Soil volatilomics uncovers tight linkage between soybean presence and soil omics profiles in agricultural fields.

Securing a stable food supply and achieving sustainable agricultural production are essential for mitigating future food insecurity. Soil metabolomics is a promising tool for capturing soil status, which is a critical issue for future sustainable food security. This study aims to provide deeper insights into the status of soybean-grown fields under varying soil conditions over three years by employing comprehensive soil volatile organic compound (VOC) profiling, also known as soil volatilomics. Profiling identified approximately 200 peaks in agricultural fields. The soil of soybean-presented plots exhibited markedly higher VOC levels than those of non-soybean plots during the flowering season. Pentanoic acid, 2,2,4-trimethyl-3-carboxyisopropyl, isobutyl ester, a discriminative soil VOC, was identified through multivariate data analysis as a distinctively present VOC in fields with or without soybean plants during the flowering period. Soil VOC profiles exhibited strong correlations with soil-related omics datasets (soil ionome, microbiome, metabolome, and physics) and no significant correlations with root microbiome and rhizosphere chemicals. These findings indicate that soil VOC profiles could serve as a valuable indicator for assessing soil status, thereby supporting efforts to ensure future global food security.

Cadmium accumulation dynamics in the rice endosperm during grain filling revealed by autoradiography.

Cadmium (Cd) is one of the environmental pollutants contaminated in our food. Several previous reports showed that rice polishing cannot be efficient to reduce Cd content in white rice, implying the characteristic Cd distribution in rice grain. However, Cd distribution has not been fully elucidated so far. Herein, 109Cd radiotracer experiment was performed using the rice seedlings at various time points after flowering to obtain autoradiographs of the brown rice to visually understand the Cd transport and distribution during the grain-filling process. It was shown that 109Cd accumulated in the outermost area of the brown rice, and also in the middle part of the starchy endosperm, resulting in the appearance of the double circle distribution pattern, which was not observed in the autoradiographs of 65Zn. The inner circle of 109Cd located around the center of the endosperm was developed particularly at around 8 and 10 days after flowering. After this period, 109Cd started to deposit at the outer part of the endosperm, which was also found in the autoradiograph of 14C-sucrose. Considering the physiology of grain development, the contribution of water transport and protein synthesis in the endosperm on the characteristic Cd distribution pattern was hypothesized.

Phosphate starvation response precedes abscisic acid response under progressive mild drought in plants.

Drought severely damages crop production, even under conditions so mild that the leaves show no signs of wilting. However, it is unclear how field-grown plants respond to mild drought. Here, we show through six years of field trials that ridges are a useful experimental tool to mimic mild drought stress in the field. Mild drought reduces inorganic phosphate levels in the leaves to activate the phosphate starvation response (PSR) in soybean plants in the field. Using Arabidopsis thaliana and its mutant plants grown in pots under controlled environments, we demonstrate that PSR occurs before abscisic acid response under progressive mild drought and that PSR plays a crucial role in plant growth under mild drought. Our observations in the field and laboratory using model crop and experimental plants provide insight into the molecular response to mild drought in field-grown plants and the relationship between nutrition and drought stress response.

Mutations in RZF1, a zinc-finger protein, reduce magnesium uptake in roots and translocation to shoots in rice.

Magnesium (Mg) homeostasis is critical for maintaining many biological processes, but little information is available to comprehend the molecular mechanisms regulating Mg concentration in rice (Oryza sativa). To make up for the lack of information, we aimed to identify mutants defective in Mg homeostasis through a forward genetic approach. As a result of the screening of 2,825 M2 seedlings mutated by ion-beam irradiation, we found a rice mutant that showed reduced Mg content in leaves and slightly increased Mg content in roots. Radiotracer 28Mg experiments showed that this mutant, named low-magnesium content 1 (LMGC1), has decreased Mg2+ influx in the root and Mg2+ translocation from root to shoot. Consequently, LMGC1 is sensitive to the low Mg condition and prone to develop chlorosis in the young mature leaf. The MutMap method identified a 7.4-kbp deletion in the LMGC1 genome leading to a loss of two genes. Genome editing using CRISPR-Cas9 further revealed that one of the two lost genes, a gene belonging to the RanBP2-type zinc-finger family that we named RanBP2-TYPE ZINC FINGER1 (OsRZF1), was the causal gene of the low Mg phenotype. OsRZF1 is a nuclear protein and may have a fundamental role in maintaining Mg homeostasis in rice plants.

Mutagenesis analysis of GMN motif in Arabidopsis thaliana Mg2+ transporter MRS2-1.

Magnesium is an important nutrient for plants, but much is still unknown about plant Mg2+ transporters. Combining with the structural prediction of AlphaFold2, we used mutagenesis and 28Mg uptake assay to study the highly conserved "GMN" motif of Arabidopsis thaliana MRS2-1 (AtMRS2-1) transporter. We demonstrated that the glycine and methionine in GMN motif are essential for AtMRS2-1 to transport Mg2+.

Distinct Functions of the Atypical Terminal Hydrophilic Domain of the HKT Transporter in the Liverwort Marchantia polymorpha.

K+/Na+ homeostasis is important for land plants, particularly under salt stress. In this study, the structure and ion transport properties of the high-affinity K+ transporter (HKT) of the liverwort Marchantia polymorpha were investigated. Only one HKT gene, MpHKT1, was identified in the genome of M. polymorpha. Phylogenetic analysis of HKT proteins revealed that non-seed plants possess HKTs grouped into a clade independent of the other two clades including HKTs of angiosperms. A distinct long hydrophilic domain was found in the C-terminus of MpHKT1. Complementary DNA (cDNA) of truncated MpHKT1 (t-MpHKT1) encoding the MpHKT_Δ596-812 protein was used to examine the functions of the C-terminal domain. Both MpHKT1 transporters fused with enhanced green fluorescent protein at the N-terminus were localized to the plasma membrane when expressed in rice protoplasts. Two-electrode voltage clamp experiments using Xenopus laevis oocytes indicated that MpHKT1 mediated the transport of monovalent alkali cations with higher selectivity for Na+ and K+, but truncation of the C-terminal domain significantly reduced the transport activity with a decrease in the Na+ permeability. Overexpression of MpHKT1 or t-MpHKT1 in M. polymorpha conferred accumulation of higher Na+ levels and showed higher Na+ uptake rates, compared to those of wild-type plants; however, phenotypes with t-MpHKT1 were consistently weaker than those with MpHKT1. Together, these findings suggest that the hydrophilic C-terminal domain plays a unique role in the regulation of transport activity and ion selectivity of MpHKT1.

Cesium-137 stored on and discharged from banks of an agricultural canal in Iitate, Fukushima.

This study aimed to investigate the dynamics of 137Cs around banks along an agricultural canal for paddy fields in Iitate, Fukushima, Japan. Five plots (2.4-12.6 m2) on the banks were monitored intermittently during six time periods from May 2018 to November 2019. We directly collected runoff water samples discharged from the banks followed by partitioning it into particulate and dissolved fractions and determining 137Cs in them. To investigate the source of 137Cs in the runoff water, we sequentially extracted 137Cs in various chemical forms from litter samples collected on the banks. The results showed that the discharge rates of the dissolved 137Cs per unit area from the plots were lower than those observed at the downstream of the agricultural canal, whereas more than 50% of the 137Cs discharged from the plots was in the dissolved fraction. Moreover, the results indicate that 137Cs stored in the standing plants and the litter was the primary source of the dissolved 137Cs discharged into the agricultural canal. The concentrations of the water-soluble 137Cs in the litter per plot area may have been retained by the sufficiently higher concentrations of 137Cs in litter in other chemical forms and those in the standing plants, which are the source of the litter.

Radiocesium transfer rates among pigs fed haylage contaminated with low levels of cesium at two differentiation stages.

The objective of this study was to determine the radiocesium transfer rates of pigs fed haylage contaminated with low levels of cesium at different growth stages. We measured the body weight of juvenile and adult pigs during the treatment period to confirm their health status. We also performed pig blood hematologic and biochemical analyses at both growth stages. To our knowledge, this is the first study to report pig radiocesium transfer coefficient rates after 1 month of chronic oral treatment, which is the period assumed to be required for body equilibrium under a diet of radiocesium-contaminated food. The results showed higher radiocesium retention rates in the kidneys, liver, spleen, genitals, psoas major, bladder, thyroid, and urine than in the blood and bone (tibia and femur) of pigs at both growth stages. The radiocesium retention levels were generally higher in juvenile pigs than in adult pigs, with the highest transfer coefficient ratio in the kidneys (16.2%).

Seasonal changes in radiocesium and potassium concentrations in current-year shoots of saplings of three tree species in Fukushima, Japan.

We studied seasonal changes in radiocesium (137Cs) activity and potassium concentrations in current-year leaves and branches of Pinus densiflora (naturally regenerated saplings), Cryptomeria japonica (planted saplings) and Quercus serrata (planted saplings and coppice shoots) in Fukushima, Japan. We collected current-year shoots from 10 individuals of each species over two growing seasons at intervals of 1-4 months, between June 2016 and December 2017. For the deciduous species Q. serrata, we also collected dead leaves that remained attached to branches in December to investigate reabsorption of 137Cs. All collected shoots were divided into leaves and branches, oven-dried, and ground; dry weights of each sample were recorded. 137Cs activity concentrations were measured using a germanium semiconductor detector. Potassium concentrations were quantified using inductively coupled plasma optical emission spectrometry (ICP-OES). Increases in dry weight were observed in both leaves and branches between May/June and August; growth then slowed considerably and virtually ceased after October. Clear seasonal changes in 137Cs activity concentrations were observed in both 2016 and 2017, regardless of tree species. Concentrations were higher in young leaves and branches during May and June, then decreased and changed relatively little from August to winter. Reduced 137Cs activity concentrations in dead leaves of Q. serrata were observed only in December 2017 (approximately 15% lower than in October). This reduction may indicate reabsorption of 137Cs in leaves prior to shedding. The changes in potassium concentrations were similar to those in 137Cs in both years. Potassium concentrations were higher in young leaves than in mature leaf and branch samples collected later in the year. A reduction of about 50% in the potassium concentrations in dead leaves of Q. serrata was also observed in December. A positive relationship between 137Cs and potassium concentrations in leaves and branches was observed in all species, except for planted Q. serrata. This relationship may indicate that 137Cs moves in tree shoots with potassium. Leaf and branch weight correlated negatively with 137Cs and potassium concentrations. Reduced concentrations may indicate dilution of these elements as a result of biomass increases over the growing season. Our results imply that irrespective of species, 137Cs exhibits seasonal variations resulting from dilution; these variations correspond with trends in potassium, with higher levels in young organs and decreased levels in older organs.

Short-Term Magnesium Deficiency Triggers Nutrient Retranslocation in Arabidopsis thaliana.

Magnesium (Mg) is essential for many biological processes in plant cells, and its deficiency causes yield reduction in crop systems. Low Mg status reportedly affects photosynthesis, sucrose partitioning and biomass allocation. However, earlier physiological responses to Mg deficiency are scarcely described. Here, we report that Mg deficiency in Arabidopsis thaliana first modified the mineral profile in mature leaves within 1 or 2 days, then affected sucrose partitioning after 4 days, and net photosynthesis and biomass production after 6 days. The short-term Mg deficiency reduced the contents of phosphorus (P), potassium, manganese, zinc and molybdenum in mature but not in expanding (young) leaves. While P content decreased in mature leaves, P transport from roots to mature leaves was not affected, indicating that Mg deficiency triggered retranslocation of the mineral nutrients from mature leaves. A global transcriptome analysis revealed that Mg deficiency triggered the expression of genes involved in defence response in young leaves.

Potassium supply reduces cesium uptake in Konara oak not by an alteration of uptake mechanism, but by the uptake competition between the ions.

Radiocesium contamination of forests has been a severe problem after the Fukushima Daiichi Nuclear Power Plant accident in 2011. Bed logs of Konara oak (Quercus serrata Murray), used for mushroom cultivation, were an economically important product from the forests prior to their contamination. One of the potential countermeasures to reduce radiocesium content in trees is potassium fertilization, but the evidence for the effect of K+ in reducing Cs+ uptake has not been obtained yet in the woody plant. Therefore, we investigated the ability of rhizospheric K+ to suppress uptake and translocation of Cs+ in Konara oak seedlings through hydroponic experiments in order to clarify the effect of K+. Elemental analysis showed that the seedlings cultivated for 4 weeks under low-K (K+ = 50 µM) contained higher amount of Cs comparing to the seedlings cultivated under high-K (K+ = 3 mM). Then, the uptake rate of Cs+ and K+ in the seedlings from the solution having 50 µM K+ and 0.1 µM Cs+ was calculated using radioactive 137Cs+ and 42K+ to evaluate the effect of growth condition on the ion uptake mechanism. The interference between Cs+ and K+ at the site of root uptake was also evaluated based on the Cs+ and K+ uptake rates at K+ concentrations of 50 µM, 200 µM, and 3 mM in the seedlings grown under the medium-K (K+ = 200 µM) condition. As a result, the Cs+ uptake rate at 50 µM K+ was not influenced by the growth condition, whereas Cs+ uptake decreased when the uptake solution itself was supplemented with 3 mM K+. In addition, the Cs/K ratio in the seedlings was found to rise to exceed the Cs/K ratio in the culture solution as the rhizospheric K+ concentration increased, which was in contrast with previous findings in herbaceous plants. Our experiments demonstrated the first direct evidence for woody plants that a high K+ concentration can suppress Cs accumulation in Konara oak and that it was derived from competition for uptake between K+ and Cs+ in the rhizosphere, not from the growth K+ condition.

Visualization of 14CO2 gas fixation by plants.

Using the real-time radioisotope imaging system (RRIS), we present the carbon dioxide gas fixation process of a soybean plant applying the 14C-labeled gas. When 14CO2 gas was supplied to the selected mature leaf, the fixed carbon, photosynthate, was transferred and accumulated to the younger leaves preferentially within 24 h. When 14CO2 gas was supplied to the younger leaves, fixed carbon was hardly moved. In the case of the pods, fixed 14CO2 gas in the leaf was preferentially transferred to the closest pod.

Magnesium uptake characteristics in Arabidopsis revealed by 28Mg tracer studies.

MAIN CONCLUSION: The Mg2+ uptake system in Arabidopsis roots is Gd3+- and Fe2+-sensitive, and responds to a changing Mg2+ concentration within 1 h with the participation of AtMRS2 transporters. Magnesium (Mg2+) absorption and the mechanism regulating its activity have not been clarified yet. To address these issues, it is necessary to reveal the characteristics of Mg2+ uptake in roots. Therefore, we first investigated the Mg2+ uptake characteristics in roots of 1-week-old Arabidopsis plants using 28Mg. The Mg2+ uptake system in roots was up-regulated within 1 h in response to the low Mg2+ condition. This induction was inhibited in Arabidopsis "mitochondrial RNA splicing 2/magnesium transport" mutants atmrs2-4/atmgt6 and atmrs2-7/atmgt7, while the expression of AtMRS2-4/AtMGT6 and AtMRS2-7/AtMGT7 genes in the Arabidopsis wild-type was not responsive to Mg2+ conditions. In addition, the Mg deficiency-induced Mg2+ uptake system was shut-down within 5 min when Mg2+ was resupplied to the environment. An inhibition study showed that the constitutive mechanism functioning in Mg2+ uptake under Mg2+ sufficient conditions was sensitive to a number of divalent and trivalent cations, particularly Gd3+ and Fe2+, but not to K+.

T-DNA Tagging-Based Gain-of-Function of OsHKT1;4 Reinforces Na Exclusion from Leaves and Stems but Triggers Na Toxicity in Roots of Rice Under Salt Stress.

The high affinity K⁺ transporter 1;4 (HKT1;4) in rice (Oryza sativa), which shows Na⁺ selective transport with little K⁺ transport activity, has been suggested to be involved in reducing Na in leaves and stems under salt stress. However, detailed physiological roles of OsHKT1;4 remain unknown. Here, we have characterized a transfer DNA (T-DNA) insertion mutant line of rice, which overexpresses OsHKT1;4, owing to enhancer elements in the T-DNA, to gain an insight into the impact of OsHKT1;4 on salt tolerance of rice. The homozygous mutant (the O/E line) accumulated significantly lower concentrations of Na in young leaves, stems, and seeds than the sibling WT line under salt stress. Interestingly, however, the mutation rendered the O/E plants more salt sensitive than WT plants. Together with the evaluation of biomass of rice lines, rhizosphere acidification assays using a pH indicator bromocresol purple and 22NaCl tracer experiments have led to an assumption that roots of O/E plants suffered heavier damages from Na which excessively accumulated in the root due to increased activity of Na⁺ uptake and Na⁺ exclusion in the vasculature. Implications toward the application of the HKT1-mediated Na⁺ exclusion system to the breeding of salt tolerant crop cultivars will be discussed.

Production of low-Cs+ rice plants by inactivation of the K+ transporter OsHAK1 with the CRISPR-Cas system.

The occurrence of radiocesium in food has raised sharp health concerns after nuclear accidents. Despite being present at low concentrations in contaminated soils (below µm), cesium (Cs+ ) can be taken up by crops and transported to their edible parts. This plant capacity to take up Cs+ from low concentrations has notably affected the production of rice (Oryza sativa L.) in Japan after the nuclear accident at Fukushima in 2011. Several strategies have been put into practice to reduce Cs+ content in this crop species such as contaminated soil removal or adaptation of agricultural practices, including dedicated fertilizer management, with limited impact or pernicious side-effects. Conversely, the development of biotechnological approaches aimed at reducing Cs+ accumulation in rice remain challenging. Here, we show that inactivation of the Cs+ -permeable K+ transporter OsHAK1 with the CRISPR-Cas system dramatically reduced Cs+ uptake by rice plants. Cs+ uptake in rice roots and in transformed yeast cells that expressed OsHAK1 displayed very similar kinetics parameters. In rice, Cs+ uptake is dependent on two functional properties of OsHAK1: (i) a poor capacity of this system to discriminate between Cs+ and K+ ; and (ii) a high capacity to transport Cs+ from very low external concentrations that is likely to involve an active transport mechanism. In an experiment with a Fukushima soil highly contaminated with 137 Cs+ , plants lacking OsHAK1 function displayed strikingly reduced levels of 137 Cs+ in roots and shoots. These results open stimulating perspectives to smartly produce safe food in regions contaminated by nuclear accidents.

Erratum: Acetate-mediated novel survival strategy against drought in plants.

This corrects the article DOI: 10.1038/nplants.2017.97.

Acetate-mediated novel survival strategy against drought in plants.

Water deficit caused by global climate changes seriously endangers the survival of organisms and crop productivity, and increases environmental deterioration1,2. Plants' resistance to drought involves global reprogramming of transcription, cellular metabolism, hormone signalling and chromatin modification3-8. However, how these regulatory responses are coordinated via the various pathways, and the underlying mechanisms, are largely unknown. Herein, we report an essential drought-responsive network in which plants trigger a dynamic metabolic flux conversion from glycolysis into acetate synthesis to stimulate the jasmonate (JA) signalling pathway to confer drought tolerance. In Arabidopsis, the ON/OFF switching of this whole network is directly dependent on histone deacetylase HDA6. In addition, exogenous acetic acid promotes de novo JA synthesis and enrichment of histone H4 acetylation, which influences the priming of the JA signalling pathway for plant drought tolerance. This novel acetate function is evolutionarily conserved as a survival strategy against environmental changes in plants. Furthermore, the external application of acetic acid successfully enhanced the drought tolerance in Arabidopsis, rapeseed, maize, rice and wheat plants. Our findings highlight a radically new survival strategy that exploits an epigenetic switch of metabolic flux conversion and hormone signalling by which plants adapt to drought.

OsHKT1;5 mediates Na+ exclusion in the vasculature to protect leaf blades and reproductive tissues from salt toxicity in rice.

Salt tolerance quantitative trait loci analysis of rice has revealed that the SKC1 locus, which is involved in a higher K+ /Na+ ratio in shoots, corresponds to the OsHKT1;5 gene encoding a Na+ -selective transporter. However, physiological roles of OsHKT1;5 in rice exposed to salt stress remain elusive, and no OsHKT1;5 gene disruption mutants have been characterized to date. In this study, we dissected two independent T-DNA insertional OsHKT1;5 mutants. Measurements of ion contents in tissues and 22 Na+ tracer imaging experiments showed that loss-of-function of OsHKT1;5 in salt-stressed rice roots triggers massive Na+ accumulation in shoots. Salt stress-induced increases in the OsHKT1;5 transcript were observed in roots and basal stems, including basal nodes. Immuno-staining using an anti-OsHKT1;5 peptide antibody indicated that OsHKT1;5 is localized in cells adjacent to the xylem in roots. Additionally, direct introduction of 22 Na+ tracer to leaf sheaths also demonstrated the involvement of OsHKT1;5 in xylem Na+ unloading in leaf sheaths. Furthermore, OsHKT1;5 was indicated to be present in the plasma membrane and found to localize also in the phloem of diffuse vascular bundles in basal nodes. Together with the characteristic 22 Na+ allocation in the blade of the developing immature leaf in the mutants, these results suggest a novel function of OsHKT1;5 in mediating Na+ exclusion in the phloem to prevent Na+ transfer to young leaf blades. Our findings further demonstrate that the function of OsHKT1;5 is crucial over growth stages of rice, including the protection of the next generation seeds as well as of vital leaf blades under salt stress.

Visualization of Uptake of Mineral Elements and the Dynamics of Photosynthates in Arabidopsis by a Newly Developed Real-Time Radioisotope Imaging System (RRIS).

Minerals and photosynthates are essential for many plant processes, but their imaging in live plants is difficult. We have developed a method for their live imaging in Arabidopsis using a real-time radioisotope imaging system. When each radioisotope,(22)Na,(28)Mg,(32)P-phosphate,(35)S-sulfate,(42)K,(45)Ca,(54)Mn and(137)Cs, was employed as an ion tracer, ion movement from root to shoot over 24 h was clearly observed. The movements of(22)Na,(42)K,(32)P,(35)S and(137)Cs were fast so that they spread to the tip of stems. In contrast, high accumulation of(28)Mg,(45)Ca and(54)Mn was found in the basal part of the main stem. Based on this time-course analysis, the velocity of ion movement in the main stem was calculated, and found to be fastest for S and K among the ions we tested in this study. Furthermore, application of a heat-girdling treatment allowed determination of individual ion movement via xylem flow alone, excluding phloem flow, within the main stem of 43-day-old Arabidopsis inflorescences. We also successfully developed a new system for visualizing photosynthates using labeled carbon dioxide,(14)CO2 Using this system, the switching of source/sink organs and phloem flow direction could be monitored in parts of whole shoots and over time. In roots,(14)C photosynthates accumulated intensively in the growing root tip area, 200-800 µm behind the meristem. These results show that this real-time radioisotope imaging system allows visualization of many nuclides over a long time-course and thus constitutes a powerful tool for the analysis of various physiological phenomena.

OsHKT1;4-mediated Na(+) transport in stems contributes to Na(+) exclusion from leaf blades of rice at the reproductive growth stage upon salt stress.

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.