The <scp>MYB41</scp> ‐ <scp>MYB74</scp> module and <scp>VOZ1</scp> transcription factor synergistically regulate aliphatic suberin monomer biosynthesis in <i>Chenopodium quinoa</i>

Suberin is a biopolymer barrier mainly deposited on roots to protect plants against drought stress. Suberin monomers are composed of long-chain and very-long-chain fatty acids and their derived compounds. The mechanisms of suberin biosynthesis are well investigated in Arabidopsis, rice, and tomato. However, it remains unclear whether these mechanisms are conserved in halophytes. Here, we performed a systematic characterization of suberin biosynthesis in Chenopodium quinoa, a typical halophyte, and found that quinoa roots produce the typical suberin monomers in endodermis as found in other species. Aliphatic suberin biosynthesis in quinoa was upregulated during development or after exogenous abscisic acid (ABA) treatment. RNA-seq analysis showed that CqMYB41a/b and CqMYB74a/b were significantly induced in quinoa roots after exogenous ABA treatment. Overexpression of these MYB genes caused an ectopic and dramatic accumulation of aliphatic suberin monomers in tobacco leaves. We found both CqMYB41a/b and CqMYB74a/b could directly regulate the expression of each other, suggesting that ABA regulates quinoa suberin biosynthesis via a CqMYB41a/b-CqMYB74a/b module-cascade with positive feedback. Additionally, we also characterized two new regulators of suberin biosynthesis in quinoa, CqVOZ1a/b, localized to both the endoplasmic reticulum (ER) and nucleus. Further analysis by stable transformation in the Arabidopsis voz1-1 voz2-1 double mutant and transient expression in tobacco revealed that CqVOZ1a/b function in the nucleus to regulate flowering and suberin monomer production. Yeast one-hybrid (Y1H) and transcription activation analysis showed that CqVOZ1a/b could directly activate the expression of suberin biosynthesis related genes and CqMYB74a/b. However, high-dose overexpression of CqVOZ1a/b in Arabidopsis inhibited suberin monomer production and MYB74 expression. Furthermore, transcription activation analysis revealed that CqVOZ1 compete with CqMYB41 to negatively regulate CqMYB74 expression. Collectively, these findings indicate that CqVOZ1a/b may play an important role in maintaining suberin monomer homeostasis.