DWARF AND LESS TILLERS ON CHROMOSOME 3 Promotes Tillering in Rice by Sustaining FLORAL ORGAN NUMBER 1 Expression.

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 1080-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.

Natural variation of TBR confers plant zinc toxicity tolerance through root cell wall pectin methylesterification.

Zinc (Zn) is an essential micronutrient but can be cytotoxic when present in excess. Plants have evolved mechanisms to tolerate Zn toxicity. To identify genetic loci responsible for natural variation of plant tolerance to Zn toxicity, we conduct genome-wide association studies for root growth responses to high Zn and identify 21 significant associated loci. Among these loci, we identify Trichome Birefringence (TBR) allelic variation determining root growth variation in high Zn conditions. Natural alleles of TBR determine TBR transcript and protein levels which affect pectin methylesterification in root cell walls. Together with previously published data showing that pectin methylesterification increase goes along with decreased Zn binding to cell walls in TBR mutants, our findings lead to a model in which TBR allelic variation enables Zn tolerance through modulating root cell wall pectin methylesterification. The role of TBR in Zn tolerance is conserved across dicot and monocot plant species.

Photosynthesis regulates tillering bud elongation and nitrogen-use efficiency via sugar-induced NGR5 in rice.

Rice tillering is one of the most important agronomical traits largely determining grain yield. Photosynthesis and nitrogen availability are two important factors affecting rice tiller bud elongation; however, underlying mechanism and their cross-talk is poorly understood. Here, we used map-based cloning, transcriptome profiling, phenotypic analysis, and molecular genetics to understand the roles of the Decreased Tiller Number 1 (DTN1) gene that encodes the fructose-1,6-bisphosphate aldolase and involves in photosynthesis required for light-induced axillary bud elongation in rice. Deficiency of DTN1 results in the reduced photosynthetic rate and decreased contents of sucrose and other sugars in both leaves and axillary buds, and the reduced tiller number in dtn1 mutant could be partially rescued by exogenous sucrose treatment. Furthermore, we found that the expression of nitrogen-mediated tiller growth response 5 (NGR5) was remarkably decreased in shoot base of dtn1-2, which can be activated by sucrose treatment. Overexpression of NGR5 in the dtn1-2 could partially rescue the reduced tiller number, and the tiller number of dtn1-2 was insensitive to nitrogen supply. This work demonstrated that the sugar level regulated by photosynthesis and DTN1 could positively regulate NGR5 expression, which coordinates the cross-talk between carbon and nitrate to control tiller bud outgrowth in rice.

Loss-of-function of PGL10 impairs photosynthesis and tolerance to high-temperature stress in rice.

High temperature (HT) affects the production of chlorophyll (Chl) pigment and inhibits cellular processes that impair photosynthesis, and growth and development in plants. However, the molecular mechanisms underlying heat stress in rice are not fully understood yet. In this study, we identified two mutants varying in leaf color from the ethylmethanesulfonate mutant library of indica rice cv. Zhongjiazao-17, which showed pale-green leaf color and variegated leaf phenotype under HT conditions. Mut-map revealed that both mutants were allelic, and their phenotype was controlled by a single recessive gene PALE GREEN LEAF 10 (PGL10) that encodes NADPH:protochlorophyllide oxidoreductase B, which is required for the reduction of protochlorophyllide into chlorophyllide in light-dependent tetrapyrrole biosynthetic pathway-based Chl synthesis. Overexpression-based complementation and CRISPR/Cas9-based knockout analyses confirmed the results of Mut-map. Moreover, qRT-PCR-based expression analysis of PGL10 showed that it expresses in almost all plant parts with the lowest expression in root, followed by seed, third leaf, and then other green tissues in both mutants, pgl10a and pgl10b. Its protein localizes in chloroplasts, and the first 17 amino acids from N-terminus are responsible for signals in chloroplasts. Moreover, transcriptome analysis performed under HT conditions revealed that the genes involved in the Chl biosynthesis and degradation, photosynthesis, and reactive oxygen species detoxification were differentially expressed in mutants compared to WT. Thus, these results indicate that PGL10 is required for maintaining chloroplast function and plays an important role in rice adaptation to HT stress conditions by controlling photosynthetic activity.

Production of grains with ultra-low heavy metal accumulation by pyramiding novel Alleles of OsNramp5 and OsLsi2 in two-line hybrid rice.

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.

An orchestrated ethylene-gibberellin signaling cascade contributes to mesocotyl elongation and emergence of rice direct seeding.

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.

OsLESV and OsESV1 promote transitory and storage starch biosynthesis to determine rice grain quality and yield.

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.

Switching action modes of miR408-5p mediates auxin signaling in rice.

MicroRNAs (miRNAs) play fundamental roles in many developmental and physiological processes in eukaryotes. MiRNAs in plants generally regulate their targets via either mRNA cleavage or translation repression; however, which approach plays a major role and whether these two function modes can shift remains elusive. Here, we identify a miRNA, miR408-5p that regulates AUXIN/INDOLE ACETIC ACID 30 (IAA30), a critical repressor in the auxin pathway via switching action modes in rice. We find that miR408-5p usually inhibits IAA30 protein translation, but in a high auxin environment, it promotes the decay of IAA30 mRNA when it is overproduced. We further demonstrate that IDEAL PLANT ARCHITECTURE1 (IPA1), an SPL transcription factor regulated by miR156, mediates leaf inclination through association with miR408-5p precursor promoter. We finally show that the miR156-IPA1-miR408-5p-IAA30 module could be controlled by miR393, which silences auxin receptors. Together, our results define an alternative auxin transduction signaling pathway in rice that involves the switching of function modes by miR408-5p, which contributes to a better understanding of the action machinery as well as the cooperative network of miRNAs in plants.

A mediator of OsbZIP46 deactivation and degradation negatively regulates seed dormancy in rice.

Preharvest sprouting (PHS) is a deleterious phenotype that occurs frequently in rice-growing regions where the temperature and precipitation are high. It negatively affects yield, quality, and downstream grain processing. Seed dormancy is a trait related to PHS. Longer seed dormancy is preferred for rice production as it can prevent PHS. Here, we map QTLs associated with rice seed dormancy and clone Seed Dormancy 3.1 (SDR3.1) underlying one major QTL. SDR3.1 encodes a mediator of OsbZIP46 deactivation and degradation (MODD). We show that SDR3.1 negatively regulates seed dormancy by inhibiting the transcriptional activity of ABIs. In addition, we reveal two critical amino acids of SDR3.1 that are critical for the differences in seed dormancy between the Xian/indica and Geng/japonica cultivars. Further, SDR3.1 has been artificially selected during rice domestication. We propose a two-line model for the process of rice seed dormancy domestication from wild rice to modern cultivars. We believe the candidate gene and germplasm studied in this study would be beneficial for the genetic improvement of rice seed dormancy.

The elite eating quality alleles Wxb and ALKb are regulated by OsDOF18 and coordinately improve head rice yield.

Head rice yield (HRY) measures rice milling quality and determines final grain yield and commercial value. Here, we report that two major quantitative trait loci for milling quality in rice, qMq-1 and qMq-2, represent allelic variants of Waxylv/Waxyb (hereafter Wx) encoding Granule-Bound Starch Synthase I (GBSSI) and Alkali Spreading Value ALKc/ALKb encoding Soluble Starch Synthase IIa (SSIIa), respectively. Complementation and overexpression transgenic lines in indica and japonica backgrounds confirmed that Wx and ALK coordinately regulate HRY by affecting amylose content, the number of amylopectin branches, amyloplast size, and thus grain filling and hardness. The transcription factor OsDOF18 acts upstream of Wx and ALK by activating their transcription. Furthermore, rice accessions with Wxb and ALKb alleles showed improved HRY over those with Wxlv and ALKc. Our study not only reveals the novel molecular mechanism underlying the formation of HRY but also provides a strategy for breeding rice cultivars with improved HRY.

YGL3 Encoding an IPP and DMAPP Synthase Interacts with OsPIL11 to Regulate Chloroplast Development in Rice.

As the source of isoprenoid precursors, the plastidial methylerythritol phosphate (MEP) pathway plays an essential role in plant development. Here, we report a novel rice (Oryza sativa L.) mutant ygl3 (yellow-green leaf3) that exhibits yellow-green leaves and lower photosynthetic efficiency compared to the wild type due to abnormal chloroplast ultrastructure and reduced chlorophyll content. Map-based cloning showed that YGL3, one of the major genes involved in the MEP pathway, encodes 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, which is localized in the thylakoid membrane. A single base substitution in ygl3 plants resulted in lower 4-hydroxy-3-methylbut-2-enyl diphosphate reductase activity and lower contents of isopentenyl diphosphate (IPP) compared to the wild type. The transcript levels of genes involved in the syntheses of chlorophyll and thylakoid membrane proteins were significantly reduced in the ygl3 mutant compared to the wild type. The phytochrome interacting factor-like gene OsPIL11 regulated chlorophyll synthesis during the de-etiolation process by directly binding to the promoter of YGL3 to activate its expression. The findings provides a theoretical basis for understanding the molecular mechanisms by which the MEP pathway regulate chloroplast development in rice.

OsLEA1b Modulates Starch Biosynthesis at High Temperatures in Rice.

High temperatures accelerate the accumulation of storage material in seeds, often leading to defects in grain filling. However, the mechanisms regulating grain filling at high temperatures remain unknown. Here, we want to explore the quality factors influenced by the environment and have identified a LATE EMBROYGENESIS ABUNDANT gene, OsLEA1b, a heat-stress-responsive gene in rice grain filling. OsLEA1b is highly expressed in the endosperm, and its coding protein localizes to the nucleus and cytoplasm. Knock-out mutants of OsLEA1b had abnormal compound starch granules in endosperm cells and chalky endosperm with significantly decreased grain weight and grain number per panicle. The oslea1b mutants exhibited a lower proportion of short starch chains with degrees of polymerization values from 6 to 13 and a higher proportion of chains with degrees from 14 to 48, as well as significantly lower contents of starch, protein, and lipid compared to the wild type. The difference was exacerbated under high temperature conditions. Moreover, OsLEA1b was induced by drought stress. The survival rate of oslea1b mutants decreased significantly under drought stress treatment, with significant increase in ROS levels. These results indicate that OsLEA1b regulates starch biosynthesis and influences rice grain quality, especially under high temperatures. This provides a valuable resource for genetic improvement in rice grain quality.

Genome-wide identification and expression analysis of 3-ketoacyl-CoA synthase gene family in rice (Oryza sativa L.) under cadmium stress.

3-Ketoacyl-CoA synthase (KCS) is the key rate-limiting enzyme for the synthesis of very long-chain fatty acids (VLCFAs) in plants, which determines the carbon chain length of VLCFAs. However, a comprehensive study of KCSs in Oryza sativa has not been reported yet. In this study, we identified 22 OsKCS genes in rice, which are unevenly distributed on nine chromosomes. The OsKCS gene family is divided into six subclasses. Many cis-acting elements related to plant growth, light, hormone, and stress response were enriched in the promoters of OsKCS genes. Gene duplication played a crucial role in the expansion of the OsKCS gene family and underwent a strong purifying selection. Quantitative Real-time polymerase chain reaction (qRT-PCR) results revealed that most KCS genes are constitutively expressed. We also revealed that KCS genes responded differently to exogenous cadmium stress in japonica and indica background, and the KCS genes with higher expression in leaves and seeds may have functions under cadmium stress. This study provides a basis for further understanding the functions of KCS genes and the biosynthesis of VLCFA in rice.

Exogenous abscisic acid represses rice flowering via SAPK8-ABF1-Ehd1/Ehd2 pathway.

INTRODUCTION: Rice flowering is a major agronomic trait, determining yield and ecological adaptability in particular regions. ABA plays an essential role in rice flowering, but the underlying molecular mechanism remains largely elusive. OBJECTIVES: In this study, we demonstrated a "SAPK8-ABF1-Ehd1/Ehd2" pathway, through which exogenous ABA represses rice flowering in a photoperiod-independent manner. METHODS: We generated abf1 and sapk8 mutants using the CRISPR-Cas9 method. Using yeast two-hybrid, Pull down, BiFC and kinase assays, SAPK8 interacted and phosphorylated ABF1. ABF1 directly bound to the promoters of Ehd1 and Ehd2 using ChIP-qPCR, EMSA, and LUC transient transcriptional activity assay, and suppressed the transcription of these genes. RESULTS: Under both long day and short day conditions, simultaneous knock-out of ABF1 and its homolog bZIP40 accelerated flowering, while SAPK8 and ABF1 over-expression lines exhibited delayed flowering and hypersensitivity to ABA-mediated flowering repression. After perceiving the ABA signal, SAPK8 physically binds to and phosphorylates ABF1 to enhance its binding to the promoters of master positive flowering regulators Ehd1 and Ehd2. Upon interacting with FIE2, ABF1 recruited PRC2 complex to deposit H3K27me3 suppressive histone modification on Ehd1 and Ehd2 to suppress these genes transcription, thereby leading to later flowering. CONCLUSION: Our work highlighted the biological functions of SAPK8 and ABF1 in ABA signaling, flowering control and the involvement of a PRC2-mediated epigenetic repression mechanism in the transcription regulation governed by ABF1 on ABA-mediated rice flowering repression.

Transcriptome and Proteome Analysis Revealed That Hormone and Reactive Oxygen Species Synergetically Regulate Dormancy of Introgression Line in Rice (Oryza sativa L.).

Dormancy is a complex agronomy phenotype controlled by multiple signaling and a key trait repressing pre-harvest sprouting (PHS). However, the signaling network of dormancy remains unclear. In this study, we used Zhonghua11 (ZH11) with a weak dormancy, and Introgression line (IL) with a strong dormancy to study the mechanism of hormones and reactive oxygen species (ROS) crosstalk regulating rice dormancy. The germination experiment showed that the germination rate of ZH11 was 76.86%, while that of IL was only 1.25%. Transcriptome analysis showed that there were 1658 differentially expressed genes (DEGs) between IL and ZH11, of which 577 were up-regulated and 1081 were down-regulated. Additionally, DEGs were mainly enriched in oxidoreductase activity, cell periphery, and plant hormone signal transduction pathways. Tandem mass tags (TMT) quantitative proteomics analysis showed 275 differentially expressed proteins (DEPs) between IL and ZH11, of which 176 proteins were up-regulated, 99 were down-regulated, and the DEPs were mainly enriched in the metabolic process and oxidation-reduction process. The comprehensive transcriptome and proteome analysis showed that their correlation was very low, and only 56 genes were co-expressed. Hormone content detection showed that IL had significantly lower abscisic acid (ABA) contents than the ZH11 while having significantly higher jasmonic acid (JA) contents than the ZH11. ROS content measurement showed that the hydrogen peroxide (H2O2) content of IL was significantly lower than the ZH11, while the production rate of superoxide anion (O2.-) was significantly higher than the ZH11. These results indicate that hormones and ROS crosstalk to regulate rice dormancy. In particular, this study has deepened our mechanism of ROS and JA crosstalk regulating rice dormancy and is conducive to our precise inhibition of PHS.

Genome-Wide Analysis and Functional Characterization of Pyruvate Kinase (PK) Gene Family Modulating Rice Yield and Quality.

Pyruvate kinase (PK) is one of the three rate-limiting enzymes of glycolysis, and it plays a pivotal role in energy metabolism. In this study, we have identified 10 PK genes from the rice genome. Initially, these genes were divided into two categories: cytoplasmic pyruvate kinase (PKc) and plastid pyruvate kinase (PKp). Then, an expression analysis revealed that OsPK1, OsPK3, OsPK4, OsPK6, and OsPK9 were highly expressed in grains. Moreover, PKs can form heteropolymers. In addition, it was found that ABA significantly regulates the expression of PK genes (OsPK1, OsPK4, OsPK9, and OsPK10) in rice. Intriguingly, all the genes were found to be substantially involved in the regulation of rice grain quality and yield. For example, the disruption of OsPK3, OsPK5, OsPK7, OsPK8, and OsPK10 and OsPK4, OsPK5, OsPK6, and OsPK10 decreased the 1000-grain weight and the seed setting rate, respectively. Further, the disruption of OsPK4, OsPK6, OsPK8, and OsPK10 through the CRISPR/Cas9 system showed an increase in the content of total starch and a decrease in protein content compared to the WT. Similarly, manipulations of the OsPK4, OsPK8, and OsPK10 genes increased the amylose content. Meanwhile, the grains of all CRISPR mutants and RNAi lines, except ospk6, showed a significant increase in the chalkiness rate compared to the wild type. Overall, this study characterizes the functions of all the genes of the PK gene family and shows their untapped potential to improve rice yield and quality traits.

Genome-Wide Association Study of Zinc Toxicity Tolerance within a Rice Core Collection (Oryza sativa L.).

Zinc (Zn) is an essential micronutrient for rice, but it is toxic at a high concentration, especially in acid soils. It is yet unknown which genes regulate Zn tolerance in rice. In the present study, a genome-wide association study (GWAS) was performed for Zn tolerance in rice at the seedling stage within a rice core collection, named Ting's core collection, which showed extensive phenotypic variations in Zn toxicity with high-density single-nucleotide polymorphisms (SNPs). A total of 7 and 19 quantitative trait loci (QTL) were detected using root elongation (RE) and relative root elongation (RRE) under high Zn toxicity, respectively. Among them, 24 QTL were novel, and qRRE15 was located in the same region where 3 QTL were reported previously. In addition, qRE4 and qRRE9 were identical. Furthermore, we found eight candidate genes that are involved in abiotic and biotic stress, immunity, cell expansion, and phosphate transport in the loci of qRRE8, qRRE9, and qRRE15. Moreover, four candidate genes, i.e., Os01g0200700, Os06g0621900, Os06g0493600, and Os06g0622700, were verified correlating to Zn tolerance in rice by quantitative real time-PCR (qRT-PCR). Taken together, these results provide significant insight into the genetic basis for Zn toxicity tolerance and tolerant germplasm for developing rice tolerance to Zn toxicity and improving rice production in Zn-contaminated soils.

Genomic footprints related with adaptation and fumonisins production in Fusarium proliferatum.

Fusarium proliferatum is the principal etiological agent of rice spikelet rot disease (RSRD) in China, causing yield losses and fumonisins contamination in rice. The intraspecific variability and evolution pattern of the pathogen is poorly understood. Here, we performed whole-genome resequencing of 67 F. proliferatum strains collected from major rice-growing regions in China. Population structure indicated that eastern population of F. proliferatum located in Yangtze River with the high genetic diversity and recombinant mode that was predicted as the putative center of origin. Southern population and northeast population were likely been introduced into local populations through gene flow, and genetic differentiation between them might be shaped by rice-driven domestication. A total of 121 distinct genomic loci implicated 85 candidate genes were suggestively associated with variation of fumonisin B1 (FB1) production by genome-wide association study (GWAS). We subsequently tested the function of five candidate genes (gabap, chsD, palA, hxk1, and isw2) mapped in our association study by FB1 quantification of deletion strains, and mutants showed the impact on FB1 production as compared to the wide-type strain. Together, this is the first study to provide insights into the evolution and adaptation in natural populations of F. proliferatum on rice, as well as the complex genetic architecture for fumonisins biosynthesis.

OPAQUE3, encoding a transmembrane bZIP transcription factor, regulates endosperm storage protein and starch biosynthesis in rice.

Starch and storage proteins are the main components of rice (Oryza sativa L.) grains. Despite their importance, the molecular regulatory mechanisms of storage protein and starch biosynthesis remain largely elusive. Here, we identified a rice opaque endosperm mutant, opaque3 (o3), that overaccumulates 57-kDa proglutelins and has significantly lower protein and starch contents than the wild type. The o3 mutant also has abnormal protein body structures and compound starch grains in its endosperm cells. OPAQUE3 (O3) encodes a transmembrane basic leucine zipper (bZIP) transcription factor (OsbZIP60) and is localized in the endoplasmic reticulum (ER) and the nucleus, but it is localized mostly in the nucleus under ER stress. We demonstrated that O3 could activate the expression of several starch synthesis-related genes (GBSSI, AGPL2, SBEI, and ISA2) and storage protein synthesis-related genes (OsGluA2, Prol14, and Glb1). O3 also plays an important role in protein processing and export in the ER by directly binding to the promoters and activating the expression of OsBIP1 and PDIL1-1, two major chaperones that assist with folding of immature secretory proteins in the ER of rice endosperm cells. High-temperature conditions aggravate ER stress and result in more abnormal grain development in o3 mutants. We also revealed that OsbZIP50 can assist O3 in response to ER stress, especially under high-temperature conditions. We thus demonstrate that O3 plays a central role in rice grain development by participating simultaneously in the regulation of storage protein and starch biosynthesis and the maintenance of ER homeostasis in endosperm cells.

Pseudouridylation of chloroplast ribosomal RNA contributes to low temperature acclimation in rice.

Ribosomal RNAs (rRNAs) undergo many modifications during transcription and maturation; homeostasis of rRNA modifications is essential for chloroplast biogenesis in plants. The chloroplast acts as a hub to sense environmental signals, such as cold temperature. However, how RNA modifications contribute to low temperature responses remains unknown. Here we reveal that pseudouridine (Ψ) modification of rice chloroplast rRNAs mediated by the pseudouridine synthase (OsPUS1) contributes to cold tolerance at seedling stage. Loss-function of OsPUS1 leads to abnormal chloroplast development and albino seedling phenotype at low temperature. We find that OsPUS1 is accumulated upon cold and binds to chloroplast precursor rRNAs (pre-rRNAs) to catalyse the pseudouridylation on rRNA. These modifications on chloroplast rRNAs could be required for their processing, as the reduction of mature chloroplast rRNAs and accumulation of pre-rRNAs are observed in ospus1-1 at low temperature. Therefore, the ribosome activity and translation in chloroplasts is disturbed in ospus1-1. Furthermore, transcriptome and translatome analysis reveals that OsPUS1 balances growth and stress-responsive state, preventing excess reactive oxygen species accumulation. Taken together, our findings unveil a crucial function of Ψ in chloroplast ribosome biogenesis and cold tolerance in rice, with potential applications in crop improvement.