing and knock out lines of AtWDS1 (encoding a WD repeat protein), indicates that AtWDS1 negatively regulates age-dependent and dark-induced leaf senescence. Furthermore, we observed ROS accumulation and altered tolerance of oxidative stress in atwds1 plants, as well as upregulated expression of oxidative stress-responsive genes. The location of an EGFP-AtWDS1 fusion protein in the nucleus of transformed cells and plants indicates that AtWDS1 is a nuclear protein, and, using a Dual-Luciferase assay, we showed that AtWDS1 can act as a transcription activator. However, the lack of a nuclear localization sequence in AtWDS1 suggests that its presence in the nucleus must depend on interactions with other proteins. Indeed, we found that AtWDS1 interacts directly with AtRanBPM, and that mutation of the AtRanBPM gene results in partial mislocalization of AtWDS1 in the cytoplasm. Together, these results suggest a role for AtWDS1 as a novel modulator of redox homeostasis, which responds to developmental and stress signals to regulate 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.

Chlorophyll catabolism precedes changes in chloroplast structure and proteome during leaf senescence.

The earliest visual changes of leaf senescence occur in the chloroplast as chlorophyll is degraded and photosynthesis declines. Yet, a comprehensive understanding of the sequence of catabolic events occurring in chloroplasts during natural leaf senescence is still missing. Here, we combined confocal and electron microscopy together with proteomics and biochemistry to follow structural and molecular changes during Arabidopsis leaf senescence. We observed that initiation of chlorophyll catabolism precedes other breakdown processes. Chloroplast size, stacking of thylakoids, and efficiency of PSII remain stable until late stages of senescence, whereas the number and size of plastoglobules increase. Unlike catabolic enzymes, whose level increase, the level of most proteins decreases during senescence, and chloroplast proteins are overrepresented among these. However, the rate of their disappearance is variable, mostly uncoordinated and independent of their inherent stability during earlier developmental stages. Unexpectedly, degradation of chlorophyll‐binding proteins lags behind chlorophyll catabolism. Autophagy and vacuole proteins are retained at relatively high levels, highlighting the role of extra‐plastidic degradation processes especially in late stages of senescence. The observation that chlorophyll catabolism precedes all other catabolic events may suggest that this process enables or signals further catabolic processes in chloroplasts.

Characterization of Somatic Embryogenesis Receptor-Like Kinase 4 as a Negative Regulator of Leaf Senescence in Arabidopsis.

Leaf senescence is a genetically controlled process that involves the perception of extracellular signals and signal transduction. The receptor-like protein kinases (RLKs) are known to act as an important class of cell surface receptors and are involved in multiple biological processes such as development and stress responses. The functions of a number of RLK members have been characterized in Arabidopsis and other plant species, but only a limited number of RLK proteins have been reported to be associated with leaf senescence. In the present study, we have characterized the role of the somatic embryogenesis receptor kinase 4 (SERK4) gene in leaf senescence. The expression of SERK4 was up-regulated during leaf senescence and by several abiotic stress treatments in Arabidopsis. The serk4-1 knockout mutant was found to display a significant early leaf senescence phenotype. Furthermore, the results of overexpression analysis and complementary analysis supported the idea that SERK4 acts as a negative regulator in the process of leaf senescence.

Circadian control of ORE1 by PRR9 positively regulates leaf senescence in Arabidopsis

The circadian clock coordinates the daily cyclic rhythm of numerous biological processes by regulating a large portion of the transcriptome. In animals, the circadian clock is involved in aging and senescence, and circadian disruption by mutations in clock genes frequently accelerates aging. Conversely, aging alters circadian rhythmicity, which causes age-associated physiological alterations. However, interactions between the circadian clock and aging have been rarely studied in plants. Here, we investigated potential roles for the circadian clock in the regulation of leaf senescence in plants. Members of the evening complex in Arabidopsis circadian clock, EARLY FLOWERING 3 (ELF3), EARLY FLOWERING 4 (ELF4), and LUX ARRHYTHMO (LUX), as well as the morning component PSEUDO-RESPONSE REGULATOR 9 (PRR9), affect both age-dependent and dark-induced leaf senescence. The circadian clock regulates the expression of several senescence-related transcription factors. In particular, PRR9 binds directly to the promoter of the positive aging regulator ORESARA1 (ORE1) gene to promote its expression. PRR9 also represses miR164, a posttranscriptional repressor of ORE1 Consistently, genetic analysis revealed that delayed leaf senescence of a prr9 mutant was rescued by ORE1 overexpression. Thus, PRR9, a core circadian component, is a key regulator of leaf senescence via positive regulation of ORE1 through a feed-forward pathway involving posttranscriptional regulation by miR164 and direct transcriptional regulation. Our results indicate that, in plants, the circadian clock and leaf senescence are intimately interwoven as are the clock and aging in animals.

Calmodulin 1 Regulates Senescence and ABA Response in Arabidopsis.

Cellular calcium acts as a second messenger and regulates diverse developmental events and stress responses. Cytosolic calcium has long been considered as an important regulator of senescence, however, the role of Ca2+ in plant senescence has remained elusive. Here we show that the Calmodulin 1 (CaM1) gene, which encodes Ca2+-binding protein calmodulin 1, positively regulates leaf senescence in Arabidopsis. Yellowing of leaves, accumulation of reactive oxygen species (ROS), and expression of the senescence-associated gene 12 (SAG12) were significantly enhanced in CaM1 overexpression plants. In contrast, abscisic acid (ABA)-triggered ROS production and stomatal closure were reduced in amiRNA-CaM1 plants. We found a positive-feedback regulation loop among three signaling components, CaM1, RPK1, and RbohF, which physically associate with each other. RPK1 positively regulates the expression of the CaM1 gene, and the CaM1 protein, in turn, up-regulates RbohF gene expression. Interestingly, the expression of CaM1 was down-regulated in rbohD, rbohF, and rbohD/F mutants. We show that CaM1 positively regulates ROS production, leaf senescence, and ABA response in Arabidopsis.

Time-evolving genetic networks reveal a NAC troika that negatively regulates leaf senescence in Arabidopsis

Senescence is controlled by time-evolving networks that describe the temporal transition of interactions among senescence regulators. Here, we present time-evolving networks for NAM/ATAF/CUC (NAC) transcription factors in Arabidopsis during leaf aging. The most evident characteristic of these time-dependent networks was a shift from positive to negative regulation among NACs at a presenescent stage. ANAC017, ANAC082, and ANAC090, referred to as a "NAC troika," govern the positive-to-negative regulatory shift. Knockout of the NAC troika accelerated senescence and the induction of other NACs, whereas overexpression of the NAC troika had the opposite effects. Transcriptome and molecular analyses revealed shared suppression of senescence-promoting processes by the NAC troika, including salicylic acid (SA) and reactive oxygen species (ROS) responses, but with predominant regulation of SA and ROS responses by ANAC090 and ANAC017, respectively. Our time-evolving networks provide a unique regulatory module of presenescent repressors that direct the timely induction of senescence-promoting processes at the presenescent stage of leaf aging.

Comparative transcriptome analysis in Arabidopsis ein2/ore3 and ahk3/ore12 mutants during dark-induced leaf senescence.

Leaf senescence involves degenerative but active biological processes that require balanced regulation of pro- and anti-senescing activities. Ethylene and cytokinin are major antagonistic regulatory hormones that control the timing and progression rate of leaf senescence. To identify the roles of these hormones in the regulation of leaf senescence in Arabidopsis, global gene expression profiles in detached leaves of the wild type, an ethylene-insensitive mutant (ein2/ore3), and a constitutive cytokinin response mutant (ahk3/ore12) were investigated during dark-induced leaf senescence. Comparative transcriptome analyses revealed that genes involved in oxidative or salt stress response were preferentially altered in the ein2/ore3 mutant, whereas genes involved in ribosome biogenesis were affected in the ahk3/ore12 mutant during dark-induced leaf senescence. Similar results were also obtained for developmental senescence. Through extensive molecular and physiological analyses in ein2/ore3 and ahk3/ore12 during dark-induced leaf senescence, together with responses when treated with cytokinin and ethylene inhibitor, we conclude that ethylene acts as a senescence-promoting factor via the transcriptional regulation of stress-related responses, whereas cytokinin acts as an anti-senescing agent by maintaining cellular activities and preserving the translational machinery. These findings provide new insights into how plants utilize two antagonistic hormones, ethylene and cytokinin, to regulate the molecular programming of leaf senescence.