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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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Comparison of wt and mutant plants grown on soil.Plants were grown for 42 days on soil under identical conditions in a controlled-environment growth room (22°C 16 h light, 20°C 8 h dark cycle). Arabidopsis thaliana (Columbia) wt, hma2-4 (SALK_034393), hma4-2 (SALK_050924) and the double hma2-4 hma4-2 mutant are shown.
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pone-0013388-g003: Comparison of wt and mutant plants grown on soil.Plants were grown for 42 days on soil under identical conditions in a controlled-environment growth room (22°C 16 h light, 20°C 8 h dark cycle). Arabidopsis thaliana (Columbia) wt, hma2-4 (SALK_034393), hma4-2 (SALK_050924) and the double hma2-4 hma4-2 mutant are shown.

Mentions: Under the soil and growth conditions used in this study neither the hma2-4 nor hma4-2 single mutants showed any distinct vegetative growth phenotype, however the hma2-4 hma4-2 double mutant was significantly stunted compared to wild-type (Figure 3). This mutant occasionally produced a bolt over the time frame of these experiments (45 days) but it was always very short. In contrast, at this stage wild type plants had bolted and produced flowers, siliques and seeds (Figure 3). After 60 days growth on soil, a small proportion of hma2 hma4 mutants had produced several small bolts and some flowers, but siliques were not produced and thus no seed could be obtained. This was similar to the phenotype already reported for hma2 hma4 in the Ws background [12] except that we did not observe that the plants in our study were chlorotic under our soil conditions as has been observed previously.


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)

Comparison of wt and mutant plants grown on soil.Plants were grown for 42 days on soil under identical conditions in a controlled-environment growth room (22°C 16 h light, 20°C 8 h dark cycle). Arabidopsis thaliana (Columbia) wt, hma2-4 (SALK_034393), hma4-2 (SALK_050924) and the double hma2-4 hma4-2 mutant are shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958113&req=5

pone-0013388-g003: Comparison of wt and mutant plants grown on soil.Plants were grown for 42 days on soil under identical conditions in a controlled-environment growth room (22°C 16 h light, 20°C 8 h dark cycle). Arabidopsis thaliana (Columbia) wt, hma2-4 (SALK_034393), hma4-2 (SALK_050924) and the double hma2-4 hma4-2 mutant are shown.
Mentions: Under the soil and growth conditions used in this study neither the hma2-4 nor hma4-2 single mutants showed any distinct vegetative growth phenotype, however the hma2-4 hma4-2 double mutant was significantly stunted compared to wild-type (Figure 3). This mutant occasionally produced a bolt over the time frame of these experiments (45 days) but it was always very short. In contrast, at this stage wild type plants had bolted and produced flowers, siliques and seeds (Figure 3). After 60 days growth on soil, a small proportion of hma2 hma4 mutants had produced several small bolts and some flowers, but siliques were not produced and thus no seed could be obtained. This was similar to the phenotype already reported for hma2 hma4 in the Ws background [12] except that we did not observe that the plants in our study were chlorotic under our soil conditions as has been observed previously.

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