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Summary: Plant cuticular wax accumulation limits nonstomatal transpiration and is regulated by external environmental stresses. DEWAX (DECREASE WAX BIOSYNTHESIS) plays a vital role in diurnal wax biosynthesis. However, how DEWAX expression is controlled and the molecular mechanism of wax biosynthesis regulated by the diurnal cycle remains largely unknown. Here, we identified two Arabidopsis MYB‐SHAQKYF transcription factors, MYS1 and MYS2, as new regulators in wax biosynthesis and drought tolerance.Mutations of both MYS1 and MYS2 caused significantly reduced leaf wax, whereas overexpression of MYS1 or MYS2 increased leaf wax biosynthesis and enhanced drought tolerance. Our results demonstrated that MYS1 and MYS2 act as transcription repressors and directly suppress DEWAX expression via ethylene response factor‐associated amphiphilic repression motifs.Genetic interaction analysis with DEWAX, SPL9 (SQUAMOSA PROMOTER BINDING PROTEIN‐LIKE 9), and CER1 (ECERIFERUM 1) in wax biosynthesis and under drought stresses demonstrated that MYS1 and MYS2 act upstream of the DEWAX‐SPL9 module, thus regulating CER1 expression. Expression analysis suggested that the diurnal expression pattern of DEWAX is partly regulated by MYS1 and MYS2.Our findings demonstrate the roles of two unidentified transcription repressors, MYS1 and MYS2, in wax biosynthesis and provide insights into the mechanism of diurnal cycle‐regulated wax biosynthesis. [ABSTRACT FROM AUTHOR]

Summary: Iso‐branched wax compounds are well known in plants, but their biosynthetic pathways are still mostly unknown. It has been speculated that branched waxes are derived from branched‐chain amino acid (BCAA) catabolism, but the evidence for this is very limited.Gas chromatography‐flame ionisation detection (GC‐FID) analysis revealed that mutations in two subunits of the branched‐chain ketoacid dehydrogenase (BCKDH) complex, a key enzyme complex in the degradation of BCAAs, significantly decreased the amounts of branched wax compounds, indicating that BCAA degradation may be integral to the synthesis of iso‐branched wax. Substrate feeding studies further revealed that the metabolic precursor of iso‐branched wax compounds is isobutyric acid (iBA), which is derived from valine degradation in Arabidopsis.We also isolated a novel mutant and found that its branched wax deficient phenotype could not be rescued by iBA. Map‐based cloning together with complementation analysis revealed that mutation in ACYL‐ACTIVATING ENZYME 9 (AAE9) is responsible for this phenotype.Genetic and enzyme activity analysis demonstrated that AAE9 is located downstream of the BCAA degradation pathway, and that it activates iBA to isobutyryl‐CoA for use on branched wax synthesis. Taken together, our study demonstrates that AAE9 is a key factor connecting BCAA catabolism with branched wax biosynthesis. [ABSTRACT FROM AUTHOR]