Rice (Oryza sativa L.) cultivation using direct seeding is susceptible to chilling stress, particularly during seed germination and early seedling growth in the early season of a double cropping system. Alternatively, seed priming with various plant growth-promoting hormones is an effective technique to promote rapid and uniform emergence under chilling stress. Therefore, we evaluated the impact of gibberellin A3 (GA₃) and brassinolide (BR) priming on rice seed emergence, examining their proteomic responses under low-temperature conditions. Results indicated that GA₃ and BR increased the seed germination rate by 22.67% and 7.33% at 72 h and 35% and 15% at 96 h compared to the control (CK), respectively. Furthermore, proteomic analysis identified 2551, 2614, and 2592 differentially expressed proteins (DEPs) in GA, BR, and CK, respectively. Among them, GA exhibited 84 upregulated and 260 downregulated DEPs, while BR showed 112 upregulated and 102 downregulated DEPs, and CK had 123 upregulated and 81 downregulated DEPs. Notably, under chilling stress, both GA₃ and BR are involved in peroxide metabolism, phenylpropanoid biosynthesis, and inositol phosphate metabolism, enhancing antioxidant capacity and providing energy substances for germination. In addition, GA₃ triggers the specific regulation of stress responsive protein activation, GTP activation, and ascorbic acid biosynthesis and promotes the stability and integrity of cell membranes, as well as the synthesis of cell walls, providing physical defense for seeds to resist low temperatures. At the same time, BR triggers specific involvement in ribosome synthesis and amino acid synthesis, promoting biosynthetic ability and metabolic regulation to maintain plant life activities under low-temperature stress. Furthermore, the various genes’ expression (OsJ_16716, OsPAL1, RINO1) confirmed GA₃ and BR involved in peroxide metabolism, phenylpropanoid biosynthesis, and inositol phosphate metabolism, enhancing antioxidant capacity and providing energy substances for germination. This study provides valuable insights into how rice seed embryo responds to and tolerates chilling stress with GA₃ seed priming.

Photosynthesis is responsible for 90–95% of organic matter in crop yield. The light energy utilization rate of high-yielding rice varieties is 1.0–1.5%, but the ideal value is about 3–5%. Yield can be further improved by improving photosynthetic function. Through the initial screening and re-screening of 220 Southeast Asian germplasm resources, we found that the net photosynthetic rate of Southeast Asian germplasm resource C1 was 36.96 μmol m⁻² S⁻¹, which is close to that of C₄ plant maize and 3.26 times higher than that of Southeast Asian germplasm resource G164 at 11.26 μmol m⁻² S⁻¹. Using C1 and G164 as materials, we compared the tissue structure, chloroplast ultrastructure, photosynthetic physiological indicators, and proteomics of sword leaves to determine the factors affecting photosynthetic function. Compared with G164, C1 exhibited increased number of vascular bundles, increased stomatal size and density, more abundant and neatly arranged chloroplasts and thylakoid grana, and higher chlorophyll fluorescence parameters. The activities and contents of the key photosynthetic enzyme Rubisco were higher in C1 than in G164. The two germplasm resources were subjected to iTRAQ analysis, and the results showed that compared with C1, nine proteins were down-regulated and one protein was up-regulated and associated with photosynthetic electron transport in G164; a total of 17 differential proteins were associated with CO₂ fixation, and nine were up-regulated and eight differential proteins were down-regulated in G164. The identified genes encode proteins in the photosynthesis and carbon fixation pathways, and the changes in gene expression were verified by real-time qPCR. The gene expression patterns were consistent with the protein expression patterns. The results suggest that most differential proteins are involved in electron transfer from PSII to PSI and in the CO₂ fixation pathway, and increasing the levels of such proteins can effectively enhance the photosynthetic efficiency. C1 can be used as a donor material for selection of high light efficiency varieties and in-depth photosynthesis studies.

Rice (Oryza sativa L.) leaf rolling enhances canopy architecture and photosynthetic efficiency, thereby improving yield potential. Through genome-wide association studies (GWAS) of 802 Xian and Geng rice accessions, we identified 32 loci associated with the leaf rolling index (LRI) and characterized OUTWARD ROLLED LEAF 4 (ORL4) as a regulatory gene from natural germplasm. CRISPR/Cas9-mediated knockout of ORL4 resulted in abaxially rolled leaves, driven by excessive bulliform cell proliferation and disrupted vascular patterning. A promoter variant (Chr04_19562737-C/T) modulates ORL4 expression by altering the binding affinity of the transcription factor RICE OUTERMOST CELL-SPECIFIC 8 (ROC8). Carriers of the ORL4C allele exhibit stronger ROC8 binding, higher ORL4 expression, and adaxially rolled or flat leaves; ORL4T carriers show the opposite phenotype. ORL4 forms a ternary complex with ROC8 and TOPLESS-RELATED PROTEIN 2(TPL2) to suppress ABAXIALLY CURLED LEAF 1(ACL1), a positive regulator of bulliform cell differentiation. Evolutionary analyses indicated that the ORL4C and ORL4T alleles originated from Oryza nivara and Oryza rufipogon, with ORL4C favored in Geng and ORL4T in Xian during domestication. Promoter editing of ORL4 in the elite cultivar “Shennong 89-366” generated lines with enhanced LRI, achieving a significant yield increase in field trials. This study elucidates the genetic and molecular basis of leaf rolling and provides a promising strategy for rice yield improvement via precise promoter editing.

Panicle architecture is a key agronomic trait determining rice yield. Identifying and characterizing the regulatory genes controlling panicle morphogenesis is essential for breeding improved rice varieties. To clarify the regulatory pathways underlying panicle architecture and identify novel candidate genes, we performed RNA sequencing (RNA-seq) of 1 cm and 15 cm young panicles from Zhonghua 11 (ZH11). Numerous differentially expressed genes were identified, primarily enriched in metabolic and cellular processes. Among the differentially expressed transcription factors, we identified a previously uncharacterized Homeodomain-Leucine Zipper (HD-Zip) family gene, PaniCle Development 8 (PCD8; LOC_Os08g37580), which was highly expressed in young panicles. CRISPR/Cas9-mediated knockout of PCD8 (PCD8-KO) significantly reduced grain number per panicle, seed setting rate, and grain width while increasing grain length, grain length-to-width ratio, and chalkiness, indicating that PCD8 regulates panicle morphogenesis. Analysis of the 3,000 Rice Genomes (3 K-RG) population identified four major PCD8 haplotypes. Haplotype4 (Hap4), a temperate japonica-specific haplotype associated with long grains, carries a G/A substitution at - 2464 bp in the promoter. Haplotype3 (Hap3) and Hap4 likely evolved from Haplotype1 (Hap1), and all four haplotypes are closely related. PCD8 showed lower nucleotide diversity in temperate japonica and indica than in wild rice. Neutrality tests indicated balancing selection in wild rice and positive selection in indica, whereas reduced diversity in temperate japonica mainly reflects domestication bottlenecks rather than strong locus-specific selection. DNA affinity purification sequencing (DAP‑seq) identified candidate downstream targets of PCD8, and electrophoretic mobility shift assays (EMSA) confirmed direct binding to selected targets. Multi-omics analyses demonstrate that PCD8 is a novel transcription factor regulating panicle morphogenesis through downstream gene networks, providing a promising candidate for high-yield rice breeding.

Background: High-temperature stress can cause serious abiotic damage that limits the yield and quality of rice. Heat tolerance during the flowering stage of rice is a key trait that can guarantee a high and stable yield under heat stress. Heat tolerance is a complex trait that is regulated by numerous quantitative trait loci (QTLs); of which, however, few underlying genes have been fine mapped and cloned.Results: In this study, the F_2:3 population derived from a cross between Huanghuazhan (HHZ), a heat-tolerant indica cultivar, and 9311, a heat-sensitive indica rice variety, were used to map the QTLs for heat tolerance during the flowering stage of rice. The F_2:3 lines were treated under 38 °C from 9:30 am to 3:30 pm continuously for 3 days in a phytotron, and spikelet fertility was assessed. A new major QTL, qHTT8, controlling heat tolerance was located on chromosome 8 using the bulked-segregant analysis (BSA)-seq method. The QTL qHTT8 was determined to be located in the 355500–4520000 bp region, with a size of 0.965 Mb. The candidate region of qHTT8 on chromosome 8 contained 65 predicted genes. Ten putative predicted genes were found to be associated with abiotic stress tolerance. In future studies, the backcross population will be constructed to fine map and validate the effect of qHTT8.Conclusion: The information obtained in this study is useful for the fine mapping and cloning of qHTT8 and breeding heat-tolerant varieties of rice using marker-assisted selection.

As the two main variety groups in improved rice, IND (indica) and JAP (temperate japonica and tropical japonica), extensive studies have utilized (single-nucleotide polymorphisms) SNPs on 233 selected improved rice cultivars to analyze the genetic basis of agronomic traits during their improvement and to compare their similarities and differences. However, the roles of other types of variations, such as insertions and deletions (INDELs) and structural variations (SVs), remain relatively underexplored. Here, using resequencing data from 233 improved rice accessions (IND: 142, JAP: 91), we identified 811,646 INDELs and 16,231 SVs in IND, and 652,793 INDELs and 10,533 SVs in JAP. The abundance of INDELs and SVs decreased as their length increased in both variety groups. INDELs and SVs also showed an uneven distribution across the chromosomes of the two variety groups. In IND, 38.92% of INDELs and 44.1% of SVs were located within the 2 kb upstream and downstream of genes; in JAP, this proportion was 38.49% for INDELs and 43.22% for SVs. By performing genome wide association studies (GWAS) using phenotypic data of six agronomic traits (heading date, flag leaf length, flag leaf width, panicle number, plant height, and thousand grain weight) along with INDELs and SVs, we identified 3,222 significant IND-INDELs, 537 significant IND-SVs, 1,996 significant JAP-INDELs, and 286 significant JAP-SVs. Comparison of significant loci revealed that IND and JAP shared only one INDEL associated with flag leaf length and one SV associated with panicle number, suggesting distinct genetic architectures determined by INDELs and SVs for these traits in the two groups. Furthermore, haplotype analysis of candidate genes demonstrated that INDELs and SVs influence key functional genes, such as the gene TAD1 (IND-INDELs) in flag leaf length, RCN2 (IND-SVs) in heading date, PLS2 (JAP-INDELs) in plant height, and OsYLC2 (JAP-SVs) in leaf development. This study analyzed the variation patterns of INDELs and SVs during the improvement of IND and JAP varieties, and identified INDELs and SVs associated with agronomic traits. These findings will provide valuable genetic and material resources for rice breeding.