OsPIP2;1 Positively Regulates Rice Tolerance to Water Stress Under Coupling of Partial Root-Zone Drying and Nitrogen Forms

The coupling of partial root-zone drying (PRD) with nitrogen forms exerts an interactive "water-promoted fertilization" effect, which enhances rice (<i>Oryza sativa</i> L.) growth and development, improves water use efficiency (WUE), mediates the expression of aquaporins (AQPs), and alters root water conductivity. In this study, gene cloning and CRISPR-Cas9 technologies were employed to construct overexpression and knockout vectors of the <i>OsPIP2;1</i> gene, which were then transformed into rice (cv. Meixiangzhan 2). Three water treatments were set: normal irrigation (CK); partial root-zone drying (PRD); and 10% PEG-simulated water stress (PEG), combined with a nitrogen form ratio of ammonium nitrogen (NH₄⁺) to nitrate nitrogen (NO₃^-) at 50:50 (A50/N50) for the coupled treatment of rice seedlings. The results showed that under the coupled treatment of PRD and the aforementioned nitrogen form, the expression level of the <i>OsPIP2;1</i> gene in roots was upregulated by 0.62-fold on the seventh day, while its expression level in leaves was downregulated by 1.84-fold. Overexpression of <i>OsPIP2;1</i> enabled Meixiangzhan 2 to maintain a higher abscisic acid (ABA) level under different water conditions, which helped rice reduce water potential and enhance water absorption. Compared with the CK treatment, overexpression of <i>OsPIP2;1</i> increased the superoxide dismutase (SOD) activity of rice under PRD by 26.98%, effectively alleviating tissue damage caused by excessive accumulation of O₂^-. The physiological and biochemical characteristics of <i>OsPIP2;1</i>-overexpressing rice showed correlations under PRD and A50/N50 nitrogen form conditions, with WUE exhibiting a significant positive correlation with transpiration rate, chlorophyll content, nitrogen content, and Rubisco enzyme activity. Overexpression of <i>OsPIP2;1</i> could promote root growth and increase the total biomass of rice plants. The application of the <i>OsPIP2;1</i> gene in rice genetic engineering modification holds great potential for improving important agricultural traits of crops. This study provides new insights into the mechanism by which the AQP family regulates water use in rice and has certain significance for exploring the role of AQP genes in rice growth and development as well as in response to water stress.