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, ²²Na⁺ 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.