Molecular Mechanism On Transcription Factor WRKY47 Participating In The Adaptation Responses Of Brassica Napus And Arabidopsis To Boron Stresses

Boron?B?is essential for plant growth and development,but the optimal range of B concentrations for plant growth is narrow.Although efficient B uptake and transport have been studied well in Arabidopsis,the molecular regulatory mechanism has long been confused in both Arabidopsis and Brassica napus?B.napus?.B.napus is hypersensitive to B deficiency and also susceptible to excess B.Accumulating evidences has highlighted the importance of WRKY genes in response to nutrient conditions.In this study,we carried out a systematic investigation on the involvement of transcription factor Bna WRKYs in the B stress adaptation of B.napus and the participation of WRKY47 in B stress regulation.The main results of this research are as follows:1. Bna WRKY family genes act as new players in response of B.napus to B deficiency and toxicityGenome-wide transcriptomic analysis in B.napus revealed numerous B stress responsive genes,including transcription factors,signal transducers and structural molecules.Among these responsive genes,64 Bna WRKYs transcription factor family genes showed more than two-fold differential expressions under B deficiency?0.25?M B?or B toxicity?1500?M B?relative to the normal B condition?25?M B?.Interestingly,the promoters of B transport-related genes,NIPs and BORs,are statistically enriched in W-box cis-element which is a well-characterized WRKY binding domain.Furthermore,Bna An.WRKY26/A9.WRKY47/A1.WKRY53/Cn.WRKY57 were tested in yeast-one hybrid assays and showed strong binding activity with the conserved sequences containing a W-box in the promoters of the B transport-related genes Bna NIP5;1s and Bna BOR1s.EMSA assay showed that Bna A1.WKRY53 and Bna Cn.WRKY57,but not Bna An.WRKY26 and Bna A9.WRKY47 bind to the conserved sequence in Bna NIP5;1s and in Bna BOR1s.These findings identified Bna WRKYs transcription factor as new players in B stress response and might function by interacting with B transport-related genes with certain diversity and specifity.2. Bna A9.WRKY47 contributes to the adaptation of B.napus to low B stress by up-regulating the boric acid channel gene Bna A3.NIP5;1Bna A9.WRKY47 was selected to be further analysed owing to its significantly high expression in roots and leaves induced by B deficiency.Green fluorescent protein fused to the target protein demonstrated the nuclear localization of Bna A9.WRKY47.CRISPR/Cas9 mediated knockout lines of Bna A9.WRKY47 in B.napus showed increased sensitivity to low B and lower contents of B than wild-type plants.In contrast,overexpression of Bna A9.WRKY47 enhanced the tolerance to low B with higher B contents in tissues than in wild-type plants.Consistent with the phenotypic response and B accumulation in these transgenic lines,the transcription activity of Bna A3.NIP5;1,a B efficiency candidate gene,decreased in the knockout lines,but significantly increased in the overexpressing lines under low B condition.Yeast one hybrid assays and electrophoretic mobility shift assays indicated that the W-box element TGAC^{-646 to-643}presumably played an important role in the binding of Bna A9.WRKY47 with Bna A3.NIP5;1.Transient expression experiments in tobacco and in situ hybridization showed that Bna A9.WRKY47 directly activated Bna A3.NIP5;1 expression through binding to the specific cis-element.Taken together,our results varifed Bna WRKYs as new players in B stress,and Bna A9.WRKY47contributes to the adaptation of B.napus to B deficiency through upregulating Bna A3.NIP5;1 expression to facilitate efficient B uptake.3. At WRKY47 in response to high B stress in ArabidopsisThe function of WRKY47 has not been identified in Arabidopsis.The wrky47mutants?wrky47-1 and wrky47-3?were isolated due to the tolerance of B toxicity.Quantitative analysis of WRKY47 m RNA abundance indicated that its transcription activity was down-regulated by high B levels.Fluorescently labeled WRKY47-GFP was localized in nucleus.The wrky47 mutants developed longer primary root length,higher chlorophyll contents,more fertile siliques in high B conditions compared to Col-0 plants.Overexpression of WRKY47 in Arabidopsis conferred less growth and development than Col-0 plants in high B conditions.More importantly,B concentrations in the shoots were lower in the wrky47 mutants and higher in the overexpressing lines than that in Col-0 plants.These results demonstrate that WRKY47 gene negatively regulates the plant tolerance in high B levels.In summary,the study revealed the molecular mechanism on the transcription factor WRKY47 contribute to the adaptation to low B by upregulating Bna A3.NIP5;1in B.napus and to high B stress in Arabidopsis.