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

The study addresses how to simultaneously enhance nitrogen (N) and carbon (C) acquisition to improve crop productivity and environmental performance. In C3 crops like rice, productivity is limited by both low intercellular CO2 for RuBisCO and inefficient N use, leading to large fertiliser inputs and environmental losses. Plasma membrane H+-ATPase energizes ion transport and has been shown to mediate light-induced stomatal opening in model plants. Given paddy rice relies predominantly on NH4+ uptake and its assimilation generates cytosolic H+, the authors hypothesised that PM H+-ATPase (specifically OSA1) coordinates NH4+ uptake/assimilation in roots and stomatal opening/photosynthesis in leaves, and that overexpressing OSA1 could increase yield and NUE in rice.

Background highlights include: PM H+-ATPase generates proton motive force across the plasma membrane, energizing nutrient uptake and guard-cell function. Overexpression in Arabidopsis guard cells enhances stomatal opening and growth. Paddy rice primarily uses NH4+ as N source; effective uptake must be matched with assimilation via the GS/GOGAT cycle, which produces cytosolic H+. PM H+-ATPase contributes to nutrient transport (nitrate, phosphate, K+) and cytosolic pH homeostasis, and is upregulated under NH4+ nutrition in rice roots; enhanced activity supports growth at high NH4+. Prior attempts to overexpress individual ammonium transporters (AMTs) or GS alone increased uptake or assimilation but caused poor growth, highlighting the need for coordinated regulation. These studies motivated testing whether manipulating OSA1 could synchronously improve NH4+ metabolism and photosynthesis in a major crop.

The authors generated OSA1-overexpressing rice lines (OSA1#1-#3; CaMV-35S promoter) and osa1 knockout mutants (TOS17 insertion lines osa1-1 to osa1-3). Plants were grown hydroponically under controlled conditions for laboratory assays. PM H+-ATPase protein levels were assessed by immunoblotting; ATP hydrolytic activity was measured using isolated plasma membranes with vanadate-sensitive assays. 15NH4+ uptake assays were performed by short-term (5–30 min) incubations across NH4+ concentrations (0.5–8 mM), including pharmacological stimulation (fusicoccin) and inhibition (vanadate). Total N and C, and mineral nutrients (K, P, Ca, S, Fe, Zn) were quantified from dried tissues. Stomatal aperture assays used epidermal fragments under defined light and ABA conditions; gas exchange (stomatal conductance, CO2 assimilation) was measured with LI-6400 under saturating white light, with light-response and A–Ci curves. RNA-seq profiled leaves and roots of WT, OSA1-ox (OSA1#2), and osa1-2 plants (NextSeq 500; EdgeR; FDR < 0.05), followed by GO enrichment. Rhizosphere acidification was visualised with bromocresol purple agar and H+ efflux measured by SIET. Field trials were conducted over two seasons at three locations in central China (Nanjing-S 2016; Nanjing-N and Fengyang 2017) with four N fertiliser levels (0, 100, 200, 300 kg N/ha). Yield components (panicle weight, panicles per hill, spikelets per panicle) and agronomic NUE (yield increase per kg N) were determined; an additional validation was performed in Hainan (tropical, short-day). Statistical analyses used two-tailed Student’s t tests and ANOVA with Tukey’s test, as appropriate.

• Pharmacological activation of PM H+-ATPase with fusicoccin increased 15NH4+ absorption by 17% (dark) and 11% (light), and light elevated transpiration, supporting a role in NH4+ uptake.
• OSA1 overexpression: OSA1 transcript increased 7.4–8.6× in roots and 3.5–5.3× in leaves; PM H+-ATPase protein ~40% higher and activity ~30% higher than WT. Biomass (dry weight) increased 18–33% at 4 weeks; mutants were 33–52% lower.
• NH4+ uptake: 15NH4+ absorption rates were significantly higher in OSA1-ox across 0.5–8 mM, affecting both high- and low-affinity systems; mutants had lower rates. Over 30 min at 2 mM 15NH4+, OSA1-ox were 20–30% higher; vanadate suppressed uptake in all lines.
• Nutrient accumulation: Total N increased 16–57% in OSA1-ox and decreased in mutants; other nutrients (K, P, Ca, S, Fe, Zn) increased in OSA1-ox and decreased in mutants. Total C increased 21–47% in OSA1-ox and decreased in mutants, indicating enhanced CO2 fixation.
• Stomata and photosynthesis: Under light, OSA1-ox had a higher open/closed stomata ratio; ABA sensitivity and stomatal morphology were unchanged. Stomatal conductance was about double in OSA1-ox; photosynthetic rates increased 26–28% (saturating WL). Light-response curves showed 15–34% higher CO2 assimilation under high irradiance; A–Ci curves were higher. Water-use efficiency decreased by 13–21%. Mutants had 22–37% lower conductance and 27–35% lower photosynthesis.
• Transcriptomics: In OSA1-ox, 1373 (leaves) and 1124 (roots) transcripts were upregulated; in osa1-2, 347 (leaves) and 3295 (roots) transcripts were downregulated (and vice versa for down/up). Enriched processes included photosynthesis, NH4+ assimilation, amino acid metabolism, carbohydrate and ion transport, and N utilisation. Multiple AMTs (AMT1;1/1;2, AMT2;1/2;3, AMT3;1/3;3) and NH4+ assimilation genes (GS1;2, GS2, NADH-GOGAT1/2, Fd-GOGAT) were upregulated in OSA1-ox and downregulated in mutants. Photosynthesis-related genes (e.g., Psb28, PsbQ, PsaH, RbcS) were induced in OSA1-ox. GRF4, a key regulator of N/C metabolism, was upregulated.
• Field performance: Across three locations and four N levels, OSA1-ox grain yield was 27–39% higher than WT (mean ~33%). Panicle weight increased 18–42%, panicles per hill 15–20%, and spikelets per panicle 8–16%; plant height, panicle length, filled grain rate, and 1000-grain weight were unchanged. Under zero N (N–N), OSA1-ox still yielded 12–20% higher. Agronomic NUE was ~46% higher at all N levels. With half the N fertiliser (100 kg/ha), OSA1-ox out-yielded WT at 200 kg/ha, achieving the same yield with half the N input. An independent Hainan trial also showed higher yield.

The findings demonstrate that OSA1, a PM H+-ATPase, coordinately enhances root NH4+ uptake/assimilation and leaf CO2 uptake/photosynthesis in rice. Elevated PM H+-ATPase activity increases proton motive force and rhizosphere acidification, stimulating NH4+ transport and broader nutrient uptake, while maintaining cytosolic pH during NH4+ assimilation by pumping excess H+ out. Parallel upregulation of AMT transporters and GS/GOGAT enzymes indicates a coordinated program that prevents ammonium toxicity and boosts N assimilation into amino acids. In leaves, OSA1 enhances light-induced stomatal opening, increasing stomatal conductance and photosynthetic capacity, which supplies carbon skeletons and energy to support N assimilation, linking C and N metabolism. The net result is increased plant C and N accumulation, vegetative growth, and significantly higher grain yield and NUE across varying N supplies. Given the conservation of PM H+-ATPase functions in plants, this strategy may generalize to other crops, offering agronomic and environmental benefits through reduced fertiliser demand.

Overexpressing the rice PM H+-ATPase gene OSA1 simultaneously improves NH4+ uptake and assimilation in roots and enhances stomatal conductance and photosynthesis in leaves, resulting in substantial gains in biomass, grain yield (~33%), and agronomic NUE (~46%) under field conditions. The approach achieves comparable yields with half the N fertiliser input. Transcriptome data suggest broad activation of transporters and metabolic pathways, including AMTs, GS/GOGAT, photosynthesis genes, and the transcription factor GRF4. Given the conserved role of PM H+-ATPase, promoting its activity (PUMP plants) could be a general strategy for increasing yield and sustainability in crops. Future work should elucidate the signaling cascade linking OSA1 activity to transcriptional reprogramming, define master regulators like GRF4 in this context, and develop non-transgenic routes (e.g., genome editing) to upregulate PM H+-ATPase for practical deployment.