Functional Analysis of the Heat-Tolerance Candidate Gene BcNCED3 in Non-Heading Chinese Cabbage (Brassica campestris ssp. chinensis).

Non-heading Chinese cabbage (Brassica campestris ssp. chinensis) is an economically important leafy vegetable whose quality and yield are severely constrained by high temperature. Abscisic acid (ABA) is a key stress hormone, and 9-cis-epoxycarotenoid dioxygenase (NCED) is the rate-limiting enzyme of its biosynthesis; however, the function of NCED in the thermotolerance of this crop remains unclear. In this study, the heat-tolerance candidate gene BcNCED3 was functionally characterized in non-heading Chinese cabbage using bioinformatics, subcellular localization, expression profiling, exogenous ABA treatment, genetic transformation, and CRISPR/Cas12a editing. BcNCED3 has an intronless 1797 bp open reading frame encoding a 598-amino-acid hydrophilic protein with a conserved RPE65 domain, is localized to the chloroplast, and is highly conserved across Brassicaceae. Its transcript was strongly induced under 40 °C heat stress, with reaching markedly higher transcript abundance at 6 h in the heat-tolerant cultivar 'Suzhou Qing' than in the heat-sensitive 'Aijiaohuang'. Exogenous ABA (10 µM) pretreatment alleviated heat injury, increased endogenous ABA content by 1.8-fold, upregulated heat-responsive genes (BcHSFA1, BcHSFA2, BcHSP101, BcDREB2A, BcMBF1c and BcGolS1) by 1.5- to 3.2-fold, enhanced POD, SOD, and CAT activities by 25-40%, and reduced malondialdehyde accumulation by 32%. BcNCED3-overexpressing plants with 12- to 30-fold increased expression reproduced these responses, whereas Cas12a knockout plants exhibited heat hypersensitivity. These results demonstrate that BcNCED3 positively regulates thermotolerance in non-heading Chinese cabbage and provide a genetic resource for heat-tolerance breeding.