Cytokinins regulate rice lamina joint development and leaf angle

Abstract

Abstract Leaf angle is determined by lamina joint inclination and is an important agronomic trait that determines plant architecture, photosynthetic efficiency, and crop yield. Cytokinins (CKs) are phytohormones involved in shaping rice (Oryza sativa L.) architecture, but their role in leaf angle remains unknown. Here, we report that CK accumulation mediated by rice CK OXIDASE/DEHYDROGENASE3 (OsCKX3) controls lamina joint development and negatively regulates leaf angle. Phenotypic analysis showed that rice osckx3 mutants had smaller leaf angles, while the overexpression lines (OsCKX3-OE) had larger leaf angles. Histological sections indicated that the leaf inclination changes in the osckx3 and OsCKX3-OE lines resulted from asymmetric proliferation of the cells and vascular bundles in the lamina joint. Reverse transcription quantitative PCR, promoter-fused β-glucuronidase expression, and subcellular localization assays indicated that OsCKX3 was highly expressed in the lamina joint, and OsCKX3-GFP fusion protein localized to the endoplasmic reticulum. The enzyme assays using recombinant protein OsCKX3 revealed that OsCKX3 prefers trans-zeatin (tZ) and isopentenyladenine (iP). Consistently, tZ and iP levels increased in the osckx3 mutants but decreased in the OsCKX3 overexpression lines. Interestingly, agronomic trait analysis of the rice grown in the paddy field indicated that osckx3 displayed a smaller leaf angle and enhanced primary branch number, grain size, 1,000-grain weight, and flag leaf size. Collectively, our results revealed that enhancing CK levels in the lamina joint by disrupting OsCKX3 negatively regulates leaf angle, highlighting that the CK pathway can be engineered to reduce leaf angle in rice and possibly in other cereals.