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

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Abstract

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.

Abstract

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.

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Data availability

The RNA sequencing data have been deposited in the NCBI BioProject database under the accession number PRJNA776912.

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Acknowledgements

This work was supported by the National Key R&D Program of China (2018YFD1000200), Huazhong Agricultural University Scientific & Technological Self–innovation Foundation, the National Natural Science Foundation of China (No. 31772368, 31972473) and China Agriculture Research System of MOF and MARA (CARS–26). We thank Prof. Zuoxiong Liu (College of Foreign Language, Huazhong Agricultural University) for advice on this manuscript.

Funding

This article was funded by National Natural Science Foundation of China (Nos. 31772368 and 31972473).

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Authors and Affiliations

  1. National R&D Center for Citrus Postharvest Technology, Wuhan, 430070, China

    Hongbin Yang, Zhifeng Zhu, Mingfei Zhang, Xin Li, Rangwei Xu, Feng Zhu, Juan Xu, Xiuxin Deng & Yunjiang Cheng

  2. Key Laboratory of Horticultural Plant Biology, Ministry of Education, Wuhan, 430070, China

    Hongbin Yang, Zhifeng Zhu, Mingfei Zhang, Xin Li, Rangwei Xu, Feng Zhu, Juan Xu, Xiuxin Deng & Yunjiang Cheng

  3. College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, 430070, China

    Hongbin Yang, Zhifeng Zhu, Mingfei Zhang, Xin Li, Rangwei Xu, Feng Zhu, Juan Xu, Xiuxin Deng & Yunjiang Cheng

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  1. Hongbin Yang
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  4. Xin Li
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Contributions

YC conceived the idea and designed the research; HY and ZZ collected the materials and analyzed the data; HY wrote the manuscript; XL, MZ, FZ, RX, JX and XD helped to improve the manuscript. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Yunjiang Cheng.

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299_2021_2826_MOESM1_ESM.tif

Fig. S1 qRT-PCR analysis of LACS genes in HJ and ZK fruit at the ripening stage. HJ, Citrus reticulata; ZK, Citrus trifoliata. The genes with p-value < 0.05 (Student’s t-test) and fold change > 2 are considered to be differentially expressed and marked with * in the figure. Values represent means of three biological replicates. Error bars = SD (TIF 628 kb)

299_2021_2826_MOESM2_ESM.tif

Fig. S2 qRT-PCR analysis of FAE complex-related genes (KCS, KCR, ECR and HCD), CER2 and CER26 in HJ and ZK fruit at the ripening stage. HJ, Citrus reticulata; ZK, Citrus trifoliata. The genes with p-value < 0.05 (Student’s t-test) and fold change > 2 are considered to be differentially expressed and marked with * in the figure. Values represent means of three biological replicates. Error bars = SD (TIF 2542 kb)

299_2021_2826_MOESM3_ESM.tif

Fig. S3 Principal component analysis for transcriptome data of HJ and ZK. The left and right show the samples of ZK and HJ fruit at different developmental stages, respectively. HJ, Citrus reticulata; ZK, Citrus trifoliata (TIF 558 kb)

299_2021_2826_MOESM4_ESM.tif

Fig. S4 Co-expression network construction. a, gene cluster dendrogram and gene modules. Different colors represent different gene modules. b, visual co-expression network (TIF 964 kb)

299_2021_2826_MOESM5_ESM.tif

Fig. S5 Dual luciferase transactivation assay. CitNAC029 could not activate the expression of CitKCS12. The figure shows a schematic diagram of the dual luciferase reporter experiment. The LUC/REN ratio represents the relative activity of the CitKCS12 promoter. Each sample has at least six biological replicates, and the error bar indicates standard deviation. Significant difference was evaluated by Student’s t-test (p-value > 0.05, ns). No significance, ns (TIF 184 kb)

299_2021_2826_MOESM6_ESM.tif

Fig. S6 qRT-PCR analysis of genes in the alcohol-forming pathway (CitFAR) and the alkane-forming pathway (CitCER1-2 and CitCER3) in HJ and ZK fruit at the ripening stage. HJ, Citrus reticulata; ZK, Citrus trifoliata. The genes with p-value < 0.05 (Student’s t-test) and fold change > 2 are considered to be differentially expressed and marked with * in the figure. Values represent means of three biological replicates. Error bars = SD (TIF 734 kb)

299_2021_2826_MOESM7_ESM.tif

Fig. S7 Identification for transgenic Arabidopsis. Genomic DNA-PCR analyses of CitKCS1, CitKCS12, CitWRKY28 and CitNAC029 in Arabidopsis wild type and the corresponding transgenic lines (TIF 914 kb)

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Yang, H., Zhu, Z., Zhang, M. et al. CitWRKY28 and CitNAC029 promote the synthesis of cuticular wax by activating CitKCS gene expression in citrus fruit. Plant Cell Rep 41, 905–920 (2022). https://doi.org/10.1007/s00299-021-02826-x

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