PLANT SIGNALING & BEHAVIOR 2016, VOL. 11, NO. 8, e1206169 (4 pages) http://dx.doi.org/10.1080/15592324.2016.1206169
ARTICLE ADDENDUM
Differential selection of sodium and potassium ions by TsHKT1;2 Akhtar Ali and Dae-Jin Yun Division of Applied Life Science (BK21plus Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, South Korea
ABSTRACT
ARTICLE HISTORY
Among abiotic stresses, soil salinity is a major threat to agriculture. To address and control the effects of high salinity on plants, it is important to understand their responses to salt stress that disturbs the homeostatic equilibrium at cellular and molecular levels. To deal and control effects of high salinity on plants, it is important to understand their responses to salt stress that disturbs the homeostatic equilibrium at cellular and molecular levels. In this regard, halophytes (salt tolerant plants) can provide superior models for the study of salt stress defense parameters compared to glycophytes (salt sensitive species). TsHKT1;2 one of the 3 copies of HKT1 in the Arabidopsis relative halophyte, Thellungiella salsuginea acts as a potassium transporter, even under salt stress. TsHKT1;2 includes a conserved Asp (D) residue in the 2nd pore-loop domain. Most other HKT1 sequences, including AtHKT1, contain Asn (N) in this position. We found that athkt1-1 plants complemented by TsHKT1;2 under native AtHKT1 promoter were more tolerant to salt stress, while substitution of Asp (D207) by Asn (N) significantly reduced resistance to salinity. We suggest that the presence of Asn or Asp is the essential feature that defines and establishes cation selectivity in dicot HKT1-type transporters.
Received 27 May 2016 Revised 20 June 2016 Accepted 21 June 2016
HKT1-type transporters play a crucial role in plant adaptation to increasing salinity in the soil. These transporters are involved in mediating the distribution of NaC within the plant by a repeated pattern of removing NaC from the xylem, particularly in the roots, such that the amount of NaC arriving in the shoot becomes more easily manageable.1-4 Homologs of HKT1 genes and proteins have been identified in a number of plant species, including Arabidopsis. Their ion selectivities were originally characterized in yeast and/or Xenopus oocytes.5-16 Based on protein structure and ion selectivity, HKT1-type transporters are generally classified into subclasses 1 and 2.17-19 Members of subclass-1 are selective sodium transporters or uniporters whereas subclass-2 members are sodium/potassium co-transporters or symporters.1820 All HKT1 proteins known from dicots include a serine at the predicted filter position in P-loop A of the protein. Like AtHKT1, TsHKT1;2 belongs to subclass-1, as it contains the conserved serine residue (S68) in the selectivity filter position in the pore-loop (P-loop) domain.14,21,22 However TsHKT1;2 shows significantly higher affinity for potassium than for sodium ions.23 The conundrum represented by the fundamentally different behavior of the TsHKT1;2 gene and protein seems to ask for a review of the HKT1 dogma. Retaining the capacity for potassium uptake is important for plants during salt stress.16,24,25 We reported earlier that salt stress leads to up-regulation of TsHKT1;2 at the transcript level, possibly highlighting a role in salinity stress.25 This pattern of regulation is different from that shown by Arabidopsis as AtHKT1 in highly saline media imports NaC instead of KC.21
KEYWORDS
Arabidopsis; glycophyte; halophyte; HKT1; salt stress; thellungiella salsuginea
However, upregulation in response to salt stress is consistent with observations for rice OsHKT1 and OsHKT2.26 Under salt stress, when the cytosolic sodium concentration reaches a toxic level, plants activate high affinity potassium transporters for potassium uptake to restore cellular NaC and KC balance.15,27,28 Up-regulation of HKT1 transcript under KC-deficient conditions seems detrimental if the HKT1 protein is sodium specific.18,26 Conversely, its activation in response to high salinity suggests that TsHKT1;2 might function as a highaffinity potassium transporter in Thellungiella salsuginea. When aligned with TaHKT1, AvHKT1 and AtHKT1, TsHKT1;2 shows much higher similarity to AtHKT1.21 AtHKT1 functions as a selective sodium transporter in yeast and Xenopus laevis oocytes.7 By contrast, when expressed in yeast and oocytes, TsHKT1;2 functions as a high-affinity potassium and sodium cotransporter. Thus, while categorized as a subclass-1 protein, TsHKT1;2 does not follow the rule established for this class.21 Cation selectivities of HKT1 transporters are interconvertible by exchanging a single amino acid in their pore-loop domain.19,21,25 The presence of an aspartic acid (Asp) replacing an asparagine residue (Asn) converts the NaC uniporter AtHKT1 into a NaC/KC symporter.25 As shown by the yeast experiments, TsHKT1;2 is a high affinity potassium, low affinity sodium co-transporter.21 Substitution of Asp (D207) in TsHKT1;2 by Asn (N) leads to the loss of potassium selectivity and enhanced sodium uptake activity (25). To analyze the effect of such manipulations in plants, TsHKT1;2 and its mutant forms, TsHKT1;2D207N, TsHKT1;2D238N and TsHKT1;22D2N (where both D207 and D238 were replaced by N207 and N238)
CONTACT Dae-Jin Yun [email protected] Department of Biochemistry, Gyeongsang National University, Jinju, South Korea Thellungiella was recently re-classified as Eutrema; all species in the genus have been converted with the suffix “um” (http://www.uniprot.org/taxonomy/72664). Thus, T. salsuginea is now E. salsugineum. We use the name Thellungiella in this article. © 2016 Taylor & Francis Group, LLC
e1206169-2
A. ALI AND D.-J. YUN
were expressed in the athkt1-1 knockout background under control of the native Arabidopsis AtHKT1 promoter. Several homozygous transgenic lines were selected, among them only similar transcript level of TsHKT1;2 containing lines (at least 3 independent lines for each genotype) were used for further experiments (Fig. 1A). As expected, replacing Asp with Asn in TsHKT1;2 abolishes potassium transport and converts TsHKT1;2 into a NaC uniporter.21 TsHKT1;2 and TpHKT1;2 (Tp for Thellungiella parvula) contain 2 conserved Asp (D) residues, one in the 2nd P-loop domain (D207/205) and the second in the adjacent transmembrane domain (D238/236). The yeast potassium transporter, ScTRK1, also contains an Asp residue in the pore-loop domain.21 As shown in Fig. 1, D207 has a very clear effect on TsHKT1;2 functionality.21 Most other HKTs, including AtHKT1, have Asn (N) in this position.25 Substitution of Asn (N211) by Asp in AtHKT1 at the same position (N211D), provided proof for the importance of this residue upon expression not only in yeast and oocytes but also in plants.25 This identifies the Asp residue in the pore-loop domain as necessary for potassium selectivity (Fig. 2). In addition, electrophysiological analysis showed that TsHKT1;2 is able to transport NaC and KC at similar levels, whereas AtHKT1 has higher affinity toward NaC than KC, that were correlated with those observed in yeast.25 Another key
difference between AtHKT1;1 and TsHKT1;2 is inward rectification. AtHKT1;1 carries both inward and outward currents of NaC, whereas TsHKT1;2 showed strong inward rectification.25 A balance between sodium and potassium ions under salt stress is crucial for plant survival, but it is not clear how such balance can be maintained under conditions of (hyper-) accumulation of NaC (and, to some degree, Cl¡) leading to osmotic stress and ionic imbalance.2,24 It is believed that high affinity potassium transporters will be active during salt stress. The presence and stress-induced activity of NaC/HC antiporters have also been shown.29 Yet other transporters are active in partitioning NaC into vacuoles which can act as ultimate sinks for sodium ions.30,31 In this scenario, the function of transporters with the same characteristics as AtHKT1 (and the majority of plants) is not reconcilable. However, the localization of AtHKT1 in xylem parenchyma cells appears to provide an answer to this conundrum because its activity in that location could reduce the flux of sodium ions to the shoot tip under condition of excess NaC. For plants that may be exposed to an excess of sodium ions, the function of HKT1 isoforms seems to have changed from a distribution that retards NaC flux throughout the plant into a supporting role as KC transporters. The ability of Thellungiella species to maintain a low cytosolic NaC/KC ratio in the presence of high
Figure 1. Phenotype of ProAtHKT1::TsHKT1;2 wild type and its mutant forms expressed in athkt1-1 background under salt stress. (A). RT-PCR was carried out with 3/microgram of total RNA extracted from 10-day-old MS media grown seedlings. (B) Ten-day-old MS media grown seedlings of Col-gl (wild-type), athkt1-1, and transgenic athkt1-1 plants expressing TsHKT1;2, TsHKT1;2D207N, TsHKT1;2D238N and TsHKT1;22D2N (where both D207 and D238 were replaced by N) driven by the AtHKT1 promoter were transferred to soil and further grown for 2/weeks followed by 300 mM NaCl treatment for another 2/weeks. Photographs were taken at the end of salt treatment. (C) Seeds of the same lines used in Fig. 1B were grown in hydroponic solution for 1/week followed by 20 or 30 mM NaCl treatment as indicated. Photographs were taken 2 weeks after the addition of salt. (D) and (E) After salt treatment, fresh weights (plants used in B and C (30 mM NaCl)) were measured, with error bars representing standard deviations from 3 independent repeats (n D 30). Significant difference from wild-type was determined by a Student’s t test, single or double asterisks indicate a P value of
ARTICLE ADDENDUM
Differential selection of sodium and potassium ions by TsHKT1;2 Akhtar Ali and Dae-Jin Yun Division of Applied Life Science (BK21plus Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, South Korea
ABSTRACT
ARTICLE HISTORY
Among abiotic stresses, soil salinity is a major threat to agriculture. To address and control the effects of high salinity on plants, it is important to understand their responses to salt stress that disturbs the homeostatic equilibrium at cellular and molecular levels. To deal and control effects of high salinity on plants, it is important to understand their responses to salt stress that disturbs the homeostatic equilibrium at cellular and molecular levels. In this regard, halophytes (salt tolerant plants) can provide superior models for the study of salt stress defense parameters compared to glycophytes (salt sensitive species). TsHKT1;2 one of the 3 copies of HKT1 in the Arabidopsis relative halophyte, Thellungiella salsuginea acts as a potassium transporter, even under salt stress. TsHKT1;2 includes a conserved Asp (D) residue in the 2nd pore-loop domain. Most other HKT1 sequences, including AtHKT1, contain Asn (N) in this position. We found that athkt1-1 plants complemented by TsHKT1;2 under native AtHKT1 promoter were more tolerant to salt stress, while substitution of Asp (D207) by Asn (N) significantly reduced resistance to salinity. We suggest that the presence of Asn or Asp is the essential feature that defines and establishes cation selectivity in dicot HKT1-type transporters.
Received 27 May 2016 Revised 20 June 2016 Accepted 21 June 2016
HKT1-type transporters play a crucial role in plant adaptation to increasing salinity in the soil. These transporters are involved in mediating the distribution of NaC within the plant by a repeated pattern of removing NaC from the xylem, particularly in the roots, such that the amount of NaC arriving in the shoot becomes more easily manageable.1-4 Homologs of HKT1 genes and proteins have been identified in a number of plant species, including Arabidopsis. Their ion selectivities were originally characterized in yeast and/or Xenopus oocytes.5-16 Based on protein structure and ion selectivity, HKT1-type transporters are generally classified into subclasses 1 and 2.17-19 Members of subclass-1 are selective sodium transporters or uniporters whereas subclass-2 members are sodium/potassium co-transporters or symporters.1820 All HKT1 proteins known from dicots include a serine at the predicted filter position in P-loop A of the protein. Like AtHKT1, TsHKT1;2 belongs to subclass-1, as it contains the conserved serine residue (S68) in the selectivity filter position in the pore-loop (P-loop) domain.14,21,22 However TsHKT1;2 shows significantly higher affinity for potassium than for sodium ions.23 The conundrum represented by the fundamentally different behavior of the TsHKT1;2 gene and protein seems to ask for a review of the HKT1 dogma. Retaining the capacity for potassium uptake is important for plants during salt stress.16,24,25 We reported earlier that salt stress leads to up-regulation of TsHKT1;2 at the transcript level, possibly highlighting a role in salinity stress.25 This pattern of regulation is different from that shown by Arabidopsis as AtHKT1 in highly saline media imports NaC instead of KC.21
KEYWORDS
Arabidopsis; glycophyte; halophyte; HKT1; salt stress; thellungiella salsuginea
However, upregulation in response to salt stress is consistent with observations for rice OsHKT1 and OsHKT2.26 Under salt stress, when the cytosolic sodium concentration reaches a toxic level, plants activate high affinity potassium transporters for potassium uptake to restore cellular NaC and KC balance.15,27,28 Up-regulation of HKT1 transcript under KC-deficient conditions seems detrimental if the HKT1 protein is sodium specific.18,26 Conversely, its activation in response to high salinity suggests that TsHKT1;2 might function as a highaffinity potassium transporter in Thellungiella salsuginea. When aligned with TaHKT1, AvHKT1 and AtHKT1, TsHKT1;2 shows much higher similarity to AtHKT1.21 AtHKT1 functions as a selective sodium transporter in yeast and Xenopus laevis oocytes.7 By contrast, when expressed in yeast and oocytes, TsHKT1;2 functions as a high-affinity potassium and sodium cotransporter. Thus, while categorized as a subclass-1 protein, TsHKT1;2 does not follow the rule established for this class.21 Cation selectivities of HKT1 transporters are interconvertible by exchanging a single amino acid in their pore-loop domain.19,21,25 The presence of an aspartic acid (Asp) replacing an asparagine residue (Asn) converts the NaC uniporter AtHKT1 into a NaC/KC symporter.25 As shown by the yeast experiments, TsHKT1;2 is a high affinity potassium, low affinity sodium co-transporter.21 Substitution of Asp (D207) in TsHKT1;2 by Asn (N) leads to the loss of potassium selectivity and enhanced sodium uptake activity (25). To analyze the effect of such manipulations in plants, TsHKT1;2 and its mutant forms, TsHKT1;2D207N, TsHKT1;2D238N and TsHKT1;22D2N (where both D207 and D238 were replaced by N207 and N238)
CONTACT Dae-Jin Yun [email protected] Department of Biochemistry, Gyeongsang National University, Jinju, South Korea Thellungiella was recently re-classified as Eutrema; all species in the genus have been converted with the suffix “um” (http://www.uniprot.org/taxonomy/72664). Thus, T. salsuginea is now E. salsugineum. We use the name Thellungiella in this article. © 2016 Taylor & Francis Group, LLC
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A. ALI AND D.-J. YUN
were expressed in the athkt1-1 knockout background under control of the native Arabidopsis AtHKT1 promoter. Several homozygous transgenic lines were selected, among them only similar transcript level of TsHKT1;2 containing lines (at least 3 independent lines for each genotype) were used for further experiments (Fig. 1A). As expected, replacing Asp with Asn in TsHKT1;2 abolishes potassium transport and converts TsHKT1;2 into a NaC uniporter.21 TsHKT1;2 and TpHKT1;2 (Tp for Thellungiella parvula) contain 2 conserved Asp (D) residues, one in the 2nd P-loop domain (D207/205) and the second in the adjacent transmembrane domain (D238/236). The yeast potassium transporter, ScTRK1, also contains an Asp residue in the pore-loop domain.21 As shown in Fig. 1, D207 has a very clear effect on TsHKT1;2 functionality.21 Most other HKTs, including AtHKT1, have Asn (N) in this position.25 Substitution of Asn (N211) by Asp in AtHKT1 at the same position (N211D), provided proof for the importance of this residue upon expression not only in yeast and oocytes but also in plants.25 This identifies the Asp residue in the pore-loop domain as necessary for potassium selectivity (Fig. 2). In addition, electrophysiological analysis showed that TsHKT1;2 is able to transport NaC and KC at similar levels, whereas AtHKT1 has higher affinity toward NaC than KC, that were correlated with those observed in yeast.25 Another key
difference between AtHKT1;1 and TsHKT1;2 is inward rectification. AtHKT1;1 carries both inward and outward currents of NaC, whereas TsHKT1;2 showed strong inward rectification.25 A balance between sodium and potassium ions under salt stress is crucial for plant survival, but it is not clear how such balance can be maintained under conditions of (hyper-) accumulation of NaC (and, to some degree, Cl¡) leading to osmotic stress and ionic imbalance.2,24 It is believed that high affinity potassium transporters will be active during salt stress. The presence and stress-induced activity of NaC/HC antiporters have also been shown.29 Yet other transporters are active in partitioning NaC into vacuoles which can act as ultimate sinks for sodium ions.30,31 In this scenario, the function of transporters with the same characteristics as AtHKT1 (and the majority of plants) is not reconcilable. However, the localization of AtHKT1 in xylem parenchyma cells appears to provide an answer to this conundrum because its activity in that location could reduce the flux of sodium ions to the shoot tip under condition of excess NaC. For plants that may be exposed to an excess of sodium ions, the function of HKT1 isoforms seems to have changed from a distribution that retards NaC flux throughout the plant into a supporting role as KC transporters. The ability of Thellungiella species to maintain a low cytosolic NaC/KC ratio in the presence of high
Figure 1. Phenotype of ProAtHKT1::TsHKT1;2 wild type and its mutant forms expressed in athkt1-1 background under salt stress. (A). RT-PCR was carried out with 3/microgram of total RNA extracted from 10-day-old MS media grown seedlings. (B) Ten-day-old MS media grown seedlings of Col-gl (wild-type), athkt1-1, and transgenic athkt1-1 plants expressing TsHKT1;2, TsHKT1;2D207N, TsHKT1;2D238N and TsHKT1;22D2N (where both D207 and D238 were replaced by N) driven by the AtHKT1 promoter were transferred to soil and further grown for 2/weeks followed by 300 mM NaCl treatment for another 2/weeks. Photographs were taken at the end of salt treatment. (C) Seeds of the same lines used in Fig. 1B were grown in hydroponic solution for 1/week followed by 20 or 30 mM NaCl treatment as indicated. Photographs were taken 2 weeks after the addition of salt. (D) and (E) After salt treatment, fresh weights (plants used in B and C (30 mM NaCl)) were measured, with error bars representing standard deviations from 3 independent repeats (n D 30). Significant difference from wild-type was determined by a Student’s t test, single or double asterisks indicate a P value of
