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.

Introduction/Background: Global warming greatly limits the productivity of rice. Rice plants are highly sensitive to heat stress at the flowering stage. The selection of heat-tolerant varieties is considered the most effective approach for ensuring global food security in the coming decades.Methods: Based on previous screening and QTL localization results, we selected tolerant varieties (Huang Huazhan, HZ) and susceptible varieties (Yang Dao6, YD) of rice and studied their physiological characteristics, gene expression responses, and proteomic differences of their anthers under heat stress. The differentially expressed proteins (DEPs) were validated by real-time PCR.Results: The activities of the antioxidant enzymes CAT, SOD, POD, and APX were 8.36%, 9.56%, 20.61%, and 25.34% higher in HZ than in YD under heat stress, respectively. Similarly, the content of proline and soluble sugar was 8.32% and 14.47% higher in HZ than in YD, respectively. The content of MDA and H₂O₂ was 8.11% and 39.5% lower in HZ than in YD, respectively. The ratio of endogenous GA₃/ABA in HZ was 10.65, which was significantly higher than that of YD (3.84). In addition, we validated the candidate genes LOC_Os08g07010 and LOC_Os08g07440 that our team located in 2021, and the result showed that the expression of these two heat-tolerant genes in the anthers was significantly higher in HH than in YH. DEPs involved in the response to heat stress were identified by TMT proteomics, five upregulated and three downregulated differential expression proteins in HH. DEPs were verified by RT-qPCR.Discussion: These results provide new insights into the physiological characteristics, dominant DEPs, and gene expression responses in both rice varieties under heat stress. Our results indicate that the antioxidant and osmoregulatory capacities, the ratio of endogenous GA₃ and ABA, these DEPs are mainly involved in the pathways of phenylpropanoid biosynthesis, ubiquitin-mediated proteolysis, carbohydrate metabolism, thiamine metabolism, protein processing in the endoplasmic reticulum, and folding, sorting, and degradation were upregulated to a greater degree in HZ than in YD. Additional studies were performed to clarify the roles of these proteins in response to heat stress.

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.