SummaryExogenous melatonin application confers abiotic stress resistance in bermudagrass through modulation of antioxidants and metabolic homeostasis, and extensive transcriptional reprogramming such as the reorientation of photorespiratory, carbohydrate, and nitrogen metabolism.

Drought stress is an important environmental factor limiting plant productivity. In this study, we screened drought-resistant transgenic plants from 65 promoter-pyrabactin resistance 1-like (PYL) abscisic acid (ABA) receptor gene combinations and discovered that pRD29A::PYL9 transgenic lines showed dramatically increased drought resistance and drought-induced leaf senescence in both Arabidopsis and rice. Previous studies suggested that ABA promotes senescence by causing ethylene production. However, we found that ABA promotes leaf senescence in an ethylene-independent manner by activating sucrose nonfermenting 1-related protein kinase 2s (SnRK2s), which subsequently phosphorylate ABA-responsive element-binding factors (ABFs) and Related to ABA-Insensitive 3/VP1 (RAV1) transcription factors. The phosphorylated ABFs and RAV1 up-regulate the expression of senescence-associated genes, partly by up-regulating the expression of Oresara 1. The pyl9 and ABA-insensitive 1-1 single mutants, pyl8-1pyl9 double mutant, and snrk2.2/3/6 triple mutant showed reduced ABA-induced leaf senescence relative to the WT, whereas pRD29A::PYL9 transgenic plants showed enhanced ABA-induced leaf senescence. We found that leaf senescence may benefit drought resistance by helping to generate an osmotic potential gradient, which is increased in pRD29A::PYL9 transgenic plants and causes water to preferentially flow to developing tissues. Our results uncover the molecular mechanism of ABA-induced leaf senescence and suggest an important role of PYL9 and leaf senescence in promoting resistance to extreme drought stress.drought stress | abscisic acid | PYL | dormancy | Arabidopsis C ell and organ senescence causes programmed cell death to regulate the growth and development of organisms. In plants, leaf senescence increases the transfer of nutrients to developing and storage tissues. Recently, studies on transgenic tobacco showed that delayed leaf senescence increases plant resistance to drought stress (1). However, the senescence and abscission of older leaves and subsequent transfer of nutrients are known to increase plant survival under abiotic stresses, including drought, low or high temperatures, and darkness (2, 3). Senescence mainly develops in an age-dependent manner and is also triggered by environmental stresses and phytohormones, such as abscisic acid (ABA), ethylene, salicylic acid, and jasmonic acid, but delayed by cytokinin (4).Senescence-associated genes (SAGs) are induced by leaf senescence. The expression of SAGs is tightly controlled by several senescence-promoting, plant-specific NAC (NAM, ATAF1, and CUC2) transcription factors, such as Oresara 1 (ORE1) (5), Oresara 1 sister 1 (ORS1) (6), and AtNAP (7). Environmental stimuli and phytohormones may regulate leaf senescence through NACs. Phytochrome-interacting factor 4 (PIF4) and PIF5 transcription factors promote dark-induced senescence by activating ORE1 expression (8). The expression of ORE1, AtNAP, and OsNAP (ortholog of AtNAP) is up-regulated by ABA by an unknown molecular m...

Arginine is an important medium for the transport and storage of nitrogen, and arginase (also known as arginine amidohydrolase, ARGAH) is responsible for catalyse of arginine into ornithine and urea in plants. In this study, the impact of AtARGAHs on abiotic stress response was investigated by manipulating AtARGAHs expression. In the knockout mutants of AtARGAHs, enhanced tolerances were observed to multiple abiotic stresses including water deficit, salt, and freezing stresses, while AtARGAH1- and AtARGAH2-overexpressing lines exhibited reduced abiotic stress tolerances compared to the wild type. Consistently, the enhanced tolerances were confirmed by the changes of physiological parameters including electrolyte leakage, water loss rate, stomatal aperture, and survival rate. Interestingly, the direct downstream products of arginine catabolism including polyamines and nitric oxide (NO) concentrations significantly increased in the AtARGAHs-knockout lines, but decreased in overexpressing lines under control conditions. Additionally, the AtARGAHs-overexpressing and -knockout lines displayed significantly reduced relative arginine (% of total free amino acids) relative to the wild type. Similarly, reactive oxygen species accumulation was remarkably regulated by AtARGAHs under abiotic stress conditions, as shown from hydrogen peroxide (H2O2), superoxide radical () concentrations, and antioxidant enzyme activities. Taken together, this is the first report, as far as is known, to provide evidence that AtARGAHs negatively regulate many abiotic stress tolerances, at least partially, attribute to their roles in modulating arginine metabolism and reactive oxygen species accumulation. Biotechnological strategy based on manipulation of AtARGAHs expression will be valuable for future crop breeding.

Melatonin (N-acetyl-5-methoxytryptamine) serves as an important signal molecule during plant developmental processes and multiple abiotic stress responses. However, the involvement of melatonin in thermotolerance and the underlying molecular mechanism in Arabidopsis were largely unknown. In this study, we found that the endogenous melatonin level in Arabidopsis leaves was significantly induced by heat stress treatment, and exogenous melatonin treatment conferred improved thermotolerance in Arabidopsis. The transcript levels of class A1 heat-shock factors (HSFA1s), which serve as the master regulators of heat stress responses, were significantly upregulated by heat stress and exogenous melatonin treatment in Arabidopsis. Notably, exogenous melatonin-enhanced thermotolerance was largely alleviated in HSFA1s quadruple knockout (QK) mutants, and HSFA1s-activated transcripts of heat-responsive genes (HSFA2, heat stress-associated 32 (HSA32), heat-shock protein 90 (HSP90), and 101 (HSP101)) might be contributed to melatonin-mediated thermotolerance. Taken together, this study provided direct link between melatonin and thermotolerance and indicated the involvement of HSFA1s-activated heat-responsive genes in melatonin-mediated thermotolerance in Arabidopsis.

Melatonin (N-acetyl-5-methoxytryptamine) is not only a widely known animal hormone, but also an important regulator in plant development and multiple abiotic stress responses. Recently, it has been revealed that melatonin alleviated cold stress through mediating several cold-related genes, including C-REPEAT-BINDING FACTORs (CBFs)/Drought Response Element Binding factors (DREBs), COR15a, and three transcription factors (CAMTA1, ZINC FINGER OF ARABIDOPSIS THALIANA 10 (ZAT10), and ZAT12). In this study, we quantified the endogenous melatonin level in Arabidopsis plant leaves and found the endogenous melatonin levels were significantly induced by cold stress (4 °C) treatment. In addition, we found one cysteine2/histidine2-type zinc finger transcription factor, ZAT6, was involved in melatonin-mediated freezing stress response in Arabidopsis. Interestingly, exogenous melatonin enhanced freezing stress resistance was largely alleviated in AtZAT6 knockdown plants, but was enhanced in AtZAT6 overexpressing plants. Moreover, the expression levels of AtZAT6 and AtCBFs were commonly upregulated by cold stress (4 °C) and exogenous melatonin treatments, and modulation of AtZAT6 expression significantly affected the induction AtCBFs transcripts by cold stress (4 °C) and exogenous melatonin treatments. Taken together, AtZAT6-activated CBF pathway might be essential for melatonin-mediated freezing stress response in Arabidopsis.

The cysteine2/histidine2-type zinc finger proteins are a large family of transcription regulators, and some of them play essential roles in plant responses to biotic and abiotic stress. In this study, we found that expression of C 2 H 2 -type ZINC FINGER OF ARABIDOPSIS THALIANA6 (AtZAT6) was transcriptionally induced by salt, dehydration, cold stress treatments, and pathogen infection, and AtZAT6 was predominantly located in the nucleus. AtZAT6-overexpressing plants exhibited improved resistance to pathogen infection, salt, drought, and freezing stresses, while AtZAT6 knockdown plants showed decreased stress resistance. AtZAT6 positively modulates expression levels of stress-related genes by directly binding to the TACAAT motifs in the promoter region of pathogen-related genes (ENHANCED DISEASE SUSCEPTIBILITY1, PHYTOALEXIN DEFICIENT4, PATHOGENESIS-RELATED GENE1 [PR1], PR2, and PR5) and abiotic stress-responsive genes (C-REPEAT-BINDING FACTOR1 [CBF1], CBF2, and CBF3). Moreover, overexpression of AtZAT6 exhibited pleiotrophic phenotypes with curly leaves and smallsized plant at vegetative stage and reduced size of floral organs and siliques at the reproductive stage. Modulation of AtZAT6 also positively regulates the accumulation of salicylic acid and reactive oxygen species (hydrogen peroxide and superoxide radical). Taken together, our findings indicated that AtZAT6 plays important roles in plant development and positively modulates biotic and abiotic stress resistance by activating the expression levels of salicylic acid-related genes and CBF genes.

Melatonin (N-acetyl-5-methoxytryptamine) functions as a ubiquitous modulator in multiple plant developmental processes and various stress responses. However, the involvement of melatonin in natural leaf senescence and the underlying molecular mechanism in Arabidopsis remain unclear. In this study, we found that the endogenous melatonin level was significantly induced in a developmental stage-dependent manner, and exogenous melatonin treatment delayed natural leaf senescence in Arabidopsis. The expression level of AUXIN RESISTANT 3 (AXR3)/INDOLE-3-ACETIC ACID INDUCIBLE 17 (IAA17) was significantly downregulated by exogenous melatonin treatment and decreased with developmental age in Arabidopsis. Further investigation indicated that AtIAA17-overexpressing plants showed early leaf senescence with lower chlorophyll content in rosette leaves compared with wild-type plants, while AtIAA17 knockout mutants displayed delayed leaf senescence with higher chlorophyll content. Notably, exogenous melatonin-delayed leaf senescence was largely alleviated in AtIAA17-overexpressing plants, and AtIAA17-activated senescence-related SENESCENCE 4 (SEN4) and SENESCENCE-ASSOCIATED GENE 12 (SAG12) transcripts might have contributed to the process of natural leaf senescence. Taken together, the results indicate that AtIAA17 is a positive modulator of natural leaf senescence and provides direct link between melatonin and AtIAA17 in the process of natural leaf senescence in Arabidopsis.

Nitric oxide (NO) plays essential roles in many physiological and developmental processes in plants, including biotic and abiotic stresses, which have adverse effects on agricultural production. However, due to the lack of findings regarding nitric oxide synthase (NOS), many difficulties arise in investigating the physiological roles of NO in vivo and thus its utilization for genetic engineering. Here, to explore the possibility of manipulating the endogenous NO level, rat neuronal NOS (nNOS) was expressed in Arabidopsis thaliana. The 35S::nNOS plants showed higher NOS activity and accumulation of NO using the fluorescent probe 3-amino, 4-aminomethyl-2', 7'-difluorescein, diacetate (DAF-FM DA) assay and the hemoglobin assay. Compared with the wild type, the 35S::nNOS plants displayed improved salt and drought tolerance, which was further confirmed by changes in physiological parameters including reduced water loss rate, reduced stomatal aperture, and altered proline and malondialdehyde content. Quantitative real-time PCR analyses revealed that the expression of several stress-regulated genes was up-regulated in the transgenic lines. Furthermore, the transgenic lines also showed enhanced disease resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 by activating the expression of defense-related genes. In addition, we found that the 35S::nNOS lines flowered late by regulating the expression of CO, FLC and LFY genes. Together, these results demonstrated that it is a useful strategy to exploit the roles of plant NO in various processes by the expression of rat nNOS. The approach may also be useful for genetic engineering of crops with increased environmental adaptations.