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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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Determination of the Fe concentration in Δccc1 cells transformed with AtVTL1, AtVTL2 or AtVTL5.(A) Shown is the Fe concentration in yeast vacuoles isolated from the Δccc1 mutant, Δccc1 transformed with an empty plasmid (pUE), AtVTL1, AtVTL2 or AtVTL5. Error bars indicate the standard error of the mean. Bar graphs labeled with “+” are significantly different for the Δccc1 mutant (p<0.05). (B) Vacuoles were isolated from Δccc1 yeast cells transformed with His-tagged AtVTL1 (VTL1-H6) or with untagged AtVTL1 (VTL1) as a control. AtVTL1-H6 was localized by dot-blots using an anti-His antibody. The positions of the dot-blots are approximate. The Fe concentrations (nmole/µg protein) in the fraction corresponding to the dot-blot are given.
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pone-0110468-g002: Determination of the Fe concentration in Δccc1 cells transformed with AtVTL1, AtVTL2 or AtVTL5.(A) Shown is the Fe concentration in yeast vacuoles isolated from the Δccc1 mutant, Δccc1 transformed with an empty plasmid (pUE), AtVTL1, AtVTL2 or AtVTL5. Error bars indicate the standard error of the mean. Bar graphs labeled with “+” are significantly different for the Δccc1 mutant (p<0.05). (B) Vacuoles were isolated from Δccc1 yeast cells transformed with His-tagged AtVTL1 (VTL1-H6) or with untagged AtVTL1 (VTL1) as a control. AtVTL1-H6 was localized by dot-blots using an anti-His antibody. The positions of the dot-blots are approximate. The Fe concentrations (nmole/µg protein) in the fraction corresponding to the dot-blot are given.

Mentions: To test this hypothesis, the Fe concentration in isolated yeast vacuoles was investigated. Intact yeast vacuoles were isolated by floatation through a Ficoll step gradient as described by Li et al. [25]. We confirmed the composition of the vacuolar fraction by re-centrifugation the fraction on a continuous sucrose gradient followed by marker enzyme analysis (Fig. S1). As expected, the vacuole marker, bafilomycin-sensitive ATPase, co-localized with membrane proteins and no detectable plasma membrane (vanadate-sensitive ATPase) or endoplasmic reticulum (NADPH cytochrome c reductase) activities were detected in the gradient. Vacuoles, isolated from log-phase Δccc1 cells or from Δccc1 cells transformed with the control vector grown in liquid SD medium containing 1 mM FeSO4, had approximately 2 nmoles Fe per µg protein, whereas Δccc1 cells expressing AtVTL1, AtVTL2 or AtVTL5 had a greater than 3-fold higher Fe content (Fig. 2A).


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

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

Determination of the Fe concentration in Δccc1 cells transformed with AtVTL1, AtVTL2 or AtVTL5.(A) Shown is the Fe concentration in yeast vacuoles isolated from the Δccc1 mutant, Δccc1 transformed with an empty plasmid (pUE), AtVTL1, AtVTL2 or AtVTL5. Error bars indicate the standard error of the mean. Bar graphs labeled with “+” are significantly different for the Δccc1 mutant (p<0.05). (B) Vacuoles were isolated from Δccc1 yeast cells transformed with His-tagged AtVTL1 (VTL1-H6) or with untagged AtVTL1 (VTL1) as a control. AtVTL1-H6 was localized by dot-blots using an anti-His antibody. The positions of the dot-blots are approximate. The Fe concentrations (nmole/µg protein) in the fraction corresponding to the dot-blot are given.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110468-g002: Determination of the Fe concentration in Δccc1 cells transformed with AtVTL1, AtVTL2 or AtVTL5.(A) Shown is the Fe concentration in yeast vacuoles isolated from the Δccc1 mutant, Δccc1 transformed with an empty plasmid (pUE), AtVTL1, AtVTL2 or AtVTL5. Error bars indicate the standard error of the mean. Bar graphs labeled with “+” are significantly different for the Δccc1 mutant (p<0.05). (B) Vacuoles were isolated from Δccc1 yeast cells transformed with His-tagged AtVTL1 (VTL1-H6) or with untagged AtVTL1 (VTL1) as a control. AtVTL1-H6 was localized by dot-blots using an anti-His antibody. The positions of the dot-blots are approximate. The Fe concentrations (nmole/µg protein) in the fraction corresponding to the dot-blot are given.
Mentions: To test this hypothesis, the Fe concentration in isolated yeast vacuoles was investigated. Intact yeast vacuoles were isolated by floatation through a Ficoll step gradient as described by Li et al. [25]. We confirmed the composition of the vacuolar fraction by re-centrifugation the fraction on a continuous sucrose gradient followed by marker enzyme analysis (Fig. S1). As expected, the vacuole marker, bafilomycin-sensitive ATPase, co-localized with membrane proteins and no detectable plasma membrane (vanadate-sensitive ATPase) or endoplasmic reticulum (NADPH cytochrome c reductase) activities were detected in the gradient. Vacuoles, isolated from log-phase Δccc1 cells or from Δccc1 cells transformed with the control vector grown in liquid SD medium containing 1 mM FeSO4, had approximately 2 nmoles Fe per µg protein, whereas Δccc1 cells expressing AtVTL1, AtVTL2 or AtVTL5 had a greater than 3-fold higher Fe content (Fig. 2A).

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