Limits...
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

Transport activity of AtHKT1;1, AtHKT1;1-S68G, SlHKT1;2 and SlHKT1;2-S70G constructs expressed in Xenopus laevis oocytes. Oocytes were injected with AtHKT1;1 and SlHKT1;2 and the indicated mutated transporters. (A,E,I,M) Currents recorded at three Na+ concentrations (1, 3, and 10 mM) with 1 mM K+ as background; (B,F,J,N) Currents recorded at three K+ concentrations (1, 3, and 10 mM) with 1 mM Na+ as background; (C,G,K,O) Reversal potential shifts as a function of ion concentration. Only transporters where the S of the 1st pore domain was mutated to a G were permeable to K+ as indicated by the large positive shifts in the reversal potential with increasing concentrations of K+ in the bath; (D,H,L,P) Absolute currents as a function of ion concentration. Transporters where the S of the 1st pore domain was mutated to a G showed an increase in current with increasing K+ concentration in the bath. Data are means ± SE (n = 3, experiments done with at least two different oocyte batches).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4219482&req=5

Figure 2: Transport activity of AtHKT1;1, AtHKT1;1-S68G, SlHKT1;2 and SlHKT1;2-S70G constructs expressed in Xenopus laevis oocytes. Oocytes were injected with AtHKT1;1 and SlHKT1;2 and the indicated mutated transporters. (A,E,I,M) Currents recorded at three Na+ concentrations (1, 3, and 10 mM) with 1 mM K+ as background; (B,F,J,N) Currents recorded at three K+ concentrations (1, 3, and 10 mM) with 1 mM Na+ as background; (C,G,K,O) Reversal potential shifts as a function of ion concentration. Only transporters where the S of the 1st pore domain was mutated to a G were permeable to K+ as indicated by the large positive shifts in the reversal potential with increasing concentrations of K+ in the bath; (D,H,L,P) Absolute currents as a function of ion concentration. Transporters where the S of the 1st pore domain was mutated to a G showed an increase in current with increasing K+ concentration in the bath. Data are means ± SE (n = 3, experiments done with at least two different oocyte batches).

Mentions: To test the hypothesis that S70 of the tomato SlHKT1;2 protein is crucial for the Na+ selectivity we replaced S70 by a G (SlHKT1;2-S70G). cRNA of SlHKT1;2-S70G was injected in Xenopus laevis oocytes. After 2 days of incubation, currents produced in the presence of Na+ and K+ ions were recorded in oocytes expressing both WT and mutated HKT1 transporters from Arabidopsis thaliana and Solanum lycopersicum (n = 3) (Figure 2). AtHKT1;1-S68G expressing oocytes (Maser et al., 2002) were used as a positive control of SlHKT1;2-S70G. Currents produced by oocytes expressing either AtHKT1;1 (Figure 2A) or SlHKT1;2 (Figure 2I) increased when the oocytes were bathed in higher Na+ concentrations (as seen by a more negative current). Increasing external K+ concentration did not result in any change in the current levels produced by AtHKT1;1 expressing oocytes (Figure 2B). In contrast, SlHKT1;2 mediated inward and outward currents were sensitive to external K+ concentration as both currents decreased with increasing bath K+ concentration (Figure 2J). Increased concentrations of K+ result in an inhibition on the transport of Na+ by SlHKT1;2 but not by AtHKT1;1. When oocytes expressing either AtHKT1;1-S68G or SlHKT1;2-S70G were bathed with either increasing Na+ concentration (Figures 2E,M) or K+ concentration (Figures 2F,N) currents increased for both cations tested. For both AtHKT1;1- and SlHKT1;2-mediated currents a higher Na+ concentration but not a higher K+ concentration resulted in positive shifts in the reversal potential (Figures 2C,K), which is indicative of Na+ permeation. Reversal potentials obtained with oocytes expressing either AtHKT1;1-S68G (Figure 2G) or SlHKT1;2-S70G (Figure 2O) showed positive shifts when both Na+ concentration or K+ concentration increased, indicating that the presence of a G residue at the filter position of the first pore domain allows the transport of both Na+ and K+ ions. Figures 2D,H,L,P show the currents recorded at −140 mV for AtHKT1;1, AtHKT1;1-S68G, SlHKT1;2 and SlHKT1;2-S70G, respectively. These results show that the Na+-mediated current of SlHKT1;2 is reduced by increased concentrations of K+ in the bath. The presence of K+ ions affects the transport of Na+ by SlHKT1;2. This effect is not observed with AtHKT1;1.


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)

Transport activity of AtHKT1;1, AtHKT1;1-S68G, SlHKT1;2 and SlHKT1;2-S70G constructs expressed in Xenopus laevis oocytes. Oocytes were injected with AtHKT1;1 and SlHKT1;2 and the indicated mutated transporters. (A,E,I,M) Currents recorded at three Na+ concentrations (1, 3, and 10 mM) with 1 mM K+ as background; (B,F,J,N) Currents recorded at three K+ concentrations (1, 3, and 10 mM) with 1 mM Na+ as background; (C,G,K,O) Reversal potential shifts as a function of ion concentration. Only transporters where the S of the 1st pore domain was mutated to a G were permeable to K+ as indicated by the large positive shifts in the reversal potential with increasing concentrations of K+ in the bath; (D,H,L,P) Absolute currents as a function of ion concentration. Transporters where the S of the 1st pore domain was mutated to a G showed an increase in current with increasing K+ concentration in the bath. Data are means ± SE (n = 3, experiments done with at least two different oocyte batches).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Transport activity of AtHKT1;1, AtHKT1;1-S68G, SlHKT1;2 and SlHKT1;2-S70G constructs expressed in Xenopus laevis oocytes. Oocytes were injected with AtHKT1;1 and SlHKT1;2 and the indicated mutated transporters. (A,E,I,M) Currents recorded at three Na+ concentrations (1, 3, and 10 mM) with 1 mM K+ as background; (B,F,J,N) Currents recorded at three K+ concentrations (1, 3, and 10 mM) with 1 mM Na+ as background; (C,G,K,O) Reversal potential shifts as a function of ion concentration. Only transporters where the S of the 1st pore domain was mutated to a G were permeable to K+ as indicated by the large positive shifts in the reversal potential with increasing concentrations of K+ in the bath; (D,H,L,P) Absolute currents as a function of ion concentration. Transporters where the S of the 1st pore domain was mutated to a G showed an increase in current with increasing K+ concentration in the bath. Data are means ± SE (n = 3, experiments done with at least two different oocyte batches).
Mentions: To test the hypothesis that S70 of the tomato SlHKT1;2 protein is crucial for the Na+ selectivity we replaced S70 by a G (SlHKT1;2-S70G). cRNA of SlHKT1;2-S70G was injected in Xenopus laevis oocytes. After 2 days of incubation, currents produced in the presence of Na+ and K+ ions were recorded in oocytes expressing both WT and mutated HKT1 transporters from Arabidopsis thaliana and Solanum lycopersicum (n = 3) (Figure 2). AtHKT1;1-S68G expressing oocytes (Maser et al., 2002) were used as a positive control of SlHKT1;2-S70G. Currents produced by oocytes expressing either AtHKT1;1 (Figure 2A) or SlHKT1;2 (Figure 2I) increased when the oocytes were bathed in higher Na+ concentrations (as seen by a more negative current). Increasing external K+ concentration did not result in any change in the current levels produced by AtHKT1;1 expressing oocytes (Figure 2B). In contrast, SlHKT1;2 mediated inward and outward currents were sensitive to external K+ concentration as both currents decreased with increasing bath K+ concentration (Figure 2J). Increased concentrations of K+ result in an inhibition on the transport of Na+ by SlHKT1;2 but not by AtHKT1;1. When oocytes expressing either AtHKT1;1-S68G or SlHKT1;2-S70G were bathed with either increasing Na+ concentration (Figures 2E,M) or K+ concentration (Figures 2F,N) currents increased for both cations tested. For both AtHKT1;1- and SlHKT1;2-mediated currents a higher Na+ concentration but not a higher K+ concentration resulted in positive shifts in the reversal potential (Figures 2C,K), which is indicative of Na+ permeation. Reversal potentials obtained with oocytes expressing either AtHKT1;1-S68G (Figure 2G) or SlHKT1;2-S70G (Figure 2O) showed positive shifts when both Na+ concentration or K+ concentration increased, indicating that the presence of a G residue at the filter position of the first pore domain allows the transport of both Na+ and K+ ions. Figures 2D,H,L,P show the currents recorded at −140 mV for AtHKT1;1, AtHKT1;1-S68G, SlHKT1;2 and SlHKT1;2-S70G, respectively. These results show that the Na+-mediated current of SlHKT1;2 is reduced by increased concentrations of K+ in the bath. The presence of K+ ions affects the transport of Na+ by SlHKT1;2. This effect is not observed with AtHKT1;1.

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