Magnesium (Mg2+) is an essential mineral nutrient for plants and is the most abundant free divalent cation in plant cells. However, our knowledge of the role of this ion in the plant cell is limited, and the mechanisms of homeostasis and transport of the ion are almost completely unknown. A. Tutone (this laboratory) identified an Arabidopsis thaliana gene by the complementation of a Mg2+-uptake yeast mutant (CM66). This gene, referred to as AtMRS2-11, was expressed as cDNA from a strong yeast promoter and allowed the growth of the CM66 yeast strain on standard media. The conceptually translated AtMRS2-11 protein sequence was used in this study to identify nine additional proteins by sequence homology searches using the BLAST algorithm. The corresponding genes have been cloned from cDNA (A. thaliana ecotype Landsberg erecta) and sequenced. Protein sequence similarity suggests that the family forms a sub-section of the CorA super-family of Mg2+ transport proteins. The mutant yeast used to identify the family initially was also used to show that two family members in addition to AtMRS2-11 were able to complement the Mg2+-dependent growth phenotype. When fused to eGFP, these proteins showed a localisation consistent with some of the protein reaching the yeast cell membrane. The other members of the family were also fused to eGFP and showed a range of localisation patterns within the yeast cell. None of the three AtMRS2 proteins previously able to complement the yeast mutant phenotype did so when fused to eGFP. RNA transcripts from the AtMRS2 family were detected by RT-PCR in organ-scale preparations of total RNA from A. thaliana. Most family members were detected in all of the organs tested. Northern analysis of AtMRS2-11 RNA transcript level showed that the gene was more highly expressed in leaf tissue, but was not affected by decreased levels of Mg2+ in the growth media. The levels of steady state AtMRS2-11 mRNA transcript were elevated two-fold in the light during the diurnal cycle, but no change was detected during light-induced greening of etiolated seedlings. A stable transgenic A. thaliana line expressing the gusA gene from the promoter region of AtMRS2-11 was used to localise the promoter's activity to cells containing chloroplasts. The expression appeared highest in younger cells. VI The AtMRS2-11 protein was predicted to contain a chloroplast targeting peptide. Western analysis demonstrated that AtMRS2-11 was enriched in the total proteins of isolated chloroplasts as compared to extracts from whole plants. The AtMRS2-11:eGFP fusion protein was also detected in chloroplasts by fluorescence microscopy. Flame atomic absorption spectroscopy was used in conjunction with isolated chloroplasts to try to determine the effects of the overaccumulation of the AtMRS2-11 protein in a transgenic A. thaliana plant line (constructed by A. Tutone). A rapid uptake or binding of Mg2+ was seen in chloroplasts isolated from both wild type and transgenic lines, but no differences were observed in either the rate of Mg2+ uptake/binding or the final Mg2+ content.
| Identifer | oai:union.ndltd.org:ADTP/274373 |
| Date | January 2004 |
| Creators | Drummond, Revel Scott MacGregor |
| Publisher | ResearchSpace@Auckland |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated., http://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm, Copyright: The author |
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