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Functional Study Of Arabidopsis WRKY Transcription Factors In Response To Abiotic Stresses

Posted on:2015-03-21Degree:DoctorType:Dissertation
Country:ChinaCandidate:Z J DingFull Text:PDF
GTID:1220330431488917Subject:Botany
Abstract/Summary:PDF Full Text Request
Drought, high-salinity and aluminum toxicity (in acid soil), which severely inhibit the productivity of the crops, comprise the main abiotic stresses. However, the signal transduction and the molecular regulatory mechanisms of plants in response to these stresses still need to be investigated. Transcription factors play essential roles in plant signaling networks. For one thing, they may sense the upstream signals and hence regulate the expression of downstream genes; for another, they may also act in the node of the regulatory networks and be involved in the cross-talk between multiple signaling pathways. Thus, the study of transcription factors in response to stresses will improve our understanding of the molecular mechanisms of plants in adaptation to unfavorable conditions. WRKY proteins, which regulate a great number of processes (e.g. development and stress responses), comprise one of the largest transcription factor families in plants. While the WRKYs have been well studied in plants responding to biotic stresses, the roles of them in abiotic stresses are poorly understood. Here, we isolated two WRKYs (WRKY41and WRKY46) that can be rapidly induced by hydrogen peroxide and cadmium, from thirty three WRKY members in Arabidopsis thaliana. The functions of the two WRKYs were further investigated. The major results are listed as follows:1. WRKY46regulates osmotic stress responses and light-dependent stomotal movementGene expression analysis revealed that WRKY46was more responsive to water stress (drought, osmotic and salt stress) when compared with other abiotic treatments. Drought and salt stress rapidly induced the expression of WRKY46at both transcriptional level and protein level. T-DNA insertion of WRKY46led to more sensitivity to drought and salt stress, as the mutants (wrky46) had worse shoot growth than the wild-type (Col-0) plants under the stress. Whereas, overexpression of WRKY46(OV46) increased tolerance to osmotic and salt stress on the agar plates (closed environment), but resulted in hypersensitivity in soil-grown plants (open environment) with a higher water loss rate. Stomatal closing of OV46line was insensitive to abscisic acid (ABA) due to the lower reactive oxygen species (ROS) accumulation in the guard cells. We further found that WRKY46could be induced by light, was expressed in guard cells, in which its expression was not affected by dehydration, and was involved in light-dependent stomatal opening. Microarray analysis revealed that WRKY46regulated a set of genes involved in cellular osmoprotection and redox homeostasis under dehydration stress. Moreover, WRKY46modulated light-dependent starch metabolism in guard cells via regulating QUA-QUINE STARCH (QQS) gene expression.Taken together, we proposed that WRKY46play dual roles in osmotic stress responses and stomotal regulation independently:on the one hand, WRKY46is involved in cellular osmoprotection and oxidative detoxification; on the other hand, it regulates stomatal opening via modulation of starch metabolism in guard cells.2. WRKY46regulates lateral root development under osmotic/salt stressWRK46was expressed in lateral root primordium (LRPs) during early LR development and in root stele of mature lateral root (LR). Lack of WRKY46significantly reduced LR formation under osmotic/salt stress, while overexpression of it enhanced LR development under the stress. Exogenous auxin could largely restore LR development on loss-of-function wrky46mutants under osmotic/salt stress, and the auxin transport inhibitor2,3,5-triiodobenzoic acid (TIBA) inhibited both wild-type (Col-0) and WRKY46-overexpressing line (OV46) LR development under the stress, suggesting that the regulation of LR development by WRKY46depends on auxin. We found that WRKY46expression was down-regulated by abscisic acid (ABA) signaling, while up-regulated by an ABA-independent signal induced by osmotic/salt stress, by using ABA related mutants. The DR5:GUS expression was reduced in roots of wrky46mutants versus Col-0, and both wrky46mutant and OV46line showed altered levels of free indole-3-acetic acid (IAA) and IAA conjugates in roots versus Col-0under osmotic/salt stress. RT-qPCR revealed that WRKY46regulated the expression of ABI4and certain auxin conjugating genes under the stress. ChIP-qPCR further confirmed that WRKY46bound directly to the promoters of these genes.In summary, our results demonstrated that WRKY46is involved in the feedforward inhibition of osmotic/salt stress-dependent LR inhibition, via regulating ABA signaling and auxin homeostasis.3. WRKY46regulates aluminum-induced malate secretionALMT1that encodes a plasma-localized malate transporter is a key aluminum (Al) resistance gene. We found that the expression of ALMT1was significantly inhibited by WRKY46from the previous microarray data. The expression of WRKY46was down-regulated after Al treatment for3h, while ALMT1expression was induced under the same treatment. Moreover, WRKY46and ALMT1were spatially co-localized in root stele, indicating the possible interaction between the two genes. Mutation of WRKY46by T-DNA insertion led to better root growth under Al stress, and lower root Al content compared with the wild type Col-0. The wrky46mutant showed increased root malate secretion, which was consistent with the higher ALMT1expression in the mutant. Transient expression analysis using truncated promoter of ALMT1showed that ALMT1expression could be inhibited by WRKY46in tobacco leaves. The yeast one-hybrid assay and ChIP-qPCR analysis further revealed that WRKY46directly bound to ALMT1promoter through specific W-boxes. Taken together, we demonstrated that WRKY46is a negative regulator of ALMTl, mutation of WRKY46leads to increased malate secretion and reduced Al accumulation in the root apices, thus confers higher Al resistance.4. WRKY41regulates seed dormancyAlthough WRKY41rapidly responded to hydrogen peroxide and salt treatment, the loss-of-function wrky41mutant had similar responses to the wild type under these stresses. Nevertheless, we found that WRKY41was preferentially expressed in seed. Lack of WRKY41conferred reduced primary seed dormancy and thermoinhibition-dependent secondary dormancy, and decreased sensitivity to ABA during germination and early seedling growth, phenotypes resembling those by lack of ABI3. Absence of WRKY41decreased ABI3transcript abundance in maturing and imbibed seeds, whilst transgenically overexpressing WRKY41increased ABI3expression. Moreover, transgenic overexpression of ABI3completely restored seed dormancy phenotypes on wrky41. ChIP-qPCR and EMS A revealed that WRKY41bound directly to the ABI3promoter through three adjacent W-boxes, and a transactivation assay indicated these W-boxes essential for ABI3expression. Besides, WRKY41did not act downstream of ABA, and the regulation of ABI3by WRKY41was not through ABA and other factors known to promote ABI3transcription during seed maturation and germination. However, we also showed that high concentrations of ABA promoted negative feedback regulation of WRKY41expression. Finally, analysis of wrky41aba2double mutant confirmed that WRKY41and ABA collaboratively regulate ABI3expression and seed dormancy.
Keywords/Search Tags:drought, salt stress, aluminum toxicity, seed dormancy, stomata, lateral root, transcription factor, WRKY, Arabidopsis thaliana
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