Functional Characterization of Rubisco Activase Genes in <em>Kandelia candel</em> Under the Stress of Flooding and Salinity

Rubisco activase (RCA) is an ATP-dependent enzyme that plays a crucial role in plant stress responses by regulating the catalytic activity of Rubisco. However, the alternative splicing and functional characteristics of the RCA gene exhibit notable species-specific diversity. The variable splice forms and functions of the RCA gene in mangrove plants remain poorly understood. We herein cloned the RCA genes in the leaves of mangrove plant Kandelia candel (L.) in response to combined flooding and salinity stress, and performed systematic expression analysis and functional validation. Our results demonstrated that the RCA gene undergoes alternative splicing to produce two isoforms, designated as KcRCAl (GenBank accession: MG492021) and KcRCAs (GenBank accession: MG492022), respectively. The KcRCAl encodes a 440-amino acid protein (42.49 kDa) belonging to the β-isoforms, while KcRCAs encodes a 474-amino acid protein (46.10 kDa) classified as the α-isoforms. Moreover, protein structure analysis revealed that both isoforms contain phosphorylation and lysine acetylation modification sites. Phylogenetic analysis indicated that KcRCA shares the closest evolutionary relationship with RCA from Cicer arietinum (chickpea) and Durio zibethinus (durian). Furthermore, RT-qPCR analysis revealed that the expression levels of KcRCAl and KcRCAs were significantly upregulated in K. Candel leaves under the combined stress condition. The following functional validation studies in transgenic Arabidopsis demonstrated that overexpression of the KcRCA genes enhances the plant&#039;s tolerance to resist flooding and salinity stress while improving antioxidant capacity, increased RCA and Rubisco activities, thereby maintaining photosynthetic efficiency under combined flooding and salinity stress. Our study not only provides new experimental evidence for understanding the molecular mechanisms of plant flooding and salinity stress, but also offers theoretical foundations for breeding flooding-and salinity-tolerant crops.