Rice (Oryza sativa L.), a thermophilic crop, is highly sensitive to cold stress, particularly during the seedling stage. Developing cold-tolerant rice varieties is a possible strategy to mitigate yield losses caused by low temperatures. However, few genes for cold tolerance have been identified. In this study, we identified OsALA4 (Aminophospholipid ATPase 4), encoding a plasma membrane-localized P4-ATPase, from a chromosomal segment substitution line (CSSL-K2832-2) harboring cold-tolerance QTL qLTS5 (Low Temperature Sensitive 5). Genetic and subcellular localization analyses revealed that OsALA4 regulates cold tolerance by maintaining plasma membrane fluidity and cellular homeostasis. Physiological assessments showed that OsALA4 reduces malondialdehyde (MDA), electrolyte leakage, reactive oxygen species (ROS), and cell death under cold stress. Promoter activity assays indicated that stronger OsALA4 expression in Nipponbare (OsALA4^Nip) correlated with enhanced cold tolerance. Further experiments demonstrated that SNP sites within the promoter regions (−1500 bp to −700 bp) of OsALA4^Nip and OsALA4⁹³¹¹ influenced their activity. This study highlights OsALA4 as a valuable genetic target for breeding cold tolerant rice.

Grain size is a key factor influencing grain weight in rice. In this study, a chromosome segment substitution line (CSSL9-17) was identified, that exhibits a significant reduction in both grain size and weight compared to its donor parent 93-11. Further investigation identified two quantitative trait loci (QTL) on chromosome 11, designated qGW11a and qGW11b, which contribute to 1000-grain weight with an additive effect. LOC_Os11g05690, encoding the amino acid permease OsCAT8, is the target gene of qGW11a. Overexpression of OsCAT8 resulted in decreased grain weight, while OsCAT8 knockout mutants exhibited increased grain weight. The 287-bp located within the OsCAT8 promoter region of 93-11 negatively regulates its activity, which is subsequently correlated with an increase in grain size and weight. These results suggest that OsCAT8 functions as a negative regulator of grain size and grain weight in rice.

Rice is one of the three most important food crops in the world. Increasing rice yield is an effective way to ensure food security. Grain size is a key factor affecting rice yield; however, the genetic and molecular mechanisms regulating grain size have not been fully investigated. In this study, we identified a rice mutant, wide grain 4-D (wg4-D), that exhibited a significant increase in grain width and a decrease in grain length. Histological analysis demonstrated that WG4 affects cell expansion thereby regulating grain size. MutMap-based gene mapping and complementary transgenic experiments revealed that WG4 encodes an alpha-tubulin, OsTubA1. A SNP mutation in WG4 affected the arrangement of cortical microtubules and caused a wide-grain phenotype. WG4 is located in nuclei and cytoplasm and expressed in various tissues. Our results provide insights into the function of tubulin in rice and identifies novel targets the regulation of grain size in crop breeding.