Cloning and characterisation of a magnesium ion transport gene from Arabidopsis thaliana

Abstract

Magnesium (Mg2+) is an essential cellular constituent, which plays a prominent role in photosynthesis and plant metabolism. However, Mg2+ uptake, transport and homeostasis in eukaryotes are poorly understood at both the physiological and the molecular level. The overall aims of this project were to isolate Mg2+ transport genes from Arabidopsis thaliana by complementation of a yeast strain lacking Mg transport genes (ALR1 and ALR2) and to use heterologous expression systems to gain insight into the biochemical, electrophysiological and biological roles of the proteins involved. An Arabidopsis gene (AtMRS2-ll) was identified as an effective suppressor of the air lair 2 mutant phenotype. AtMRS2-ll encodes a protein that contains structural features similar to bacterial (CorA) and yeast (ALR1 and ALR2) Mg2+ transport genes, including possession of an absolutely conserved GMN motif (Li L., Tutone A. F., Drummond R. S .M., Gardner R. C., and Luan S. 2001. Plant Cell 13, 2761-2755). It is most similar to the yeast MRS2 gene, recently identified as encoding a Mg2+ uptake system in yeast mitochondria. The MRS2 family in Arabidopsis has at least 10 members, most of which are expressed in a range of plant tissues. One member of this family, AtMRS2-10 functionally complemented a bacterial mutant lacking Mg2+ transport capability (Li et al., 2001, ibid.). A second member, AtMRS2-l, complemented the yeast mrs2 mutant lacking mitochondrial Mg2+transport capability (Schock et al., 2000). These results, together with the ability of AtMRS2-l 1 to complement the yeast alrlalr2 mutant, suggest that the AtMRS2-l 1 family plays a role in the Mg2+ transport in plants. The ability of AtMRS2-ll to suppress the growth defect of the air lair 2 mutant strain suggested that this gene might encode a plant Mg2+ transport system. Direct evidence for this hypothesis came from measurement of yeast cellular Mg content over a 60-min period. Results demonstrated that the air lair2 yeast mutant overexpressing AtMRS2-ll increased the i cellular Mg content of starved cells four-fold during the uptake period. Western analysis with AtMRS2-ll antibody showed that AtMRS2-ll co-purified with the plasma membrane suggesting that the protein is located at the yeast plasma membrane. These results are consistent with a direct involvement of AtMRS2-l 1 in the influx of Mg2+ ions into yeast cells. Overexpression of AtMRS2-ll in yeast alters toxicity to a range of metal ions similar to those transported by other members of the corA family. These results suggest that AtMRS2-ll is capable of transporting other divalent cations in addition to Mg2+. AAS measurement also showed that Al3+ is a potent inhibitor of Mg2+ transport by AtMRS2-11. Xenopus oocytes were used to characterise the transport properties of AtMRS2-ll protein in detail. Conditions for the analysis of AtMRS2-ll in this system were first established and protocol was confirmed with the functional expression of a control transport gene. However, analysis of oocytes injected with AtMRS2-ll transcript identified Mg2+-dependent current in only one batch of oocytes. In the vast majority of cases, "death" of Xenopus oocytes injected with AtMRS2-l 1 was observed. While several explanations could account for the results, this work supports the hypothesis that the oocytes death and the significant inward current identified were both due to expression of AtMRS2-l 1. To evaluate the physiological roles of the AtMRS2-ll gene in plants, transgenic Arabidopsis lines were generated that were homozygous for a single copy of the T-DNA and stably express the sense-oriented AtMRS2-ll cDNA under the control CaMV 35S promoter. The homozygous transgenic lines showed increased expression of the AtMRS2-ll transcript although the level of expression varied across the transgenic lines. However, no phenotypicI change in plant growth or development was detected and no differences were seen in Mg content, the response of the plants to extremes of Mg concentration or sensitivity to a range of metal cations. More carefully designed experiments focusing on Mg2+ uptake and transport may be required to detect the effects of this gene. In conclusion, the identification of a plant gene family that transports Mg2+ will help develop our understanding of molecular mechanisms underlying Mg uptake, transport and homeostasis in higher plants. The work described in this thesis provides a starting point for understanding the biochemical and biological function of the family of MRS2 genes in plants.