Molecular cloning and functional characterization of mitochondrial malate dehydrogenase (mMDH) is involved in exogenous GABA increasing root hypoxia tolerance in muskmelon plants

Abstract

The gamma-aminobutyrate (GABA) shunt, which is closely associated with tricarboxylic acid (TCA) cycle anaplerosis in mitochondria, is important for increasing plant tolerance to environmental stress. Proteomic studies have demonstrated that mitochondrial malate dehydrogenase (mMDH) is involved in the GABA-mediated alleviation of root hypoxia-induced injury; however, the regulatory mechanisms of mMDH in plant cells remain largely unknown. In this study, a full-length 1422-bp sequence, designated CmmMDH and submitted to the GenBank database (accession number KU934189), was cloned from muskmelon plants. Sequence comparisons indicated that CmmMDH shares high similarity with other plant mMDHs, the highest being with watermelon mMDH. Hypoxic stress induced a significant increase in CmmMDH gene expression and in the roots, stems and leaves, MDH protein content and activity initially significantly increased but then decreased, which may be attributed to the stress response under hypoxic conditions. Exogenous GABA treatment strongly promoted CmmMDH gene expression, protein content and MDH activity under both normoxic and hypoxic conditions, but this effect was more obvious under the latter. Furthermore, to compensate for the energy deficiency caused by hypoxia stress, the contents of key TCA cycle metabolites significantly decreased, although some increases occurred during the initial period, including increased succinic acid, fumaric acid, malic acid, oxaloacetic acid (OAA) and citric acid levels. However, GABA supplementation significantly increased the contents of five key metabolites in the TCA cycle (especially succinic acid, malic acid and OAA), and this effect was more prominent under normoxic conditions than under hypoxic conditions. This study provides strong evidence that CmmMDH may play an important role in GABA-induced tolerance to root hypoxia due to increasing TCA cycle intermediates through a GABA shunt anaplerotic role, which is related to MDH gene expression, protein content and activity in the roots and leaves of muskmelon seedlings.