WRKY9 transcription factor regulates cytochrome P450 genes CYP94B3 and CYP86B1, leading to increased root suberin and salt tolerance in Arabidopsis

Abstract

Salinity affects crop productivity worldwide and mangroves growing under high salinity exhibit adaptations such as enhanced root apoplastic barrier to survive under such conditions. We have identified two cytochrome P450 family genes, AoCYP94B3 and AoCYP86B1 from the mangrove tree Avicennia officinalis and characterized them using atcyp94b3 and atcyp86b1, which are mutants of their putative Arabidopsis orthologs and the corresponding complemented lines with A. officinalis genes. CYP94B3 and CYP86B1 transcripts were induced upon salt treatment in the roots of both A. officinalis and Arabidopsis. Both AoCYP94B3 and AoCYP86B1 were localized to the endoplasmic reticulum. Heterologous expression of 35S::AoCYP94B3 and 35S:: AoCYP86B1 in their respective Arabidopsis mutants (atcyp94b3 and atcyp86b1) increased the salt tolerance of the transgenic seedlings by reducing the amount of Na + accumulation in the shoots. Moreover, the reduced root suberin phenotype of atcyp94b3 was rescued in the 35S::AoCYP94B3;atcyp94b3 transgenic Arabidopsis seedlings. Gas-chromatography and mass spectrometry analyses showed that the amount of suberin monomers (C-16 ω-hydroxy acids, C-16 α, ω-dicarboxylic acids and C-20 eicosanol) were increased in the roots of 35S::AoCYP94B3;atcyp94b3 Arabidopsis seedlings. Using chromatin immunoprecipitation and electrophoretic mobility shift assays, we identified AtWRKY9 as the upstream regulator of AtCYP94B3 and AtCYP86B1 in Arabidopsis. In addition, atwrky9 showed suppressed expression of AtCYP94B3 and AtCYP86B1 transcripts, and reduced suberin in the roots. These results show that AtWRKY9 controls suberin deposition by regulating AtCYP94B3 and AtCYP86B1, leading to salt tolerance. Our data can be used for generating salttolerant crop plants in the future.

Transcriptional Regulators of Suberin Biosynthesis in Arabidopsis thaliana

“…CYP94B1 overexpression, complementation (with Arabidopsis officinalis CYP94B1), and mutant experiments demonstrated a role for CYP94B1 in providing 1-21 ω-hydroxy fatty acid (C16 and C22) and α,ω-dicarboxylic fatty acids (C16) that are important for the diffusion barrier properties of the suberin polymer, and ultimately has a positive role in Arabidopsis' response to salt-stress (Krishnamurthy et al, 2020). The same role was found for CYP94B3 (Krishnamurthy et al, 2021). It appears that these two CYP94 genes complement the CYP86 suberin biosynthesis genes in the root endodermis to enhance suberin deposition under salt-stress, and it is possible that this same concept can be applied to the other genes involved in suberin biosynthesis.…”

“…Two other CYP genes, CYP94B1 and CYP94B3, are also involved in suberin biosynthesis (Krishnamurthy et al, 2020;Krishnamurthy et al, 2021). Under non-stress conditions CYP94B1 was expressed throughout the entire Arabidopsis plant.…”

“…ChIP and EMSA revealed WRKY9 could bind directly to the CYP94B3 and CYP86B1 promoters. CYP94B3 was connected to the production of root endodermis suberin lamellae, more specifically the 20:0 fatty alcohol, 16:0 ω-hydroxy acids, and 16:0 α, 1-27 ω-dicarboxylic acids that make up the suberin polymer (Krishnamurthy et al, 2021). WRKY56 is of interest as it was substantially upregulated in roots over-expressing MYB39 (Cohen et al, 2020) and MYB53, and was downregulated in roots of myb53 myb92 myb93 triple knockouts (Klein, 2019).…”

“…R2R3-MYB, WRKY, and NAC transcription factors have previously been shown to influence suberin biosynthesis (Cohen et al, 2020;Kosma et al, 2014;Krishnamurthy et al, 2020Krishnamurthy et al, , 2021Mahmood et al, 2019;To et al, 2020). With five R2R3-MYB TFs transcriptionally activating suberin biosynthesis genes (Figure 5) and WRKY56 (Klein, 2019)(Figure 7), MYB and WRKY TF DNA-binding motifs were the focus of cis-element search of the suberin biosynthesis gene promoters.…”

“…Rather, MYB92 could bind and activate the promoter of a fatty acid 3-58 synthesis gene, BCCP2, and these actions required the presence of two adjacent AC-rich motifs (To et al, 2020). WRKY33 and WRKY9 were found to bind to W-boxes with a sequence of TTGAC in the promoters of CYP86B1, CYP94B1, and CYP94B3 (Krishnamurthy et al, 2020(Krishnamurthy et al, , 2021. Since some R2R3-MYB TFs are known to interact with bHLH TFs and a WD40-repeat protein to form a MBW complex, bHLH DNAbinding motifs were also searched.…”