CRISPR/Cas knockout of the NADPH oxidase gene OsRbohB reduces ROS overaccumulation and enhances heat stress tolerance in rice

Heat stress (HS) has become a major factor limiting crop yields worldwide. HS inhibits plant growth by ROS accumulation, and NADPH oxidases (Rbohs) are major ROS producers in plants. Here, we show that CRISPR/Cas knockout of the OsRbohB (OsRbohB-KO) significantly increased rice tolerance to HS imposed at various different growth stages. We produced OsRbohB-KO and OsRbohB-overexpression (OsRbohB-OE) lines in a japonica cultivar, Nipponbare. Compared with nontransgenic wild-type (WT) plants, the OsRbohB-KO lines showed a significant increase in chlorophyll contents (5.2%-58.0%), plant growth (48.2%-65.6%) and grain yield (8.9%-20.5%), while reducing HS-induced ROS accumulation in seeds (21.3%-33.0%), seedlings (13.0%-30.4%), anthers (13.1%-20.3%) and grains (9.7%-22.1%), under HS conditions. Analysis of yield components revealed that the increased yield of OsRbohB-KO plants was due to increased starch synthetase activity, spikelets per panicle (2.0%-9.3%), filled spikelets (4.8%-15.5%), percentage of filled spikelets (2.4%-6.8%) and 1000-grain weight (2.9%-7.4%) under HS conditions during the reproductive stage. Grain milling and appearance quality, and starch content were also significantly increased in OsRbohB-KO plants under HS conditions during the mature stage. Furthermore, OsRbohB-KO significantly upregulated the expression levels of heat shock-related genes, OsHSP23.7, OsHSP17.7, OsHSF7 and OsHsfA2a, in rice seedlings and grains under long-term HS conditions. Conversely, OsRbohB-OE resulted in phenotypes that were opposite to OsRbohB-KO in most cases. Our results suggest that suppression of OsRbohB provides an effective approach for alleviating heat damage and improving grain yield and quality of rice under long-term HS conditions.