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SUMMARY: The plant cuticle is composed of cuticular wax and cutin polymers and plays an essential role in plant tolerance to diverse abiotic and biotic stresses. Several stresses, including water deficit and salinity, regulate the synthesis of cuticular wax and cutin monomers. However, the effect of wounding on wax and cutin monomer production and the associated molecular mechanisms remain unclear. In this study, we determined that the accumulation of wax and cutin monomers in Arabidopsis leaves is positively regulated by wounding primarily through the jasmonic acid (JA) signaling pathway. Moreover, we observed that a wound‐ and JA‐responsive gene (CYP96A4) encoding an ER‐localized cytochrome P450 enzyme was highly expressed in leaves. Further analyses indicated that wound‐induced wax and cutin monomer production was severely inhibited in the cyp96a4 mutant. Furthermore, CYP96A4 interacted with CER1 and CER3, the core enzymes in the alkane‐forming pathway associated with wax biosynthesis, and modulated CER3 activity to influence aldehyde production in wax synthesis. In addition, transcripts of MYC2 and JAZ1, key genes in JA signaling pathway, were significantly reduced in cyp96a4 mutant. Collectively, these findings demonstrate that CYP96A4 functions as a cofactor of the alkane synthesis complex or participates in JA signaling pathway that contributes to cuticular wax biosynthesis and cutin monomer formation in response to wounding. Significance Statement: Plant cuticle is a lipidic barrier composed of cuticular wax and cutin polymers. Cuticle plays a critical role in protecting plants from various biotic and abiotic stresses. Wounds due to mechanical damage or insect feeding are common and are potentially harmful to plants. However, the effect of wounding on plant cuticular wax and cutin monomer biosynthesis remains unclear. In our study, the findings present the validation that the Arabidopsis cuticle development was positively regulated by wounding mainly through the jasmonic acid (JA) signaling pathway. We also characterized the wound‐ and JA‐responsive gene CYP96A4, which is required for wound‐ and JA‐induced synthesis of cuticular wax and cutin monomer. [ABSTRACT FROM AUTHOR]

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]