Generally, knockdown of OsHKT1;4 resulted in the accumulating more Na+ in every organ tested . In particular, the largest influence from the reduction in OsHKT1;4 expression was observed in the Na+ content of flag leaf blades, where approximately 3.5–4-fold increases in Na+ content in RNAi plants was detected on average compared with that of wild-type plants . Flag leaf sheaths of the RNAi plants also exhibited 2.5–3-fold increases inNa+ accumulation compared with control plants . In contrast, no marked difference in K+ accumulation between the wild-type and RNAi plants was found in all organs tested . After the completion of NaCl stress treatment with the soil-grown rice plants, a proportion of the plants were subsequently maintained by watering with normal tap water to investigate the Na+ content in the mature rice grains. The Na+ content of ripening grains was 25–34 % higher in RNAi plants compared with wild-type plants . In contrast, the Na+ contents of non-ripening grains and rachis-branches tended to be highly variable, but no noticeable difference was observed between wild-type and OsHKT1;4 RNAi plants . We also conducted a 22Na+ -tracer analysis on OsHKT1;4 RNAi and Nipponbare WT plants. The inflorescence including the peduncle and ear excised from the edge of node I was soaked in a solution containing 22Na+ for the direct absorption. As a result,hydroponic barley fodder system peduncles from OsHKT1;4 RNAi plants tended to allow the transfer of a larger amount of Na+ from the cut-end to the upper regions in comparison with WT with an exception of an independent plant from the OsHKT1;4 RNAi-II line, which exhibited a similar tracer profile to that of WT .
Na+ -selective transport mediated by some class I HKT transporters have been indicated to play a crucial role in Na+ exclusion from leaves of salt-stressed plants. The HKT1;4-A2 locus in durum wheat, which was derived from a wild wheat relative Triticum monococcum, was highlighted as a strong candidate for a salt tolerance QTL named Nax1 . In rice, a role of OsHKT1;4 in controlling Na+ concentrations in leaf blades was suggested by comparative analyses of Na+ contents in leaf blades and the level of OsHKT1;4 transcripts in sheaths using salt tolerant indica rice varieties and a japonica rice cultivar Nipponbare. However, the ion transport properties and physiological functions of OsHKT1;4 remain to be elucidated. Stable and constitutive expression of OsHKT1;4 in a salt hypersensitive strain of S. cerevisiae G19 led to an increase in sensitivity to increases in extracellular NaCl concentration, with significant increases in Na+ accumulation in the cells . Plasma membrane-targeted OsHKT1;4 was found to elicit large currents stimulated by Na+ in X. laevis oocytes with shifts in zero-current potentials toward a more depolarized status, dependent on increases in the Na+ concentration in the bath solution . A 10-fold increase in the Na+ concentration in the bath resulted in the shift of the reversal potential of 34.5 ± 1.3 mV on average , which was smaller than the theoretical Nernstian shift of 58–59 mV. Note however that the reversal potential shifts of OsHKT1;4-expressing oocytes can be less than the theoretical value because the cytoplasmic Na+ concentrations of the oocytes may also shift due to the function of OsHKT1;4.
Further experiments will be needed to characterize the ion selectivity of OsHKT1;4 in detail. In addition, investigation of monovalent cation selectivity of OsHKT1;4 expressed in oocytes bathed in solutions containing solo cation-chloride salts further revealed that this transporter is highly selective for Na+ amongst Li+ , K+ , Rb+ , Cs+ , Na+ , and NH4 + . These results indicate that OsHKT1;4 is a Na+ transporter. HKT proteins have been suggested to contain four selectivity-filter-pore domains that are distantly related to a bacterial K+ channel. HKT1 transporters have been found to be highly selective for Na+ , and in general a serine residue at the key amino acid position for K+ selectivity in the first p-loop domain is conserved instead of a glycine residue, which corresponds to the first glycine in the GYG motif of the shaker-type K+ channel. Corresponding amino acid positions in the three other p-loop domains of OsHKT1;4 were reported to be glycine residues, resulting in a SGGG type for the p-loop domains of OsHKT1;4 as typical HKT1 transporters. The property of Na+ selective transport by OsHKT1;4 was consistent with the prediction of Na+ selectivity of HKT transporters based on the p-loop hypothesis.QTL analyses for salt tolerance of durum wheat plants have led to the identification of the salt tolerance determining Nax1 locus, which was deduced to be the TmHKT1;4-A2 gene. The Nax1 locusmediated xylem Na+ unloading in roots and leaf sheaths of durum wheat plants has been suggested to avoid Na+ over-accumulation in leaf blades during salinity stress. Relatively steady expression in leaf sheaths throughout growth stages is a distinctive feature of the OsHKT1;4 gene in Nipponbare plants . In 3-weekold Nipponbare plants, grown in hydroponic culture, the expression of OsHKT1;4 was also observed in roots .
However, the level of OsHKT1;4 expression mostly showed significant decreases in tissues/organs of salt-stressed Nipponbare plants at the vegetative growth stage under salinity stress . OsHKT1;4 RNAi plants in the vegetative growth stage did not show any noticeable difference either in visual phenotype or in Na+ content after the imposition of 50 mM NaCl stress compared with Nipponbare wild-type plants . These results suggested a possibility that OsHKT1;4- mediated Na+ transport does not provide a profound contribution to vital Na+ homeostasis during the vegetative growth phase of the japonica rice cultivar during salinity stress. Another characteristic feature of OsHKT1;4 gene expression was its high expression in the stem, including the peduncle and the internode II, of rice plants at the reproductive growth stage . The observed expression profile of OsHKT1;4 was consistent with that found in the RiceXPro database, in which OsHKT1;4 is highly up-regulated in the stem of rice plants in heading and ripening stages. Long term salinity stress treatment with gradual increases in NaCl concentration in soil-grown Nipponbare plants from heading to ripening stages led to significant increases in OsHKT1;4 expression in the peduncle, with relatively steady expression levels in the flag leaf blade and internode II independent of salt treatments . Significant up-regulation of OsHKT1;4 expression was also observed in node I, although the basal expression level in this tissue was far less than that in the peduncle . Similar long-term salinity stress treatments of soil-grown OsHKT1;4 RNAi and wild-type plants resulted in significantly higher Na+ contents in aerial tissues of RNAi plants, with the highest impact on the Na+ content of flag leaf blades compared with wild-type plants . Together with Na+ selective transport mediated by plasma membrane-targeted OsHKT1;4 , these results suggested that OsHKT1;4 contributes to the prevention of Na+ over-accumulation in aerial parts, in particular leaf blades of Nipponbare plants that are in the reproductive growth stage,livestock fodder system during salinity stress. HKT1;4 transporters in wheat have been suggested to function in xylem Na+ unloading in roots and leaf sheaths upon salinity stress to reduce Na+ transfer into leaf blades . On the other hand, HKT1-mediated Na+ recirculation via the downward stream of the phloem has been argued as a potential working model for HKT1 transporters . OsHKT1;4 RNAi plants in the reproductive growth stage accumulated more Na+ not only in leaves but also in tissues of the stem investigated under salinity stress . The reason for the phenotype is not clear yet. In a previous study, analyses on athkt1;1 mutants of Arabidopsis indicated that the dysfunction of AtHKT1;1- mediated Na+ unloading from xylem caused the impairment of Na+ recirculation via phloem as well, which could together be attributed to Na+ over-accumulation in shoots of athkt1;1 mutants upon salinity stress.
Na+ -imaging analysis indicated a higher amount of the Na+ transfer in peduncles of OsHKT1;4 RNAi plants than WT plants with an exception of an independent OsHKT1;4 RNAi-II plant , suggesting that OsHKT1;4 mediates Na+ unloading from xylem and reduced activity of OsHKT1;4 leads to an increase in Na+ accumulation in this tissue. Together, Na+ over accumulation in aerial parts of OsHKT1;4 RNAi plants upon salinity might be due to the insufficient activity of OsHKT1;4 in Na+ unloading from xylem, which in turn could also bring about inhibition of Na+ recirculation. To elucidate the precise function of OsHKT1;4 in Na+ exclusion in rice, a detailed investigation into whether OsHKT1;4 predominantly mediates xylem Na+ unloading or phloem-involved Na+ recirculation or both will be an essential question to be addressed in future research. In this respect, it will be also interesting to study the in planta localization of OsHKT1;4 in order to investigate possible regulatory mechanisms that can control its PM localization, and hence Na+ transport and salt tolerance. Indeed, our subcellular localization analyses leave room for some speculation. Using rice protoplasts, we could clearly observe the PM localization of OsHKT1;4 , but also the presence of the protein in unidentified vesicles that were neither ER nor GA structures . This vesicle could represent a means to control the abundance of OsHKT1;4 in the PM. Recently it has been demonstrated that another member of the HKT family, OsHKT1;3, is targeted to the GA and undergoes strict control of its trafficking. Note that the subcellular localization of OsHKT1;4 was investigated using rice protoplasts over-expressing the chimeric EGFPOsHKT1;4 protein. The hypothesis that endocytotic mechanisms regulate the amount of OsHKT1;4 in the PM requires further investigation using rice plants. In addition to aerial tissues, increases in the Na+ content of ripening grains from OsHKT1;4 RNAi plants were found during salinity stress conditions compared with wild-type plants . The expression of OsHKT1;4 was up-regulated by salt stress in the peduncle and node I . In Node I, it was also found by LMD-combined qPCR analysis that the OsHKT1;4 gene was predominantly expressed in the DVB, which is connected to the ear of rice. Taken together, these results suggested a potential contribution of OsHKT1;4 in protecting reproductive organs and seeds from Na+ toxicity in rice plants in addition to the leaf blades upon salinity stress .Symbiotic plant–microbe interactions benefit both participants. While plants provide nutrition, their associated microbes provide essential metabolites for plant growth and stress tolerance. However, these metabolisms are not yet well understood. Elucidating microbial metabolisms and their regulation is therefore critical for realizing new opportunities in sustainable agriculture. Plant–microbe interactions help plants in many ways. For example, diazotrophic microbes convert dinitrogen gas into plant-available nitrogen , which promotes plant growth . Some microbes produce plant hormones and/or metabolites similar to plant hormones that support plant growth and development . Additionally, microbe-produced volatile organic compounds can improve plants’ ability to tolerate both biotic and abiotic stressors, including insects, pathogens, drought, and extreme temperature. Microbe–microbe interactions also play vital, although indirect, roles in plant–microbe interactions. Microbial metabolites mediate microbe–microbe interactions through metabolic exchanges and complementation, as well as by functioning as chemical signals. For example, quorum-sensing molecules promote formation of biofilms that enhance microbe survival, production of other specialized metabolites, cell motility, and root development.Overall, symbiotic relationships among microbes and plants form the core of biomes. Modulating these relationships is emerging as a potential strategy for making agriculture more sustainable. Many research efforts have focused on discovering plant growth-promoting rhizomicrobes . Application of PGPRs to agricultural fields, however, has yielded inconsistent benefits for various crop species and cultivation conditions, perhaps because the pre-existing microbiomes are resilient and soon overgrow the introduced species. This limitation is extremely hard to overcome because of the complex interactions involved. Synthetic biology has emerged as a promising strategy because it facilitates targeted engineering of key microbial strains, thereby harnessing the microbiome’s power to increase sustainability in agriculture. Both new strains that can robustly colonize diverse plant species and strains from original communities may be engineered to improve plant growth without chemical fertilizers and pesticides . Introducing plant growth-promoting traits to these strains may provide consistent benefits, regardless of changes in crop species or cultivation conditions. Researchers often attempt to understand metabolic networks, as well as microbe–microbe and plant–microbe interactions and their regulation, by investigating carbon metabolism. However, N metabolism has been explored less, despite the versatile roles of N metabolites in cellular signaling and physiology.