Cloning And Functional Analysis Of The Al Tolerance Genes MATE And ALMT In Cabbage

Low pH is a major soil constraint to agricultural production, reducing yield on almost25%of the world’s arable lands. On highly acidic soils (pH<5.0), the rhizotoxic aluminum species, A13+, is solubilized,inhibiting root growth and function. A major physiological mechanism of plant aluminum tolerance involves aluminum activation of membrane transporters that mediateOrganic acid (such as malate and citrate) release from the root apex. ALMT gene is responsible for malate exudation and MATE gene is responsible for citrate exudation. Transgenic approaches could significantly increase A1resistance and yields. Cabbage (Brassica oleracea) is one of the most important vegetable crops around the world. ALMT and MATE homologous genes BoMATE (NCBI GenBank accession no. KF031944) and BoALMT1(NCBI GenBank accession no. KF322104) are isolated from cabbage. Compared with the wild-type Arabidopsis, the transgenic lines constitutively overexpressing BoMATE and BoALMT1could enhance A1tolerance.The results are shown as follows:1. The coding region of BoMATE was1554bp in length, with the deduced protein consisting of517amino acid residues. The predicted protein sequence was57%identical to TaMATE from Triticum aestivum,51%identical to SbMATE from Sorghum bicolor, and89%identical to AtMATE from Arabidopsis thaliana. Similar to other citrate-transporting MATE proteins, BoMATE consisted of12predicted transmembrane domains and a characteristic highly conserved amino acid sequence in the loop between the second and third transmembrane domains. Phylogenetic analysis of BoMATE and other MATEs with known functions in plant species showed that BoMATE was most closely clustered with AtMATE from Arabidopsis thaliana.2. We measured the efflux of radioactively labeled citrate from cells loaded with14C-labeled citrate in BoMATE expressing cells. The14C efflux from cells expressing BoMATE showed a higher efflux activity for citrate than that observed in control cells. Electrophysiological analysis and SIET analysis ofXenopus oocytes expressing BoMATE indicated BoMATE is activated by Al.3. The expression of BoMATE was more abundant in roots than in shoots, and it was highly induced by A1treatment. BoMATE is specifically induced by elevated Al levels, but not other metals. BoMATE expression was no significant difference between different Al concentration and time of A1exposure. Citrate exudation from cabbage roots increased durations of Al exposure. Transient expression of BoMATE in onion epidermal cells demonstrated that it localized to the plasma membrane.4. Compared with the wild-type Arabidopsis, the transgenic lines constitutively overexpressing BoMATE enhanced A1tolerance and increased citrate secretion. In addition, Arabidopsis transgenic lines had a lower K+efflux and higher H+efflux, in the presence of Al, than control wild type in the distal elongation zone (DEZ).5. The coding region of BoALMT1was1497bp in length, with the deduced protein consisting of498amino acid residues. The predicted protein sequence was33%identical to TaMATE from Triticum aestivum,73%identical to AtALMT1from Arabidopsis thaliana, and99%identical to BnALMT1from Brassica napus. Similar to other malate-transporting ALMT1proteins, BoALMT1consisted of5predicted transmembrane domains. Phylogenetic analysis of BoALMT1and other ALMTls with known functions in plant species showed that BoALMT1was most closely clustered with BnALMT1from Brassica napus.6. The expression of BoALMT1was more abundant in roots than in shoots, and it was highly induced by A1treatment. BoALMT1is specifically induced by elevated A1levels, but not other metals. BoALMT1expression was no significant difference between different A1concentration and time of A1exposure. Citrate exudation from cabbage roots increased durations of A1exposure. Transient expression of BoALMT1in onion epidermal cells demonstrated that it localized to the plasma membrane.7. SIET analysis of Xenopus oocytes expressing BoALMT1indicated overexpressing BoALMT1enhanced A1tolerance. Expression of BoALMT1in transgenic Arabidopsis plants results in enhanced citrate release and A1tolerance.