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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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Arabidopsis hma2 hma4 plants are expressing AtHMA4-FL, AtHMA4-trunc or AtHMA4-C-term.Semi-quantitative RT-PCR shows expression of AtHMA4 versions in 5 independent transformant lines for each construct in the Arabidopsis hma2 hma4 mutant. A, RT-PCR for lines expressing AtHMA4 FL (plants 1–5) or AtHMA4-trunc (plants 1–5) using primers that detect a region before the C-terminus.. B, RT-PCR for lines expressing AtHMA4 FL (plants 1–5) or AtHMA4-C-term (plants 1–5) using primers that detect a region within the C-terminus. Wild type plant (wt) and hma2 hma4 mutant are shown as well as hma2 hma4 mutant expressing vector alone (hma2 hma4 V). Actin was used as a control.
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pone-0013388-g002: Arabidopsis hma2 hma4 plants are expressing AtHMA4-FL, AtHMA4-trunc or AtHMA4-C-term.Semi-quantitative RT-PCR shows expression of AtHMA4 versions in 5 independent transformant lines for each construct in the Arabidopsis hma2 hma4 mutant. A, RT-PCR for lines expressing AtHMA4 FL (plants 1–5) or AtHMA4-trunc (plants 1–5) using primers that detect a region before the C-terminus.. B, RT-PCR for lines expressing AtHMA4 FL (plants 1–5) or AtHMA4-C-term (plants 1–5) using primers that detect a region within the C-terminus. Wild type plant (wt) and hma2 hma4 mutant are shown as well as hma2 hma4 mutant expressing vector alone (hma2 hma4 V). Actin was used as a control.

Mentions: The hma2 hma4 double knockout mutant was transformed with AtHMA4-FL, AtHMA4-trunc or AtHMA4-C-term expressed under control of the 35S promoter. We used RT-PCR to confirm disruption of the endogenous genes, and expression of the introduced constructs (Figure 2). One pair of primers was used to amplify a region in the first half of the AtHMA4 cDNA (see Figure S2 for primer positions). These primers do not amplify any product from hma2 hma4 or hma2 hma4 vector control cDNA, showing that full length AtHMA4 is not expressed in these plants. They amplify a product of the predicted size from cDNA of the hma2 hma4 double knockout transformed either with the 35S-AtHMA4-FL or the 35S-AtHMA4-trunc construct (Figure 2A). This shows that AtHMA4-FL or AtHMA4-trunc constructs are being expressed in these hma2 hma4 transformants. A second pair of primers was used to amplify AtHMA4 cDNA within the region corresponding to the C-terminus of the protein. These primers amplified a product of the predicted size from wt A. thaliana and from the hma2 hma4 double knockout transformed with 35S-AtHMA4-FL or 35S-AtHMA4-C-term (Figure 2B). A faint product from this region was also detected in hma2 hma4 and hma2 hma4 transformed with the empty vector, indicating low expression of a partial AtHMA4 transcript from this mutant (Figure 2B). The T-DNA insertion in this gene occurs after the fourth transmembrane domain, in the cytoplasmic ‘A’ domain (see Figures S1 and S2 for insertion position). A partial transcript could be initiated downstream of the insertion. This would not be predicted to have any transport activity however it could mean that there are very low levels of the C-terminal region expressed in these plants which could have an effect on metal chelation. Overall the results show that the constructs are being expressed in these lines, but expression levels vary slightly between lines. AtHMA4 FL line 2 showed low amplification levels for AtHMA4 but actin was also low in this sample. If the level of actin is taken into account then this line has similar expression levels to the other lines. AtHMA4-C-term line 3 also did not seem to show enhanced expression as the amplified product was comparable to that seen in the hma2 hma4 mutant (Figure 2B). In this case actin levels were only slightly lower.


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)

Arabidopsis hma2 hma4 plants are expressing AtHMA4-FL, AtHMA4-trunc or AtHMA4-C-term.Semi-quantitative RT-PCR shows expression of AtHMA4 versions in 5 independent transformant lines for each construct in the Arabidopsis hma2 hma4 mutant. A, RT-PCR for lines expressing AtHMA4 FL (plants 1–5) or AtHMA4-trunc (plants 1–5) using primers that detect a region before the C-terminus.. B, RT-PCR for lines expressing AtHMA4 FL (plants 1–5) or AtHMA4-C-term (plants 1–5) using primers that detect a region within the C-terminus. Wild type plant (wt) and hma2 hma4 mutant are shown as well as hma2 hma4 mutant expressing vector alone (hma2 hma4 V). Actin was used as a control.
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Related In: Results  -  Collection

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pone-0013388-g002: Arabidopsis hma2 hma4 plants are expressing AtHMA4-FL, AtHMA4-trunc or AtHMA4-C-term.Semi-quantitative RT-PCR shows expression of AtHMA4 versions in 5 independent transformant lines for each construct in the Arabidopsis hma2 hma4 mutant. A, RT-PCR for lines expressing AtHMA4 FL (plants 1–5) or AtHMA4-trunc (plants 1–5) using primers that detect a region before the C-terminus.. B, RT-PCR for lines expressing AtHMA4 FL (plants 1–5) or AtHMA4-C-term (plants 1–5) using primers that detect a region within the C-terminus. Wild type plant (wt) and hma2 hma4 mutant are shown as well as hma2 hma4 mutant expressing vector alone (hma2 hma4 V). Actin was used as a control.
Mentions: The hma2 hma4 double knockout mutant was transformed with AtHMA4-FL, AtHMA4-trunc or AtHMA4-C-term expressed under control of the 35S promoter. We used RT-PCR to confirm disruption of the endogenous genes, and expression of the introduced constructs (Figure 2). One pair of primers was used to amplify a region in the first half of the AtHMA4 cDNA (see Figure S2 for primer positions). These primers do not amplify any product from hma2 hma4 or hma2 hma4 vector control cDNA, showing that full length AtHMA4 is not expressed in these plants. They amplify a product of the predicted size from cDNA of the hma2 hma4 double knockout transformed either with the 35S-AtHMA4-FL or the 35S-AtHMA4-trunc construct (Figure 2A). This shows that AtHMA4-FL or AtHMA4-trunc constructs are being expressed in these hma2 hma4 transformants. A second pair of primers was used to amplify AtHMA4 cDNA within the region corresponding to the C-terminus of the protein. These primers amplified a product of the predicted size from wt A. thaliana and from the hma2 hma4 double knockout transformed with 35S-AtHMA4-FL or 35S-AtHMA4-C-term (Figure 2B). A faint product from this region was also detected in hma2 hma4 and hma2 hma4 transformed with the empty vector, indicating low expression of a partial AtHMA4 transcript from this mutant (Figure 2B). The T-DNA insertion in this gene occurs after the fourth transmembrane domain, in the cytoplasmic ‘A’ domain (see Figures S1 and S2 for insertion position). A partial transcript could be initiated downstream of the insertion. This would not be predicted to have any transport activity however it could mean that there are very low levels of the C-terminal region expressed in these plants which could have an effect on metal chelation. Overall the results show that the constructs are being expressed in these lines, but expression levels vary slightly between lines. AtHMA4 FL line 2 showed low amplification levels for AtHMA4 but actin was also low in this sample. If the level of actin is taken into account then this line has similar expression levels to the other lines. AtHMA4-C-term line 3 also did not seem to show enhanced expression as the amplified product was comparable to that seen in the hma2 hma4 mutant (Figure 2B). In this case actin levels were only slightly lower.

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