Molecular characterization of a cytosolic malate dehydrogenase gene(GhcMDH1) from cotton

Abstract

Malate dehydrogenase(MDH) is a key enzyme that catalyzes the reversible oxidation of oxaloacetate to malate and plays an important role in the physiological processes of plant growth and development. However, cyto-solic malate dehydrogenase(cMDH), which is crucial for malate synthesis in the cytosol, still has not been extensively characterized in plants. Here, we isolated a cytosolic malate dehydrogenase gene, designated as GhcMDH1, from Gossypium hirsutum and characterized its possible molecular function in cotton fiber. The cloned cDNA of GhcMDH1 is 1520 base pairs in length, and has an open reading frame of 999 base pairs, encoding for 332 amino acid residues with an estimated molecular weight of 35580 and pI of 6.35. Sequence alignment showed that the de-duced amino acid sequence of GhcMDH1 protein shared a high similarity to other plant cMDHs. Confocal and im-munological analysis confirmed that GhcMDH1 protein was subcellularly localized to the cytosol. Quantitative real-time polymerase chain reaction(PCR) revealed that GhcMDH1 was constitutively expressed in all vegetative cotton tissues, with slightly lower levels in roots than stems and leaves. Interestingly, the transcripts of GhcMDH1 were detected in 5―25 d post anthesis(DPA) fibers and highly abundant at 15 DPA fibers. The total MDH activities and malate contents of cotton fibers were positively correlated with the fiber elongation rates, suggesting that GhcMDH1 may function in malate synthesis in fast fiber elongation. In agreement with this suspicion, the recombi-nant His-GhcMDH1 protein mainly drives the reaction towards malate generation in vitro. In conclusion, our mole-cular characterization of the GhcMDH1 gene provides valuable insights to further investigate the malate equilibrium in cotton fiber development.