CitWRKY28 and CitNAC029 promote the synthesis of cuticular wax by activating CitKCS gene expression in citrus fruit.

KEY MESSAGE: CitWRKY28 and CitNAC029 are involved in cuticular wax synthesis as indicated by the comparative analysis of fruit aliphatic wax content between Citrus reticulata and Citrus trifoliata and gene co-expression analysis. Cuticular wax covers the fruit surface, playing important roles in reduction of fruit water loss and resistance to pathogen invasion. However, there is limited research on the synthesis and transcriptional regulation of cuticular wax in citrus fruit. In this study, we characterized the variations of aliphatic wax in HJ (Citrus reticulata) and ZK (Citrus trifoliata) from young fruit to mature fruit, as well as performed transcriptome sequencing on 27 samples at different fruit developmental stages. The results revealed that the ZK fruit always had a higher aliphatic wax content than the HJ fruit during development. qRT-PCR analysis demonstrated that two KCS genes, CitKCS1 and CitKCS12, had the most significant difference in expression between HJ and ZK. Furthermore, a heterologous expression assay in Arabidopsis indicated that CitKCS1 and CitKCS12 are involved in cuticular wax synthesis. Subsequently, gene co-expression network analysis screened CitWRKY28 and CitNAC029. Dual luciferase and EMSA assays indicated that CitWRKY28 might bind to the promoter of CitKCS1 and CitKCS12 and CitNAC029 might bind to that of CitKCS1 to activate their expression. Moreover, CitWRKY28 and CitNAC029 could promote the accumulation of cuticular wax in Arabidopsis leaves. Our findings provide new insights into the synthesis and regulation of cuticular wax and valuable information for further mining of wax-related genes in citrus fruit.

Genome-Wide Identification of Peanut KCS Genes Reveals That AhKCS1 and AhKCS28 Are Involved in Regulating VLCFA Contents in Seeds.

The peanut (Arachis hypogaea L.) is an important oilseed crop worldwide. Compared to other common edible vegetable oils, peanut oil contains a higher content of saturated fatty acids (SFAs), approximately 20-40% of which are very long chain fatty acids (VLCFAs). To understand the basis for this oil profile, we interrogated genes for peanut ß-ketoacyl-CoA synthase (KCS), which is known to be a key enzyme in VLCFA biosynthesis. A total of 30 AhKCS genes were identified in the assembled genome of the peanut. Based on transcriptome data, nine AhKCS genes with high expression levels in developing seeds were cloned and expressed in yeast. All these AhKCSs could produce VLCFAs but result in different profiles, indicating that the AhKCSs catalyzed fatty acid elongation with different substrate specificities. Expression level analysis of these nine AhKCS genes was performed in developing seeds from six peanut germplasm lines with different VLCFA contents. Among these genes, the expression levels of AhKCS1 or AhKCS28 were, 4-10-fold higher than that of any other AhKCS. However, only the expression levels of AhKCS1 and AhKCS28 were significantly and positively correlated with the VLCFA content, suggesting that AhKCS1 and AhKCS28 were involved in the regulation of VLCFA content in the peanut seed. Further subcellular localization analysis indicated that AhKCS1 and AhKCS28 were located at the endoplasmic reticulum (ER). Overexpression of AhKCS1 or AhKCS28 in Arabidopsis increased the contents of VLCFAs in the seed, especially for very long chain saturated fatty acids (VLCSFAs). Taken together, this study suggests that AhKCS1 and AhKCS28 could be key genes in regulating VLCFA biosynthesis in the seed, which could be applied to improve the health-promoting and nutritional qualities of the peanut.