Cloning and functional characterization of a citrate synthase gene of Rhododendron micranthum Turcz

Abstract

Citrate synthase (CS) is a rate-limiting enzyme in tricarboxylic acid (TCA) cycle, which is related to numerous plant physiological events, like mitochondrial energy metabolism and stress resistance, and has a key function in aluminum (Al) stress response. Rhododendron micranthum Turcz. has been used as ornamental plants worldwide. This species prefers acidic soils, and thus its growth and ornamental quality are often limited by Al toxicity. In the present work, the full-length cDNA encoding a CS (here designated RmCS) was isolated and characterized. The RmCS contained the conserved CS domain and a N-terminal PWPN-box. RmCS protein was predicted to be localized to the cytoplasm and mitochondria. A neighbor-joining tree indicated that RmCS was similar to CS proteins of Actinidia chinensis var. chinensis and Vitis riparia the most. RmCS was highly expressed in stems; particularly, it shows predominant expression in flowers in the first flowering period, whereas in leaves peak expression levels were detected in September. Southern blotting analysis demonstrated that one to three copies of RmCS were successfully integrated in the genome of transgenic tobacco. Overexpression of RmCS significantly increased salt, alkali, drought, and Al tolerance, but reduced the plant height and leaf area, of transgenic tobacco. Roots of RmCS-overexpressing tobacco plants secreted higher concentrations of malic acid, citric acid, and tartaric acid. Decreased malonaldehyde while elevated proline contents in the leaf suggested that the improved abiotic stress tolerance conferred by RmCS was associated with decreased membrane damage and improved osmotic adjustment. The above findings indicated the potentially vital function of RmCS in abiotic stress responses of R. micranthum, and lays certain theoretical foundation for improving stress resistance of R. micranthum.