Molecular characterization of Tetragonia tetragonoides (Pall.) aquaporin (AQP) members and their roles in the response to combined high salinity-alkalinity-drought and heat stress

Tetragonia tetragonoides (Pall.) Kuntze (Aizoaceae, 2n = 2x = 32) is a secretohalophyte with excellent tolerance to high salinity–alkalinity, drought, and heat stress. As a medicinal and edible vegetable, T. tetragonoides is widely distributed in the coastal tropics and subtropics worldwide. Aquaporin (AQP) proteins, belonging to the major intrinsic protein (MIP) superfamily, are water channel proteins that facilitate the transport of water and other substrates across cell membranes. AQPs play important roles in physiological processes, including water transport, nutrient acquisition (carbon, nitrogen, and micronutrients), and abiotic stress responses. However, knowledge of AQPs in the special habitat plant T. tetragonoides is limited. In this study, we identified 58 candidate TtAQP genes in the T. tetragonoides genome and classified them into five subfamilies based on phylogenetic analysis with 15 plasma membrane intrinsic proteins (PIPs), 18 tonoplast intrinsic proteins (TIPs), 19 NOD26-like intrinsic proteins (NIPs), 4 small basic intrinsic proteins (SIPs) and 2 uncategorized X intrinsic proteins (XIPs). Gene structure and protein conserved domain analyses showed that the majority of the deduced TtAQPs contained signature transmembrane domains, NPA motifs, the ar/R selectivity filter, and Froger’s positions, suggesting substrate specificity for these TtAQPs. Analyses of cis -acting elements (CEs) in TtAQP s’ promoter regions revealed the presence of stress-responsive and hormone responsive elements, indicating complex regulatory mechanisms for the expression of TtAQP s. TtAQP s exhibited different expression patterns among tissues and under different stress conditions based on RNA sequencing and quantitative reverse transcription PCR assays. Several TtAQP s were cloned and heterologously expressed in yeast to confirm the stress tolerance conferred by the overexpression of these genes. Our findings provide a comprehensive framework for further functional studies of TtAQP s and their potential applications in crop genetic improvement. The results also enhance our understanding of the ecological adaptability of T. tetragonoides to extremely harsh environments and offer valuable insights for developing stress-tolerant transgenic plants through genetic engineering techniques.