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BMC Plant Biol
2016 Jan 19;16:22. doi: 10.1186/s12870-016-0709-4.
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OsHKT1;4-mediated Na(+) transport in stems contributes to Na(+) exclusion from leaf blades of rice at the reproductive growth stage upon salt stress.
Suzuki K
,
Yamaji N
,
Costa A
,
Okuma E
,
Kobayashi NI
,
Kashiwagi T
,
Katsuhara M
,
Wang C
,
Tanoi K
,
Murata Y
,
Schroeder JI
,
Ma JF
,
Horie T
.
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BACKGROUND: Na(+) exclusion from leaf blades is one of the key mechanisms for glycophytes to cope with salinity stress. Certain class I transporters of the high-affinity K(+) transporter (HKT) family have been demonstrated to mediate leaf blade-Na(+) exclusion upon salinity stress via Na(+)-selective transport. Multiple HKT1 transporters are known to function in rice (Oryza sativa). However, the ion transport function of OsHKT1;4 and its contribution to the Na(+) exclusion mechanism in rice remain to be elucidated.
RESULTS: Here, we report results of the functional characterization of the OsHKT1;4 transporter in rice. OsHKT1;4 mediated robust Na(+) transport in Saccharomyces cerevisiae and Xenopus laevis oocytes. Electrophysiological experiments demonstrated that OsHKT1;4 shows strong Na(+) selectivity among cations tested, including Li(+), Na(+), K(+), Rb(+), Cs(+), and NH4 (+), in oocytes. A chimeric protein, EGFP-OsHKT1;4, was found to be functional in oocytes and targeted to the plasma membrane of rice protoplasts. The level of OsHKT1;4 transcripts was prominent in leaf sheaths throughout the growth stages. Unexpectedly however, we demonstrate here accumulation of OsHKT1;4 transcripts in the stem including internode II and peduncle in the reproductive growth stage. Moreover, phenotypic analysis of OsHKT1;4 RNAi plants in the vegetative growth stage revealed no profound influence on the growth and ion accumulation in comparison with WT plants upon salinity stress. However, imposition of salinity stress on the RNAi plants in the reproductive growth stage caused significant Na(+) overaccumulation in aerial organs, in particular, leaf blades and sheaths. In addition, (22)Na(+) tracer experiments using peduncles of RNAi and WT plants suggested xylem Na(+) unloading by OsHKT1;4.
CONCLUSIONS: Taken together, our results indicate a newly recognized function of OsHKT1;4 in Na(+) exclusion in stems together with leaf sheaths, thus excluding Na(+) from leaf blades of a japonica rice cultivar in the reproductive growth stage, but the contribution is low when the plants are in the vegetative growth stage.
Fig. 1. OsHKT1;4 expression increases Na+ hypersensitivity of yeast cells promoting Na+ over-accumulation. The OsHKT1;4 cDNA from rice cultivar Nipponbare was constitutively expressed in strain G19 of S. cerevisiae under the control of the GAP promoter. a, G19 cells harboring the empty vector or expressing OsHKT1;4 were grown on the arginine phosphate (AP) medium containing 1 mM KCl with or without the addition of 50 mM NaCl. 1:10 serial dilutions of each G19 line with a starting OD600 of 0.1 were prepared and spotted on AP plates as described previously [51]. All plates were incubated at 30 °C, and photographs were taken after 5 days. b, Na+ content of G19 lines that were incubated in synthetic complete (SC) medium supplemented with 25 mM NaCl for the indicated time period (n = 6, ±SD). The Welch’s-t test was used for the statistical analysis and asterisks indicate a significant difference compared with vector-harboring control cells at each time point (P < 0.001)
Fig. 2. Analyses of OsHKT1;4-mediated ion transport by two electrode voltage clamp experiments using X. laevis oocytes. a, A representative confocal microscopic image of green fluorescence from oocytes injected with 3 ng of EGFP-OsHKT1;4 cRNA. b, Red fluorescence of the same oocyte shown in a, treated with the plasma membrane marker FM4-64. c, Overlay image of a and b. d, A plot profile of EGFP (green trace) and FM4-64 (red trace) fluorescence, corresponding to the boxed region in white in panel c and the white line shown in the inset image. Cyt and Ext represent the cytosolic side and the external side of the plasma membrane of the oocyte, respectively. e, Current profiles obtained using an oocyte injected with either 3 ng of EGFP-OsHKT1;4 cRNA (cell shown in a) or water in the presence of 2 mM Na+ with a step pulse protocol described below. Zero current levels are shown by arrows. f, Current profiles obtained using an oocyte injected with either 3 ng of OsHKT1;4 cRNA or water in the presence of 2 mM or 20 mM Na+ with a step pulse protocol described below. g, Current–voltage relationships of oocytes injected with 3 ng of OsHKT1;4 cRNA or water, bathed in solutions supplemented with 2 mM or 20 mM Na+ (n = 6-7 for OsHKT1;4 cRNA-injected oocytes and n = 3-4 for water injected oocytes, ±SE). Voltage steps from +30 to −150 mV were applied with a holding potential of −40 mV
Fig. 3. Monovalent cation selectivity of OsHKT1;4 expressed in X. laevis oocytes. Current–voltage relationships of oocytes injected with 3 ng of OsHKT1;4 cRNA or water (inset), bathed in solutions containing each 10 mM chloride salt indicated in the graph (n = 9-11, ±SE). Voltage steps from +30 to −150 mV were applied with a holding potential of −40 mV
Fig. 4. Subcellular localization of EGFP-OsHKT1;4 in rice protoplasts. EGFP-OsHKT1;4 protein was transiently expressed in protoplasts of rice seedlings under the control of the cauliflower mosaic virus 35S promoter. Fluorescence was analyzed by confocal microscopy. a, EGFP fluorescence (green) from a single focal plane of a representative rice protoplast co-expressing chimeric EGFP-OsHKT1;4 protein and CBL1n-OFP. b, OFP fluorescence (red) from the same protoplast as shown in a. c, Overlay image of a and b. d, Bright field image of the protoplast shown in a. e, EGFP fluorescence (green) from a single focal plane of a representative rice protoplast expressing free EGFP protein. f, OFP fluorescence from CBL1n-OFP, co-expressed in the same protoplast as shown in e. g, Overlay image of e and f. h, Bright field image of the protoplast shown in e. i, EGFP fluorescence (green) from a single focal plane of a representative rice protoplast co-expressing chimeric EGFP-OsHKT1;4 protein and ER-mCherry. j, mCherry fluorescence (red) marking the endoplasmic reticulum (ER) of the same protoplast as shown in i. k, Overlay image of i and j. l, Bright field image of the protoplast shown in i. m, EGFP fluorescence (green) from an internal single focal plane of a representative rice protoplast co-expressing chimeric EGFP-OsHKT1;4 protein and Golgi-mCherry (white arrows indicate punctate structures labeled by EGFP). n, mCherry fluorescence marking the Golgi apparatus (GA) of the same protoplast as shown in m (white arrows indicate typical GA structures). o, Overlay image of m and n showing partial co-localization of EGFP and mCherry fluorescence (corresponding to GA structures marked by arrows in N) with some punctate-like structures labeled with the EGFP alone. p, Bright field image of the protoplast shown in m
Fig. 5. Growth stage-dependent expression of OsHKT1;4 in various tissues of a japonica rice cultivar Nipponbare. RNA samples from various tissues were prepared from rice plants at the indicated growth stages as described previously [38], and quantitative real-time PCR analysis was performed using specific primers for OsHKT1;4 (n = 3, ±SD). Relative expression of OsHKT1;4 is shown, with its relative expression in the lower leaf sheath of 14-week-old (flowering) plants to 1
Fig. 6. Expression profile of OsHKT1;4 in various tissues of Nipponbare rice plants. Quantitative real-time PCR analyses were performed using RNA samples derived from various tissues. The expression of OsHKT1;4 and an internal control OsSMT3 was determined. The level of OsHKT1;4 expression was normalized using OsSMT3 expression. a, Relative expression of OsHKT1;4 in tissues of hydroponically grown Nipponbare plants in the vegetative growth phase (3-weeks-old) with or without 50 mM NaCl treatments for 3 days is shown setting its expression in the basal node without stress to 1 (n = 6, ±SD). LB6: 6th leaf blade; LB5: 5th leaf blade; LS6: 6th leaf sheath; LS5: 5th leaf sheath; BN: basal node; R: root. b, Relative expression of OsHKT1;4 in tissues of soil-grown Nipponbare plants in the reproductive growth phase with or without NaCl treatments (25–100 mM) for more than 1 month is shown setting its expression in the flag leaf sheath to 1 (n = 5-6, ±SD). FLB: flag leaf blade; FLC: flag leaf sheath; P: peduncle; N I: node I; IN II: internode II; N II: node II. Note that insets in a and b show the data sets from some tissues in a smaller scale than that in the main graphs. c, Relative OsHKT1;4 expression in enlarged vascular bundles (EBVs) of node I, diffuse vascular bundles (DVBs) of node I, and the basal stem (BS) is shown setting its expression in the basal stem to 1 (n = 6, ±SD). EVBs and DVBs were excised from node I by laser microdissection (LMD). N.D. indicates “not detected”. The Welch’s-t test was used for the statistical analysis: * P < 0.05, ** P < 0.01, *** P < 0.001 vs. no stress condition (a, b) or basal stem (c)
Fig. 7. Phenotypic analysis of OsHKT1;4 RNAi plants in the reproductive growth stage. Nipponbare wild-type and two independent OsHKT1;4 RNAi plants were planted in the same pot filled with paddy-filed soil and grown for approximately 3 months. When the plants started heading, NaCl treatment was initiated by gradually increasing the concentration of Na+ in tap water from 25 mM to 100 mM for more than a month. Tissues of the upper parts were excised and washed briefly by the ultrapure water. Ion contents were determined using an inductively coupled plasma optical emission spectrometer (n = 23-28, ±SD). a, Na+ content in each tissue. b, K+ content in each tissue. FLB: flag leaf blade; FLC: flag leaf sheath; P: peduncle; N I: node I; IN II: internode II; N II: node II. The Welch’s-t test was used for the statistical analysis: * P < 0.01, ** P < 0.001 vs. Nipponbare wild-type
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