Characterization of selected members of clade A protein phosphatase 2Cs (PP2Cs) family in Brassica juncea var. tumida

Background and aims: Abscisic acid (ABA) is essential for plant stress adaptation and growth regulation, and clade A PP2Cs function as core negative regulators in ABA signal transduction. Nevertheless, the biological functions of clade A PP2C family genes remain largely uncharacterized in tuber mustard (Brassica juncea var. tumida). This study aimed to systematically characterize clade A BjuPP2C genes and explore their potential roles in abiotic stress response, stem swelling development, and resistance to Plasmodiophora brassicae. Methods: We profiled the expression patterns of clade A BjuPP2C genes under multiple abiotic stresses and P. brassicae inoculation, as well as across successive stem swelling developmental stages. Four candidate genes (BjuPP2C56_B1, BjuPP2C37_A1, BjuPP2C37_B1, and BjuPP2C24_O1) with prominent expression changes were selected for CDS cloning. Subcellular localization assays were performed to determine their intracellular distribution, and yeast two-hybrid (Y2H) analysis was used to verify physical interactions between these PP2C proteins and PYR1-Like ABA receptors (BjuPYLs). Results: Most clade A BjuPP2C genes were significantly induced by abiotic stresses and pathogen infection, and the transcript abundance of several clade A BjuPP2C members was upregulated during stem swelling development. In particular, BjuPP2C56_B1, BjuPP2C37_A1, BjuPP2C37_B1, and BjuPP2C24_O1 were markedly upregulated both at the early stage of P. brassicae infection and the initial phase of stem swelling. Subcellular localization demonstrated that BjuPP2C37_A1, BjuPP2C37_B1, and BjuPP2C24_O1 localized to nucleus and BjuPP2C56_B1 localized to nucleus and cytoplasm. Y2H results revealed that BjuPP2C56_B1, BjuPP2C37_A1, and BjuPP2C37_B1 interact with BjuPYLs in tuber mustard, whereas no interaction was detected between BjuPP2C24_O1 and BjuPYLs. Conclusions: The clade A BjuPP2C genes participate in ABA-mediated stress response, clubroot resistance, and stem swelling development in tuber mustard. Four key PP2C members differ in subcellular distribution and their capacity to bind ABA receptors, laying a foundation for further dissecting the ABA regulatory network governing stem swelling development and pathogen defense in tuber mustard.