Amylose content (AC) is a key determinant of rice eating and cooking quality (ECQ). Lower AC is generally associated with improved palatability and is therefore a desirable trait in rice breeding;however,effective manipulation of AC remains a challenge.In this study,we identified AC6,a novel endosperm-specific early nodulin-like (ENODL) gene,belonging to a 32-member ENODL family.

Seed germination is a complex, multistep developmental process that is critical for plant growth and regulated by intricate polygenic networks. Despite its agronomic significance, the precise mechanism underlying this process in rice remains incompletely understood. In this study, we determined a seed-specific gene OsDOG1L1, the ortholog of Arabidopsis DOG1, as an essential regulator that inhibits germination while promoting seed dormancy in rice. Overexpression of OsDOG1L1 led to delayed germination and enhanced dormancy, whereas loss-of-function mutants showed accelerated germination and reduced dormancy. Temporal analysis showed a progressive decline in OsDOG1L1 transcript and protein levels during germination. Furthermore, we identified two C2H2-type zinc finger transcription factors, ZFP36 (also known as Bsr-d1) and ZFP252, as direct regulators of OsDOG1L1, functioning upstream to repress OsDOG1L1 expression. Molecular and genetic analyses demonstrated that OsDOG1L1 interacts with and suppresses the phosphatase activity of clade A protein phosphatase 2Cs (PP2Cs), OsPP2C09 and OsPP2C30, thereby enhancing ABA signaling to inhibit seed germination. Together, our findings uncover a ZFP36/ZFP252-OsDOG1L1-OsPP2Cs regulatory module that governs rice seed germination, providing insights into the molecular regulation of germination control in rice.

Efficient seed germination and seedling establishment are critical for achieving high rice yields. This process is regulated by numerous metabolic pathways, including the mobilization of stored starch and the utilization of sugars. The Waxy (Wx) gene, which controls amylose content (AC) in rice grains, plays a key role in this process. Premature transcription termination of Wx determines whether the seed exhibits a waxy or non-waxy phenotype. While Wx is well established as a critical regulator of eating and cooking quality (ECQ) in rice, the present study identifies a novel role for Wx in mediating brassinosteroid (BR)-regulated seed germination. Analysis of Wx-related near-isogenic and transgenic lines revealed that seeds carrying the Wxa allele showed faster germination and superior post-germinative growth than those carrying the wx allele. This enhanced post-germinative growth was associated with increased Wx expression, which significantly boosted amylase activity and led to extensive starch degradation. Additionally, the higher expression and activity of α-amylase in Wxa seeds resulted in greater efficiency of starch-to-sugar conversion. In vitro embryo culture assays demonstrated that the Wxa allele and glucose acted synergistically to promote plumule growth. Moreover, differential Wx expression influenced ABA biosynthesis and catabolism in germinated seeds. Collectively, these findings suggest that appropriately modifying Wx expression to optimize starch composition could achieve the dual goal of improving both ECQ and germination-related traits in rice breeding.

Elevated temperatures are a major environmental stress factor that impairs rice productivity and compromises grain quality. This study investigated the effects of natural allelic variation in soluble starch synthase I (SSI) on grain quality and starch structural properties under high-temperature (HT) conditions. Four near-isogenic lines (NILs) differing in SSI alleles and carrying either the Wxb or wx backgrounds were cultivated under HT stress. A range of analytical techniques revealed that HT significantly reduced grain appearance quality, protein content, apparent amylose content (AAC), and total starch content across all NILs. In addition, rapid visco-analysis (RVA) profiles of rice flour showed a marked reduction in viscosity under HT. Starch fine structure analysis demonstrated a decrease in short-chain amylopectin and an increase in long-chain amylopectin under HT, which was associated with enhanced starch crystallinity and elevated gelatinization temperatures. Notably, rice lines carrying the SSIi allele, in both Wxb and wx backgrounds, exhibited milder declines in grain quality traits compared to those with the SSIj allele. These findings provide new insights into the role of SSI allelic variation in maintaining rice grain quality under HT stress and offer a genetic basis for breeding heat-tolerant rice varieties.

Pre-harvest sprouting (PHS), caused by weak seed dormancy and environmental stimuli, leads to significant losses in both crop yield and grain quality. Breeding crop cultivars with enhanced PHS resistance represents a promising strategy to address this challenge. However, limited useful genetic resources has hindered the progress in rice molecular breeding. Through screening of a rice mutant library, we identify the ethylene response factor115 (erf115) mutant, which exhibits enhanced PHS resistance. Genetic analysis reveals that ERF115 functions as a negative regulator of seed dormancy. Mechanistic assays show that the E3 ubiquitin-protein ligase Grain Width 2 (GW2) interacts with and ubiquitinates ERF115, thereby promoting its proteasomal degradation. Accordingly, gw2 mutants display increased PHS susceptibility. ERF115 also interacts with the transcription factor SLR1-like 2 (SLRL2) and represses its transcriptional activation activity, consequently reducing the expression of the dormancy gene Mother of FT and TFL1 like 2 (MFT2). Haplotype analysis identifies three major ERF115 haplotypes (HapI-HapIII), among which ERF115Hapl represents an elite allele associated with reduced PHS. Collectively, our findings reveal a GW2-ERF115-SLRL2 regulatory module that integrates ubiquitin-mediated regulation and hormone signaling to fine-tune rice seed dormancy, providing valuable genetic resources for breeding PHS-resistant rice varieties.

Seed germination initiates the plant life cycle, but it exhibits high sensitivity to salt stress, which is a significant environmental factor limiting rice production. Brassinosteroid (BR) is a growth-promoting phytohormone that mitigates various stresses in rice including salt, drought, and extreme temperatures. However, the mechanisms by which BR alleviates salt stress during seed germination remain inadequately characterized. This study demonstrates that seed-specific overexpression of OsDWF4, a rate-limiting gene in BR biosynthesis, enhances rice germination. The DWF4-OX lines, which have greater endogenous BR content in the seeds, showed better germination under salt stress, corroborating the results obtained through exogenous BR application. Antioxidant enzyme analyses demonstrated that BR enhances the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Metabolomic analysis revealed that BR mitigates salt stress primarily through the biosynthesis of phenylpropanoids and secondary metabolites. Transcriptomic analysis indicated that both endogenous and exogenous BR share five co-regulated target genes and utilize a common biosynthetic pathway for stilbenoids, diarylheptanoids, and gingerols. These findings confirm the capacity of BR to enhance seed germination under salt stress and identified several BR-mediated targets for developing salt-tolerant rice varieties that are suitable for direct seeding cultivation.

China has a long history of rice cultivation and a rich rice-farming culture. As the world’s largest producer and consumer of rice, continuously advancing rice research and production is vital to ensuring food security. In this review, we systematically summarize the progress in rice research and production over the past decade (2015–2025) and highlight emerging challenges in China. This review synthesizes national and regional characteristics of rice production, consumption, trade, along with advances in the conservation and utilization of rice germplasm resources, the molecular mechanisms of domestication and genomics. It provides an in-depth elaboration of the molecular and genetic basis governing rice agronomic traits, including yield components, grain quality, hybrid fertility, nutrient-use efficiency, and abiotic/biotic resistance. This is followed by a comprehensive overview of rice cultivar improvement adapted to the major ecological zones. This work further highlights the transformative shift in rice cultivation management from traditional labor-intensive farming to simplified, smart, and unmanned cultivation systems. Future directions for rice breeding are discussed, with a focus on the integration of molecular design, multi-omics, and artificial intelligence technologies to build high-efficiency breeding systems that are conducive to sustainable and resilient rice production in China.

Grain shape is an important trait affecting yield and quality(Ren et al.2023).Indica and japonica rice have differences in quality and yield-related traits,including grain shape.Indica rice generally has a slender grain that decreases the head rice rate and yield loss,while japonica has shorter and wider grains with high yield(Jiang et al.2022).However,grain shape and chalkiness are often correlated.Grain width is a significant factor influencing grain filling.Wider grain is often accompanied by more chalkiness,whereas slender grains can optimize grain-filling pathways,reducing the chalkiness without yield loss.

High quality stands as a pivotal competitive edge in the rice industry. Optimizing amylose content (AC) and the physicochemical properties of endosperm starch by regulating the Wx gene is crucial for enhancing rice grain quality. In this study, we created a novel Wxb-d25 allele by deleting a 25 bp segment (−26 to −2) within the Wx core promoter using CRISPR/Cas9. Compared with the wild type and the previously reported Wxb-i1, Wxb-d25 exhibited no significant changes in agronomic traits. However, its grains displayed temperature-dependent variations in AC and altered transparency and viscosity characteristics, holding the potential to synergistically improve both the eating and cooking quality (ECQ) and appearance quality (AQ) of rice. Further studies demonstrated that this promoter modification, by partially disrupting the transcription initiator, significantly downregulated the original Wx-01 transcript and generated a novel Wx transcript (ONT.7395.1) in Wxb-d25 grains. Despite its low expression abundance, the ONT.7395.1 transcript could be completely processed into mature Wx mRNA. The combined effects of the dual transcripts resulted in significantly increased Wx gene expression and AC in Wxb-d25 grains under conventional cultivation conditions. These findings provide a genetic resource and a theoretical foundation for utilizing the Wxb-d25 allele to improve rice grain quality.

Brassinosteroid acts via the BZR1-SLRL2-Wx module to regulate amylose content in rice, making BZR1 a potential target for breeding rice with both superior quality and high yield.

Since 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing COVID-19, has been spreading and mutating globally despite the expedited approval of many commercial vaccines. Therefore, developing safe, effective and affordable vaccines remains essential to meet the global demand, particularly in developing countries. Transgenic plants have emerged as a promising platform to express recombinant proteins for pharmaceutical and vaccine applications. Two binary vectors, pCAMBIA1300Gt1-S1 and pCAMBIA1300Actin-S1, containing distinct promoters, were constructed and transformed into rice via Agrobacterium. Overall, 56 independent transgenic rice lines were regenerated. Expression analysis revealed that the rice-derived S1 (rS1) protein could be expressed in pGt1::S1 transgenic rice seeds. rS1 protein expression levels reached up to 282 μg/g dry weight, with S1 gene insertion having no effect on grain size and weight. The rS1 protein exhibited a high affinity for human angiotensin-converting enzyme 2 (ACE2) in vitro. Moreover, the immunogenicity of purified rS1 protein co-administered with various adjuvants demonstrated that mice vaccinated with Alum-adjuvant rS1 generated enhanced humoral immune responses with high serum IgG, IgG1 and neutralizing antibody levels. Salmonella Typhimurium flagellin (FliC)-adjuvanted rS1 elicited stronger S1-specific IgG2a levels, promoted splenocyte proliferation and induced mixed Th1/Th2/Th17 cytokine responses. This was evidenced by increased proportions of antigen-specific interferon (IFN)-γ, interleukin-4 (IL-4) and IL-17A-positive CD4+ T lymphocytes, suggesting its potential to induce both humoral and cellular immune responses. These findings suggest that rS1 protein offers a promising approach for affordable COVID-19 subunit vaccine production, and this strategy can be universally applied to other viral vaccines.

DNA methylation plays a crucial role in the regulation of gene expression, ensuring normal growth and development and enabling responses to biotic and abiotic stresses in plants. Carbon dots (CDs) can participate in the entire plant life cycle and improve yield, however, the understanding of how CDs affect plant growth remains limited. Here, we demonstrate that CDs induce subtle but significant global DNA hypermethylation in rice, particularly in CHG and CHH contexts. This hypermethylation may be driven by the upregulation of OsCMTs triggered by CDs. CDs can stabilize i-motifs-potential regulators of active genes-in the promoter regions of OsCMT1 and OsCMT2, leading to increased CHG and CHH methylation. Our findings suggest that CDs modulate global DNA methylation changes, influencing the transcription of genes associated with phenotypic alterations in plants.

DELLA degradation is controlled not only by gibberellic acid (GA) but also by various GA/GID1-independent factors such as light, temperature, and shade. New insights on the evolution of DELLA family proteins and the biological role of DELLA-like proteins in seed traits provides valuable directions for future crop breeding programs.

As the staple food for more than half of the world's population, rice requires elite varieties with superior quality and high yield to ensure food security. Agronomic traits, such as grain size, leaf angle, seed dormancy, and germination, will affect rice yield. Identification and cloning of key genes and elucidation of molecular mechanisms regulating these traits expedite rice breeding. The OVATE Family Proteins (OFPs), a unique family of transcription regulators, play critical roles in regulating grain or fruit size, plant morphology, and stress responses. Here, we have successfully identified OsOFP9, an uncharacterized OFP member in rice, and demonstrated its irreplaceable role in controlling several key agronomic traits. Mutation of OsOFP9 results in severe pre-harvest sprouting, promoted seed germination, smaller grains, and reduced leaf angle. Mechanistic studies revealed that the OsOFP9 mutation reduced abscisic acid (ABA) levels and increased gibberellin (GA) levels, thereby affecting the ABA/GA ratio and α-amylase activity. In addition, OsOFP9 directly interacts with GS9 and DLT, key transcriptional regulators involved in the BR signaling pathway controlling grain size and leaf angle, respectively. Functional assays showed that OsOFP9 inhibited the transcriptional activation activity of GS9, but enhanced the transcriptional repression activity of DLT. Genetic evidence showed that GS9 and DLT function downstream of OsOFP9, consistent with the results of the transcriptional activity assay. In conclusion, this study reveals the crucial role of OsOFP9 in regulating several important agronomic traits and elucidates its molecular mechanism in coordinating multiple plant hormones, thus providing valuable insights and genetic resources for improving rice yield.

Rice seeds are an important energy source for humans. Seed traits are difficult to observe and controlled by complex networks. Therefore, mutant libraries enriched in seed traits are vital for interpreting gene functions during seed development as well as grain yield and quality formation. Using the simple and efficient genomic editing tool, several CRISPR/Cas9-based mutant libraries have been generated in rice (Chen et al., 2022; Lu et al., 2017; Meng et al., 2017), and other crops (Bai et al., 2020; Jacobs et al., 2017; Liu et al., 2020). Genome-wide mutants have some disadvantages (Gaillochet et al., 2021), whereas appropriate-scale mutants may help focus on the special study, such as seed traits. Thus, screening specific gene sets as targets is crucial (Liu et al., 2023). Besides, traditional individual editing may be beneficial for appropriate-scale population compared with the reported pooled transformation (Liu et al., 2023), and has advantages when studying genes related to seed lethality. In this study, we first identified 3288 genes with significantly differential expression using RNA sequencing (expression in seeds and twofold than leaf, hull and inflorescence, p < 0.05), which were defined as seed differentially expressed genes (Table S1), which may be important for seed function. Furthermore, we identified a stringent group of 1206 genes with a strong preference for seed expression, which was defined as the seed-preferred gene (expression in seeds and 10-fold than leaf, hull and inflorescence, p < 0.05, Table S2), and examined their functions. They are further subdivided into two categories, the 1160 specific (I, II and III) and the 46 dominant, based on the degree of tissue-restricted expression (Table S2). However, we also included 374 genes whose expression did not meet the 10-fold threshold but were hypothesized to involve in seed development from other literatures (Table S2). We also used public databases RiceXPro, RGAP and TENOR to classify the integrated 1580 genes into three subgroups, 794 endosperm-preferred genes (OsEnP), 291 embryo-preferred genes (OsEmP) and 495 others (Tables S2 and S3). These results provided more information regarding the expression of seed-preferred genes. As an initial attempt to establish a seed-preferred gene knockout (KO) mutant library, we chose 244 genes from the above 1580 ones, including 174 OsEnPs, 56 OsEmPs and 14 others (Table S2), covering various types of expression patterns but with an emphasis on endosperm. Besides, other 66 genes of interest were used as controls. Finally, a total of 310 genes were selected for KO trial (Table S2; Figure S1a), and their expression patterns are presented through a clustering heat map as shown in Figure 1a and Table S4 and S5. To reduce the possibility of off-target, we used strict criteria to design the guide RNAs (gRNAs) (Figure S2) and produced 375 gRNAs (Figure S1b; Table S6–S8), of which 99 were obtained from an existed library (Lu et al., 2017). Notably, to ensure the acquisition of mutants and analyse the importance of gRNA design within same one gene, two gRNAs were designed for 65 genes. Then, the mutant library was constructed by transforming the design gRNAs one by one (Figure S1c,d). A total of 2688 stable T0 transgenic seedlings were generated and genotyped, which covers all 375 gRNAs (Figure S3; Table S9). Therein, 2184 of the 2598 seedlings targeted 367 gRNAs, successfully mutated. The mutation frequency was 84.06%. The remaining 90 seedlings for the eight gRNAs had no mutations (Figure 1b). Correspondingly, 302 genes got mutants and eight genes did not get mutants (Table S9). However, the mutation rate of each gRNA differed significantly (Figures S4 and S5). Therein, 96% of gRNAs yielded 3–8 mutant seedlings (Figure S6). All the mutation sites were caused by short sequence insertion and/or deletion (Figure S7), and the seedlings for most gRNAs contained frameshift mutations (Figure S3). Subsequently, we investigated the transmission of mutations from T0 to T1 generations in the planted T1 lines, and found that all 1002 decodable T0 transformants produced the expected genotypes in T1 lines following classic Mendelian law, and 92.74% of the 427 undecodable T0 transformants could be decoded in T1 lines might due to the generational reduction in sequence complexity (Figure S8). Most T0 transformants (72.16%) contained a single copy transgene region (T-DNA) through separation ratio analysis of hygromycin resistance gene, and it was easy to obtain transgene-free mutants (Table S11), which are essential for further using these novel germplasm resources. Off-target effects is a major concern for CRISPR/cas9 system. Therefore, the first putative off-target sites of each gRNA were screened using off-target tools, and probable off-target sites for the 18 gRNAs were sequenced in the T1 lines (off-score >0.6). No off-target mutations were observed in these selected gRNAs (Table S7). To mine novel gene resources related to seed development, we then carefully measured several important seed traits for all these mutants. Specifically, at least 74 candidate genes were identified to have a significant influence on taste quality, such as apparent amylose content, protein content, and starch viscosity (Tables S12 and S13). For the grain appearance quality, 14 candidates were differed, including nine unknown OsEnP genes (Figure 1c; Table S12). In conclusion, a high proportion of genes or mutants (>50%) exhibited altered grain phenotypes, even within the limited scope of the investigation (Table S12), implying that the approach is effective for mining seed mutants. Chalkiness is susceptible to genetic and environmental factors, resulting in difficulty to cloning the caused genes. Using this library, several chalky mutants from the unknown genes were successfully identified (Figure 1c), as one example by Chalk3/LOC_Os03g45210 (Figure 1). The expression of Chalk3 gene was preferentially high in the developing endosperm, with the highest in the middle stage during seed development (Figure 1d; Table S2). The chalk3 mutant, SG6280, exhibited no visible differences in plant architecture or grain size (Figure 1e,f; Figure S9); however, the grain chalkiness significantly increased, with a pronounced core and belly white endosperm (Figure 1g–j). Another mutant from the Chalk3 gene, SG6281, exhibited the same increased chalkiness (Figure S10). These results confirmed that Chalk3 indeed plays a specific role in regulation of grain chalkiness. The chalky area of chalk3 had notably different starch grains from those of wild type (Figure 1j). Total starch and protein contents were lower in chalk3 grains, whereas soluble sugar levels were higher (Figure S11a–c). The chalkiness of chalk3 mutants is susceptible to environmental influences (Figure S11d). RNA-sequencing analysis revealed that the chalk3 mutation resulted in a number of differentially expressed genes (DEGs; Table S14). Additionally, these DEGs were significantly (p < 0.05) enriched for carbon and nitrogen metabolism, and plant hormone signal transduction (Figure S12). These results indicated that chalk3 mutation alters the accumulation of stored substances in seeds, causing chalky endosperm, which might be involved in plant hormone, providing a new clue for regulating chalkiness (Zhao et al., 2022). In conclusion, we gave the expression of seed-preferred genes in rice, and established a seed-preferred mutant library on an appropriate scale based on CRISPR/Cas9 individual editing. As an example for mining novel genes using these mutants, we further elucidated the function of Chalk3/LOC_Os03g45210 on the regulation of grain appearance quality. This manageable seed-preferred mutant library provides a resource for identifying unknown genes involved in seed development. And the batch approach may be feasible for gradually generating an individual gene mutant library that covers all genes. This work was supported by grants from the National Key Research and Development Program of China (2022YFD1200103), the National Natural Science Foundation of China (32230074 and 31971914) and the Government of Jiangsu Province (BE2022336, BM2022008-02, 23KJA210001 and PAPD). The authors declare no competing interests. Q. Li, C. C. and Q. Liu designed and supervised this study. D. Z., S. C., Y. H., G. L., J.L., Q.Y., T.Z. and J.S. performed the experiments. D. Z., S. C., X. F., C. Z., T. Z. and Q. L. analysed the data. D. Z., S. C. and Y. H. wrote this article. T. Z., Q. Li., C. C. and Q. Liu revised this article. All data in this study are available in this article or supplementary information. RNA sequencing data were as follows: SRA, PRJNA817295. Figure S1-S12 Supplementary Figures. Table S1-S14 Supplementary Tables. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Brassinosteroids (BRs) are phytohormones that regulate stomatal development. In this study, we report that BR represses stomatal development in etiolated Arabidopsis (Arabidopsis thaliana) cotyledons via transcription factors BRASSINAZOLE RESISTANT 1 (BZR1) and bri1-EMS SUPPRESSOR1 (BES1), which directly target MITOGEN-ACTIVATED PROTEIN KINASE KINASE 9 (MKK9) and FAMA, 2 important genes for stomatal development. BZR1/BES1 bind MKK9 and FAMA promoters in vitro and in vivo, and mutation of the BZR1/BES1 binding motif in MKK9/FAMA promoters abolishes their transcription regulation by BZR1/BES1 in plants. Expression of a constitutively active MKK9 (MKK9DD) suppressed overproduction of stomata induced by BR deficiency, while expression of a constitutively inactive MKK9 (MKK9KR) induced high-density stomata in bzr1-1D. In addition, bzr-h, a sextuple mutant of the BZR1 family of proteins, produced overabundant stomata, and the dominant bzr1-1D and bes1-D mutants effectively suppressed the stomata-overproducing phenotype of brassinosteroid insensitive 1-116 (bri1-116) and brassinosteroid insensitive 2-1 (bin2-1). In conclusion, our results revealed important roles of BZR1/BES1 in stomatal development, and their transcriptional regulation of MKK9 and FAMA expression may contribute to BR-regulated stomatal development in etiolated Arabidopsis cotyledons.

Background Rice is one of the major staples that feeds about one half of the global populations, and it is important to identify the genetic loci for the traits related to yield improvement. Lodging will cause severe yield loss when it happens, and stem diameter has been characterized as an important trait for lodging resistance. However, most QTLs for stem diameter have not been finely dissected due to their sensitivity to environmental fluctuation. Result In this study, we performed QTL analysis for stem diameter using populations derived from Nipponbare (NIP) and strong culm variety YYP1, and confirmed the single and combined effect of three major QTLs by recombinant inbred lines (RILs). Based on the QTL location, we found that qWS5 is a novel QTL not well characterized before. To finely dissect the novel locus, several recombinant heterogeneous inbred families (HIFs) were selected from the RILs for linkage analysis and their derived nearly isogenic lines (NILs) were subjected to detailed trait investigation throughout different years. The HIF-NILs strategy confined the QTL to about 380 kb region supported by repeated genotype and phenotype data, and it lays the foundation for QTL cloning in the future. In addition, introgression of the QTL to an elite japonica variety SD785 was performed by successive backcrossing, and it confirmed the value of qWS5 in increasing stem diameter and other agronomic traits during rice breeding. Conclusions We prove that qWS5 is a novel QTL with relatively stable effect for stem diameter and the QTL can be finely mapped to small region by the HIF-NILs strategy. The result will facilitate the improvement of rice lodging resistance by molecular marker assisted selection breeding.