Transcription factor BnaA9.WRKY47 contributes to the adaptation of Brassica napus to low boron stress by up-regulating the boric acid channel gene BnaA3.NIP5;1

Abstract

Boron (B) deficiency is one of the major causes of growth inhibition and yield reduction in Brassica napus (B. napus). However, the molecular mechanisms of low B adaptation in B. napus are largely unknown. Here, fifty-one BnaWRKY transcription factors were identified as responsive to B deficiency in B. napus, in which BnaAn.WRKY26, BnaA9.WRKY47, BnaA1.WKRY53 and BnaCn.WRKY57 were tested in yeast one-hybrid assays and showed strong binding activity with conserved sequences containing a W box in the promoters of the B transport-related genes BnaNIP5;1s and BnaBOR1s. Green fluorescent protein fused to the target protein demonstrated the nuclear localization of BnaA9.WRKY47. CRISPR/Cas9-mediated knockout lines of BnaA9.WRKY47 in B. napus had increased sensitivity to low B and lower contents of B than wild-type plants. In contrast, overexpression of BnaA9.WRKY47 enhanced the adaptation 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 BnaA3.NIP5;1, a B efficiency candidate gene, was decreased in the knockout lines but was significantly increased in the overexpressing lines under low B conditions. Electrophoretic mobility shift assays, transient expression experiments in tobacco and in situ hybridizations showed that BnaA9.WRKY47 directly activated BnaA3.NIP5;1 expression through binding to the specific cis-element. Taken together, our findings support BnaWRKYs as new participants in response to low B, and BnaA9.WRKY47 contributes to the adaptation of B. napus to B deficiency through up-regulating BnaA3.NIP5;1 expression to facilitate efficient B uptake.

Keywords: Brassica napus; W box; WRKY; boric acid channel; low B adaptation; transcription factor.