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.

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.

Seed germination and flowering initiation are both transitions responding to similar seasonal cues. This study shows that ABSCISIC ACID-INSENSITIVE MUTANT 5 (ABI5), a bZIP transcription factor, which plays an important role in the abscisic acid (ABA)-arrested seed germination, is robustly associated with the floral transition in Arabidopsis. Under long-day conditions, overexpression of ABI5 could delay floral transition through upregulating FLOWERING LOCUS C (FLC) expression. In contrast, ectopically overexpressing FLC in an abi5 mutant reversed the earlier flowering phenotype. Further analysis indicated that transactivation of FLC could be promoted by ABI5 and/or other abscisic acid-responsive element (ABRE)-binding factors (ABFs). The expression of FLC that was promoted by ABI5 and/or other ABFs could be blocked in a triple SNF1-related protein kinase (SnRK) mutant, snrk2.2/2.3/2.6, despite the presence of ABA. In sharp contrast, when SnRK2.6 was coexpressed, the reduction of transactivity of FLC was reverted in mesophyll protoplasts of snrk2.2/2.3/2.6. Additional results from analysing transgenic plants carrying mutations of phosphoamino acids (ABI5 S42AS145AT201A), which are conserved in ABI5, suggested that SnRK2-mediated ABI5 and/or ABF phosphorylation may be crucial for promoting FLC expression. The transgenic plants ABI5 S42AS145AT201A were insensitive to ABA in seed germination, in addition to having an earlier flowering phenotype. Direct binding of ABI5 to the ABRE/G-box promoter elements existing in FLC was demonstrated by chromatin immunoprecipitation. Mutations at the ABRE/G-box regions in FLC promoter sequences abolished the ABI5-promoted transactivation of FLC. In summary, these results may decipher the inhibitory effect of ABA on floral transition in Arabidopsis.

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.

SUMMARYAbscisic acid (ABA) and cytokinin are key hormones controlling plant development. How ABA and cytokinin interplay to control the transition from a dry seed into a young seedling remains elusive. Here we undertook a gain-of-function genetic screen to identify ABA-insensitive mutants during seed germination in Arabidopsis using an estradiol-inducible approach. In the presence of estradiol, one of these mutants gim1 (germination insensitive to ABA mutant 1) exhibited an elevated level of cytokinin that was attributed to the estradiolinduced expression of AtIPT8 that encodes an isopentenyltransferase for the biosynthesis of cytokinins. Our data on OE-2 and Com-1 transgenic plants carrying the ectopically expressing AtIPT8 gene indicated that the elevation of cytokinin level was responsible for the ABA-insensitivity of gim1 seed germination. Further analyses on alterations of gene transcriptomes in the gim1 mutant demonstrated that the expression of some ABA-inducible genes, including ABI5, was reduced, and could not be restored by exogenous ABA treatment. Moreover, we also failed to observe the ABA-mediated repression of a family of cytokinin signal transducers and transcription repressors called type-A ARR4, ARR5 and ARR6 in the gim1 seedlings. Further analysis demonstrated that type-A ARR4, ARR5 and ARR6 could negatively regulate ABI5 expression, and the physical interaction of ABI5 and type-A ARR4, ARR5 and ARR6 proteins was detected. In summary, our study suggests that the interaction of ABA and cytokinin during seed germination and seedling growth can be mediated by the interplay of transcriptional regulators 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.

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Bermudagrass (Cynodon dactylon) is a widely used warm-season turfgrass and one of the most drought tolerant species. Dissecting the natural variation in drought tolerance and physiological responses will bring us powerful basis and novel insight for plant breeding. In the present study, we evaluated the natural variation of drought tolerance among nine bermudagrass varieties by measuring physiological responses after drought stress treatment through withholding water. Three groups differing in drought tolerance were identified, including two tolerant, five moderately tolerant and two susceptible varieties. Under drought stress condition, drought sensitive variety (Yukon) showed relative higher water loss, more severe cell membrane damage (EL), and more accumulation of hydrogen peroxide (H2O2) and malondialdehyde (MDA), while drought tolerant variety (Tifgreen) exhibited significantly higher antioxidant enzymes activities. Further results indicated that drought induced cell injury in different varieties (Yukon, SR9554 and Tifgreen) exhibited liner correlation with leaf water content (LWC), H2O2 content, MDA content and antioxidant enzyme activities. Additionally, Tifgreen plants had significantly higher levels of osmolytes (proline level and soluble sugars) when compared with Yukon and SR9554 under drought stress condition. Taken together, our results indicated that natural variation of drought stress tolerance in bermudagrass varieties might be largely related to the induced changes of water status, osmolyte accumulation and antioxidant defense system.