RISBZ1 encodes the transcription factor bZIP58, which regulates grain filling; intron editing of RISBZ1 to eliminate aberrant alternatively spliced transcripts increased grain weight without compromising key agronomic traits under normal conditions, and enhanced grain weight and quality under heat stress conditions.

Rice (Oryza sativa L.) is a vital cereal crop in the world. With societal development, the demand for superior rice quality has increased annually. Chalkiness refers to the white opaque regions in rice endosperm caused by disorganized starch granule structure, which diminishes rice appearance, cooking quality, and processing properties. The chalky grain rate (CGR), defined as the proportion of chalky grains relative to total grains, quantifies chalkiness severity. As a polygenic quantitative trait, chalkiness has been investigated through genome-wide association study (GWAS)—an established method for identifying quantitative trait loci (QTL) using natural populations and high-density single nucleotide polymorphism (SNP) markers. Recent advances in high-throughput genotyping have significantly enhanced GWAS capabilities for dissecting polygenic traits. In this study, we analyzed CGR in 168 representative rice cultivars and conducted GWAS with 3.83 million SNP markers, identifying three associated loci. Through integrated candidate gene analysis, expression profile assay, haplotype assessment, and linkage disequilibrium analysis of locus qPGWC-4-1, we identified CGR1 as a key candidate gene. Mechanistically, CGR1 may regulate ribosome biogenesis and assembly to influence chalkiness development. The four major haplotypes of CGR1 exhibit significant divergence between indica and japonica subspecies. A natural variation at the terminal coding region of CGR1 results in the loss of a stop codon. These findings advance our understanding of the genetic regulation of rice chalkiness and provide genetic resources for future quality improvement.

Phytochrome B (phyB), the predominant red-light photoreceptor, interacts with various proteins, initiating a set of molecular events in plant cells that promote light responses. In this study, we identified AHL21, a member of the AT-hook motif nuclear-localized (AHL) family, as a phyB-interacting protein in Arabidopsis. AHL21 positively regulates photomorphogenesis by directly binding to PHYTOCHROME INTERACTING FACTOR 4 (PIF4) promoter and downregulating its expression. Moreover, AHL21 physically interacts with PIF4 and represses its transcriptional activation activity. Hence, AHL21 acts genetically upstream of PIF4. In addition, phyB directly interacts with both AHL21 and PIF4 and functions as a molecular glue that enhances the AHL21-PIF4 interaction. Collectively, AHL21 functions as a key component of the phyB-PIF4 signaling module to enable plants to adapt to fluctuating light conditions.

Rice grain yield is primarily determined by three key agronomic traits: panicle number, grain number per panicle, and grain weight(GW). However, the inherent tradeoffs among these yield components remain a persistent challenge in rice breeding programs. Notably, compared with GW, brown rice weight(BRW) provides a more direct metric associated with actual grain yield potential. In this study, we conducted a two-year replicated genome-wide association study to elucidate the genetic architecture of BRW and identify new loci regulating GW. Among seven consistently detected loci across experimental replicates, four were not co-localized with previously reported genes associated with BRW or GW traits. BRW1.1, one of the four newly identified loci, was found to encode a novel RNA-binding protein. Functional characterization revealed that BRW1.1 acts as a negative regulator of BRW, potentially through modulating m RNA translation processes. Intriguingly, through integrated analysis of mutant phenotypes and haplotype variations, we demonstrated that BRW1.1 mediates the physiological tradeoff between GW and panicle number. This study not only delineates the genetic determinants of BRW but also identifies BRW1.1 as a promising molecular target for breaking the yield component tradeoff in precision rice breeding.

Intron retention (IR) is the most common alternative splicing event in Arabidopsis. An increasing number of studies have demonstrated the major role of IR in gene expression regulation. The impacts of IR on plant growth and development and response to environments remain underexplored. Here, we found that IR functions directly in gene expression regulation on a genome-wide scale through the detainment of intron-retained transcripts (IRTs) in the nucleus. Nuclear-retained IRTs can be kept away from translation through this mechanism. COP1-dependent light modulation of the IRTs of light signaling genes, such as PIF4, RVE1, and ABA3, contribute to seedling morphological development in response to changing light conditions. Furthermore, light-induced IR changes are under the control of the spliceosome, and in part through COP1-dependent ubiquitination and degradation of DCS1, a plant-specific spliceosomal component. Our data suggest that light regulates the activity of the spliceosome and the consequent IRT nucleus detainment to modulate photomorphogenesis through COP1.

Cytoplasmic male sterility (CMS) is an agronomically significant trait that causes dysfunction in pollen and anther development. It is often observed during successive backcrossing between distantly related species. Here, we show that Asian japonica cultivars (Oryza sativa) exhibit CMS when the nucleus is replaced with that of the African rice Oryza glaberrima. The CMS line produced stunted anthers and did not set any seeds. Mitochondrial orf288 RNA was detected in the anthers of CMS lines but not in fertility restorer lines. The mitochondrial genome-edited japonica rice that was depleted of orf288 did not exhibit male sterility when backcrossed with O. glaberrima. These results demonstrate that orf288 is a CMS-causing gene. As orf288 commonly occurs in the mitochondrial genomes of japonica rice, these results indicate that common japonica rice cultivars possess a cryptic CMS-causing gene hidden in their mitochondrial genomes.

Preharvest sprouting (PHS) is an undesirable trait that decreases yield and quality in rice production. Understanding the genes and regulatory mechanisms underlying PHS is of great significance for breeding PHS-resistant rice. In this study, we identified a mutant, preharvest sprouting 39 (phs39), that exhibited an obvious PHS phenotype in the field. MutMap+ analysis and transgenic experiments demonstrated that OsAAH, which encodes allantoate amidohydrolase, is the causal gene of phs39 and is essential for PHS resistance. OsAAH was highly expressed in roots and leaves at the heading stage and gradually increased and then weakly declined in the seed developmental stage. OsAAH protein was localized to the endoplasmic reticulum, with a function of hydrolyzing allantoate in vitro. Disruption of OsAAH increased the levels of ureides (allantoate and allantoin) and activated the tricarboxylic acid (TCA) cycle, and thus increased energy levels in developing seeds. Additionally, the disruption of OsAAH significantly increased asparagine, arginine, and lysine levels, decreased tryptophan levels, and decreased levels of indole-3-acetic acid (IAA) and abscisic acid (ABA). Our findings revealed that the OsAAH of ureide catabolism is involved in the regulation of rice PHS via energy and hormone metabolisms, which will help to facilitate the breeding of rice PHS-resistant varieties.

Soil salinity has a major impact on rice seed germination, severely limiting rice production. Herein, a rice germination defective mutant under salt stress (gdss) was identified by using chemical mutagenesis. The GDSS gene was detected via MutMap and shown to encode potassium transporter OsHAK9. Phenotypic analysis of complementation and mutant lines demonstrated that OsHAK9 was an essential regulator responsible for seed germination under salt stress. OsHAK9 is highly expressed in germinating seed embryos. Ion contents and non-invasive micro-test technology results showed that OsHAK9 restricted K+ efflux in salt-exposed germinating seeds for the balance of K+/Na+. Disruption of OsHAK9 significantly reduced gibberellin 4 (GA4) levels, and the germination defective phenotype of oshak9a was partly rescued by exogenous GA3 treatment under salt stress. RNA sequencing (RNA-seq) and real-time quantitative polymerase chain reaction analysis demonstrated that the disruption of OsHAK9 improved the GA-deactivated gene OsGA2ox7 expression in germinating seeds under salt stress, and the expression of OsGA2ox7 was significantly inhibited by salt stress. Null mutants of OsGA2ox7 created using clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 approach displayed a dramatically increased seed germination ability under salt stress. Overall, our results highlight that OsHAK9 regulates seed germination performance under salt stress involving preventing GA degradation by mediating OsGA2ox7, which provides a novel clue about the relationship between GA and OsHAKs in rice.

Mutualistic interactions between host plants and their microbiota have the potential to provide disease resistance. Most research has focused on the rhizosphere, but it is unclear how the microbiome associated with the aerial surface of plants protects against infection. Here we identify a metabolic defence underlying the mutualistic interaction between the panicle and the resident microbiota in rice to defend against a globally prevalent phytopathogen, Ustilaginoidea virens, which causes false-smut disease. Analysis of the 16S ribosomal RNA gene and internal transcribed spacer sequencing data identified keystone microbial taxa enriched in the disease-suppressive panicle, in particular Lactobacillus spp. and Aspergillus spp. Integration of these data with primary metabolism profiling, host genome editing and microbial isolate transplantation experiments revealed that plants with these taxa could resist U. virens infection in a host branched-chain amino acid (BCAA)-dependent manner. Leucine, a predominant BCAA, suppressed U. virens pathogenicity by inducing apoptosis-like cell death through H2O2 overproduction. Additionally, preliminary field experiments showed that leucine could be used in combination with chemical fungicides with a 50% reduction in dose but similar efficacy to higher fungicide concentrations. These findings may facilitate protection of crops from panicle diseases prevalent at a global scale.

IntroductionRice genomes contain endogenous viral elements homologous to rice tungro bacilliform virus (RTBV) from the pararetrovirus family Caulimoviridae. These viral elements, known as endogenous RTBV-like sequences (eRTBVLs), comprise five subfamilies, eRTBVL-A, -B, -C, -D, and -X. Four subfamilies (A, B, C, and X) are present to a limited degree in the genomes of the Asian cultivated rice Oryza sativa (spp. japonica and indica) and the closely related wild species Oryza rufipogon.MethodsThe eRTBVL-D sequences are widely distributed within these and other Oryza AA-genome species. Fifteen eRTBVL-D segments identified in the japonica (Nipponbare) genome occur mostly at orthologous chromosomal positions in other AA-genome species. The eRTBVL-D sequences were inserted into the genomes just before speciation of the AA-genome species.Results and discussionTen eRTBVL-D segments are located at six loci, which were used for our evolutionary analyses during the speciation of the AA-genome species. The degree of genetic differentiation varied among the eRTBVL-D segments. Of the six loci, three showed phylogenetic trees consistent with the standard speciation pattern (SSP) of the AA-genome species (Type A), and the other three represented phylogenies different from the SSP (Type B). The atypical phylogenetic trees for the Type B loci revealed chromosome region–specific evolution among the AA-genome species that is associated with phylogenetic incongruences: complex genome rearrangements between eRTBVL-D segments, an introgression between the distant species, and low genetic diversity of a shared eRTBVL-D segment. Using eRTBVL-D as an indicator, this study revealed the phylogenetic incongruence of local chromosomal regions with different topologies that developed during speciation.

KEY MESSAGE:Two causal OsTTL and OsSAPK1 genes of the key locus qNL3.1 significantly associated with seed germination under salt stress were identified via a genome-wide association study, which could improve rice seed germination under salt stress. Rice is a salt-sensitive crop, and its seed germination determines subsequent seedling establishment and yields. In this study, 168 accessions were investigated for the genetic control of seed germination under salt stress based on the germination rate (GR), germination index (GI), time at which 50% germination was achieved (T50) and mean level (ML). Extensive natural variation in seed germination was observed among accessions under salt stress. Correlation analysis showed significantly positive correlations among GR, GI and ML and a negative correlation with T50 during seed germination under salt stress. Forty-nine loci significantly associated with seed germination under salt stress were identified, and seven of these were identified in both years. By comparison, 16 loci were colocated with the previous QTLs, and the remaining 33 loci might be novel. qNL3.1, colocated with qLTG-3, was simultaneously identified with the four indices in two years and might be a key locus for seed germination under salt stress. Analysis of candidate genes showed that two genes, the similar to transthyretin-like protein OsTTL and the serine/threonine protein kinase OsSAPK1, were the causal genes of qNL3.1. Germination tests indicated that both Osttl and Ossapk1 mutants significantly reduced seed germination under salt stress compared to the wild type. Haplotype analysis showed that Hap.1 of OsTTL and Hap.1 of OsSAPK1 genes were excellent alleles, and their combination resulted in high seed germination under salt stress. Eight accessions with elite performance of seed germination under salt stress were identified, which could improve rice seed germination under salt stress.

Bacteria equipped with virulence systems based on highly bioactive small molecules can circumvent their host's defense mechanisms. Pathogens employing this strategy are currently threatening global rice production. In the present study, variations in the virulence of the highly destructive Burkholderia plantarii were observed in different rice-producing regions. The environment-linked variation was not attributable to any known host-related or external factors. Co-occurrence analyses indicated a connection between reduced virulence and 5-Amino-1,3,4-thiadiazole-2-thiol (ATT), a non-bactericidal organic compound. ATT, which accumulates in rice plants during metabolization of specific agrochemicals, was found to reduce virulence factor secretion by B. plantarii up to 88.8% and inhibit pathogen virulence by hijacking an upstream signaling cascade. Detailed assessment of the newly discovered virulence inhibitor resulted in mechanistic insights into positive effects of ATT accumulation in plant tissues. Mechanisms of virulence alleviation were deciphered by integrating high-throughput data, gene knockout mutants, and molecular interaction assays. TroK, a histidine protein kinase in a two-component system that regulates virulence factor secretion, is likely the molecular target antagonized by ATT. Our findings provide novel insights into virulence modulation in an important plant-pathogen system that relies on the host's metabolic activity and subsequent signaling interference.

The cultivation of rice varieties with high seed vigour is vital for the direct seeding of rice, and the molecular basis of regulation of seed vigour remains elusive. Here, we cloned a new gene OsHIPL1, which encodes hedgehog-interacting protein-like 1 protein as a causal gene of the major QTL qSV3 for rice seed vigour. OsHIPL1 was mainly localized in the plasma membrane and nucleus. RNA sequencing (RNA-seq) revealed that the ABA-related genes were involved in the OsHIPL1 regulation of seed vigour in rice. The higher levels of endogenous ABA were measured in germinating seeds of OsHIPL1 mutants and NIL-qsv3 line compared to IR26 plants, with two up-regulated ABA biosynthesis genes (OsZEP and OsNCED4) and one down-regulated ABA catabolism gene OsABA8ox3. The expression of abscisic acid-insensitive 3 (OsABI3), OsABI4 and OsABI5 was significantly up-regulated in germinating seeds of OsHIPL1 mutants and NIL-qsv3 line compared to IR26 plants. These results indicate that the regulation of seed vigour of OsHIPL1 may be through modulating endogenous ABA levels and altering OsABIs expression during seed germination in rice. Meanwhile, we found that OsHIPL1 interacted with the aquaporin OsPIP1;1, then affected water uptake to promote rice seed germination. Based on analysis of single-nucleotide polymorphism data of rice accessions, we identified a Hap1 haplotype of OsHIPL1 that was positively correlated with seed germination. Our findings showed novel insights into the molecular mechanism of OsHIPL1 on seed vigour.

Seed germination plays a pivotal role in the plant life cycle, and its precise regulatory mechanisms are not clear. In this study, 19 quantitative trait loci (QTLs) associated with rice seed germination were identified through genome-wide association studies (GWAS) of the following traits in 2016 and 2017: germination rate (GR) at 3, 5, and 7 days after imbibition (DAI) and germination index (GI). Two major stable QTLs, qSG4 and qSG11.1, were found to be associated with GR and GI over 2 continuous years. Furthermore, OsPK5, encoding a pyruvate kinase, was shown to be a crucial regulator of seed germination in rice, and might be a causal gene of the key QTL qSG11.1, on chromosome 11. Natural variation in OsPK5 function altered the activity of pyruvate kinase. The disruption of OsPK5 function resulted in slow germination and seedling growth during seed germination, blocked glycolytic metabolism, caused glucose accumulation, decreased energy levels, and affected the GA/ABA balance. Taken together, our results provide novel insights into the roles of OsPK5 in seed germination, and facilitate its application in rice breeding to improve seed vigour.

Nitrogen-use efficiency (NUE) in rice is a complex quantitative trait involved in multiple biological processes and agronomic traits; however, the genetic basis and regulatory network of NUE remain largely unknown. We constructed a high-resolution microarray-based genetic map for 261 recombinant inbred lines derived from two indica parents. Using 2,345 bin markers, comprehensive analyses of quantitative trait loci (QTLs) of seven key agronomic traits under two different N levels were performed. A total of 11 non-redundant QTLs for effective panicle number (EPN), 7 for grain number per panicle, 13 for thousand-grain weight, 2 for seed-setting percentage, 15 for plant height, 12 for panicle length, and 6 for grain yield per plant were identified. The QTL regions were as small as 512 kb on average, and more than half spanned an interval smaller than 100 kb. Using this advantage, we identified possible candidate genes of two major EPN-related QTLs. One QTL detected under both N levels possibly encodes a DELLA protein SLR1, which is known to regulate NUE, although the natural variations of this protein have not been reported. The other QTL detected only under a high N level could encode the transcription factor OsbZIP59. We also predicted the possible candidate genes for another three of the NUE-related QTLs. Our results provide a reference for improving NUE-related QTL cloning and promote our understanding of NUE regulation in indica rice.

水稻（Oryza sativa）的高产优质是我国粮食安全的重要保障,也是育种家一直追求的目标。水稻籽粒灌浆速率（GFR）是一个重要而复杂的农艺性状,直接影响籽粒充实度、粒重和米质。目前,快速灌浆的优良水稻品种缺乏,可供育种利用的相关优异基因资源有限,已成为制约水稻产量和品质进一步提高的瓶颈。相对于水稻的其它农艺性状, GFR具有复杂的时空动态和环境可变性,相关研究长期围绕灌浆过程的生理生化特性和栽培措施展开,而分子机制和遗传调控研究启动较晚。该文以近年来国内外发现的水稻GFR相关基因为主线,从糖类代谢和运输相关基因对GFR的影响、转录和翻译调控基因对GFR的调节、粒型和粒重等相关数量性状位点（QTL）对GFR的作用,以及GFR相关QTL的分析和克隆4个方面,对GFR分子机制与遗传调控进行综述;并对GFR的研究策略特别是表型组学相关技术的应用前景进行展望,以期推动该领域的基础研究和育种应用。

Cereal crop production is severely affected by seed-borne bacterial diseases across the world. Locally occurring disease resistance in various crops remains elusive. Here, we have observed that rice plants of the same cultivar can be differentiated into disease-resistant and susceptible phenotypes under the same pathogen pressure. Following the identification of a seed-endophytic bacterium as the resistance-conferring agent, integration of high-throughput data, gene mutagenesis and molecular interaction assays facilitated the discovery of the underlying mode of action. Sphingomonas melonis that is accumulated and transmitted across generations in disease-resistant rice seeds confers resistance to disease-susceptible phenotypes by producing anthranilic acid. Without affecting cell growth, anthranilic acid interferes with the sigma factor RpoS of the seed-borne pathogen Burkholderia plantarii, probably leading to impairment of upstream cascades that are required for virulence factor biosynthesis. The overall findings highlight the hidden role of seed endophytes in the phytopathology paradigm of 'disease triangles', which encompass the plant, pathogens and environmental conditions. These insights are potentially exploitable for modern crop cultivation threatened by globally widespread bacterial diseases.

The sugar status of a plant acts as a signal affecting growth and development. The phenomenon by which high levels of sugars inhibit seedling establishment has been widely used to gain insight into sugar-signaling pathways. Natural allelic variation has been identified at the ANAC060 locus. The Arabidopsis Columbia ecotype produces a short ANAC060 protein without a transmembrane domain that is constitutively located to the nucleus, causing sugar insensitivity when overexpressed. In this study, we generated a genome-wide DNA-binding map of ANAC060 via chromatin immunoprecipitation sequencing using transgenic lines that express a functional ANAC060-GFP fusion protein in an anac060 background. A total of 3282 genes associated with ANAC060-binding sites were identified. These genes were enriched in biotic and abiotic stress responses, and the G-box binding motif was highly enriched in ANAC060-bound genomic regions. Expression microarray analysis resulted in the identification of 8350 genes whose activities were altered in the anac060 mutant and upon sugar treatment. Cluster analysis revealed that ANAC060 attenuates sugar-regulated gene expression. Direct target genes of ANAC060 included equivalent numbers of genes that were upregulated or downregulated by ANAC060. The various functions of these target genes indicate that ANAC060 has several functions. Our results demonstrate that ANAC060 directly binds to the promoter of ABI5 and represses the sugar-induced transcription of ABI5. Genetic data indicate that ABI5 is epistatic to ANAC060 in both sugar and abscisic acid responses.

Accumulation of toxic heavy metals and metalloids (THMMs) in crop grain remarkably affects food safety and human health. Reducing the content of THMMs in grain requires the identification and manipulation of the genes regulating their accumulation. This study aimed to determine the genetic variations affecting grain THMM accumulation in rice by using association mapping. We used 276 accessions with 416 K single nucleotide polymorphisms (SNPs) and performed genome-wide association analysis of grain THMM concentrations in rice grown in heavily multi-contaminated farmlands. We detected 22, 17, and 21 quantitative trait loci (QTLs) for grain arsenic, cadmium, and lead concentrations, respectively. Both inter- and intra-subpopulation variants accounted for these QTLs. Most QTLs contained no known THMM-related genes and represented unidentified novel genes. We examined the candidate genes in qGAS1, a QTL for grain arsenic concentration with the best P-value detected for the entire population. We speculated that a transport protein of the multidrug and toxin extrusion family could be the candidate gene for this QTL. Our study suggested that the genetic regulation of grain THMM accumulation is very complex and largely unknown. The QTLs and SNPs identified in this study might help in the identification of new genes regulating THMM accumulation and aid in marker-assisted breeding of rice with low grain THMM content.

Rice tungro disease (RTD) is a serious threat to rice production in South and Southeast Asia. RTD is caused by Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). Co-infection of RTSV and RTBV results in more severe symptoms in African rice Oryza glaberrima than in Asian rice O. sativa . In this study, we examined whether RTBV alone causes more severe symptoms in O. glaberrima than in O. sativa . The examination of 18 accessions of O. glaberrima for RTBV infection indicated that all the O. glaberrima accessions examined were susceptible to RTBV. The reactions to RTBV of three O. glaberrima accessions and two RTBV-susceptible varieties of O. sativa , Taichung Native 1 (TN1) and IR 64, were compared. RTBV accumulation varied depending on the plants and differences in RTBV accumulation were not evident between the two species. However, the O. glaberrima accessions were significantly more stunted by RTBV than IR 64 was. Discoloration of leaves by RTBV was evident in the O. glaberrima plants, but not in the O. sativa plants. Collectively, these results presumably indicated that O. glaberrima is generally more vulnerable to RTBV than O. sativa is.