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• W2115928499 abstract "Article31 May 2007free access Rice OsHKT2;1 transporter mediates large Na+ influx component into K+-starved roots for growth Tomoaki Horie Tomoaki Horie Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Alex Costa Alex Costa Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Tae Houn Kim Tae Houn Kim Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Min Jung Han Min Jung Han National Research Laboratory of Plant Functional Genomics, Department of Life Science, Pohang University of Science and Technology, Kyungbuk, Republic of Korea Search for more papers by this author Rie Horie Rie Horie Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Ho-Yin Leung Ho-Yin Leung Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Akio Miyao Akio Miyao Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences, Ibaraki, Japan Search for more papers by this author Hirohiko Hirochika Hirohiko Hirochika Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences, Ibaraki, Japan Search for more papers by this author Gynheung An Gynheung An National Research Laboratory of Plant Functional Genomics, Department of Life Science, Pohang University of Science and Technology, Kyungbuk, Republic of Korea Search for more papers by this author Julian I Schroeder Corresponding Author Julian I Schroeder Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Tomoaki Horie Tomoaki Horie Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Alex Costa Alex Costa Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Tae Houn Kim Tae Houn Kim Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Min Jung Han Min Jung Han National Research Laboratory of Plant Functional Genomics, Department of Life Science, Pohang University of Science and Technology, Kyungbuk, Republic of Korea Search for more papers by this author Rie Horie Rie Horie Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Ho-Yin Leung Ho-Yin Leung Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Akio Miyao Akio Miyao Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences, Ibaraki, Japan Search for more papers by this author Hirohiko Hirochika Hirohiko Hirochika Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences, Ibaraki, Japan Search for more papers by this author Gynheung An Gynheung An National Research Laboratory of Plant Functional Genomics, Department of Life Science, Pohang University of Science and Technology, Kyungbuk, Republic of Korea Search for more papers by this author Julian I Schroeder Corresponding Author Julian I Schroeder Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Author Information Tomoaki Horie1, Alex Costa1, Tae Houn Kim1, Min Jung Han2, Rie Horie1, Ho-Yin Leung1, Akio Miyao3, Hirohiko Hirochika3, Gynheung An2 and Julian I Schroeder 1 1Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA, USA 2National Research Laboratory of Plant Functional Genomics, Department of Life Science, Pohang University of Science and Technology, Kyungbuk, Republic of Korea 3Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences, Ibaraki, Japan *Corresponding author. Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, Room 5214, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0116, USA. Tel.: +1 858 534 7759; Fax: +1 858 534 7108; E-mail: [email protected] The EMBO Journal (2007)26:3003-3014https://doi.org/10.1038/sj.emboj.7601732 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Excessive accumulation of sodium in plants causes toxicity. No mutation that greatly diminishes sodium (Na+) influx into plant roots has been isolated. The OsHKT2;1 (previously named OsHKT1) transporter from rice functions as a relatively Na+-selective transporter in heterologous expression systems, but the in vivo function of OsHKT2;1 remains unknown. Here, we analyzed transposon-insertion rice lines disrupted in OsHKT2;1. Interestingly, three independent oshkt2;1-null alleles exhibited significantly reduced growth compared with wild-type plants under low Na+ and K+ starvation conditions. The mutant alleles accumulated less Na+, but not less K+, in roots and shoots. OsHKT2;1 was mainly expressed in the cortex and endodermis of roots. 22Na+ tracer influx experiments revealed that Na+ influx into oshkt2;1-null roots was dramatically reduced compared with wild-type plants. A rapid repression of OsHKT2;1-mediated Na+ influx and mRNA reduction were found when wild-type plants were exposed to 30 mM NaCl. These analyses demonstrate that Na+ can enhance growth of rice under K+ starvation conditions, and that OsHKT2;1 is the central transporter for nutritional Na+ uptake into K+-starved rice roots. Introduction Sodium (Na+) is an alkali cation, which is not accumulated at large concentrations in most plant cells, in contrast to the alkali cation potassium (K+), which is an essential macronutrient for plant growth. High concentrations of external Na+ inhibit K+ absorption (Rains and Epstein, 1965, 1967b) and elevated Na+ concentrations in plant cells disturb functions of vital enzymes (Murguía et al, 1995) and photosynthesis (Tsugane et al, 1999), leading to Na+ toxicity and cell death. On the other hand, classical plant physiological studies have reported a positive effect of low Na+ concentrations on the growth of many plant species, but the underlying Na+ uptake mechanisms remain unknown (Flowers and Läuchli, 1983). Several genes that encode Na+ permeable transporters/channels have been identified in plants. Biochemical analyses of vacuoles revealed a mechanism for the sequestration of Na+ into vacuoles under salt stress via Na+/H+ antiporters (Blumwald and Poole, 1985, 1987). Genome sequencing of Arabidopsis thaliana led to the identification of the corresponding plant Na+/H+ antiporter genes, AtNHX1 to 6 (Apse et al, 1999; Gaxiola et al, 1999; Yokoi et al, 2002; Aharon et al, 2003). An essential salt tolerance gene of Arabidopsis, salt overly sensitive 1 (SOS1), also belongs to the same H+ exchanger family of transporters (Shi et al, 2000). The SOS1 transporter has been reported to function in long-distance Na+ transport from roots to shoots in xylem parenchyma cells during salt stress (Shi et al, 2002). Electrophysiological analyses using root cortex cells and suspension cultured cells of monocot plants suggest that toxic Na+ influx into roots under high external Na+ concentrations is mediated by voltage-independent channels (VIC) or non-selective cation channels (NSC) (Amtmann et al, 1997; Roberts and Tester, 1997; Tyerman et al, 1997; Buschmann et al, 2000; Davenport and Tester, 2000). VIC/NSC currents have also been reported in Arabidopsis roots and were shown to be downregulated by addition of cAMP and cGMP (Maathuis and Sanders, 2001). Recently, the cyclic nucleotide-gated channel 3 (CNGC3) gene was proposed to mediate non-selective cation uptake in the root of Arabidopsis plants (Gobert et al, 2006). The detailed molecular identities of VIC/NSC, however, remain unknown and no genetic mutations have been isolated in any plant species, to date, that show a strong reduction in Na+ influx into roots. HKT-type transporters have been characterized in several plant and bacterial species (Schachtman and Schroeder, 1994; Rubio et al, 1995; Fairbairn et al, 2000; Uozumi et al, 2000; Horie et al, 2001; Golldack et al, 2002; Garciadeblás et al, 2003; Su et al, 2003; Ren et al, 2005; Tholema et al, 2005). TaHKT2;1 (previously named TaHKT1) (Platten et al, 2006), from wheat, has been shown to mediate at least two transport modes in heterologous expression systems, K+–Na+ co-uptake and Na+ influx at high Na+ concentrations (Rubio et al, 1995; Gassmann et al, 1996). Analyses of the ion transport specificity of AtHKT1;1, previously named AtHKT1, in Arabidopsis and OsHKT2;1 (OsHKT1) in rice revealed that these HKT1 transporters/channels showed a relatively Na+-selective transport activity relative to K+ transport activity in yeast and Xenopus oocytes (Uozumi et al, 2000; Horie et al, 2001; Garciadeblás et al, 2003). Null mutations in AtHKT1;1 (Mäser et al, 2002a; Gong et al, 2004) and a reduced-function mutant allele of AtHKT1;1 (Berthomieu et al, 2003) cause Na+ over-accumulation in shoots, resulting in leaf chlorosis. Detailed 22Na+ influx studies showed that AtHKT1;1 does not mediate Na+ influx into Arabidopsis roots (Berthomieu et al, 2003; Essah et al, 2003). Recent studies have shown that AtHKT1;1 in Arabidopsis and its closest homologue, SKC1 or OsHKT1;5 in rice, function by removing Na+ from the xylem sap, thus reducing Na+ accumulation in leaves (Ren et al, 2005; Sunarpi et al, 2005; Horie et al, 2006). Unlike AtHKT1;1, which is a single-copy gene in Arabidopsis thaliana, seven full-length OsHKT genes were identified in the japonica rice genome based on the completed genome sequence (Garciadeblás et al, 2003). Molecular genetic identification of transporters that mediate Na+ influx into plant roots is crucial for developing models of Na+ uptake mechanisms into plant roots. In this study, we have isolated and characterized OsHKT2;1-disrupted rice plants from a Tos17-tagged population (Hirochika, 1997, 2001; Yamazaki et al, 2001), to determine the physiological role of the OsHKT2;1 transporter in rice. Here, we demonstrate that oshkt2;1 loss-of-function mutant alleles cause a dramatic reduction in Na+ influx into K+-starved rice roots. We further demonstrate that OsHKT2;1 plays an exclusive role in nutritional Na+ uptake into K+-starved rice roots, with an apparent Na+ affinity (Km) of ≈280 to 330 μM. Interestingly, OsHKT2;1-mediated Na+ influx does not cause Na+ toxicity, owing to a rapid downregulation of the OsHKT2;1 transporter upon Na+ stress. Results Isolation of oshkt2;1 mutants and corresponding TosWT plants Ion selectivity analyses of OsHKT2;1 expressed in yeast and Xenopus oocytes showed that OsHKT2;1 functions as a Na+ transporter in these heterologous expression systems (Horie et al, 2001; Mäser et al, 2002b; Garciadeblás et al, 2003). But the in vivo function of OsHKT2;1 in rice remains unknown. We searched the rice Tos17 insertion mutant database (Hirochika, 1997, 2001; Miyao et al, 2003) for a putative OsHKT2;1 gene-disrupted line to be used for uncovering the physiological role of OsHKT2;1 in rice plants. The retrotransposon Tos17 undergoes local transposition events only during tissue culture, but is stable and non-motile in rice plants. We identified five putative OsHKT2;1 insertion alleles. Among them, three oshkt2;1 alleles, named oshkt2;1-1, oshkt2;1-2 and oshkt2;1-3 (Figure 1A), were chosen for further characterization, based on the fertility of plants and the germination rate of seeds of the next generation. In addition, we pursued isolation of related OsHKT2;1 wild-type (WT) control plants, named ‘TosWT’. Each Tos17 mutant line comprises an average of 8–10 insertions, which demands additional WT controls for characterization of mutants. TosWT control lines were isolated from the same seed populations as oshkt2;1-1, oshkt2;1-2 and oshkt2;1-3 by screening for individuals that show no insertion in the OsHKT2;1 gene. Southern hybridization showed polymorphisms in autoradiographs, confirming the insertion in OsHKT2;1 in each line (data not shown). RT–PCR analyses using the primer set shown in Figure 1A showed that mature OsHKT2;1 mRNA is missing in oshkt2;1-1, oshkt2;1-2 plants (Figure 1B) and oshkt2;1-3 (Supplementary Figure 1A). These data show the disruption of the OsHKT2;1 gene in the oshkt2;1-1, oshkt2;1-2 and oshkt2;1-3 mutant lines. Figure 1.Isolation of homozygous Tos17 insertion mutants in the OsHKT2;1 gene. (A) A schematic diagram of oshkt2;1-1, oshkt2;1-2 and oshkt2;1-3 alleles. White boxes represent exons. oshkt2;1-3 has an insertion at the exon–intron boundary. s1 and as3 are primers used for RT–PCR. (B) RT–PCR analyses using cDNA derived from whole plant tissues of oshkt2;1 mutant and WT plants (10-day old plants), which were grown under K+ starvation conditions. Download figure Download PowerPoint oshkt2;1 mutant plants exhibit reduced growth under low Na+ and K+-starved conditions oshkt2;1 and TosWT plants showed normal growth and were indistinguishable from WT cv. Nipponbare plants, either on soil or on ordinary nutrient media (Figure 2A and data not shown). A low-affinity Na+ uptake mode of OsHKT2;1 has been hypothesized based on functional analyses, using heterologous expression systems (Horie et al, 2001). We thus first imposed NaCl stress (50–200 mM) on both soil grown and hydroponically grown plants. However, no notable difference in the visual phenotypes was observed between oshkt2;1 and WT plants (data not shown). Figure 2.oshkt2;1 mutant plants show reduced growth under low Na+ and K+-starved growth conditions. (A) Fresh weights of 25-day-old WT, TosWT2;1-1, TosWT2;1-2, oshkt2;1-1 and oshkt2;1-2 plants, which were grown under 1.25 mM K+ conditions for the last 15 days (n=12; ±s.d.). (B) Fresh weights of WT, TosWT2;1-1, TosWT2;1-2, oshkt2;1-1 and oshkt2;1-2 plants, which were germinated and grown in 1 mM CaSO4 solution for 10 days (n=18; ±s.d.). (C, D) Twenty-five-day-old plants, which were grown under hydroponic conditions for the last 15 days in the presence of 0.5 mM Na+ in a K+-free medium. oshkt2;1 mutant plants showed reduced growth (C) and chlorotic withering of the oldest leaves (D). Representative photographs of plants are shown and were observed in three independent experiments with over 18 total plants of each line. (E) Fresh weights of 25 day-old WT, TosWT2;1-1, TosWT2;1-2, oshkt2;1-1 and oshkt2;1-2 plants, which were grown under the same 0.5 mM Na+ condition for the last 15 days as in (C) and (D) (n=12; ±s.d.). Download figure Download PowerPoint The accumulation of OsHKT2;1 mRNA was reported to dramatically increase in response to K+ starvation (Horie et al, 2001; Garciadeblás et al, 2003). Ten-day old oshkt2;1 plants grown in a 1 mM CaSO4 (K+ deprived) solution showed no visible growth defects and no differences in the fresh weight compared with TosWT plants (Figure 2B, P>0.36 for TosWT2;1-1 versus oshkt2;1-1; P>0.08 for TosWT2;1-2 versus oshkt2;1-2; Supplementary Figure 2A, P>0.22 for TosWT2;1-3 versus oshkt2;1-3). The plants were subsequently grown for 15 additional days in hydroponic culture medium containing 0.5 mM NaNO3 (see Materials and methods). Fifteen days later, oshkt2;1-1, oshkt2;1-2 and oshkt2;1-3 mutant plants showed remarkably reduced growth, accompanied with a withering of the oldest leaf compared with TosWT2;1-1, TosWT2;1-2, TosWT2;1-3 and WT plants (Figure 2C and D and Supplementary Figure 2B and C). Fresh weights of oshkt2;1 mutant plants were reduced approximately by 30–40% compared with TosWT plants (Figure 2E, P<1.6 × 10−5 for TosWT2;1-1 versus oshkt2;1-1; P<7.1 × 10−7 for TosWT2;1-2 versus oshkt2;1-2; Supplementary Figure 2D, P<5.7 × 10−6 for TosWT2;1-3 versus oshkt2;1-3). These data show that the OsHKT2;1 transporter allows improved growth of WT plants under low nutrient K+-starved conditions when 0.5 mM NaNO3 was added to the growth solution. oshkt2;1 mutant plants accumulate less Na+ in both shoots and roots We determined Na+ contents of oshkt2;1 mutant, TosWT and WT plants. Ten-day-old plants grown in 1 mM CaSO4 solution were transferred onto minimal medium containing 0.5 mM Na+ and grown for nine additional days. Interestingly, oshkt2;1-1, oshkt2;1-2 and oshkt2;1-3 mutant plants were found to accumulate considerably less Na+ in both roots and shoots compared with TosWT and WT plants (Figure 3 and Supplementary Figure 3). These results show that OsHKT2;1 functions in Na+ accumulation in roots and also in shoots. Figure 3.oshkt2;1 mutant plants accumulate less Na+ in roots and shoots. Nineteen-day-old plants, which were hydroponically cultured under 0.5 mM Na+- and K+-free conditions for the last 9 days, were used. Na+ contents of roots (A) and shoots (B) were measured by ICP-OES. Error bars represent standard deviations (n=6). Download figure Download PowerPoint OsHKT2;1 expression in cortical and endodermal cells in roots and in vascular bundle regions in leaves Transgenic rice plants carrying the 1.6 kb OsHKT2;1 promoter-β-glucuronidase (GUS) or green fluorescence protein (GFP) gene constructs were produced in order to determine the expression pattern of OsHKT2;1. Strong GUS signals were found in the main root but not in the root tip region (Figure 4A and B). Sections of GUS-stained roots further showed that cortex cells and endodermis cells were strongly stained (Figure 4C, see red stain). Sections of GUS-stained leaves showed the expression of OsHKT2;1 in vascular bundle regions (Figure 4D and E). Figure 4.OsHKT2;1 gene expression in the cortex and endodermis of K+-starved roots and in leaf vascular bundles. Transgenic rice plants expressing GUS or GFP reporter genes under the control of a 1.6 kb OsHKT2;1 promoter were grown in 1 mM CaSO4 solution in the presence of hygromycin. (A, B) Strong GUS staining was detected in main roots (A) but not in root tip regions (B). (C) A dark-field microscopic image of sections derived from GUS-stained main roots. Sections of GUS-stained (red staining) main roots showed strong signals in cortical and endodermal cells. (D, E) Dark-field microscopic images of sections derived from GUS-stained leaves. The sections showed strong GUS signals (red stain) in vascular bundle regions. In (E) is an enlarged image of a section of the region shown in (D). Note that the reddish staining in (C–E) shows GUS signals, as these were obtained using dark-field microscopy. (F) A 3D reconstruction image of GFP fluorescence derived from K+-starved roots of OsHKT2;1 promoter-GFP plants, showing strong GFP fluorescence in root cortex cells. (G) A 3D reconstruction image of propidium iodide fluorescence derived from K+-starved roots of the same plant shown in (F). (H) Combined images of GFP and propidium iodide fluorescence shown in (F) and (G). (I) An enlarged image of K+-starved roots shown in (H). (J, K) Combined images of GFP and propidium iodide fluorescence derived from 10-day-old OsHKT2;1 promoter-GFP plants grown in 1 mM CaSO4 solution supplemented with either 30 mM K+ (J) or 30 mM Na+ (K). (L) GFP fluorescence from three different conditions were quantified and normalized relative to propidium iodide fluorescence. Fluorescence intensities of GFP and propidium iodide in (J) were measured using three independent plants for each ionic condition (±s.d.). Download figure Download PowerPoint Tissue expression of OsHKT2;1 was further analyzed using transgenic plants expressing GFP driven by the 1.6 kb OsHKT2;1 promoter. Roots of hygromycin B-resistant plants were briefly stained with propidium iodide (Swarup et al, 2004). Fluorescence was analyzed by confocal microscopy. A strong GFP fluorescence was found in root cortex cells of K+-starved main roots (Figure 4 F, H and I). The tissue specificity of OsHKT2:1 expression in K+-starved roots of japonica rice presented in this study overlaps with, but also slightly differs from the expression pattern in indica rice varieties (Golldack et al, 2002). The reason for this discrepancy could be the difference in the regulation of OsHKT2;1 expression between the two rice varieties and experimental conditions. To analyze the transcriptional regulation of OsHKT2;1 in response to external K+ and Na+ concentrations, OsHKT2;1 promoter-GFP plants were grown in 1 mM CaSO4 solution supplemented with either 30 mM K+ or 30 mM Na+. Addition of 30 mM K+ or 30 mM Na+ led to substantial reductions in GFP fluorescence of root cells (Figure 4J and K) compared with those of K+-starved roots (Figure 4H). GFP and propidium iodide fluorescence intensities were quantified based on 3D reconstruction images obtained by z-series stacking and GFP fluorescence intensities and normalized to propidium iodide fluorescence intensities. The average GFP fluorescence intensity of K+-starved roots was approximately five times higher than that of 30 mM K+- or 30 mM Na+-treated roots (Figure 4L). Taken together, these results demonstrate that the OsHKT2;1 gene is mainly expressed at the cortex cell layer and the endodermal cell layer of K+-starved japonica rice roots, and that OsHKT2;1 gene expression is controlled by external K+ and Na+ concentrations at the transcriptional level. oshkt2;1 mutant plants accumulate less Na+ in the xylem sap We next determined Na+ contents of the xylem sap. Ten-day-old oshkt2;1 mutant and TosWT plants grown in 1 mM Ca2+ solution were transferred to hydroponic nutrient medium containing 0.5 mM Na+ and cultured for 2 days. Xylem sap was collected and Na+ concentrations were determined by inductively coupled plasma-optic emission spectroscopy (ICP-OES). The results clearly showed that oshkt2;1-1 and oshkt2;1-2 mutant plants contained much less Na+ compared with TosWT plants (Figure 5). Na+ levels in the xylem sap of oshkt2;1-1 were reduced by approximately 81.0% compared with TosWT2;1-1 (Figure 5A, P<0.0008 for oshkt2;1-1 versus TosWT2;1-1) and those of oshkt2;1-2 were reduced by approximately 89% compared with TosWT2;1-2 (Figure 5B, P<0.001 for oshkt2;1-2 versus TosWT2;1-2). Figure 5.oshkt2;1 mutant plants accumulate less Na+ in the xylem sap. Ten-day-old seedlings grown in 1 mM CaSO4 solution were subsequently grown in 0.5 mM Na+- and K+-free conditions two more days. Xylem sap was collected from WT, TosWT2;1-1, TosWT2;1-2, oshkt2;1-1 and oshkt2;1-2. Na+ contents were measured by ICP-OES. (A) TosWT2;1-1 versus oshkt2;1--1. (B) TosWT2;1-1 versus oshkt2;1-1. Xylem sap extractions for (A) were performed at a different time of year than (B). Error bars represent standard error (n=8). Download figure Download PowerPoint OsHKT2;1 localization at the plasma membrane To gain insight into the subcellular localization of the OsHKT2;1 protein in plant cells, a chimeric EGFP-OsHKT2;1 cDNA was constructed and placed downstream of the CaMV 35S promoter. Transient expression analyses were performed using tobacco leaf mesophyll cells. A bright fluorescence bordering the cell was found in mesophyll cells expressing EGFP-OsHKT2;1 (Figure 6A). Cells were subsequently stained with the plasma membrane marker FM4-64 (Bolte et al, 2004). As shown in Figure 6B, FM4-64-derived red fluorescence was detected at the border of cells. Combined images showed an overlap of the two different fluorescence emissions at the border of cells (Figure 6C and E), indicating that the EGFP-OsHKT2;1 protein localizes at the plasma membrane. Fluorescence images derived from EGFP and FM4-64 were analyzed in control experiments. In controls, green fluorescence was more broadly observed in the cell, including in the nucleus (Figure 6D), compared with EGFP-OsHKT2;1-derived fluorescence (Figure 6A). Native red chloroplast fluorescence showed the localization of chloroplasts, in addition to the plasma membrane staining with FM4-64 (Figure 6D). Combined images showed that EGFP-derived fluorescence and FM4-64-derived fluorescence did not overlap at the plasma membrane (Figure 6D and F), indicating that the EGFP control protein does not localize at the plasma membrane. Figure 6.OsHKT2;1 localizes at the plasma membrane of plant cells. EGFP-OsHKT2;1 cDNA and EGFP were transiently expressed in protoplasts of tobacco mesophyll cells (A–F) and Arabidopsis epidermal cells (G, H) under the control of the 35S promoter. Fluorescence was analyzed by confocal microscopy. (A) GFP fluorescence of tobacco mesophyll cell protoplast expressing EGFP-OsHKT2;1. (B) FM4-64 plasma membrane marker fluorescence emitted from the same protoplast shown in (A). (C) Combined images of GFP and FM4-64 fluorescence shown in (A) and (B). (D) Combined images of GFP control and FM4-64 fluorescence, derived from cells harboring a control construct. Note that native red chloroplast autofluorescence was observed in addition to red FM4-64 fluorescence at the plasma membrane (D, F). (E) An enlarged image of (C). (F) An enlarged image of (D). (G) GFP fluorescence of Arabidopsis epidermal cells expressing EGFP-OsHKT2;1. (H) GFP fluorescence of Arabidopsis epidermal cells harboring a GFP control construct. Download figure Download PowerPoint Subcellular localization of EGFP-OsHKT2;1 was also tested using Arabidopsis leaf epidermal cells. An EGFP-OsHKT2;1 and a control construct were introduced into epidermal cells by particle bombardment. Epidermal cells expressing the EGFP-OsHKT2;1 construct showed fluorescence at the border of cells (Figure 6G). In contrast, cells harboring a control construct showed ubiquitous fluorescence in the cytoplasm and in the nucleus (Figure 6H). Taken together, these transient expression analyses provide evidence that the OsHKT2;1 transporter localizes at the plasma membrane of plant cells. Severe disruption of Na+ influx in oshkt2;1 mutant roots Less Na+ accumulation in shoots and roots of oshkt2;1 mutants (Figure 3) and localization analyses of OsHKT2;1 (Figures 4 and 6) indicated that OsHKT2;1 may mediate Na+ influx into rice roots. In order to test this hypothesis, we performed short-term tracer influx experiments using 22Na+. Time-dependent Na+ influx into roots of intact rice plants was monitored at 0.1 mM external Na+ using 10-day-old oshkt2;1-1, oshkt2;1-2, oshkt2;1-3, TosWT2;1-1, TosWT2;1-2, TosWT2;1-3 and WT plants, which were grown in 1 mM CaSO4 solution. Interestingly, short-term Na+ influx into rice roots at 0.1 mM external Na+ was almost completely abolished in oshkt2;1-1, oshkt2;1-2 and oshkt2;1-3 plants, while TosWT2;1-1, TosWT2;1-2, TosWT2;1-3 and WT plants exhibited similar Na+ influx time courses (Figure 7A and Supplementary Figure 4A). Figure 7.Short-term 22Na+ influx analyses show that OsHKT2;1 functions as a major Na+ uptake pathway in K+-starved rice roots. Plants were grown in 1 mM CaSO4 solution for 10 days. Time-dependent and concentration-dependent 22Na+ influx experiments were performed using 22NaCl as a tracer. (A) Time-dependent Na+ influx into roots of WT, TosWT2;1-1, TosWT2;1-2, oshkt2;1-1 and oshkt2;1-2 at 0.1 mM external Na+ (n=3; ±s.d.). (B) Concentration-dependent Na+ influx into roots of TosWT2;1-1, TosWT2;1-2, oshkt2;1-1 and oshkt2;1-2 at 1, 5, 10, 25, 50, 100 and 200 μM external Na+ (n=3; ±s.d.). (C) Concentration-dependent Na+ influx into roots of TosWT2;1-1, TosWT2;1-2, oshkt2;1-1 and oshkt2;1-2 at 1–5000 μM external Na+ (n=3; ±s.d.). Curves in (C) are plotted separately for each WT and oshkt2;1 mutant allele derived from Michaelis–Menten analyses of the data (Table Ia). (D) OsHKT2;1-dependent Na+ influx rates of rice roots. Differences in Na+ influx rates between TosWT and oshkt2;1 mutant plants are shown (±s.d.). Curves in (D) were derived from Michaelis–Menten analyses (Table Ib). (E) Time-dependent Rb+ (K+) influx into roots of WT, TosWT2;1-1, TosWT2;1-2, oshkt2;1-1 and oshkt2;1-2 at 0.1 mM external Rb+ (n=6; ±s.d.). Download figure Download PowerPoint Detailed concentration-dependent Na+ influx studies were performed in the ‘high-affinity’ Na+ uptake range, at 1, 5, 10, 25, 50, 100 and 200 μM external Na+, using oshkt2;1 mutant plants and TosWT plants. oshkt2;1-1 and oshkt2;1-2 plants showed severe Na+ influx reductions compared with TosWT2;1-1 and TosWT2;1-2 plants (Figure 7B; 92.7% reduction at 200 μM). We further characterized 22Na+ influx kinetics of oshkt2;1 mutants and TosWT plants at higher external Na+ concentrations of up to 5000 μM." @default.
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• W2115928499 title "Rice OsHKT2;1 transporter mediates large Na+ influx component into K+-starved roots for growth" @default.
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