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Functional significance of AtHMA4 C-terminal domain in planta.

Mills RF, Valdes B, Duke M, Peaston KA, Lahner B, Salt DE, Williams LE - PLoS ONE (2010)

Bottom Line: When the AtHMA4 C-terminal domain (AtHMA4-C-term) was expressed in hma2 hma4 it had no marked effect.When expressed in yeast, AtHMA4-C-term and AtHMA4-trunc conferred greater Cd and Zn tolerance than AtHMA4-FL.AtHMA4-FL is more effective in restoring shoot metal accumulation in this mutant than a C-terminally truncated version of the pump, indicating that the C-terminal domain is important in the functioning of AtHMA4 in planta.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Southampton, Southampton, Hampshire, United Kingdom.

ABSTRACT

Background: Enhancing the upward translocation of heavy metals such as Zn from root to shoot through genetic engineering has potential for biofortification and phytoremediation. This study examined the contribution of the heavy metal-transporting ATPase, AtHMA4, to the shoot ionomic profile of soil-grown plants, and investigated the importance of the C-terminal domain in the functioning of this transporter.

Principal findings: The Arabidopsis hma2 hma4 mutant has a stunted phenotype and a distinctive ionomic profile, with low shoot levels of Zn, Cd, Co, K and Rb, and high shoot Cu. Expression of AtHMA4 (AtHMA4-FL) under the CaMV-35S promoter partially rescued the stunted phenotype of hma2 hma4; rosette diameter returned to wild-type levels in the majority of lines and bolts were also produced, although the average bolt height was not restored completely. AtHMA4-FL expression rescued Co, K, Rb and Cu to wild-type levels, and partially returned Cd and Zn levels (83% and 28% of wild type respectively). In contrast, expression of AtHMA4-trunc (without the C-terminal region) in hma2 hma4 only partially restored the rosette diameter in two of five lines and bolt production was not rescued. There was no significant effect on the shoot ionomic profile, apart from Cd, which was increased to 41% of wild-type levels. When the AtHMA4 C-terminal domain (AtHMA4-C-term) was expressed in hma2 hma4 it had no marked effect. When expressed in yeast, AtHMA4-C-term and AtHMA4-trunc conferred greater Cd and Zn tolerance than AtHMA4-FL.

Conclusion: The ionome of the hma2 hma4 mutant differs markedly from wt plants. The functional relevance of domains of AtHMA4 in planta can be explored by complementing this mutant. AtHMA4-FL is more effective in restoring shoot metal accumulation in this mutant than a C-terminally truncated version of the pump, indicating that the C-terminal domain is important in the functioning of AtHMA4 in planta.

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Direct comparison of Zn and Cd tolerance conferred by AtHMA4 and truncated versions in yeast.AtHMA4-FL, AtHMA4-trunc and AtHMA4-C-term were expressed in zrc1 cot1 yeast mutant (A), wt yeast (BY4741) (B) or ycf1 mutant (C). Growth of yeast expressing these AtHMA4 versions were compared to vector (p426)-transformed controls and to either of two transport  mutants: Athma4(D401A) or AtHMA4(C357G). Plates contained SC minus uracil with 2% (w/v) galactose pH 5-5.5 and varying concentrations of Cd as CdSO4 or Zn as ZnSO4.
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pone-0013388-g001: Direct comparison of Zn and Cd tolerance conferred by AtHMA4 and truncated versions in yeast.AtHMA4-FL, AtHMA4-trunc and AtHMA4-C-term were expressed in zrc1 cot1 yeast mutant (A), wt yeast (BY4741) (B) or ycf1 mutant (C). Growth of yeast expressing these AtHMA4 versions were compared to vector (p426)-transformed controls and to either of two transport mutants: Athma4(D401A) or AtHMA4(C357G). Plates contained SC minus uracil with 2% (w/v) galactose pH 5-5.5 and varying concentrations of Cd as CdSO4 or Zn as ZnSO4.

Mentions: AtHMA4-FL, AtHMA4-trunc and AtHMA4-C-term were expressed in various yeast strains to determine their relative effectiveness in conferring Zn and Cd tolerance. We also included either of two transport mutants: Athma4(D401A), mutated in the conserved aspartate phosphorylated during the reaction cycle of all P-type ATPases or Athma4(C357G), mutated in the conserved CPC motif [11]. In these experiments, yeast were grown at pH 5–5.5 on a minimal medium with galactose to induce expression. Consistent with previous studies, full-length AtHMA4 confers Zn tolerance to the Zn-sensitive zrc1 cot1 mutant yeast when grown under these conditions (Figure 1A) and deletion of the C-terminal 459 amino acids results in greater Zn tolerance [11]. No tolerance is observed when the transport Athma4(D401A) mutant is expressed (Figure 1A). Here we show that expression of the 473 amino acid C-terminus alone (AtHMA4-C-term) conferred greater Zn tolerance to zrc1 cot1 yeast than AtHMA4-FL, although the tolerance was not as great as that conferred by AtHMA4-trunc (Figure 1A). Also demonstrated here are the relative abilities of the AtHMA4 variants in conferring Cd tolerance (Figure 1B and 1C). AtHMA4-FL expressed in wild-type yeast, confers Cd tolerance while the Athma4(C357G) mutant does not. AtHMA4-trunc conferred greater Cd tolerance than AtHMA4-FL whereas AtHMA4-C-term confers the greatest Cd tolerance (Figure 1B). In the ycf1 mutant, as shown previously expression of AtHMA4-FL confers Cd tolerance [11], [20] and in this study (Figure 1C) we show that the truncated version confers greater tolerance. The difference between the C-term and truncated versions is not as clear but the AtHMA4-C-term appears slightly more tolerant at the highest concentration tested.


Functional significance of AtHMA4 C-terminal domain in planta.

Mills RF, Valdes B, Duke M, Peaston KA, Lahner B, Salt DE, Williams LE - PLoS ONE (2010)

Direct comparison of Zn and Cd tolerance conferred by AtHMA4 and truncated versions in yeast.AtHMA4-FL, AtHMA4-trunc and AtHMA4-C-term were expressed in zrc1 cot1 yeast mutant (A), wt yeast (BY4741) (B) or ycf1 mutant (C). Growth of yeast expressing these AtHMA4 versions were compared to vector (p426)-transformed controls and to either of two transport  mutants: Athma4(D401A) or AtHMA4(C357G). Plates contained SC minus uracil with 2% (w/v) galactose pH 5-5.5 and varying concentrations of Cd as CdSO4 or Zn as ZnSO4.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958113&req=5

pone-0013388-g001: Direct comparison of Zn and Cd tolerance conferred by AtHMA4 and truncated versions in yeast.AtHMA4-FL, AtHMA4-trunc and AtHMA4-C-term were expressed in zrc1 cot1 yeast mutant (A), wt yeast (BY4741) (B) or ycf1 mutant (C). Growth of yeast expressing these AtHMA4 versions were compared to vector (p426)-transformed controls and to either of two transport mutants: Athma4(D401A) or AtHMA4(C357G). Plates contained SC minus uracil with 2% (w/v) galactose pH 5-5.5 and varying concentrations of Cd as CdSO4 or Zn as ZnSO4.
Mentions: AtHMA4-FL, AtHMA4-trunc and AtHMA4-C-term were expressed in various yeast strains to determine their relative effectiveness in conferring Zn and Cd tolerance. We also included either of two transport mutants: Athma4(D401A), mutated in the conserved aspartate phosphorylated during the reaction cycle of all P-type ATPases or Athma4(C357G), mutated in the conserved CPC motif [11]. In these experiments, yeast were grown at pH 5–5.5 on a minimal medium with galactose to induce expression. Consistent with previous studies, full-length AtHMA4 confers Zn tolerance to the Zn-sensitive zrc1 cot1 mutant yeast when grown under these conditions (Figure 1A) and deletion of the C-terminal 459 amino acids results in greater Zn tolerance [11]. No tolerance is observed when the transport Athma4(D401A) mutant is expressed (Figure 1A). Here we show that expression of the 473 amino acid C-terminus alone (AtHMA4-C-term) conferred greater Zn tolerance to zrc1 cot1 yeast than AtHMA4-FL, although the tolerance was not as great as that conferred by AtHMA4-trunc (Figure 1A). Also demonstrated here are the relative abilities of the AtHMA4 variants in conferring Cd tolerance (Figure 1B and 1C). AtHMA4-FL expressed in wild-type yeast, confers Cd tolerance while the Athma4(C357G) mutant does not. AtHMA4-trunc conferred greater Cd tolerance than AtHMA4-FL whereas AtHMA4-C-term confers the greatest Cd tolerance (Figure 1B). In the ycf1 mutant, as shown previously expression of AtHMA4-FL confers Cd tolerance [11], [20] and in this study (Figure 1C) we show that the truncated version confers greater tolerance. The difference between the C-term and truncated versions is not as clear but the AtHMA4-C-term appears slightly more tolerant at the highest concentration tested.

Bottom Line: When the AtHMA4 C-terminal domain (AtHMA4-C-term) was expressed in hma2 hma4 it had no marked effect.When expressed in yeast, AtHMA4-C-term and AtHMA4-trunc conferred greater Cd and Zn tolerance than AtHMA4-FL.AtHMA4-FL is more effective in restoring shoot metal accumulation in this mutant than a C-terminally truncated version of the pump, indicating that the C-terminal domain is important in the functioning of AtHMA4 in planta.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Southampton, Southampton, Hampshire, United Kingdom.

ABSTRACT

Background: Enhancing the upward translocation of heavy metals such as Zn from root to shoot through genetic engineering has potential for biofortification and phytoremediation. This study examined the contribution of the heavy metal-transporting ATPase, AtHMA4, to the shoot ionomic profile of soil-grown plants, and investigated the importance of the C-terminal domain in the functioning of this transporter.

Principal findings: The Arabidopsis hma2 hma4 mutant has a stunted phenotype and a distinctive ionomic profile, with low shoot levels of Zn, Cd, Co, K and Rb, and high shoot Cu. Expression of AtHMA4 (AtHMA4-FL) under the CaMV-35S promoter partially rescued the stunted phenotype of hma2 hma4; rosette diameter returned to wild-type levels in the majority of lines and bolts were also produced, although the average bolt height was not restored completely. AtHMA4-FL expression rescued Co, K, Rb and Cu to wild-type levels, and partially returned Cd and Zn levels (83% and 28% of wild type respectively). In contrast, expression of AtHMA4-trunc (without the C-terminal region) in hma2 hma4 only partially restored the rosette diameter in two of five lines and bolt production was not rescued. There was no significant effect on the shoot ionomic profile, apart from Cd, which was increased to 41% of wild-type levels. When the AtHMA4 C-terminal domain (AtHMA4-C-term) was expressed in hma2 hma4 it had no marked effect. When expressed in yeast, AtHMA4-C-term and AtHMA4-trunc conferred greater Cd and Zn tolerance than AtHMA4-FL.

Conclusion: The ionome of the hma2 hma4 mutant differs markedly from wt plants. The functional relevance of domains of AtHMA4 in planta can be explored by complementing this mutant. AtHMA4-FL is more effective in restoring shoot metal accumulation in this mutant than a C-terminally truncated version of the pump, indicating that the C-terminal domain is important in the functioning of AtHMA4 in planta.

Show MeSH