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Vacuolar-Iron-Transporter1-Like proteins mediate iron homeostasis in Arabidopsis.

Gollhofer J, Timofeev R, Lan P, Schmidt W, Buckhout TJ - PLoS ONE (2014)

Bottom Line: Isolated vacuoles from yeast expressing either of the VTL genes in the Δccc1 background had a three- to four-fold increase in Fe concentration compared to vacuoles isolated from the untransformed mutant.Seedling root growth of the Arabidopsis nramp3/nramp4 and vit1-1 mutants was decreased compared to the wild type when seedlings were grown under Fe deficiency.We conclude that the VTL proteins catalyze Fe transport into vacuoles and thus contribute to the regulation of Fe homeostasis in planta.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biology, Humboldt University Berlin, Berlin, Germany.

ABSTRACT
Iron deficiency is a nutritional problem in plants and reduces crop productivity, quality and yield. With the goal of improving the iron (Fe) storage properties of plants, we have investigated the function of three Arabidopsis proteins with homology to Vacuolar Iron Transporter1 (AtVIT1). Heterologous expression of Vacuolar Iron Transporter-Like1 (AtVTL1; At1g21140), AtVTL2 (At1g76800) or AtVTL5 (At3g25190) in the yeast vacuolar Fe transport mutant, Δccc1, restored growth in the presence of 4 mM Fe. Isolated vacuoles from yeast expressing either of the VTL genes in the Δccc1 background had a three- to four-fold increase in Fe concentration compared to vacuoles isolated from the untransformed mutant. Transiently expressed GFP-tagged AtVTL1 was localized exclusively and AtVTL2 was localized primarily to the vacuolar membrane of onion epidermis cells. Seedling root growth of the Arabidopsis nramp3/nramp4 and vit1-1 mutants was decreased compared to the wild type when seedlings were grown under Fe deficiency. When expressed under the 35S promoter in the nramp3/nramp4 or vit1-1 backgrounds, AtVTL1, AtVTL2 or AtVTL5 restored root growth in both mutants. The seed Fe concentration in the nramp3/nramp4 mutant overexpressing AtVTL1, AtVTL2 or AtVTL5 was between 50 and 60% higher than in non-transformed double mutants or wild-type plants. We conclude that the VTL proteins catalyze Fe transport into vacuoles and thus contribute to the regulation of Fe homeostasis in planta.

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RT-qPCR of AtVTL1, AtVTL2 and AtVTL5 in response to Fe and Zn supply.Seedlings were grown on agarose petri plates as described in the Materials and Methods. Growth conditions were: ES medium (ES); “0”, ES without Fe or Zn or at the concentrations indicated. RT-qPCR was standardized to the expression of the ACTIN2. Symbols indicate significant differences to expression in the ES medium in three biological replicates: **, p<0.001; *, p<0.01; and +, p<0.05.
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pone-0110468-g007: RT-qPCR of AtVTL1, AtVTL2 and AtVTL5 in response to Fe and Zn supply.Seedlings were grown on agarose petri plates as described in the Materials and Methods. Growth conditions were: ES medium (ES); “0”, ES without Fe or Zn or at the concentrations indicated. RT-qPCR was standardized to the expression of the ACTIN2. Symbols indicate significant differences to expression in the ES medium in three biological replicates: **, p<0.001; *, p<0.01; and +, p<0.05.

Mentions: Finally, we investigated the response of AtVTL1, AtVTL2 and AtVTL5 to nutrient supply using quantitative qRT-PCR. Previously, we reported that the transcriptional activity of these genes positively correlated with the Fe supply [20]. Quantitative RT-PCR analyses confirmed these results (Fig. 7). Compared to Fe-sufficient controls (40 µM Fe), the transcript abundance of AtVTL1, AtVTL2 and AtVTL5 was decreased under Fe deficiency and increased in plants grown on media containing 120 µM Fe. Similarly, transcript abundance also decreased under Zn deficiency for AtVTL1, AtVTL2 and AtVTL5 (Fig. 7). However, when the Zn concentration was increased to 5 µM, the transcript abundance for AtVTL1, AtVTL2 and AtVTL5 was not significantly different from that of control plants grown on standard media (Fig. 7). The VTL genes were unable to restore growth in the Zn sensitive Δzrc1 mutant (Fig. S7). Thus, a function of AtVTL1, AtVTL2 or AtVTL5 in vacuolar Zn transport is unlikely. Since AtVTL1, AtVTL2 and AtVTL5 responded positively to Fe supply and since their over-expression complemented vacuolar Fe transport mutations in yeast and Arabidopsis mutants, we conclude that VTL genes encode protein involved in Fe homeostasis, presumably as Fe vacuolar transporters.


Vacuolar-Iron-Transporter1-Like proteins mediate iron homeostasis in Arabidopsis.

Gollhofer J, Timofeev R, Lan P, Schmidt W, Buckhout TJ - PLoS ONE (2014)

RT-qPCR of AtVTL1, AtVTL2 and AtVTL5 in response to Fe and Zn supply.Seedlings were grown on agarose petri plates as described in the Materials and Methods. Growth conditions were: ES medium (ES); “0”, ES without Fe or Zn or at the concentrations indicated. RT-qPCR was standardized to the expression of the ACTIN2. Symbols indicate significant differences to expression in the ES medium in three biological replicates: **, p<0.001; *, p<0.01; and +, p<0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215979&req=5

pone-0110468-g007: RT-qPCR of AtVTL1, AtVTL2 and AtVTL5 in response to Fe and Zn supply.Seedlings were grown on agarose petri plates as described in the Materials and Methods. Growth conditions were: ES medium (ES); “0”, ES without Fe or Zn or at the concentrations indicated. RT-qPCR was standardized to the expression of the ACTIN2. Symbols indicate significant differences to expression in the ES medium in three biological replicates: **, p<0.001; *, p<0.01; and +, p<0.05.
Mentions: Finally, we investigated the response of AtVTL1, AtVTL2 and AtVTL5 to nutrient supply using quantitative qRT-PCR. Previously, we reported that the transcriptional activity of these genes positively correlated with the Fe supply [20]. Quantitative RT-PCR analyses confirmed these results (Fig. 7). Compared to Fe-sufficient controls (40 µM Fe), the transcript abundance of AtVTL1, AtVTL2 and AtVTL5 was decreased under Fe deficiency and increased in plants grown on media containing 120 µM Fe. Similarly, transcript abundance also decreased under Zn deficiency for AtVTL1, AtVTL2 and AtVTL5 (Fig. 7). However, when the Zn concentration was increased to 5 µM, the transcript abundance for AtVTL1, AtVTL2 and AtVTL5 was not significantly different from that of control plants grown on standard media (Fig. 7). The VTL genes were unable to restore growth in the Zn sensitive Δzrc1 mutant (Fig. S7). Thus, a function of AtVTL1, AtVTL2 or AtVTL5 in vacuolar Zn transport is unlikely. Since AtVTL1, AtVTL2 and AtVTL5 responded positively to Fe supply and since their over-expression complemented vacuolar Fe transport mutations in yeast and Arabidopsis mutants, we conclude that VTL genes encode protein involved in Fe homeostasis, presumably as Fe vacuolar transporters.

Bottom Line: Isolated vacuoles from yeast expressing either of the VTL genes in the Δccc1 background had a three- to four-fold increase in Fe concentration compared to vacuoles isolated from the untransformed mutant.Seedling root growth of the Arabidopsis nramp3/nramp4 and vit1-1 mutants was decreased compared to the wild type when seedlings were grown under Fe deficiency.We conclude that the VTL proteins catalyze Fe transport into vacuoles and thus contribute to the regulation of Fe homeostasis in planta.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biology, Humboldt University Berlin, Berlin, Germany.

ABSTRACT
Iron deficiency is a nutritional problem in plants and reduces crop productivity, quality and yield. With the goal of improving the iron (Fe) storage properties of plants, we have investigated the function of three Arabidopsis proteins with homology to Vacuolar Iron Transporter1 (AtVIT1). Heterologous expression of Vacuolar Iron Transporter-Like1 (AtVTL1; At1g21140), AtVTL2 (At1g76800) or AtVTL5 (At3g25190) in the yeast vacuolar Fe transport mutant, Δccc1, restored growth in the presence of 4 mM Fe. Isolated vacuoles from yeast expressing either of the VTL genes in the Δccc1 background had a three- to four-fold increase in Fe concentration compared to vacuoles isolated from the untransformed mutant. Transiently expressed GFP-tagged AtVTL1 was localized exclusively and AtVTL2 was localized primarily to the vacuolar membrane of onion epidermis cells. Seedling root growth of the Arabidopsis nramp3/nramp4 and vit1-1 mutants was decreased compared to the wild type when seedlings were grown under Fe deficiency. When expressed under the 35S promoter in the nramp3/nramp4 or vit1-1 backgrounds, AtVTL1, AtVTL2 or AtVTL5 restored root growth in both mutants. The seed Fe concentration in the nramp3/nramp4 mutant overexpressing AtVTL1, AtVTL2 or AtVTL5 was between 50 and 60% higher than in non-transformed double mutants or wild-type plants. We conclude that the VTL proteins catalyze Fe transport into vacuoles and thus contribute to the regulation of Fe homeostasis in planta.

Show MeSH
Related in: MedlinePlus