Regulation of CYP94B1 by WRKY33 controls apoplastic barrier formation in the roots leading to salt tolerance

Salinity is an environmental stress that causes decline in crop yield. Avicennia officinalis and other mangroves have adaptations such as ultrafiltration at the roots aided by apoplastic cell-wall barriers to thrive in saline conditions. We studied a Cytochrome P450 gene, AoCYP94B1 from A. officinalis and its Arabidopsis ortholog AtCYP94B1 that are involved in apoplastic barrier formation, and are induced by 30 minutes of salt treatment in the roots. Heterologous expression of AoCYP94B1 in atcyp94b1 Arabidopsis mutant and wild-type rice conferred increased NaCl tolerance to seedlings by enhancing root suberin deposition. Histochemical staining and GC-MS/MS quantification of suberin precursors confirmed the role of CYP94B1 in suberin biosynthesis. Using chromatin immunoprecipitation, yeast one-hybrid and luciferase assays, we identified AtWRKY33 as the upstream regulator of AtCYP94B1 in Arabidopsis. In addition, atwrky33 mutants exhibited reduced suberin and salt sensitive phenotypes, which were rescued by expressing 35S::AtCYP94B1 in atwrky33 mutant. This further confirms that the regulation of AtCYP94B1 by AtWRKY33 is part of the salt tolerance mechanism, and our findings can help in generating salt tolerant crops. One sentence summaryAtWRKY33 transcription factor regulates AtCYP94B1 to increase plant salt tolerance by enhanced suberin deposition in the endodermal cells of Arabidopsis roots