Knocking out OsPht1;9-1;10 genes decreases arsenic accumulation in rice (Oryza sativa) grains

Research Background

Arsenic is a class of carcinogens, threatening hundreds of millions of people worldwide due to arsenic-contaminated soil and groater. As the staple food in Asia, rice is a major source of dietary arsenic exposure. Arsenate (AsV) is a chemical analog of phosphate and shares transporterfrom the Pht1 family in rice; previous attempts to regulate related genes have disrupted phosphate uptake and led to yield reductions. The cultivation of genetically modified low-arsenic rice is restrictednal policies, and existing gene editing targets (such as OsLsi2) suffer from defects including impaired silicon uptake and reduced stress tolerance. Meanwhile, a certain amount of Axists in the rice rhizosphere due to oxygen release, but its specific transport mechanism remains unclear. There is an urgent need to identify key genes that mediate AsV uptake with minimon phosphate absorption.

Experimental Methods

Using the rice variety Zhonghua 11 as the material, the expression patterns f OsPht1;9/1;10 under AsV exposure and phosphorus stress were analyzed via transcriptome sequencing and qRT-PCR. The gene was expressed in phosphorus transporter-deficint yeast EY917 to verify its AsV transport capacity. CRISPR-Cas9 was used to construct OsPht1;9/1;10 double mutants, while simutants were screened as controls. Hydroponic experiments were conducted to detect arsenic uptake, translocation efficiency, and phosphorus content in the mutants. Multi-year and multi-site  trials were carried out in Guangzhou and Sanya to measure grain arsenic content and agronomic traits of the mutants. Additionally, natural variation of the gn 3,000 rice accessions was analyzed to explore its potential for conventional breeding.

Key Results

OsPht1;10 expression was significantly upregulated under AsV exposure, and both ge mediated AsV uptake and translocation in rice only under sufficient phosphorus conditions. Yeast experiments confirmed that their AsV transport capacity was higher than that of the kown OsPht1;4. Under hydroponic conditions, arsenic accumulation in the shoots of the double mutant decreased by 46.2-65.7%, and arsenic tent in xylem sap decreased by 16.5-34.8%, with no significant change in phosphorus content. In multi-year field trials, grain arsenic con the double mutant decreased by 19.2-47.3%, while yield and mineral nutrients such as zinc, iron, and manganese were unaffected; single mutants showed no sigificant low-arsenic effect. Meanwhile, natural sequence variations in OsPht1;9/1;10 were identified, providing a basis for convention

Research Conclusions

OsPht1;9/1;10 are key genes for arsenite (AsV) uptake and translocatcontributing minimally to phosphorus utilization, and exhibit functional redundancy in arsenic absorption; the double knockout of these genes is a feasible strategy for breeding low-arsenic rice, acombining it with existing targets for regulating AsIII accumulation can comprehensively reduce arsenic content in rice grains; the natural variation of these genes offers potential for non-transgenic low-ice breeding. This study not only elucidates the molecular mechanism of arsenic accumulation in rice but also provides important theoretical and practical support for addressing the global issue of rice arsenic co