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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

Differences in ion accumulation between all plants analyzed. Na+(A) and K+(B) accumulation and Na+/K+(C) ratio in the shoot of WT, athkt1;1 mutant (N6531) and transgenic lines expressing different HKT1 genes. Na+/K+ ratio was normalized for WT. The effect of the mutation in the AtHKT1;1 gene is very clear, showing a strong increase in shoot Na+ accompanied by a decrease in K+, which resulted in a very strong increase in the Na+/K+ ratio. Both lines expressing AtHKT1;1::AtHKT1;1 or AtHKT1;1::SlHKT1;2 were able to reduce the accumulation of Na+ and increase the accumulation of K+ in comparison to athkt1;1. Lines expressing AtHKT1;1::AtHKT1;1-S68G or AtHKT1;1::SlHKT1;2-S70G were not able to ameliorate the phenotype of the athkt1;1 plants. Different letters above bars indicate statistically significant differences. Values indicate the means ± SE of three to seven biological replicates.
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Figure 5: Differences in ion accumulation between all plants analyzed. Na+(A) and K+(B) accumulation and Na+/K+(C) ratio in the shoot of WT, athkt1;1 mutant (N6531) and transgenic lines expressing different HKT1 genes. Na+/K+ ratio was normalized for WT. The effect of the mutation in the AtHKT1;1 gene is very clear, showing a strong increase in shoot Na+ accompanied by a decrease in K+, which resulted in a very strong increase in the Na+/K+ ratio. Both lines expressing AtHKT1;1::AtHKT1;1 or AtHKT1;1::SlHKT1;2 were able to reduce the accumulation of Na+ and increase the accumulation of K+ in comparison to athkt1;1. Lines expressing AtHKT1;1::AtHKT1;1-S68G or AtHKT1;1::SlHKT1;2-S70G were not able to ameliorate the phenotype of the athkt1;1 plants. Different letters above bars indicate statistically significant differences. Values indicate the means ± SE of three to seven biological replicates.

Mentions: There was a marked difference in K+-sensitivity of Na+-transport mediated by the AtHKT1;1 and SlHKT1;2 proteins, as analyzed in Xenopus laevis oocytes. To see whether this difference was reflected in Na+/K+ homeostasis in planta, we expressed the AtHKT1;1 and SlHKT1;2 genes driven by a 5 kb long AtHKT1;1 promoter in the athkt1;1 mutant plants and studied how they complemented the mutant phenotype. Since the analysis of SlHKT1;2-S70G and AtHKT1;1-S68G in Xenopus oocytes showed interesting effects on transport activity and ion selectivity we transformed athkt1;1 plants with SlHKT1;2-S70G and AtHKT1-S68G, and tested the functioning of these mutated genes in planta during salinity stress. Figure 4 shows the effect of salinity treatment on the leaf fresh weight of WT plants, athkt1;1 mutants and transformed plants. athkt1;1-mutant plants were more sensitive to salt than the WT plants. Both the AtHKT1;1 and the SlHKT1;2 gene were able to complement the athkt1;1 mutant growth-phenotype on salt (Figure 4). Plants transformed with SlHKT1;2-S70G or AtHKT1-S68G were just as sensitive to salt as the athkt1;1-mutant plants. The analysis of the relative water content of the leaves did not show statistically significant differences between control and salt treated plants (data not shown) nor within transformed lines. The effect of the athkt1;1 mutation on Na+ and K+ homeostasis was pronounced (Figure 5). The athkt1;1 plants accumulated almost four-fold more Na+ and two-fold less K+, resulting in an eight-fold higher Na+/K+-ratio in the shoot of the mutant plants as compared to the WT plants. The transgenic lines that complemented the athkt1;1 growth-phenotype (AtHKT1;1 and SlHKT1;2) showed effects on ion accumulation: the athkt1;1 K+-phenotype during salt stress (i.e., strong reduction in K+ accumulation) was completely restored, but Na+ accumulation in the leaves of these transgenic lines was still significantly higher than that of the WT plants.


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)

Differences in ion accumulation between all plants analyzed. Na+(A) and K+(B) accumulation and Na+/K+(C) ratio in the shoot of WT, athkt1;1 mutant (N6531) and transgenic lines expressing different HKT1 genes. Na+/K+ ratio was normalized for WT. The effect of the mutation in the AtHKT1;1 gene is very clear, showing a strong increase in shoot Na+ accompanied by a decrease in K+, which resulted in a very strong increase in the Na+/K+ ratio. Both lines expressing AtHKT1;1::AtHKT1;1 or AtHKT1;1::SlHKT1;2 were able to reduce the accumulation of Na+ and increase the accumulation of K+ in comparison to athkt1;1. Lines expressing AtHKT1;1::AtHKT1;1-S68G or AtHKT1;1::SlHKT1;2-S70G were not able to ameliorate the phenotype of the athkt1;1 plants. Different letters above bars indicate statistically significant differences. Values indicate the means ± SE of three to seven biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Differences in ion accumulation between all plants analyzed. Na+(A) and K+(B) accumulation and Na+/K+(C) ratio in the shoot of WT, athkt1;1 mutant (N6531) and transgenic lines expressing different HKT1 genes. Na+/K+ ratio was normalized for WT. The effect of the mutation in the AtHKT1;1 gene is very clear, showing a strong increase in shoot Na+ accompanied by a decrease in K+, which resulted in a very strong increase in the Na+/K+ ratio. Both lines expressing AtHKT1;1::AtHKT1;1 or AtHKT1;1::SlHKT1;2 were able to reduce the accumulation of Na+ and increase the accumulation of K+ in comparison to athkt1;1. Lines expressing AtHKT1;1::AtHKT1;1-S68G or AtHKT1;1::SlHKT1;2-S70G were not able to ameliorate the phenotype of the athkt1;1 plants. Different letters above bars indicate statistically significant differences. Values indicate the means ± SE of three to seven biological replicates.
Mentions: There was a marked difference in K+-sensitivity of Na+-transport mediated by the AtHKT1;1 and SlHKT1;2 proteins, as analyzed in Xenopus laevis oocytes. To see whether this difference was reflected in Na+/K+ homeostasis in planta, we expressed the AtHKT1;1 and SlHKT1;2 genes driven by a 5 kb long AtHKT1;1 promoter in the athkt1;1 mutant plants and studied how they complemented the mutant phenotype. Since the analysis of SlHKT1;2-S70G and AtHKT1;1-S68G in Xenopus oocytes showed interesting effects on transport activity and ion selectivity we transformed athkt1;1 plants with SlHKT1;2-S70G and AtHKT1-S68G, and tested the functioning of these mutated genes in planta during salinity stress. Figure 4 shows the effect of salinity treatment on the leaf fresh weight of WT plants, athkt1;1 mutants and transformed plants. athkt1;1-mutant plants were more sensitive to salt than the WT plants. Both the AtHKT1;1 and the SlHKT1;2 gene were able to complement the athkt1;1 mutant growth-phenotype on salt (Figure 4). Plants transformed with SlHKT1;2-S70G or AtHKT1-S68G were just as sensitive to salt as the athkt1;1-mutant plants. The analysis of the relative water content of the leaves did not show statistically significant differences between control and salt treated plants (data not shown) nor within transformed lines. The effect of the athkt1;1 mutation on Na+ and K+ homeostasis was pronounced (Figure 5). The athkt1;1 plants accumulated almost four-fold more Na+ and two-fold less K+, resulting in an eight-fold higher Na+/K+-ratio in the shoot of the mutant plants as compared to the WT plants. The transgenic lines that complemented the athkt1;1 growth-phenotype (AtHKT1;1 and SlHKT1;2) showed effects on ion accumulation: the athkt1;1 K+-phenotype during salt stress (i.e., strong reduction in K+ accumulation) was completely restored, but Na+ accumulation in the leaves of these transgenic lines was still significantly higher than that of the WT plants.

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