Although heat-shock transcription factors are well characterized in the heat stress-related pathway, they are poorly understood in other stress responses. Here, we functionally characterized AtHsfA6a in the presence of exogenous abscisic acid (ABA) and under high salinity and dehydration conditions. AtHsfA6a expression under normal conditions is very low, but was highly induced by exogenous ABA, NaCl and drought. Unexpectedly, the levels of AtHsfA6a transcript were not significantly altered under heat and cold stresses. Electrophoretic mobility shift assays and transient transactivation assays indicated that AtHsfA6a is transcriptionally regulated by ABA-responsive element binding factor/ABAresponsive element binding protein, which are key regulators of the ABA signalling pathway. Additionally, fractionation and protoplast transient assays showed that AtHsfA6a was in cytoplasm and nucleus simultaneously; however, under conditions of high salinity the majority of AtHsfA6A was in the nucleus. Furthermore, at both seed germination and seedlings stage, plants overexpressing AtHsfA6a were hypersensitive to ABA and exhibited enhanced tolerance against salt and drought stresses. Finally, the microarray and qRT-PCR analyses revealed that many stress-responsive genes were up-regulated in the plants overexpressing AtHsfA6a. Taken together, the data strongly suggest that AtHsfA6a acts as a transcriptional activator of stress-responsive genes via the ABA-dependent signalling pathway.
SummaryPhosphoinositides (PIs) are essential metabolites which are generated by various lipid kinases and rapidly respond to a variety of environmental stimuli in eukaryotes. One of the precursors of important regulatory PIs, phosphatidylinositol (PtdIn) 4-phosphate, is synthesized by PtdIns 4-kinases (PI4K). Despite its wide distribution in eukaryotes, its role in plants remains largely unknown. Here, we show that the activity of AtPI4Kc3 gene, an Arabidopsis (Arabidopsis thaliana) type II PtdIn 4-kinase, is regulated by DNA demethylation and some abiotic stresses. AtPI4Kc3 is targeted to the nucleus and selectively bounds to a few PtdIns. It possessed autophosphorylation activity but unexpectedly had no detectable lipid kinase activity. Overexpression of AtPI4Kc3 revealed enhanced tolerance to high salinity or ABA along with inducible expression of a host of stress-responsive genes and an optimal accumulation of reactive oxygen species. Furthermore, overexpressed AtPI4Kc3 augmented the salt tolerance of bzip60 mutants. The ubiquitin-like domain of AtPI4Kc3 is demonstrated to be essential for salt stress tolerance. Besides, AtPI4Kc3-overexpressed plants showed a late-flowering phenotype, which was caused by the regulation of some flowering pathway integrators. In all, our results indicate that AtPI4Kc3 is necessary for reinforcement of plant response to abiotic stresses and delay of the floral transition.
Arabidopsis CTD-PHOSPHATASE-LIKE 1 (CPL1) is a protein phosphatase that can dephosphorylate RNA polymerase II C-terminal domain (CTD). Unlike typical CTD-phosphatases, CPL1 contains a double-stranded (ds) RNA-binding motif (dsRBM) and has been implicated for gene regulation mediated by dsRNA-dependent pathways. We investigated the role of CPL1 and its dsRBMs in various gene silencing pathways. Genetic interaction analyses revealed that cpl1 was able to partially suppress transcriptional gene silencing and DNA hypermethylation phenotype of ros1 suggesting CPL1 is involved in the RNA-directed DNA methylation pathway without reducing siRNA production. By contrast, cpl1 reduced some miRNA levels at the level of processing. Indeed, CPL1 protein interacted with proteins important for miRNA biogenesis, suggesting that CPL1 regulates miRNA processing. These results suggest that CPL1 regulates DNA methylation via a miRNA-dependent pathway.
In eukaryotes, RNA silencing, mediated by small interfering RNAs, is an evolutionarily widespread and versatile silencing mechanism that plays an important role in various biological processes. Increasing evidences suggest that various components of RNA silencing pathway are involved in plant defense machinery against microbial pathogens in Arabidopsis thaliana. Here, we show genetic and molecular evidence that Arabidopsis SDE5 is required to generate an effective resistance against the biotrophic bacteria Pseudomonas syringae pv. tomato DC3000 and for susceptibility to the necrotrophic bacteria Erwinia caratovora pv. caratovora. SDE5, encodes a putative mRNA export factor that is indispensable for transgene silencing and the production of trans-acting siRNAs. SDE5 expression is rapidly induced by exogenous application of phytohormone salicylic acid (SA), methyl jasmonate (MeJA), phytopathogenic bacteria, and flagellin. We further report that SDE5 is involved in basal plant defense and mRNA export. Our genetic data suggests that SDE5 and Nonexpressor of PR Gene1 (NPR1) may contribute to the same SA-signaling pathway. However, SDE5 over-expressing transgenic plant exhibits reduced defense responsive phenotype after flagellin treatment. Taken together, these results support the conclusion that SDE5 contributes to plant innate immunity in Arabidopsis.
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