Function and transcriptional regulation of CsKCS20 in the elongation of very-long-chain fatty acids and wax biosynthesis in Citrus sinensis flavedo

Abstract

Cuticular wax on plant aerial surfaces plays a vital role in the defense against various stresses, and the genes related to wax metabolism have been well documented in several model plants. However, there is very limited research on the key enzymes and transcription factors (TFs) associated with carbon chain distribution and wax biosynthesis in citrus fruit. In this study, an analysis of wax metabolites indicated that even carbon-chain (C24-C28) metabolites are the dominant wax components in citrus fruit, and a 3-ketoacyl-CoA synthase (KCS) family gene (CsKCS20) plays an important role in the carbon chain distribution during wax biosynthesis in a wax-deficient mutant (MT). Expression of CsKCS20 in yeast indicated that CsKCS20 can catalyze the biosynthesis of C22 and C24 very-long-chain fatty acids (VLCFAs). In addition, transcriptome and sequence analysis indicated that the differential expression of CsKCS20 between the wild-type (WT) and MT fruit can be partly attributed to the regulation of CsMYB96, which was further confirmed by yeast one-hybrid (Y1H) assays, electrophoretic mobility shift assays (EMSAs) and dual luciferase assays. The functions of CsMYB96 and CsKCS20 in wax biosynthesis were further validated by heterologous expression in Arabidopsis. In summary, this study elucidates the important roles of CsKCS20 and CsMYB96 in regulating VLCFA elongation and cuticular wax biosynthesis, which provides new directions for the improvement of citrus fruit wax quality in genetic breeding programs.

Figure 1

Comprehensive descriptions of the appearance and internal quality of fruit. (a) Phenotypic comparison between WT and MT fruit from the same garden. (b) SEM result of WT and MT fruit flavedo. (c) Water loss of WT and MT fruit. (d) Total soluble solids (TSS). (e) Total acid (TA). (f) Maturity index (TSS TA⁻¹). Values shown are the mean ± SD (n = 12 in water loss analysis and n = 3 in TSS, TA and TSS TA⁻¹ analysis). The asterisks (*) indicate significant differences (* P < 0.05 with Student’s t-test).

Figure 2

Cuticular wax profiles of WT and MT fruit flavedo. (a) Total wax content. (b) Cuticular wax chemical components. (c) Distribution of carbon chain lengths. Values shown are mean ± SD (n = 3–4). The asterisks (*) indicate significant differences (* P < 0.05 with Student’s t-test).

Figure 3

Transcription levels of candidate KCSs and subcellular localization. (a) Transcription levels of candidate genes related to the very-long-chain fatty acid elongation pathway in the two genotypes in 2020. (b) Subcellular localization of CsKCS20 in N. benthamiana leaves transformed by agroinfiltration. RFP-HDEL was used as an endoplasmic reticulum marker. Values shown are the mean ± SD (n = 3–4). The asterisks (*) represent significant differences (* P < 0.05 with Student’s t-test).

Figure 4

Heterologous expression of CsKCS20 in the BY4741 Δfah1 yeast strain. Total fatty acids were extracted from the BY4741 Δfah1 yeast strain expressing the empty vector or CsKCS20. (a) GC–MS map. (b) Fatty acid production. Values shown are the mean ± SD (n = 4). The asterisks (*) indicate significant differences (* P < 0.05 with Student’s t-test).

Figure 5

Subcellular localization and interaction of CsKCS20 and CsMYB96. (a) Subcellular localization of CsMYB96 in N. benthamiana leaves transformed by agroinfiltration. DAPI staining was used as a nuclear marker. (b) Interaction of CsMYB96 with the promoter of CsKCS20 in the yeast one-hybrid assay. (c) Electrophoretic mobility shift assay (EMSA) indicating the binding of CsMYB96 to the promoter of CsKCS20. (d) Activation effect of CsMYB96 on the promoter of CsKCS20 detected by the dual-luciferase assay. Values shown are the mean ± SD (n = 3–4). The asterisks (*) represent significant differences (* P < 0.05 with Student’s t-test). Three independent replications were performed.

Figure 6

Cuticular wax results for the inflorescence stems of Arabidopsis Col-0 and CsKCS20 OE and CsMYB96 OE transgenic lines. (a) Total cuticular wax contents. (b) Cuticular wax compositions. Values shown are the mean ± SD (n = 3–4). The asterisks (*) indicate significant differences relative to wild-type (* P < 0.05 with Student’s t-test).