Plasma membrane H+-ATPase overexpression increases rice yield via simultaneous enhancement of nutrient uptake and photosynthesis

Nitrogen (N) and carbon (C) are crucial for plant growth and crop yields. Efficient N and C utilization is vital for boosting agricultural productivity and reducing fertilizer dependence. Current limitations include inefficient N use by crops, resulting in environmental pollution from excess N loss, and inefficient CO2 fixation in C3 plants like rice and wheat due to low intercellular CO2 concentrations. Improving N and CO2 uptake is therefore a key goal in agriculture. Plasma membrane (PM) H+-ATPase, a P-type ATPase, generates a membrane potential and H+ gradient across the PM, driving various ion channels and transporters essential for physiological processes, including light-induced stomatal opening. Previous research showed that PM H+-ATPase overexpression enhances stomatal opening, photosynthesis, and growth in *Arabidopsis*. This study investigates whether manipulating PM H+-ATPase in rice, a crucial food crop, can similarly improve N and C utilization and enhance yield. Rice, unlike many terrestrial plants, grows in flooded soils where ammonium (NH4+) is the primary N source. Efficient NH4+ uptake and assimilation are essential, as high NH4+ accumulation is detrimental to growth. PM H+-ATPase aids in nutrient transport and cytosolic H+ homeostasis. Previous studies indicated a link between NH4+ nutrition and PM H+-ATPase activity in rice roots, leading to the hypothesis that PM H+-ATPase plays a role in NH4+ metabolism. This research explores the role of PM H+-ATPase in NH4+ uptake by rice roots and stomatal opening for CO2 uptake and photosynthesis in leaves, aiming to improve rice yield and N use efficiency (NUE) via OSA1 overexpression.

The research draws on previous findings demonstrating the importance of efficient nitrogen (N) and carbon (C) utilization for enhanced crop yields and sustainable agriculture. The limited ability of crops to utilize N efficiently, leading to environmental pollution from excess N fertilizer, is highlighted. Similarly, the limitations of C3 plants' photosynthesis due to low intercellular CO2 concentration are discussed. The critical role of plasma membrane (PM) H+-ATPase in generating membrane potential and H+ gradients crucial for various physiological processes is emphasized, particularly its role in light-induced stomatal opening and nutrient transport. Studies showing enhanced growth in *Arabidopsis* following PM H+-ATPase overexpression serve as a foundation for investigating its impact on rice. The unique characteristics of rice cultivation in flooded soils, where ammonium (NH4+) is the dominant N source, are noted, along with the importance of efficient NH4+ uptake and assimilation in rice. Prior research establishing the relationship between NH4+ nutrition and PM H+-ATPase activity in rice roots supports the hypothesis explored in the current study.

The study employed a multi-faceted approach involving both laboratory and field experiments. Laboratory experiments utilized hydroponic cultivation of wild-type (WT) rice, *OSA1*-overexpressing lines (*OSA1*-oxs), and *osa1* knockout mutants (*osal* mutants). The impact of PM H+-ATPase on NH4+ uptake was assessed by treating rice roots with fusicoccin (FC), a PM H+-ATPase stimulator, and measuring 15NH4+ absorption rates under light and dark conditions. Phenotypic analysis of *OSA1*-oxs and *osal* mutants included measurements of plant growth (dry weight), PM H+-ATPase protein levels and activity, and nutrient accumulation (N, C, and other elements). 15NH4+ absorption rates were measured under various NH4+ concentrations to evaluate the effects on both high- and low-affinity transport systems. Stomatal phenotypes were analyzed by comparing the ratio of open to closed stomata in WT and modified rice lines under different light and ABA treatments. Photosynthetic properties, including stomatal conductance and CO2 assimilation rates, were also examined using a LI-6400 system, and light and CO2 response curves were generated. RNA-sequencing (RNA-seq) analysis provided comprehensive gene expression profiles in leaves and roots of WT, *OSA1*-ox, and *osa1* mutant plants to identify differentially expressed genes (DEGs) and associated pathways. Field experiments were conducted over two growing seasons at three locations in China, employing various levels of nitrogen fertilization. Grain yield, panicle weight, number of panicles per hill, spikelets per panicle, and agronomic NUE were measured. Additional field trials were conducted in Hainan, China, to verify the results under different climatic conditions. Various techniques, including quantitative reverse transcription PCR, immunodetection, measurement of PM H+-ATPase activity, and detection of rhizosphere acidification, were utilized to support the findings.

Treatment of rice roots with fusicoccin (FC), a PM H+-ATPase stimulator, significantly increased 15NH4+ absorption rates, indicating the involvement of PM H+-ATPase in NH4+ uptake. *OSA1*-ox lines showed significantly higher *OSA1* expression, PM H+-ATPase protein levels, and PM H+-ATPase activity compared to WT plants, while these values were reduced in *osal* mutants. *OSA1*-ox lines exhibited enhanced plant growth (18-33% greater dry weight) and significantly higher 15NH4+ absorption rates across a wide range of NH4+ concentrations. Total N and C accumulation were considerably higher in *OSA1*-ox lines and lower in *osal* mutants than in WT plants. *OSA1*-ox plants showed increased stomatal opening, stomatal conductance, and photosynthetic rates under light conditions, while *osal* mutants exhibited reduced rates. RNA-seq analysis revealed upregulation of multiple genes associated with NH4+ transport, assimilation, and photosynthesis in *OSA1*-ox lines, and downregulation in *osal* mutants. Field trials consistently demonstrated a 27-39% increase in grain yield in *OSA1*-ox lines compared to WT plants across different locations and nitrogen fertilization levels. The increase in yield was associated with increased panicle weight, number of panicles per hill, and spikelets per panicle. *OSA1*-ox lines showed approximately 46% higher nitrogen use efficiency (NUE) than WT plants. Even under low nitrogen fertilization, *OSA1*-ox lines achieved grain yields comparable to WT under moderate fertilization levels. Independent field trials in Hainan confirmed the yield improvements under a different climate.

The findings strongly support the hypothesis that OSA1, encoding PM H+-ATPase, plays a crucial role in enhancing both nitrogen use efficiency (NUE) and photosynthesis in rice. The simultaneous increase in NH4+ uptake and assimilation in roots, coupled with the enhanced photosynthetic activity in leaves, demonstrates a coordinated improvement in both N and C metabolism. The upregulation of NH4+ transporter genes and genes involved in NH4+ assimilation, along with the increased expression of genes related to photosynthesis, provides a molecular basis for the observed improvements in yield and NUE. The increased H+ extrusion by *OSA1*-ox rice roots potentially enhances nutrient uptake by creating a higher proton motive force. The upregulation of GRF4, a key regulator of N and C metabolism, in *OSA1*-ox lines further supports the coordinated regulation of these processes. The remarkable improvement in NUE in *OSA1*-ox rice holds significant implications for sustainable agriculture, as it reduces the need for N fertilizers while maintaining high yields. The conserved nature of PM H+-ATPase in plants suggests the potential for applying this manipulation strategy to other valuable crops.

This study demonstrates that overexpression of the *OSA1* gene, encoding a PM H+-ATPase, significantly increases rice yield and NUE by concurrently enhancing NH4+ uptake and assimilation in roots and boosting photosynthesis in leaves. The coordinated upregulation of genes related to nutrient transport, assimilation, and photosynthesis underscores the integrative role of OSA1 in plant metabolism. The substantial yield increase and improved NUE under various nitrogen fertilization levels highlight the potential of this approach for sustainable agriculture. Future research could focus on exploring the regulatory mechanisms underlying OSA1-mediated improvements and investigating the applicability of this strategy to other crops.

The study primarily focused on ammonium as the nitrogen source, which may limit the generalizability of findings to other nitrogen forms. The field trials were conducted in specific regions of China, and further research is needed to confirm the results in diverse geographical locations and soil types. While RNA-seq analysis provided valuable insights into gene expression changes, further investigation of the underlying regulatory networks and signaling pathways is warranted to comprehensively understand the mechanisms involved.