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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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Complementation of the nramp3/nramp4 double mutant by over-expression of AtVTLs.The three VTL genes were over-expressed in the nramp3/nramp4 double mutant background under the control of the 35S promoter and root length was determined after 5 days of growth. Seedlings were grown without added Fe. Seedlings were grown on agarose Petri plates as described in the Materials and Methods. Asterisks indicate significant differences from the wild-type (p<0.001).
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pone-0110468-g004: Complementation of the nramp3/nramp4 double mutant by over-expression of AtVTLs.The three VTL genes were over-expressed in the nramp3/nramp4 double mutant background under the control of the 35S promoter and root length was determined after 5 days of growth. Seedlings were grown without added Fe. Seedlings were grown on agarose Petri plates as described in the Materials and Methods. Asterisks indicate significant differences from the wild-type (p<0.001).

Mentions: Over-expression of AtVTL1 in Arabidopsis did not greatly alter Fe content of shoots and roots [21], nor were changes observed in the kinetics of the response to Fe deficiency, as determined by the root plasma membrane Fe3+-chelate reductase and chlorophyll content (Figs. S3A and B). In an attempt to characterize their functions, we have ectopically expressed the AtVTL genes in the Arabidopsis nramp3/nramp4 double mutant background (17). Over-expression was confirmed by semi-quantitative RT-PCR (Fig. S4). Both NRAMP3 and NRAMP4 have been shown to be Fe vacuolar efflux carriers. The double mutant had no obvious phenotype when grown on soil; however, growth of mutant seedlings was retarded shortly after germination on an Fe-deficient substrate [17]. The phenotype was not persistent and disappeared after a few days of growth or with supplemental Fe in the media. Under Fe deficiency root growth of the nramp3/nramp4 double mutant and of the double mutant transformed with the empty vector (GL1), was greatly inhibited compared to the wild-type control at 5 d following germination (Fig. 4 and Fig. S5; p<0.001) [17]. Importantly, over-expression of AtVTL1, AtVTL2 or AtVTL5 in nramp3/nramp4 complemented this mutant growth phenotype. The early seedling root length was significantly increased (Fig. 4 and Fig. S5; p<0.001) compared to the double mutant; however, the roots were still significantly shorter than those of the wild type (p<0.001). Thus, over-expression of AtVTL1, AtVTL2 or AtVTL5 partially restored the wild-type phenotype in the nramp3/nramp4 double mutant.


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

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

Complementation of the nramp3/nramp4 double mutant by over-expression of AtVTLs.The three VTL genes were over-expressed in the nramp3/nramp4 double mutant background under the control of the 35S promoter and root length was determined after 5 days of growth. Seedlings were grown without added Fe. Seedlings were grown on agarose Petri plates as described in the Materials and Methods. Asterisks indicate significant differences from the wild-type (p<0.001).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110468-g004: Complementation of the nramp3/nramp4 double mutant by over-expression of AtVTLs.The three VTL genes were over-expressed in the nramp3/nramp4 double mutant background under the control of the 35S promoter and root length was determined after 5 days of growth. Seedlings were grown without added Fe. Seedlings were grown on agarose Petri plates as described in the Materials and Methods. Asterisks indicate significant differences from the wild-type (p<0.001).
Mentions: Over-expression of AtVTL1 in Arabidopsis did not greatly alter Fe content of shoots and roots [21], nor were changes observed in the kinetics of the response to Fe deficiency, as determined by the root plasma membrane Fe3+-chelate reductase and chlorophyll content (Figs. S3A and B). In an attempt to characterize their functions, we have ectopically expressed the AtVTL genes in the Arabidopsis nramp3/nramp4 double mutant background (17). Over-expression was confirmed by semi-quantitative RT-PCR (Fig. S4). Both NRAMP3 and NRAMP4 have been shown to be Fe vacuolar efflux carriers. The double mutant had no obvious phenotype when grown on soil; however, growth of mutant seedlings was retarded shortly after germination on an Fe-deficient substrate [17]. The phenotype was not persistent and disappeared after a few days of growth or with supplemental Fe in the media. Under Fe deficiency root growth of the nramp3/nramp4 double mutant and of the double mutant transformed with the empty vector (GL1), was greatly inhibited compared to the wild-type control at 5 d following germination (Fig. 4 and Fig. S5; p<0.001) [17]. Importantly, over-expression of AtVTL1, AtVTL2 or AtVTL5 in nramp3/nramp4 complemented this mutant growth phenotype. The early seedling root length was significantly increased (Fig. 4 and Fig. S5; p<0.001) compared to the double mutant; however, the roots were still significantly shorter than those of the wild type (p<0.001). Thus, over-expression of AtVTL1, AtVTL2 or AtVTL5 partially restored the wild-type phenotype in the nramp3/nramp4 double mutant.

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