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Assessment of natural variation in the first pore domain of the tomato HKT1;2 transporter and characterization of mutated versions of SlHKT1;2 expressed in Xenopus laevis oocytes and via complementation of the salt sensitive athkt1;1 mutant.

Almeida PM, de Boer GJ, de Boer AH - Front Plant Sci (2014)

Bottom Line: In this work, we analyzed the natural variation present in the first pore domain of the HKT1;2 coding sequence of 93 different tomato accessions, which revealed that this region was conserved among all accessions analyzed.The study of the transport characteristics of SlHKT1;2 revealed that Na(+)-transport by the tomato SlHKT1;2 protein was inhibited by the presence of K(+) at the outside of the membrane.Both AtHKT1;1-S68G and SlHKT1;2-S70G were not able to restore the phenotype of athkt1;1 mutant plants.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Biology, Faculty Earth and Life Sciences, Vrije Universiteit Amsterdam Amsterdam, Netherlands.

ABSTRACT
Single Nucleotide Polymorphisms (SNPs) within the coding sequence of HKT transporters are important for the functioning of these transporters in several plant species. To unravel the functioning of HKT transporters analysis of natural variation and multiple site-directed mutations studies are crucial. Also the in vivo functioning of HKT proteins, via complementation studies performed with athkt1;1 plants, could provide essential information about these transporters. In this work, we analyzed the natural variation present in the first pore domain of the HKT1;2 coding sequence of 93 different tomato accessions, which revealed that this region was conserved among all accessions analyzed. Analysis of mutations introduced in the first pore domain of the SlHKT1;2 gene showed, when heterologous expressed in Xenopus laevis oocytes, that the replacement of S70 by a G allowed SlHKT2;1 to transport K(+), but also caused a large reduction in both Na(+) and K(+) mediated currents. The study of the transport characteristics of SlHKT1;2 revealed that Na(+)-transport by the tomato SlHKT1;2 protein was inhibited by the presence of K(+) at the outside of the membrane. GUS expression under the AtHKT1;1 promoter gave blue staining in the vascular system of transgenic Arabidopsis. athkt1;1 mutant plants transformed with AtHKT1;1, SlHKT1;2, AtHKT1;1S68G, and SlHKT1;2S70G indicated that both AtHKT1;1 and SlHKT1;2 were able to restore the accumulation of K(+) in the shoot, although the low accumulation of Na(+) as shown by WT plants was only partially restored. The inhibition of Na(+) transport by K(+), shown by the SlHKT1;2 transporter in oocytes (and not by AtHKT1;1), was not reflected in Na(+) accumulation in the plants transformed with SlHKT1;2. Both AtHKT1;1-S68G and SlHKT1;2-S70G were not able to restore the phenotype of athkt1;1 mutant plants.

No MeSH data available.


Related in: MedlinePlus

Tomato accessions show SNPs close to the S at the filter position of the first pore domain, but not in the nucleotides coding the S residue. (A)Solanum HKT1;2 melting curve derivative plots from 93 tomato accessions showing the two different SNP's identified close to the 1st pore domain. (B) Nucleotide sequences of the HKT1;2 gene of different Solanum accessions show that both SNPs (red letters) situate close to, but do not coincide with the S70 residue of the first pore domain (green letters). Black colored letters represent conserved nucleotides.
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Figure 1: Tomato accessions show SNPs close to the S at the filter position of the first pore domain, but not in the nucleotides coding the S residue. (A)Solanum HKT1;2 melting curve derivative plots from 93 tomato accessions showing the two different SNP's identified close to the 1st pore domain. (B) Nucleotide sequences of the HKT1;2 gene of different Solanum accessions show that both SNPs (red letters) situate close to, but do not coincide with the S70 residue of the first pore domain (green letters). Black colored letters represent conserved nucleotides.

Mentions: From all 93 tomato accessions tested only three (PI 126435, LA 2931 and LA 1401) showed different melting curves (Figure 1A). To identify whether the SNPs responsible for these different melting curves were located at our target region, we amplified and sequenced the region of the first pore domain of these three accessions (Figure 1B). Sequencing results revealed that each of these three accessions have a single SNP (PI 126435 and LA 1401 have the same SNP and LA 2931 has a different SNP), although none of them in the position of interest. The SNP in the accession LA 2931 resulted in an amino acid change from valine (V) to isoleucine (I); however, both amino acids are hydrophobic. In the case of accessions PI 126435 and LA 1401 the SNP did not result in any amino acid change, as both ACA and ACT code for a threonine (T) residue.


Assessment of natural variation in the first pore domain of the tomato HKT1;2 transporter and characterization of mutated versions of SlHKT1;2 expressed in Xenopus laevis oocytes and via complementation of the salt sensitive athkt1;1 mutant.

Almeida PM, de Boer GJ, de Boer AH - Front Plant Sci (2014)

Tomato accessions show SNPs close to the S at the filter position of the first pore domain, but not in the nucleotides coding the S residue. (A)Solanum HKT1;2 melting curve derivative plots from 93 tomato accessions showing the two different SNP's identified close to the 1st pore domain. (B) Nucleotide sequences of the HKT1;2 gene of different Solanum accessions show that both SNPs (red letters) situate close to, but do not coincide with the S70 residue of the first pore domain (green letters). Black colored letters represent conserved nucleotides.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219482&req=5

Figure 1: Tomato accessions show SNPs close to the S at the filter position of the first pore domain, but not in the nucleotides coding the S residue. (A)Solanum HKT1;2 melting curve derivative plots from 93 tomato accessions showing the two different SNP's identified close to the 1st pore domain. (B) Nucleotide sequences of the HKT1;2 gene of different Solanum accessions show that both SNPs (red letters) situate close to, but do not coincide with the S70 residue of the first pore domain (green letters). Black colored letters represent conserved nucleotides.
Mentions: From all 93 tomato accessions tested only three (PI 126435, LA 2931 and LA 1401) showed different melting curves (Figure 1A). To identify whether the SNPs responsible for these different melting curves were located at our target region, we amplified and sequenced the region of the first pore domain of these three accessions (Figure 1B). Sequencing results revealed that each of these three accessions have a single SNP (PI 126435 and LA 1401 have the same SNP and LA 2931 has a different SNP), although none of them in the position of interest. The SNP in the accession LA 2931 resulted in an amino acid change from valine (V) to isoleucine (I); however, both amino acids are hydrophobic. In the case of accessions PI 126435 and LA 1401 the SNP did not result in any amino acid change, as both ACA and ACT code for a threonine (T) residue.

Bottom Line: In this work, we analyzed the natural variation present in the first pore domain of the HKT1;2 coding sequence of 93 different tomato accessions, which revealed that this region was conserved among all accessions analyzed.The study of the transport characteristics of SlHKT1;2 revealed that Na(+)-transport by the tomato SlHKT1;2 protein was inhibited by the presence of K(+) at the outside of the membrane.Both AtHKT1;1-S68G and SlHKT1;2-S70G were not able to restore the phenotype of athkt1;1 mutant plants.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Biology, Faculty Earth and Life Sciences, Vrije Universiteit Amsterdam Amsterdam, Netherlands.

ABSTRACT
Single Nucleotide Polymorphisms (SNPs) within the coding sequence of HKT transporters are important for the functioning of these transporters in several plant species. To unravel the functioning of HKT transporters analysis of natural variation and multiple site-directed mutations studies are crucial. Also the in vivo functioning of HKT proteins, via complementation studies performed with athkt1;1 plants, could provide essential information about these transporters. In this work, we analyzed the natural variation present in the first pore domain of the HKT1;2 coding sequence of 93 different tomato accessions, which revealed that this region was conserved among all accessions analyzed. Analysis of mutations introduced in the first pore domain of the SlHKT1;2 gene showed, when heterologous expressed in Xenopus laevis oocytes, that the replacement of S70 by a G allowed SlHKT2;1 to transport K(+), but also caused a large reduction in both Na(+) and K(+) mediated currents. The study of the transport characteristics of SlHKT1;2 revealed that Na(+)-transport by the tomato SlHKT1;2 protein was inhibited by the presence of K(+) at the outside of the membrane. GUS expression under the AtHKT1;1 promoter gave blue staining in the vascular system of transgenic Arabidopsis. athkt1;1 mutant plants transformed with AtHKT1;1, SlHKT1;2, AtHKT1;1S68G, and SlHKT1;2S70G indicated that both AtHKT1;1 and SlHKT1;2 were able to restore the accumulation of K(+) in the shoot, although the low accumulation of Na(+) as shown by WT plants was only partially restored. The inhibition of Na(+) transport by K(+), shown by the SlHKT1;2 transporter in oocytes (and not by AtHKT1;1), was not reflected in Na(+) accumulation in the plants transformed with SlHKT1;2. Both AtHKT1;1-S68G and SlHKT1;2-S70G were not able to restore the phenotype of athkt1;1 mutant plants.

No MeSH data available.


Related in: MedlinePlus