Rice high in resistant starch is a valuable food for human health, especially for individuals with type 2 diabetes, as it supports effective blood sugar control and provides cardiovascular and intestinal benefits. However, developing rice varieties with a high resistant starch content remains a major challenge. In this study, we identified a mutant with increased chalkiness, chalk2, from the mutant library of indica rice Zhongjian 100. The chalk2 mutants exhibited significantly higher amylose and protein contents, while the total starch and lipid contents were reduced. An analysis of resistant starch in chalk2 revealed substantial increases in two resistant starch (RS) types, RS2 and RS3. Electron microscopy revealed abnormal starch granule development in the endosperm. The chalk2 mutant also showed reduced grain length, width, and thickness, as well as a lower seed-setting rate, which ultimately led to a significant reduction in grain yield. Through physical localization, Mut-Map analysis, and transgene complementation, we found that SBEIIb was responsible for the chalk2 phenotypes, and it is a member of the starch branching enzyme (SBE) family specifically expressed in the endosperm. Furthermore, the expression levels, enzymatic activity, and protein abundance of SBEIIb were significantly reduced in chalk2 mutants. These findings suggest that SBEIIb plays a crucial role in regulating the composition of starch and resistant starch formation in indica rice.

The brown planthopper (Nilaparvata lugens Stål, BPH) is a major rice pest that feeds on sieve tubes, where plants respond by depositing callose to restrict phloem sap ingestion. However, the molecular basis of how rice stabilizes callose at plasmodesmata and how BPH overcomes this defense remains poorly understood. Here, we identify OsPDCB1, a plasmodesmal callose-binding protein that positively regulates BPH resistance by anchoring callose through its X8 domain. Loss- and gain-of-function analyses demonstrate that OsPDCB1 is essential for callose accumulation and effective phloem defense. We further identified NlVRSP1, a BPH salivary effector that is highly conserved across rice planthopper species. This effector directly interacts with OsPDCB1 and disrupts its callose-binding activity, revealing a previously uncharacterized effector-host interaction module at the plasmodesmal interface. Importantly, haplotype analysis uncovered a resistance-associated allele (OsPDCB1Hap1), enriched in Indica rice, which enhances resistance when introgressed into susceptible Japonica backgrounds. Collectively, these findings identify OsPDCB1 as a key mediator of callose-based defense and a promising genetic target for breeding BPH-resistant rice cultivars, while providing mechanistic insight into how insect effectors subvert plasmodesmal immunity.

Starch is the primary component of the endosperm and plays a crucial role in rice quality. Although the enzymes involved in starch synthesis have been extensively studied, the transcription factors that regulate these enzymes remain largely unknown. Here, we identified a MYB family transcription factor, OsMYBR1, that regulates starch biosynthesis in rice. OsMYBR1 is highly expressed during endosperm development. Mutations of OsMYBR1 result in reduced grain thickness and a decrease in 1000-grain weight. The endosperm of osmybr1 mutants exhibit rounded and loosely packed starch granules, decreased amylose content, altered fine structure of amylopectin, and modified physicochemical properties. The analysis of RT-qPCR showed that the expression of several starch-synthesis enzyme-coding genes (SSEGs), including OsGBSSⅠ, OsAGPL1, OsAGPL2, OsBEⅡb, OsISA1, PHOL, and OsSSⅢa, is altered in osmybr1 mutants. Further experiments indicated that OsMYBR1 directly binds to the promoters of OsGBSSⅠ, OsAGPL1, OsAGPL2, OsISA1, OsBEⅡb, and PHOL, resulting in an increase in the expression of OsGBSSⅠ but a decrease in the expression of OsAGPL2, OsISA1, and OsSSⅢa. In contrast, OsMYBR1-overexpressing endosperm appears normal, with starch granule morphology, increased amylopectin content, and improved alkali spreading value, indicating enhanced rice eating and cooking quality (ECQ). These findings suggest that the overexpression of OsMYBR1 could be a promising strategy for improving rice ECQ.

Transitory starch in rice leaves is critical for temporary carbon storage, plant growth, and subsequent grain filling, yet the regulatory mechanisms underlying its biosynthesis remain poorly understood. Here, we found that Leaf Starch Deficiency 3 (LSD3), a rice glutamyl-tRNA reductase (GluTR), interacts with GluTR-binding protein (GluTRBP), which in turn associates with granule-bound starch synthase II (GBSSII), the key enzyme responsible for amylose synthesis in leaves. The LSD3-GluTRBP module maintains both the protein stability and enzymatic activity of GBSSII. The lsd3 and glutrbp mutants exhibited markedly reduced GBSSII activity, resulting in significant decreases in leaf starch accumulation. Moreover, these mutants displayed severe defects in carbon-source transport from leaves to developing grains, accompanied by strong downregulat ion of genes associated with carbon-source allocation. Consequently, the lsd3 and glutrbp mutants showed reduced endosperm-storage starch content and severely compromised grain quality and yield. Notably, overexpression of GBSSII partially rescued defective phenotypes in both leaves and grains of the mutants. In summary, our findings establish that the LSD3-GluTRBP module acts upstream of GBSSII and plays a central role in transitory starch biosynthesis as well as in determining grain quality and yield in rice. These findings provide new molecular targets for simultaneously improving both grain yield and quality in rice.

Rice is a poor source of folate, an essential micronutrient for the body. Biofortification offers an effective way to enhance the folate content of rice and alleviate folate deficiencies in humans. In this study, we confirmed that OsADCS and OsGTPCHI, encoding the initial enzymes necessary for folate synthesis, positively regulate folate accumulation in knockout mutants of both japonica and indica rice backgrounds. The folate content in the low-folate japonica variety was slightly increased by the expression of the indica alleles driven by the endosperm-specific promoter. We further obtained co-expression lines by stacking OsADCS and OsGTPCHI genes; the folate accumulation in brown rice and polished rice reached 5.65 μg/g and 2.95 μg/g, respectively, representing 37.9-fold and 26.5-fold increases compared with the wild type. Transcriptomic analysis of rice grains from six transgenic lines showed that folate changes affected biological pathways involved in the synthesis and metabolism of rice seed storage substances, while the expression of other folate synthesis genes was weakly regulated. In addition, we identified Aus rice as a high-folate germplasm carrying superior haplotypes of OsADCS and OsGTPCHI through natural variation. This study provides an alternative and effective complementary strategy for rice biofortification, promoting the rational combination of metabolic engineering and conventional breeding to breed high-folate varieties.

Crop improvement remains central in addressing global challenges related to food security, climate change, and sustainable agriculture [...]

Taste is crucial for the economic value of rice (Oryza sativa L.) and determines consumer preference. However, the mechanisms underlying taste formation have remained unclear. Here, we show that OsGATA7 contributes to desirable taste quality by affecting the swelling properties, texture, and taste value of cooked rice. OsGATA7 binds to the promoter of SMOS1, and activates its expression, thereby regulating taste quality. Furthermore, SMOS1 binds to the promoter of the protein biosynthesis gene OsGluA2, and recruits the PRC2 complex to repress its expression, leading to increased protein content. The overexpression of both OsGATA7 and SMOS1 reduces protein content and enhances taste quality. The haplotypes OsGATA7Hap1 and SMOS1Hap1 maintain low protein content and improve taste scores. Collectively, these findings reveal a regulatory mechanism for taste quality formation mediated by the OsGATA7-SMOS1 protein content module, and identify the elite haplotypes OsGATA7Hap1 and SMOS1Hap1 as a means to improve taste quality.

>Sucrose transporters (SUTs) contain multiple transmembrane domains that mediate sucrose transport and provide energy for plant growth and development. However, the role of OsSUTs in regulating rice quality and grain yield remains unclear. In this study, we identified five rice SUT genes (OsSUT1–OsSUT5) and examined their molecular characteristics and biological functions.

The trend of global warming is becoming increasingly evident, with frequent extreme high-temperature events posing a severe challenge to food security. Rice (Oryza sativa L.), the world’s primary food crop, is highly susceptible to the adverse effects of high-temperature stress throughout its growth cycle. High temperatures, defined as ambient temperatures exceeding 35 °C during reproductive stages and 33 °C during vegetative stages, can impair seed germination, reduce tillering, disrupt pollination, and diminish grain quality. Notably, heat stress during the grain-filling stage accelerates grain maturation, leading to increased chalkiness, a higher degree of chalky formation, deteriorated cooking and eating quality, and decreased grain weight. To cope with high-temperature stress, rice activates a series of complex physiological and biochemical responses, including heat-related signaling pathways and transcriptional regulatory networks. Although some agronomic practices and genetic improvement methods have been developed to enhance rice’s heat tolerance, the regulatory mechanisms of rice’s response to high-temperature stress, especially the molecular mechanisms during the grain-filling stage, remain poorly understood. This review identifies knowledge gaps in understanding rice’s response mechanisms, emphasizing molecular pathways during the grain-filling stage and provides an outlook on future rice high-temperature defense measures.

Rice (Oryza sativa L.) is one of the most important food crops. Starch is the main substance of rice endosperm and largely determines the grain quality and yield. Starch biosynthesis in endosperm is very complex, requiring a series of enzymes which are also regulated by many transcription factors (TFs). But until now, the large-scale regulatory network for rice endosperm starch biosynthesis has not been established. Here, we constructed a rice endosperm starch biosynthesis regulatory network comprised of 277 TFs and 15 starch synthesis enzyme-encoding genes (SSEGs) using DNA affinity chromatography/pull-down combined with liquid chromatography-mass spectrometry (DNA pull-down and LC-MS). In this regulatory network, each SSEG is directly regulated by 7-46 TFs. Based on this network, we found a new pathway 'ABA-OsABI5-OsERF44-SSEGs' that regulates rice endosperm starch biosynthesis. We also knocked out five TFs targeting the key amylose synthesis enzyme gene OsGBSSI in japonica rice 'Nipponbare' background and found that all mutants had moderately decreased amylose content (AC) in endosperm and improved eating and cooking quality (ECQ). Notably, the knockout of OsSPL7 and OsB3 improves the ECQ without compromising the rice appearance quality, which was further validated in the indica rice 'Zhongjiazao17' background. In summary, this gene regulatory network for rice endosperm starch biosynthesis established here will provide important theoretical and practical guidance for the genetic improvement of rice quality.

In rice (Oryza sativa), defects in endosperm development can give grains a chalky texture, which decreases grain quality and is thus undesirable for breeding and marketing. However, the molecular basis of chalkiness remains largely unknown. Here, we identified CHALK10, which encodes an F-box protein that negatively regulates rice chalkiness. The chalk10 mutant exhibited abnormal starch granule development, decreased starch content, and altered starch physicochemical properties compared with the wild type. CHALK10 interacts with the gibberellin (GA) oxidase SEMIDWARF-1 (SD1) and promotes the ubiquitination and degradation of SD1 through the 26S proteasome pathway. The grains of SD1-overexpressing plants exhibited increased chalkiness; introduction of the sd1 mutant allele into the chalk10 background largely suppressed the enhanced chalkiness observed in the chalk10 mutant. GA levels were higher in the chalk10 mutant than in the wild type, and application of the bioactive GA form GA3 increased grain chalkiness. The expression of genes related to starch degradation or biosynthesis was altered in the chalk10 mutant, resulting in reduced starch production and increased metabolizable sugar content in the endosperm. In summary, our findings reveal a novel regulatory mechanism of chalkiness and provide potential targets for improving rice quality.

The chemical compound 2-acetyl-1-pyrroline (2-AP) is a major contributor to the fragrance of rice. This report presents the first efforts towards determining the quantities of 2-AP in rice by grating-based visible–near-infrared reflectance spectroscopy using a diverse set of rice varieties. Calibration models generated by preprocessing treatments using Design of Experiment (DoE) and 'one-variable-at-atime' (OVAT) methods were compared and optimized. A modified partial least squares model with "2, 3, 1, 1"/ SNV and 174 variables yielded a coefficient of determination for calibration values of 0.978. The results showed that a DoE-based preprocessing selection could efficiently and accurately optimize 2-AP calibration models, with advantages over OVAT. The findings of this study provide high-throughput screening technology for high-precision fragrance quality breeding, monitoring and control in the rice industry. The method demonstrated here also has potential for application in many other disciplines, including identification of plant fragrances and quality control of cereal products.

A mechanized direct seeding of rice with less labor and water usage, has been widely adopted. However, this approach requires varieties that exhibit uniform seedling emergence. Mesocotyl elongation (ME) offers the main drive of fast emergence of rice seedlings from soils; nevertheless, its genetic basis remains unknown. Here, we identify a major rice quantitative trait locus Mesocotyl Elongation1 (qME1), an allele of the Green Revolution gene Semi-Dwarf1 (SD1), encoding GA20-oxidase for gibberellin (GA) biosynthesis. ME1 expression is strongly induced by soil depth and ethylene. When rice grains are direct-seeded in soils, the ethylene core signaling factor OsEIL1 directly promotes ME1 transcription, accelerating bioactive GA biosynthesis. The GAs further degrade the DELLA protein SLENDER RICE 1 (SLR1), alleviating its inhibition of rice PHYTOCHROME-INTERACTING FACTOR-LIKE13 (OsPIL13) to activate the downstream expansion gene OsEXPA4 and ultimately promote rice seedling ME and emergence. The ancient traits of long mesocotyl and strong emergence ability in wild rice and landrace were gradually lost in company with the Green Revolution dwarf breeding process, and an elite ME1-R allele (D349H) is found in some modern Geng varieties (long mesocotyl lengths) in northern China, which can be used in the direct seeding and dwarf breeding of Geng varieties. Furthermore, the ectopic and high expression of ME1 driven by mesocotyl-specific promoters resulted in rice plants that could be direct-seeded without obvious plant architecture or yield penalties. Collectively, we reveal the molecular mechanism of rice ME, and provide useful information for breeding new Green Revolution varieties with long mesocotyl suitable for direct-seeding practice.

Exposure to high temperatures can impair the grain-filling process in rice (Oryza sativa L.), potentially leading to the formation of chalky endosperm, but the molecular regulation mechanism remains largely elusive. Here, we reported that high-temperature (HT) stress (day/night, 35 °C/30 °C) reduces both the grain-filling rate and grain weight of Ningjing 1 variety compared to normal temperatures (NT, day/night, 28 °C/23 °C). Grains under HT stress exhibited an opaque, milky-white appearance, alongside significant alterations in starch physicochemical properties. An integrated transcriptomic analysis of grains under HT revealed up-regulation of genes related to defense mechanisms and oxidoreductase activity, while genes involved in sucrose and starch synthesis were down-regulated, and α-amylase genes were up-regulated. Proteomic analysis of grains under HT echoed this pattern. These results demonstrate that high temperature during the grain-filling stage significantly increases rice chalkiness by down-regulating genes related to sucrose and starch synthesis, while up-regulating those involved in starch degradation.

Heat shock proteins (Hsps), acting as molecular chaperones, play a pivotal role in plant responses to environmental stress. In this study, we found a total of 192 genes encoding Hsps, which are distributed across all 12 chromosomes, with higher concentrations on chromosomes 1, 2, 3, and 5. These Hsps can be divided into six subfamilies (sHsp, Hsp40, Hsp60, Hsp70, Hsp90, and Hsp100) based on molecular weight and homology. Expression pattern data indicated that these Hsp genes can be categorized into three groups: generally high expression in almost all tissues, high tissue-specific expression, and low expression in all tissues. Further analysis of 15 representative genes found that the expression of 14 Hsp genes was upregulated by high temperatures. Subcellular localization analysis revealed seven proteins localized to the endoplasmic reticulum, while others localized to the mitochondria, chloroplasts, and nucleus. We successfully obtained the knockout mutants of above 15 Hsps by the CRISPR/Cas9 gene editing system. Under natural high-temperature conditions, the mutants of eight Hsps showed reduced yield mainly due to the seed setting rate or grain weight. Moreover, the rice quality of most of these mutants also changed, including increased grain chalkiness, decreased amylose content, and elevated total protein content, and the expressions of starch metabolism-related genes in the endosperm of these mutants were disturbed compared to the wild type under natural high-temperature conditions. In conclusion, our study provided new insights into the HSP gene family and found that it plays an important role in the formation of rice quality and yield.

Ensuring rice yield and grain safety quality are vital for human health. In this study, we developed two-line hybrid rice (TLHR) with ultra-low grain cadmium (Cd) and arsenic (As) accumulation by pyramiding novel alleles of OsNramp5 and OsLsi2. We first generated low Cd accumulation restorer (R) lines by editing OsNramp5, OsLCD, and OsLCT in japonica and indica. After confirming that OsNramp5 was most efficient in reducing Cd, we edited this gene in C815S, a genic male sterile line (GMSL), and screened it for alleles with low Cd accumulation. Next, we generated R and GMSL lines with low As accumulation by editing OsLsi2 in a series of YK17 and C815S lines. When cultivated in soils that were heavily polluted with Cd and As, the edited R, GMSL, and TLHR plants showed significantly reduced heavy metal accumulation, while maintaining a relatively stable yield potential. This study provides an effective scheme for the safe production of grains in As- and/or Cd-polluted paddy fields.

Transitory starch is an important carbon source in leaves, and its biosynthesis and metabolism are closely related to grain quality and yield. The molecular mechanisms controlling leaf transitory starch biosynthesis and degradation and their effects on rice (Oryza sativa) quality and yield remain unclear. Here, we show that OsLESV and OsESV1, the rice orthologs of AtLESV and AtESV1, are associated with transitory starch biosynthesis in rice. The total starch and amylose contents in leaves and endosperms are significantly reduced, and the final grain quality and yield are compromised in oslesv and osesv1 single and oslesv esv1 double mutants. Furthermore, we found that OsLESV and OsESV1 bind to starch, and this binding depends on a highly conserved C-terminal tryptophan-rich region that acts as a starch-binding domain. Importantly, OsLESV and OsESV1 also interact with the key enzymes of starch biosynthesis, granule-bound starch synthase I (GBSSI), GBSSII, and pyruvate orthophosphote dikiase (PPDKB), to maintain their protein stability and activity. OsLESV and OsESV1 also facilitate the targeting of GBSSI and GBSSII from plastid stroma to starch granules. Overexpression of GBSSI, GBSSII, and PPDKB can partly rescue the phenotypic defects of the oslesv and osesv1 mutants. Thus, we demonstrate that OsLESV and OsESV1 play a key role in regulating the biosynthesis of both leaf transitory starch and endosperm storage starch in rice. These findings deepen our understanding of the molecular mechanisms underlying transitory starch biosynthesis in rice leaves and reveal how the transitory starch metabolism affects rice grain quality and yield, providing useful information for the genetic improvement of rice grain quality and yield.

Three key factors determine yield in rice (Oryza sativa): panicle number, grain number, and grain weight. Panicle number is strongly associated with tiller number. Although many genes regulating tillering have been identified, whether Dof proteins are involved in controlling plant architecture remains unknown. The dwarf and less tillers on chromosome 3 (dlt3) rice mutant produces fewer tillers than the wild type. We cloned DLT3, which encodes a Dof protein that interacts with MONOCULM 3 (MOC3) in vivo and in vitro and recruits MOC1, forming a DLT3-MOC3-MOC1 complex. DLT3 binds to the promoter of FLORAL ORGAN NUMBER 1 (FON1) to activate its transcription and positively regulate tiller number. The overexpression of MOC1, MOC3, or FON1 in the dlt3 mutant increased tiller number. Collectively, these results suggest a model in which DLT3 regulates tiller number by maintaining the expression of MOC1, MOC3, and FON1. We discovered that DLT3 underwent directional selection in the Xian/indica and Geng/japonica populations during rice domestication. To provide genetic resources for breeding varieties with optimal panicle numbers, we performed large-scale diversity sequencing of the 1,080-bp DLT3 coding region of 531 accessions from different countries and regions. Haplotype analysis showed that the superior haplotype, DLT3H1, produced the most tillers, while haplotype DLT3H6 produced the fewest tillers. Our study provides important germplasm resources for breeding super high-yielding rice varieties with combinations of superior haplotypes in different target genes, which will help overcome the challenge of food and nutritional security in the future. A Dof protein controls the expression of 3 regulatory proteins that promote tiller number in rice.

Enhanced grain yield and quality traits are everlasting breeding goals. It is therefore of great significance to uncover more genetic resources associated with these two important agronomic traits. Plant MYB family transcription factors play important regulatory roles in diverse biological processes. However, studies on genetic functions of MYB in rice yield and quality are rarely to be reported. Here, we investigated a nucleus-localized transcription factor OsMYB73 which is preferentially expressed in the early developing pericarp and endosperm. We generated targeted mutagenesis of OsMYB73 in rice, and the mutants had longer grains with obvious white-belly chalky endosperm appearance phenotype. The mutants displayed various changes in starch physicochemical characteristics and lipid components. Transcriptome sequencing analysis showed that OsMYB73 was chiefly involved in cell wall development and starch metabolism. OsMYB73 mutation affects the expression of genes related to grain size, starch and lipid biosynthesis and auxin biosynthesis. Moreover, inactivation of OsMYB73 triggers broad changes in secondary metabolites. We speculate that rice OsMYB73 and OsNF-YB1 play synergistic pivotal role in simultaneously as transcription activators to regulate grain filling and storage compounds accumulation to affect endosperm development and grain chalkiness through binding OsISA2, OsLTPL36 and OsYUC11. The study provides important germplasm resources and theoretical basis for genetic improvement of rice yield and quality. In addition, we enriches the potential biological functions of rice MYB family transcription factors.