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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

Model depicting the difference in K+-sensitivity of SlHKT1;2 from S. esculentum and AtHKT1;1 from Arabidopsis. When the K+-concentration in the xylem sap is high, Na+-uptake by the SlHKT1;2 transporter is reduced. As a result the amount of Na+ in the xylem stream reaching the shoot of tomato is, at least partially, controlled by SlHKT1;2, which in turn depends on the concentration of K+ present in the xylem sap. In Arabidopsis, Na+-uptake in the XPCs by the AtHKT1;1 transporter is not affected by high K+.
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Figure 6: Model depicting the difference in K+-sensitivity of SlHKT1;2 from S. esculentum and AtHKT1;1 from Arabidopsis. When the K+-concentration in the xylem sap is high, Na+-uptake by the SlHKT1;2 transporter is reduced. As a result the amount of Na+ in the xylem stream reaching the shoot of tomato is, at least partially, controlled by SlHKT1;2, which in turn depends on the concentration of K+ present in the xylem sap. In Arabidopsis, Na+-uptake in the XPCs by the AtHKT1;1 transporter is not affected by high K+.

Mentions: The analysis of the properties of the heterologous expressed SlHKT1;2 showed that the transport characteristics were in accordance with the presence of an S in the first pore domain of the transporter (Figure 2). This is reflected in the SlHKT1;2 transport characteristics as measured in heterologous expression, where it was shown that tomato HKT1;2 transports Na+ but not K+ (Almeida et al., 2014). However, a striking difference between the transport properties of AtHKT1;1 and SlHKT1;2 expressed in oocytes was observed when currents were measured at constant Na+ in the bath (1 mM) and increasing K+ (1, 3, and 10 mM). Whereas the AtHKT1;1 mediated current was virtually insensitive to higher K+, the SlHKT1;2 mediated current decreased by 60% at 10 mM K+ (Figure 2). A similar inhibitory action of K+ on HKT-mediated currents was reported for OsHKT2;1 (Jabnoune et al., 2009) and for TmHKT1;5-A (Munns et al., 2012). It was proposed that this inhibition is caused by the association of K+ to the Na+ binding region within the pore region of HKT transporters (Rubio et al., 1995; Gassmann et al., 1996). This inhibition has not been observed with AtHKT1;1 (Uozumi et al., 2000) nor OsHKT1;5 (Ren et al., 2005) in Xenopus oocytes, which indicates that the K+-sensitivity of these transporters is different from OsHKT2;1, TmHKT1;5-A and tomato SlHKT1;2. Physiologically, this K+-induced reduction of Na+-influx might mean that the tomato plants maintain a certain K+/Na+-homeostasis in the transpiration sap. High concentrations of K+ in the xylem sap might imply a reduced Na+-uptake into the XPC, as the Na+/K+ ratio is in favor of Na+. On the other hand, low concentrations of K+ and high concentrations of Na+ in the xylem sap imply an induced Na+-uptake into the XPC, as the Na+/K+ ratio is in favor of K+ in the xylem sap. In this model, the Na+/K+ ratio is more important than the absolute concentration of Na+ in the xylem sap in determining the Na+ uptake into the XPC by HKT1 (Figure 6). Although the S → G mutation in the first pore domain of both the AtHKT1;1 and SlHKT1;2 protein had an effect on the ion selectivity of both transporters, as deduced from the shift in reversal potential at increasing external K+ (Figure 2), a major difference observed was the reduction in total current transported by SlHKT1;2-S70G and AtHKT1-S68G, which was 95 and 78% respectively of that transported by the WT proteins at 10 mM Na+ and 1 mM K+ in the bath (Figure 2). The reason for this difference is unclear as the results we obtained do not match the results of the Na+ currents produced by AtHKT1;1-S68G reported by Maser et al. (2002). Their results did not show this reduction in total currents. Interestingly, when Maser et al. (2002) mutated AtHKT1;1 in M69L, a reduction in outward currents with increasing K+ concentrations in the bath was observed, whereas in TaHKT1;2 the reverse mutation, L92M, abolished this inhibitory effect of K+ on outward currents (Maser et al., 2002). In their work, the leucine (L) adjacent to the G of the pore domain seems to confer K+ sensitivity to outward currents in contrast with the methionine (M) at that same position, which abolished the effect of K+ on Na+ currents (Maser et al., 2002). Since in our study SlHKT1;2 has a M adjacent to the G of the pore domain, no sensitivity of outward currents was expected due to increasing K+ concentrations in the bath. Nevertheless, in our study we observed outward currents, mediated by SlHKT1;2, sensitive to external K+. In a future study it would be interesting to mutate SlHKT1;2-M71L, to check whether the presence of a L adjacent to the S of the first pore domain of SlHKT1;2 enhances or decreases this inhibitory effect of external K+ on the outward Na+-currents mediated by SlHKT1;2.


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)

Model depicting the difference in K+-sensitivity of SlHKT1;2 from S. esculentum and AtHKT1;1 from Arabidopsis. When the K+-concentration in the xylem sap is high, Na+-uptake by the SlHKT1;2 transporter is reduced. As a result the amount of Na+ in the xylem stream reaching the shoot of tomato is, at least partially, controlled by SlHKT1;2, which in turn depends on the concentration of K+ present in the xylem sap. In Arabidopsis, Na+-uptake in the XPCs by the AtHKT1;1 transporter is not affected by high K+.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: Model depicting the difference in K+-sensitivity of SlHKT1;2 from S. esculentum and AtHKT1;1 from Arabidopsis. When the K+-concentration in the xylem sap is high, Na+-uptake by the SlHKT1;2 transporter is reduced. As a result the amount of Na+ in the xylem stream reaching the shoot of tomato is, at least partially, controlled by SlHKT1;2, which in turn depends on the concentration of K+ present in the xylem sap. In Arabidopsis, Na+-uptake in the XPCs by the AtHKT1;1 transporter is not affected by high K+.
Mentions: The analysis of the properties of the heterologous expressed SlHKT1;2 showed that the transport characteristics were in accordance with the presence of an S in the first pore domain of the transporter (Figure 2). This is reflected in the SlHKT1;2 transport characteristics as measured in heterologous expression, where it was shown that tomato HKT1;2 transports Na+ but not K+ (Almeida et al., 2014). However, a striking difference between the transport properties of AtHKT1;1 and SlHKT1;2 expressed in oocytes was observed when currents were measured at constant Na+ in the bath (1 mM) and increasing K+ (1, 3, and 10 mM). Whereas the AtHKT1;1 mediated current was virtually insensitive to higher K+, the SlHKT1;2 mediated current decreased by 60% at 10 mM K+ (Figure 2). A similar inhibitory action of K+ on HKT-mediated currents was reported for OsHKT2;1 (Jabnoune et al., 2009) and for TmHKT1;5-A (Munns et al., 2012). It was proposed that this inhibition is caused by the association of K+ to the Na+ binding region within the pore region of HKT transporters (Rubio et al., 1995; Gassmann et al., 1996). This inhibition has not been observed with AtHKT1;1 (Uozumi et al., 2000) nor OsHKT1;5 (Ren et al., 2005) in Xenopus oocytes, which indicates that the K+-sensitivity of these transporters is different from OsHKT2;1, TmHKT1;5-A and tomato SlHKT1;2. Physiologically, this K+-induced reduction of Na+-influx might mean that the tomato plants maintain a certain K+/Na+-homeostasis in the transpiration sap. High concentrations of K+ in the xylem sap might imply a reduced Na+-uptake into the XPC, as the Na+/K+ ratio is in favor of Na+. On the other hand, low concentrations of K+ and high concentrations of Na+ in the xylem sap imply an induced Na+-uptake into the XPC, as the Na+/K+ ratio is in favor of K+ in the xylem sap. In this model, the Na+/K+ ratio is more important than the absolute concentration of Na+ in the xylem sap in determining the Na+ uptake into the XPC by HKT1 (Figure 6). Although the S → G mutation in the first pore domain of both the AtHKT1;1 and SlHKT1;2 protein had an effect on the ion selectivity of both transporters, as deduced from the shift in reversal potential at increasing external K+ (Figure 2), a major difference observed was the reduction in total current transported by SlHKT1;2-S70G and AtHKT1-S68G, which was 95 and 78% respectively of that transported by the WT proteins at 10 mM Na+ and 1 mM K+ in the bath (Figure 2). The reason for this difference is unclear as the results we obtained do not match the results of the Na+ currents produced by AtHKT1;1-S68G reported by Maser et al. (2002). Their results did not show this reduction in total currents. Interestingly, when Maser et al. (2002) mutated AtHKT1;1 in M69L, a reduction in outward currents with increasing K+ concentrations in the bath was observed, whereas in TaHKT1;2 the reverse mutation, L92M, abolished this inhibitory effect of K+ on outward currents (Maser et al., 2002). In their work, the leucine (L) adjacent to the G of the pore domain seems to confer K+ sensitivity to outward currents in contrast with the methionine (M) at that same position, which abolished the effect of K+ on Na+ currents (Maser et al., 2002). Since in our study SlHKT1;2 has a M adjacent to the G of the pore domain, no sensitivity of outward currents was expected due to increasing K+ concentrations in the bath. Nevertheless, in our study we observed outward currents, mediated by SlHKT1;2, sensitive to external K+. In a future study it would be interesting to mutate SlHKT1;2-M71L, to check whether the presence of a L adjacent to the S of the first pore domain of SlHKT1;2 enhances or decreases this inhibitory effect of external K+ on the outward Na+-currents mediated by SlHKT1;2.

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