Not just a cycle: mitochondrial CsgabD is involved in GABA metabolism during citrus defense against biotic stress

While the role of succinic semialdehyde (SSA) dehydrogenase (SSADH; also known as gabD) is well-reported from model plants, the lack of functionality and structure of SSADH from citrus represents a significant knowledge gap. Herein, genome-wide analyses identified 17 high-confidence SSADH-like proteins from Citrus sinensis, among which three putative SSADHs have potential GABA dehydrogenase function. Sequence alignment, phylogenetic analyses, and domain architecture demonstrated high conservation among CsSSADHs (aka CsgabD) and their homologs across diverse plant taxa. Notably, CsSSADH-2 lacked a conserved QGIVC motif found in CsSSADH-1/-3. Secondary structure analyses indicated conserved aldehyde dehydrogenase domains. Homology-based 3D modeling predicted CsSSADH-1 and 2 as homo-tetramers; however, AlphaFold2-based modeling suggested their full-length monomer structures. PPI networks revealed CsSSADH-1 interacts with 10 proteins, primarily involved in GABA/succinate metabolism and the TCA cycle. Docking studies indicated that CsSSADH-1 displayed acceptable affinity and binding modes with GABA, SSA, and succinate. GABA supplementation enhances CsSSADH expression, GABA, and succinate content in a dose-dependent manner in both healthy and infected citrus plants under greenhouse conditions. CsSSADH was involved in citrus responses to 'Candidatus Liberibacter asiaticus' and/or its vector, Diaphorina citri. Nevertheless, GABA accumulation under biotic stress leads to condition-specific rerouting of GABA metabolism. Chemical inhibition of CsSSADH resulted in increased GABA accumulation but reduced succinate levels in both healthy and infected plants. This study offers the first comprehensive characterization of C. sinensis SSADH isoforms, providing insights into their evolutionary divergence, structural features, and potential functions, and enhancing our understanding of their possible roles in GABA metabolism and citrus defense responses.