Monochoria vaginalis is a serious weed in rice paddies, which is harvested as a vegetable crop, an ornamental, a forage plant and a plant for local medicinal use. In the summer of 2024, when weeding a rice breeding experimental field located at [Latitude: 23°02′53″N, Longitude: 108°19′22″E] in Wuming District, Guangxi, China, root-knot nematodes (RKNs) were found, with galls prevalent in the roots of M. vaginalis and the plants exhibiting symptoms of stunted growth and reduced vigor. Females and eggs were observed in dissected root-knots under a microscope. Morphological measurements of females (n = 20) included body length (L) = 545.3 ± 51.6 (462.2 to 628.4) μm; body width (BW) = 371.5 ± 35.9 (298.6 to 438.4) μm; stylet = 12.2 ± 1.2 (9.4 to 14.1) μm; dorsal pharyngeal gland orifice to stylet base (DGO) = 4.2 ± 0.4 (3.5 to 4.8) μm. The perineal patterns of females were dorsoventrally oval-shaped with low and round dorsal arches, a few well-marked, irregular, short, zig-zag striae, and indistinct lateral lines. Measurements of second-stage juveniles (J2s) (n = 20) were L = 467.9 ± 17.9 (432.5 to 501.2) μm; BW = 21.5 ± 2.1 (17.8 to 25.1) μm; stylet = 13.7 ± 0.6 (12.5 to 14.8) μm; DGO = 3.4 ± 0.3 (2.8 to 4.1) μm; tail = 72.8 ± 3.4 (68.7 to 80.6) μm; hyaline tail length = 19.7 ± 1.9 (16.5 to 22.8) μm. The J2s were vermiform and had a long and slender tail with a tapering hyaline tail terminus. These morphological features closely resemble the previously described characteristics of Meloidogyne graminicola (Golden and Birchfield. 1965). Individual J2s (n = 3) DNA was further explored using the primers 18S/26S (Vrain et al. 1992), C2F3/1108 (Powers and Harris. 1993), and Cox1F/Cox1R (Trinh et al. 2019). Three fragments (790 bp [GenBank accession no. PQ345083], 531 bp [PQ456744], and 413 bp [PQ350374]) were amplified and had 100% sequence homology with M. graminicola (MG773553.1, KY020413.1, and MN017128.1, respectively). The species-specific primers Mg-F3/Mg-R2 (TTATCGCATCATTTTATTTG/CGCTTTGTTAGAAAATGACCCT) were used to amplify the internal transcribed spacer (ITS) region (Htay et al. 2016), that an expected PCR fragment of approximately 369 bp was obtained. Therefore, the unknown nematode was further identified as M. graminicola. For the pathogenicity test, 10 four-leaf stage M. vaginalis seedlings planted in pots with sterilized sandy soil were inoculated with 500 freshly hatched J2s/pot from the original population, and noninoculated seedlings (n=10) were used as controls. All plants were grown in an illuminating incubator at 28°C with a 12 h light/12 h dark photoperiod. After 7 weeks, all inoculated plants showed gall symptoms on the roots identical to those observed in the fields. The nematode reproduction factor (final population/initial population) was 17.8 ± 2.3. No galls were observed on noninoculated plants. These results confirmed the pathogenicity of M. graminicola on M. vaginalis. Although the early literature mentioned that the plant was a host for M. graminicola, this was not confirmed (Bridge et al. 2005). To our knowledge, this is the first report of a natural infection of M. graminicola on M. vaginalis in China. As an annual herb with a well-developed root system, M. vaginalis supports a high population density of nematodes. Our study showed the nematode's rapid population growth on this plant, severely threatening rice production. Thus, promptly controlling M. vaginalis is essential for ecological management of M. graminicola, ensuring the protection of crop yields.

Rice leaf angle, a major determinant of plant architecture and yield potential, is tightly regulated by brassinosteroid (BR) signaling. Although the core BR pathway is well characterized, how BR responses are precisely controlled in space and time remains unclear. Here, we identify OsvWA36, a novel regulator required to fine-tune BR signaling. The osvwa36 mutant exhibits typical BR-deficient phenotypes, including decreased leaf angle and reduced BR responsiveness, whereas ubiquitin promoter-driven expression of OsvWA36 confers BR hypersensitivity. We further show that the OsvWA36 protein undergoes intrinsically disordered region (IDR)-driven liquid-liquid phase separation to form dynamic condensates associated with the endoplasmic reticulum. These condensates facilitate a direct interaction between the OsvWA36 protein and the KNOX transcription factor OSH1. Genetic analyses indicate that OsvWA36 is necessary for full activation of the OSH1-regulated transcriptional program, including induction of BR catabolic genes (e.g., CYP734A2/4/6). In parallel, OsvWA36 broadly influences the expression of key activators of BR signaling and biosynthesis. Collectively, OsvWA36 integrates both positive and negative regulatory branches of the BR network to optimize leaf angle. These findings reveal a phase separation-based mechanism underlying hormone signaling and suggest that OsvWA36 is a promising target for the engineering of plant architecture.

Soil acidification poses a significant threat to rice production, yet the molecular mechanisms underlying acid tolerance remain poorly understood. Employing a combination of comprehensive phenotypic screening with multi-omics analysis, this study investigates acid stress responses in rice. Screening of 81 cultivars identified contrasting acid-tolerant and acid-sensitive genotypes from both conventional and hybrid backgrounds. Acid stress induced pronounced alterations in root architecture and triggered extensive transcriptional reprogramming. Root transcriptomes were profiled under a gradient of acidic pH (3.5 to 6.0, with pH 6.0 as control). Physiological analyses confirmed that tolerant genotypes maintained superior membrane stability and antioxidant enzyme activities under stress. Transcriptome analysis revealed 7,451 acid-responsive differentially expressed genes (FDR < 0.05, |log₂FC| > 1), with 381 core genes commonly responsive across all genotypes. These core genes were enriched in transmembrane transport, iron homeostasis, and reactive nitrogen species metabolism. Variety-specific analysis indicated that acid-tolerant cultivars were enriched for protein repair and transport functions, while sensitive genotypes predominantly activated glutathione and phenylpropanoid pathways. Co-expression network analysis (Pearson’s |r| > 0.6) identified key modules involved in signal transduction, oxidative homeostasis, and ion balance. Transcription factor analysis revealed that conventional rice exhibited stronger activation of ERF, WRKY, and MYB families, while hybrids displayed a pattern of ion transporter and ROS-related genes, validated by qRT-PCR, aligned with physiological measurements: conventional rice maintained better ion homeostasis and antioxidant capacity, whereas the hybrid genotypes examined in this study exhibited contrasting responses, the tolerant hybrid DXY901 maintained robust defense activation, while the sensitive hybrid RFY838 showed a concurrent downregulation of proton pumps and peroxidase systems under severe stress. Collectively, our findings elucidate the transcriptional framework and physiological basis of acid stress adaptation in rice, providing potential targets for breeding acid-tolerant rice varieties.

Grain length is a crucial determinant of rice yield and quality. Although von Willebrand factor type A (VWA) domain proteins have recently emerged as regulators of plant architecture in cereals, their roles in controlling rice grain length remain largely unexplored. Here, we report the functional characterization of OsvWA36, a VWA-domain protein selected from a TMT-based quantitative proteomic screen of indica rice varieties differing in grain length. Loss-of-function OsvWA36 mutants exhibited significantly reduced grain length and thousand-grain weight, while complementation and overexpression assays confirmed its positive regulatory role in these traits. Cytological analysis revealed that the shortened grain phenotype was due to suppressed longitudinal elongation of hull epidermal cells, which was accompanied by aberrantly enhanced lignin deposition. Transcriptomic profiling and Gene Ontology (GO) enrichment analysis demonstrated that OsvWA36 is essential for the expression of a comprehensive suite of cell wall biosynthesis and modification genes, including those involved in cellulose, pectin, and lignin metabolism. Furthermore, OsvWA36 localizes to punctate structures on the endoplasmic reticulum (ER). Our study establishes OsvWA36 as a novel VWA-domain protein that positively regulates grain length by orchestrating cell wall remodeling programs, thereby bridging VWA protein function with the transcriptional regulation of cell wall dynamics in rice. This work not only identifies a promising genetic target for molecular breeding but also provides a new molecular framework for understanding grain size regulation in cereal crops.

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.

Grain length is a critical agronomic trait that directly determines rice yield. In this study, we identified OsvWA36, a von Willebrand factor A (VWA) domain protein containing intrinsically disordered regions (IDRs). We showed that it is a novel positive regulator of grain length and that its function is achieved through liquid-liquid phase separation (LLPS) and subsequent modulation of cell wall remodeling.OsvWA36 was discovered through proteomic screening, and its abundance was positively correlated with grain length. It preferentially accumulated in developing panicles and formed liquid-like condensates via IDR-mediated LLPS, as indicated by in vitro/in vivo assays and fluorescence recovery after photobleaching analysis. CRISPR-Cas9-generated osvwa36 mutants developed shorter grains due to reductions in glume cell length and the aberrant accumulation of lignin. Transcriptomic and qRT-PCR analyses revealed that deficiency in OsvWA36 suppressed the expression of genes associated with cell wall dynamics, including those involved in cellulose synthesis ( OsCESA4, OsCESA7 , and OsCSLE1 ), pectin metabolism ( OsPME68, OsPME1 ), and lignin modification ( OsCAD2, OsMYB58 , and OsExo70H3 ). Genetic complementation restored the wild-type phenotype, whereas overexpression of OsvWA36 further elongated grains. Deletion of the IDR domain abolished LLPS and resulted in short grains, phenocopying the osvwa36 mutants and underscoring the functional necessity of phase separation. Furthermore, haplotype analysis revealed that natural variation in OsvWA36 was correlated with grain length diversity in rice cultivars.In conclusion, our findings indicate that OsvWA36 regulates grain length by orchestrating cell wall remodeling through IDR-mediated LLPS and could be a useful target for molecular breeding strategies aimed at improving yield.

High temperature significantly impacts grain appearance quality, yet few studies have focused on identifying new quantitative trait loci (QTLs)/genes related to these traits under heat stress during the flowering stage in rice. In this study, a natural population of 525 rice accessions was used to identify QTLs and candidate genes associated with grain appearance quality using a Genome-Wide Association Study under heat stress. We identified 25 QTLs associated with grain length (GL), grain width (GW), and grain chalkiness (GC) under heat stress across 10 chromosomes in the three rice populations (full, indica, and japonica). Notably, three sets of overlapping QTLs were identified (set 1: qHTT-L3 and qHTT-XL3; set 2: qHTT-C5 and qHTT-XC5; set 3: qHTT-L11.1 and qHTT-GL11), located on chromosomes 3, 5, and 11, respectively. Haplotype analysis indicated that Hap1 is the superior haplotype, and pyramiding more than two superior alleles improved rice grain appearance quality (longer GL, wider GW, and lower GC) in high-temperature environments. Based on RNA-seq, qRT-PCR and functional annotations analysis, LOC_Os05g06920, LOC_Os05g06970, and LOC_Os11g28104 were highly expressed, identifying them as the high-priority candidate genes for QTLs linked to grain appearance quality (GL, GW, and GC) under heat stress. Expression analysis revealed that LOC_Os05g06920, which encodes a relA-SpoT-like protein RSH4, and LOC_Os11g28104, which encodes a protein kinase with a DUF26 domain, were highly expressed in seeds, leaves, and shoots. And LOC_Os05g06970, encoding a peroxidase precursor, exhibited high expression levels in roots. Compared to the wild-type (WT) plants, the mutants of LOC_Os05g06920, LOC_Os05g06970, and LOC_Os11g28104 exhibited increased GL and grain length-to-width ratio, but reduced GW under both natural and heat stress conditions, while the LOC_Os05g06970 and LOC_Os11g28104 mutants significantly increased the chalky grain rate and grain chalkiness degree under natural conditions. Furthermore, the LOC_Os05g06920, LOC_Os05g06970, and LOC_Os11g28104 mutants showed a lower decline in grain appearance quality traits than the WT after high-temperature treatment. These findings suggest that LOC_Os05g06920, LOC_Os05g06970, and LOC_Os11g28104 play crucial roles in regulating both grain development and heat tolerance under heat stress at anthesis, thus affecting grain appearance quality in rice. Our results provide a promising genetic resource for improving rice grain appearance quality under heat stress.

Rice blast disease, caused by Magnaporthe oryzae (M. oryzae), poses a major threat to global rice production annually. The Pi1 gene is a key determinant of resistance to this pathogen. However, the proteomic responses of rice to M. oryzae infection in both Pi1-containing and Pi1-deficient backgrounds remain poorly understood. This study investigated Pi1-mediated protein responses in rice using quantitative proteomics to compare the susceptible line MeiB and its Pi1-introgression line 96B. Comparative analysis of 4-day post-infection samples versus untreated controls identified 121 differentially expressed proteins (DEPs) in 96B and 126 in MeiB. Functional classification showed that DEPs related to cellular processes, metabolic processesprocesses, and responses to stimuli were significantly enriched in Gene Ontology (GO) analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed enrichment in metabolic pathways, secondary metabolite biosynthesis, and phenylpropanoid biosynthesis in both lines. Notably, Pi1 modulated proteins associated with apoptosis and purine metabolism during M. oryzae infection in 96B. Network analysis revealed 44 DEPs, including the pathogenesis-related protein PR10a, forming protein–protein interaction networks in 96B, compared to only 22 DEPs in MeiB. The key findings include the specific regulation of biotic stress-related proteins such as Gnk2-homologous domain-containing protein and AT-hook motif nuclear-localized protein in 96B; apoptosis and purine metabolism pathways being unique to DEPs from 96B; and the presence of pathogenesis-related protein 10a and stress-responsive proteins (e.g., Bet_v_1 domain-containing and Gnk2-homologous domain-containing proteins) in the 96B interaction network. Therefore, these results suggest that Pi1 contributes to rice blast resistance by modulating apoptosis/purine metabolism pathways and protein–protein interaction networks.

Temperature is a critical abiotic factor affecting rice (Oryza sativa L.) yields, and cold stress at the seedling stage can inhibit plant growth or even be fatal. Antioxidants such as anthocyanins accumulate in a variety of plants during cold stress, but the underlying mechanisms are not well understood. Here, we report that rice TRANSPARENT TESTA GLABRA 1 (OsTTG1), a major regulator of anthocyanin biosynthesis in rice, responds to short- and long-term cold stress at both the transcriptional and protein levels. Metabolomic and transcriptomic data indicate that OsTTG1 activates the expression of anthocyanidin synthase (OsANS) genes under cold stress. Our data also suggest that OsTTG1 forms a MYB-bHLH-WD (MBW) complex with Basic helix-loop-helix 148 (OsbHLH148) and Myb-related S3 (OsMYBS3), and this complex activates the expression of Dehydration-responsive element-binding protein 1 (OsDREB1) and OsANS genes. Together, our findings reveal the mechanisms by which OsTTG1 coordinates both anthocyanin biosynthesis and the expression of cold-responsive genes in colored rice, providing genetic resources for future cold resistance breeding in rice.

Key messageGenetic editing of grain size genes quickly improves three-line hybrid rice parents to increase the appearance quality and yield of hybrid rice.AbstractGrain size affects rice yield and quality. In this study, we used CRISPR/Cas9 to edit the grain size gene GW8 in the maintainer line WaitaiB (WTB) and restorer line Guanghui998 (GH998). The new slender sterile line WTEA (gw8) was obtained in the BC2F1 generation by transferring the grain mutation of the maintainer plant to the corresponding sterile line WantaiA (WTA, GW8) in the T1 generation. Two slender restorer lines, GH998E1 (gw8(II)) and GH998E2 (gw8(I)), were obtained in T1 generation. In the early stage, new sterile and restorer lines in grain mutations were created by targeted editing of GS3, TGW3, and GW8 genes. These parental lines were mated to detect the impact of grain-type mutations on hybrid rice yield and quality. Mutations in gs3, gw8, and tgw3 had a minimal impact on agronomic traits except the grain size and thousand-grain weight. The decrease in grain width in the combination mainly came from gw8/gw8, gs3/gs3 increased the grain length, gs3/gs3-gw8/gw8 had a more significant effect on the grain length, and gs3/gs3-gw8/gw8(I) contributed more to grain length than gs3/gs3-gw8/gw8(II). The heterozygous TGW3/tgw3 may not significantly increase grain length. Electron microscopy revealed that the low-chalky slender-grain variety had a cylindrical grain shape, a uniform distribution of endosperm cells, and tightly arranged starch grains. Quantitative fluorescence analysis of endospermdevelopment-related genes showed that the combination of slender grain hybrid rice caused by gs3 and gw8 mutations promoted endosperm development and improved appearance quality. An appropriate grain size mutation resulted in hybrid rice varieties with high yield and quality.

BACKGROUND:The brown planthopper (BPH) is an economically significant pest of rice. Bph3 is a key BPH resistance gene. However, the proteomic response of rice to BPH infestation, both in the presence and absence of Bph3, remains largely unexplored. RESULTS:In this study, we employed tandem mass tag labeling in conjunction with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to identify differentially expressed proteins (DEPs) in rice samples. We detected 265 and 125 DEPs via comparison of samples infected with BPH for 2 and 4 days with untreated samples of the BPH-sensitive line R582. For the Bph3 introgression line R373, we identified 29 and 94 DEPs in the same comparisons. Bioinformatic analysis revealed that Bph3 significantly influences the abundance of proteins associated with metabolic pathways, secondary metabolite biosynthesis, microbial metabolism in diverse environments, and phenylpropanoid biosynthesis. Moreover, Bph3 regulates the activity of proteins involved in the calcium signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, and plant hormone signal transduction. CONCLUSIONS:Our results indicate that Bph3 enhances the resistance of rice to BPH mainly by inhibiting the down-regulation of proteins associated with metabolic pathways; calcium signaling, the MAPK signaling pathway, and plant hormone signal transduction might also be involved in BPH resistance induced by Bph3.

Rice is one of the most important staple crops in the world; therefore, the improvement of rice holds great significance for enhancing agricultural production and addressing food security challenges. Although there have been numerous studies on the role of single-nucleotide polymorphisms (SNPs) in rice improvement with the development of next-generation sequencing technologies, research on the role of presence/absence variations (PAVs) in the improvement of rice is limited. In particular, there is a scarcity of studies exploring the traits and genes that may be affected by PAVs in rice. Here, we extracted PAVs utilizing resequencing data from 148 improved rice varieties distributed in Asia. We detected a total of 33,220 PAVs and found that the number of variations decreased gradually as the length of the PAVs increased. The number of PAVs was the highest on chromosome 1. Furthermore, we identified a 6 Mb hotspot region on chromosome 11 containing 1091 PAVs in which there were 29 genes related to defense responses. By conducting a genome-wide association study (GWAS) using PAV variation data and phenotypic data for five traits (flowering time, plant height, flag leaf length, flag leaf width, and panicle number) across all materials, we identified 186 significantly associated PAVs involving 20 cloned genes. A haplotype analysis and expression analysis of candidate genes revealed that important genes might be affected by PAVs, such as the flowering time gene OsSFL1 and the flag leaf width gene NAL1. Our work investigated the pattern in PAVs and explored important PAV key functional genes associated with agronomic traits. Consequently, these results provide potential and exploitable genetic resources for rice breeding.

Improving rice quality remains a crucial breeding objective, second only to enhancing yield, yet progress in quality improvement lags behind yield. The high temperature and ripening conditions in Southern China often result in poor rice quality, impacting hybrid rice production and utilization. Therefore, to address this challenge, analyzing the molecular basis of high-quality traits is essential for molecular design breeding of high-quality hybrid rice varieties. In this study, we investigated the molecular basis of grain shape, amylose content, gel consistency, gelatinization temperature, and aroma, which influence rice quality. We discovered that quality related alleles gs3, GW7TFA, gw8, chalk5, Wxb, ALKTT, and fgr can enhance rice quality when applied in breeding programs. Polymerization of gs3, GW7TFA, gw8, and chalk5 genes improves rice appearance quality. The gs3 and GW7TFA allele polymerization increasing the grain’s length-width ratio, adding the aggregation of gw8 allele can further reducing grain width. The chalk5 gene regulates low chalkiness, but low correlation to chalkiness was exhibited with grain widths below 2.0 mm, with minimal differences between Chalk5 and chalk5 alleles. Enhancing rice cooking and eating quality is achieved through Wxb and ALKTT gene polymerization, while introducing the fgr(E7) gene significantly improved rice aroma. Using molecular marker-assisted technology, we aggregated these genes to develop a batch of indica hybrid rice parents with improved rice quality are obtained. Cross-combining these enhanced parents can generate new, high-quality hybrid rice varieties suitable for cultivation in Southern China. Therefore, our findings contribute to a molecular breeding model for grain quality improvement in high-quality indica hybrid rice. This study, along with others, highlights the potential of molecular design breeding for enhancing complex traits, particularly rice grain quality.

BackgroundHeat stress threatens rice yield and quality at flowering stage. In this study, average relative seed setting rate under heat stress (RHSR) and genotypes of 284 varieties were used for a genome-wide association study.ResultsWe identified eight and six QTLs distributed on chromosomes 1, 3, 4, 5, 7 and 12 in the full population and indica, respectively. qHTT4.2 was detected in both the full population and indica as an overlapping QTL. RHSR was positively correlated with the accumulation of heat-tolerant superior alleles (SA), and indica accession contained at least two heat-tolerant SA with average RHSR greater than 43%, meeting the needs of stable production and heat-tolerant QTLs were offer yield basic for chalkiness degree, amylose content, gel consistency and gelatinization temperature. Chalkiness degree, amylose content, and gelatinization temperature under heat stress increased with accumulation of heat-tolerant SA. Gel consistency under heat stress decreased with polymerization of heat-tolerant SA. The study revealed qHTT4.2 as a stable heat-tolerant QTL that can be used for breeding that was detected in the full population and indica. And the grain quality of qHTT4.2-haplotype1 (Hap1) with chalk5, wx, and alk was better than that of qHTT4.2-Hap1 with CHALK5, WX, and ALK. Twelve putative candidate genes were identified for qHTT4.2 that enhance RHSR based on gene expression data and these genes were validated in two groups. Candidate genes LOC_Os04g52830 and LOC_Os04g52870 were induced by high temperature.ConclusionsOur findings identify strong heat-tolerant cultivars and heat-tolerant QTLs with great potential value to improve rice tolerance to heat stress, and suggest a strategy for the breeding of yield-balance-quality heat-tolerant crop varieties.

Grain qualities including milling quality, appearance quality, eating and cooking quality, and nutritional quality are important indicators in rice breeding. Significant achievements in genetic improvement of rice quality have been made. In this study, we analyzed the variation patterns of 16 traits in 1570 rice varieties and found significant improvements in appearance quality and eating and cooking quality, particularly in hybrid rice. Through genome-wide association study and allelic functional nucleotide polymorphisms analysis of quality trait genes, we found that ALK, FGR1, FLO7, GL7/GW7, GLW7, GS2, GS3, ONAC129, OsGRF8, POW1, WCR1, and Wx were associated with the genetic improvement of rice quality traits in Southern China. Allelic functional nucleotide polymorphisms analysis of 13 important rice quality genes, including fragrance gene fgr, were performed using the polymerase chain reaction amplification refractory mutation system technology. The results showed that Gui516, Gui569, Gui721, Ryousi, Rsimiao, Rbasi, and Yuehui9802 possessed multiple superior alleles. This study elucidates the phenotypic changes and molecular basis of key quality traits of varieties in Southern China. The findings will provide guidance for genetic improvement of rice quality and the development of new varieties.

Grain length is one of the most important rice grain appearance components. To better understand the protein regulated by grain length in indica rice, the tandem mass tag (TMT) labeling combined with LC-MS/MS analysis was used for quantitative identification of differentially regulated proteins by comparing six long-grain cultivars (MeiB, LongfengB, YexiangB, FengtianB, WantaiB, and DingxiangB) to the short-grain cultivar BoB, respectively. A total of 6622 proteins were detected for quantitative analysis by comparing protein content of six long-grain cultivars to the short-grain cultivar, and 715 proteins were significantly regulated, consisting of 336 uniquely over-accumulated proteins and 355 uniquely down-accumulated proteins. KEGG pathway analysis revealed that most of accumulated proteins are involved in metabolic pathways, biosynthesis of secondary metabolites and phenylpropanoid biosynthesis. Four down-accumulated proteins maybe involved in the signaling pathways for grain length regulation. LC-PRM/MS quantitative analysis was used to analyze 10 differentially expressed proteins. The results were almost consistent with the TMT quantitative analysis. qRT-PCR analysis results showed that the transcription level was not always parallel to the protein content. This study identified many novel grain length accumulated proteins through the quantitative proteomics approach, providing candidate genes for further study of grain size regulatory mechanisms. SIGNIFICANCE: Rice grain length is one of the most important characteristics influencing appearance and yield. Six long-grain cultivars (MeiB, LongfengB, YexiangB, FengtianB, WantaiB, and DingxiangB obtained in Guangxi province of China from the 2000s to 2020s) and one short-grain cultivar (BoB obtained in Guangxi province of China in 1980s) were used for comparative analyses. Totally, 715 differentially expressed proteins (DEPs) were identified using TMT-base proteomic analysis. The numbers of DEPs increased as the grain length increased. 4 DEPs may be related to rice's signaling pathways for grain size regulation. A total of 85 DEPs regulated in at least four long-grain cultivars compared with the short-grain cultivar BoB, and 7 proteins were over-accumulated, and 3 proteins were down-accumulated in six long-grain cultivars. These findings provide valuable information to better understand the mechanisms of protein regulation by grain length in rice.

Grain size is one of the most important agronomic traits for grain yield determination in rice. To better understand the proteins that are regulated by the grain size regulatory gene OsMKK3, this gene was knocked out using the CRISPR/Cas9 system, and tandem mass tag (TMT) labeling combined with liquid chromatograph-tandem mass spectrometry analysis was performed to study the regulation of proteins in the panicle. Quantitative proteomic screening revealed a total of 106 differentially expressed proteins (DEPs) via comparison of the OsMKK3 mutant line to the wild-type YexiangB, including 15 and 91 up-regulated and down-regulated DEPs, respectively. Pathway analysis revealed that DEPs were enriched in metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and photosynthesis. Strong interactions were detected among seven down-regulated proteins related to photosystem components in the protein-protein interaction network, and photosynthetic rate was decreased in mutant plants. The results of the liquid chromatography-parallel reaction monitoring/mass spectromery analysis and western blot analysis were consistent with the results of the proteomic analysis, and the results of the quantitative reverse transcription polymerase chain reaction analysis revealed that the expression levels of most candidate genes were consistent with protein levels. Overall, OsMKK3 controls grain size by regulating the protein content in cells. Our findings provide new candidate genes that will aid the study of grain size regulatory mechanisms associated with the mitogen-activated protein kinase (MAPK) signaling pathway.

[目的]在大肠杆菌中过表达、纯化水稻冷胁迫响应蛋白OsCML16,同时制备OsCML16蛋白抗体提供抗原,为采用该抗体研究水稻OsCML16蛋白的生物学功能提供参考依据.[方法]采用TMHMM 2.0在线分析软件对目的蛋白OsCML16进行跨膜区分析,利用高保真PCR扩增目的基因片段,以基因重组方法构建原核表达载体pET30a-OsCML16,并将重组质粒转入大肠杆菌表达菌株BL21(DE3)中;通过IPTG诱导OsCML16蛋白表达,然后采用亲和层析方法纯化获得目的蛋白,最后运用质谱验证法分析验证蛋白序列的真实性.[结果]通过对OsCML16蛋白跨膜区分析,发现该蛋白不包含跨膜结构,适合外源表达全长蛋白.将OsC-ML16基因链接至pET30a载体能成功构建原核表达载体pET30a-OsCML16,目的基因序列无突变位点,可用于表达目标蛋白.外源表达的OsCML16蛋白经过SDS-PAGE电泳分析,结果表明,OsCML16蛋白在28 ℃下可诱导表达,采用亲和层析方法纯化融合蛋白His6-OsCML16-His6,能成功获得分子量约30kD的目的蛋白.对纯化的蛋白进行质谱验证分析,结果鉴定到3条多肽属于OsC-ML16蛋白,表明外源表达的蛋白即为OsCML16蛋白.[结论]采用原核表达方法将水稻冷胁迫响应基因OsCML16在大肠杆菌中表达,运用亲和层析技术根据融合蛋白携带组氨酸标签纯化获得融合表达蛋白His6-OsCML16-His6,并以蛋白质谱技术验证该蛋白的序列完全正确,后续可将水稻OsCML16蛋白用于抗体制备及蛋白功能研究.

BACKGROUND:Cold damage stress significantly affects rice growth (germination and seedling) and causes serious losses in yield in temperate and high-altitude areas around the globe. OBJECTIVE:This study aimed to explore the cold tolerance (CT) locus of rice and create new cold-tolerant germplasm. We constructed a chromosome segment substitution line (CSSL) with strong CT and fine mapped quantitative trait loci (QTLs) associated with CT by performing the whole-genome resequencing of CSSL with phenotypes under cold treatment. METHODS:A chromosome CSSL, including 271 lines from a cross between the cold-tolerant wild rice Y11 (Oryza rufipogon Griff.) and the cold-sensitive rice variety GH998, was developed to map QTLs conferring CT at the germination stage. The whole-genome resequencing was performed on CSSL for mapping QTLs of associated with CT at the germination stage. RESULTS:A high-density linkage map of the CSSLs was developed using the whole-genome resequencing of 1484 bins. The QTL analysis using 615,466 single-nucleotide polymorphisms (SNPs) led to the identification of 2 QTLs related to germination rate at low-temperature on chromosome 8 (qCTG-8) and chromosome 11 (qCTG-11). The qCTG-8 and qCTG-11 explained 14.55% and 14.31% of the total phenotypic variation, respectively. We narrowed down qCTG-8 and qCTG-11 to 195.5 and 78.83-kb regions, respectively. The expression patterns of important candidate genes in different tissues, and of RNA-sequencing (RNA-seq) in CSSLs, were identified based on gene sequences in qCTG-8 and qCTG-11 cold-induced expression analysis. LOC_Os08g01120 and LOC_Os08g01390 were identified as candidate genes in qCTG-8, and LOC_Os11g32880 was identified as a candidate gene in qCTG-11. CONCLUSIONS:This study demonstrated a general method that could be used to identify useful loci and genes in wild rice and aid in the future cloning of candidate genes of qCTG-8 and qCTG-11. The CSSLs with strong CT were supported for breeding cold-tolerant rice varieties.

The NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) gene family plays a critical role in plant development. However, our understanding of the mechanisms of how NB-ARC genes regulate plant development in the plant panicle is still limited. Here, we subjected 258 NB-ARC genes in rice to genome-wide analysis to characterize their structure, function, and expression patterns. The NB-ARC genes were classified into three major groups, and group II included nine subgroups. Evolutionary analysis of NB-ARC genes in a dicotyledon plant (Arabidopsis thaliana) and two monocotyledonous plants (Oryza sativa L. and Triticum aestivum) indicated that homologous genome segments were conserved in monocotyledons and subjected to weak positive selective pressure during evolution. Dispersed and proximal replication events were detected. Expression analysis showed expression of most NB-ARC genes in roots, panicles, and leaves, and regulation at the panicle development stage in rice Ce253. The GNP12 gene encodes RGH1A protein, which regulates rice yield according to panicle length, grain number of panicle, and grain length, with eight major haplotypes. Most members of NB-ARC protein family are predicted to contain P-loop conserved domains and localize on the membrane. The results of this study will provide insight into the characteristics and evolution of NB-ARC family and suggest that GNP12 positively regulates panicle development.