Pleiotropic Effects of SEPALLATA2 Knock-out on Fruit and Wart Development in Cucumber

doi:10.13560/j.cnki.biotech.bull.1985.2025-0704

Abstract

Abstract:

Objective SEPALLATA2 (SEP2) encodes MADS family transcription factor. This study aims to decipher the role of CsSEP2 in the development of cucumber (Cucumis sativus L.) fruits and warty structures by using Cssep2 mutants and investigate the regulatory mechanism. It provides theoretical support for the in-depth analysis of important traits in cucumber. Method Real-time quantitative PCR was used to analyze the expression pattern of CsSEP2. Subcellular localization was employed to determine the specific intracellular location of CsSEP2. Cssep2 knockout mutants was carried out through stable genetic transformation, and the molecular mechanism underlying their phenotypic changes was explored at the cytological and transcriptomic levels. Yeast two-hybrid assay and firefly luciferase complementation assay were utilized to analyze the interactions between CsSEP2 and other proteins. Result CsSEP2 mRNA accumulates specifically in flowers and fruits, and CsSEP2 was localized in the nucleus. The loss-of-function mutants were generated using CRISPR/Cas9 technology. The Cssep2 mutants produced short fruits resulting from reduced cell division and cell expansion, and the fruits tended to dehisce. The warty traits of Cssep2 mutants were substantially altered, with dense spine, reduced fruit spine base and shrunken tubercules. RNA-seq analysis of the fruit epicarp and placenta revealed that loss-of-function of CsSEP2 had a considerable influence on the gene expression profile. Of note, genes that have been reported to function in fruit size and warty traits presented substantially altered expression. CsSEP2 interacted with several proteins that are known to regulate the development of warty structures. Conclusion Cssep2 mutation results in pleiotropic effects on the development of fruits and warty structures. CsSEP2 regulates fruit length and warty development by influencing the expressions of a series of genes and protein interactions.

Key words: cucumber, CsSEP2, mutants, fruit development, fruit cracking, spine, wart

LUO Wei, GONG Ao, ZHONG Yang, HU Di, ZHOU Hong-yuan, ZHANG Hong-xin, AI Ju, LUO You-wei, GAO Dong-li. Pleiotropic Effects of SEPALLATA2 Knock-out on Fruit and Wart Development in Cucumber[J]. Biotechnology Bulletin, 2026, 42(3): 283-293.

Figures/Tables 6

Fig. 1 Expression analyses (A) and subcellular localization (B) of gene CsSEP2In Fig. A, the expressions of CsSEP2 in thefruits of -3, 0, 5, 10, and 16 DAA and epicarp, mesocarp and placenta from the central parts of fruits at 16 DAA were examined. Data are means ± SD, n = 3. DAA refers to days after anthesis, the same below

Fig. 2 Knock-out of gene CsSEP2 resulted in abnormal fruit developmentA: Structure of the CsSEP2 gene and the mutated loci in the Cssep2 mutants. Exons and introns are denoted by black boxes and lines, respectively. The red nucleotide sequences are the editing target site of the gene, and the three underlined bases indicate the protospacer adjacent motif (PAM). B, C: Representative plants (B) and fruits (C) of WT and Cssep2 mutants. D: Measurement of the fruit lengths of WT and Cssep2 mutants. Data are means ± SD, n = 5. E: Macroscopic observation of fruit dehiscence of the Cssep2 mutants. F: Comparative observation of the fruit placentas of WT and Cssep2 mutants. G: The ratio of placenta areas to fruit cross areas of WT and Cssep2 mutants. Values are means ± SD, n ≥ 5

Fig. 3 Cellular dissection of the fruit and wart phenotype of the Cssep2 mutantsA, B: Cell number and size of fruit mesocarp along the longitudinal axis. Values are means ± SD, n = 540. C, D: Longitudinal sections of the fruit mesocarp at 16 DAA. E, F: Cell number and size of fruit mesocarp along the transversal axis. Values are means ± SD, n = 540. G, H: Transversal sections of the fruit mesocarp at 16 DAA. I: Fruit spine of WT and Cssep2 mutants at -3 DAA. J: Macroscopic observation of the tubercule at -3 DAA. K: Measurement of the spine base of WT and Cssep2 mutants at -3 DAA. TD: Traverse diameter, LD: longitudinal diameter; * P < 0.05; **** P < 0.000 1. Values are means ± SD, n = 18. The same below

Fig. 4 GO enrichment and RT-qPCR analysis of differentially expressed genesA: GO enrichment analysis of DEGs at 5T (placenta at 5 DAA), 16T (placenta at 16 DAA), 0W (epicarp at 0 DAA) and 16W (epicarp at 16 DAA). GO terms related with DNA binding and regulation of transcription are highlighted with red. B: Expression analysis of genes in the epicarp tissues of WT and Cssep2 mutants. C: Expression analysis of genes in the placenta tissues of WT and Cssep2 mutants; **** P < 0.000 1; ** P < 0.01; * P < 0.05; ns: not significant difference

Fig. 5 Identification of proteins interacting with CsSEP2A: Yeast two-hybrid assay results. QDO: Quadruple dropout, SD/-Leu-Trp-His-Ade. A600: Addition of Aureobasidin A with a final concentration of 600 ng/mL. B: Fireﬂy luciferase complementation imaging analysis

Fig. 6 Molecular network of fruit-related traits regulated by CsSEP2A, B: Schematic diagram of the CsSEP2-invovled molecular network of fruit development (A) and warty traits (B). The captions of the figure are in the upper right corner. CsSEP2 inhibits the lengths of cucumber fruits by suppressing the expression of CRABS CLAW[17]. CsFUL regulates fruit length by inhibiting the expressions of CsPIN1, CsPIN7 and CsSUP, and can also form protein complex with CsSEP2 to exert its functions[21]. Notably, the expressions of both CsFUL and CsSUP changed significantly in Cssep2. Compared to WT, the expressions of CsGL3[22], CsTS[23], CsGL1[24], CsTTG1[25], CsTu[26-27], and ns[28] were lower in Cssep2 mutants. These genes have been confirmed to be involved in the regulation of the formation and development of fruit spines and warts. Among them, ns encodes an auxin transporter and inhibits the density of fruit spines[28]. CsGL1 can not only inhibit the expression of CsGA20OX1, affecting the synthesis of GA and altering the density of fruit spines and the size of the fruit spine base[24], but also interacts with CsTTG1 to form a complex to regulate the density of fruit spines[25]. CsTu promotes the expressions of CsCTK[26] and CsTS1[27], altering the synthesis of cytokinin and auxin and facilitating the initiation of fruit spines and the formation of fruit warts. In Cssep2 mutants, the expressions of CsGA20OX1, CsCTK, and CsTS1 also changed to varying degrees. In addition, this study further revealed that CsSEP2 interacts with CsGL3, CsGL1, CsTTG,1 and CsTu

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