Leaf senescence is regulated by diverse developmental and environmental factors. Exogenous jasmonic acid (JA) can induce leaf senescence, whereas auxin suppresses this physiological process. Crosstalk between JA and auxin signaling has been well studied, but not during JA-induced leaf senescence. Here, we found that upon methyl jasmonate treatment, Arabidopsis thaliana wrky57 mutants produced typical leaf senescence symptoms, such as yellowing leaves, low chlorophyll content, and high cell death rates. Further investigation suggested that senescence-associated genes were upregulated in the wrky57 mutants. Chromatin immunoprecipitation experiments revealed that WRKY57 directly binds to the promoters of SENESCENCE4 and SENESCENCE-ASSOCIATED GENE12 and represses their transcription. In vivo and in vitro experiments suggested that WRKY57 interacts with JASMONATE ZIM-DOMAIN4/8 (JAZ4/8) and the AUX/IAA protein IAA29, repressors of the JA and auxin signaling pathways, respectively. Consistent with the opposing functions of JA and auxin in JA-induced leaf senescence, JAZ4/8 and IAA29 also displayed opposite functions in JA-induced leaf senescence and competitively interacted with WRKY57. Our results suggested that the JA-induced leaf senescence process can be antagonized by auxin via WRKY57. Moreover, WRKY57 protein levels were downregulated by JA but upregulated by auxin. Therefore, as a repressor in JA-induced leaf senescence, WRKY57 is a common component of the JA-and auxin-mediated signaling pathways.

No abstract

microRNAs (miRNAs) are a class of negative regulators that take part in many processes such as growth and development, stress responses, and metabolism in plants. Recently, miRNAs were shown to function in plant nutrient metabolism. Moreover, several miRNAs were identified in the response to nitrogen (N) deficiency. To investigate the functions of other miRNAs in N deficiency, deep sequencing technology was used to detect the expression of small RNAs under N-sufficient and -deficient conditions. The results showed that members from the same miRNA families displayed differential expression in response to N deficiency. Upon N starvation, the expression of miR169, miR171, miR395, miR397, miR398, miR399, miR408, miR827, and miR857 was repressed, whereas those of miR160, miR780, miR826, miR842, and miR846 were induced. miR826, a newly identified N-starvation-induced miRNA, was found to target the AOP2 gene. Among these N-starvation-responsive miRNAs, several were involved in cross-talk among responses to different nutrient (N, P, S, Cu) deficiencies. miR160, miR167, and miR171 could be responsible for the development of Arabidopsis root systems under N-starvation conditions. In addition, twenty novel miRNAs were identified and nine of them were significantly responsive to N-starvation. This study represents comprehensive expression profiling of N-starvation-responsive miRNAs and advances our understanding of the regulation of N homeostasis mediated by miRNAs.

SUMMARYSulfur is a macronutrient that is necessary for plant growth and development. Sulfate, a major source of sulfur, is taken up by plant roots and transported into various tissues for assimilation. During sulfate limitation, expression of miR395 is significantly up-regulated. miR395 targets two families of genes, ATP sulfurylases (encoded by APS genes) and sulfate transporter 2;1 (SULTR2;1, also called AST68), both of which are involved in the sulfate metabolism pathway. Their transcripts are suppressed strongly in miR395-over-expressing transgenic Arabidopsis, which over-accumulates sulfate in the shoot but not in the root. APS1 knockdown mutants accumulate twice as much sulfate as the wild-type. By constructing APS4-RNAi transgenic plants, we found that silencing the APS4 gene also results in over-accumulation of sulfate. Even though miR395-overexpressing transgenic plants over-accumulate sulfate in the shoot, they display sulfur deficiency symptoms. Additionally, the distribution of sulfate from older to younger leaves is impaired in miR395-over-expressing plants, similar to a SULTR2;1 loss-of-function mutant. The aps1-1 sultr2;1 APS4-RNAi triply repressed mutants phenocopied miR395-over-expressing plants. Our research showed that miR395 is involved in the regulation of sulfate accumulation and allocation by targeting APS genes and SULTR2;1, respectively.

No abstract

Drought is one of the most serious environmental factors that limit the productivity of agricultural crops worldwide. However, the mechanism underlying drought tolerance in plants is unclear. WRKY transcription factors are known to function in adaptation to abiotic stresses. By screening a pool of WRKY-associated T-DNA insertion mutants, we isolated a gain-of-function mutant, acquired drought tolerance (adt), showing improved drought tolerance. Under drought stress conditions, adt accumulated higher levels of ABA than wild-type plants. Stomatal aperture analysis indicated that adt was more sensitive to ABA than wild-type plants. Molecular genetic analysis revealed that a T-DNA insertion in adt led to activated expression of a WRKY gene that encodes the WRKR57 protein. Constitutive expression of WRKY57 also conferred similar drought tolerance. Consistently with the high ABA content and enhanced drought tolerance, three stress-responsive genes (RD29A, NCED3, and ABA3) were up-regulated in adt. ChIP assays demonstrated that WRKY57 can directly bind the W-box of RD29A and NCED3 promoter sequences. In addition, during ABA treatment, seed germination and early seedling growth of adt were inhibited, whereas, under high osmotic conditions, adt showed a higher seed germination frequency. In summary, our results suggested that the activated expression of WRKY57 improved drought tolerance of Arabidopsis by elevation of ABA levels. Establishment of the functions of WRKY57 will enable improvement of plant drought tolerance through gene manipulation approaches.

HighlightbHLH115 functions downstream of the E3 ligase BRUTUS and positively controls the expression of the iron-deficiency responsive bHLH transcription factors POPEYE and bHLH38/39/100/101 for iron homeostasis in Arabidopsis.

The CRISPR/Cas9-sgRNA system has been developed to mediate genome editing and become a powerful tool for biological research. Employing the CRISPR/Cas9-sgRNA system for genome editing and manipulation has accelerated research and expanded researchers’ ability to generate genetic models. However, the method evaluating the efficiency of sgRNAs is lacking in plants. Based on the nucleotide compositions and secondary structures of sgRNAs which have been experimentally validated in plants, we instituted criteria to design efficient sgRNAs. To facilitate the assembly of multiple sgRNA cassettes, we also developed a new strategy to rapidly construct CRISPR/Cas9-sgRNA system for multiplex editing in plants. In theory, up to ten single guide RNA (sgRNA) cassettes can be simultaneously assembled into the final binary vectors. As a proof of concept, 21 sgRNAs complying with the criteria were designed and the corresponding Cas9/sgRNAs expression vectors were constructed. Sequencing analysis of transgenic rice plants suggested that 82% of the desired target sites were edited with deletion, insertion, substitution, and inversion, displaying high editing efficiency. This work provides a convenient approach to select efficient sgRNAs for target editing.