GW9 determines grain size and floral organ identity in rice.

Grain weight is an important determinant of grain yield. However, the underlying regulatory mechanisms for grain size remain to be fully elucidated. Here, we identify a rice mutant grain weight 9 (gw9), which exhibits larger and heavier grains due to excessive cell proliferation and expansion in spikelet hull. GW9 encodes a nucleus-localized protein containing both C2H2 zinc finger (C2H2-ZnF) and VRN2-EMF2-FIS2-SUZ12 (VEFS) domains, serving as a negative regulator of grain size and weight. Interestingly, the non-frameshift mutations in C2H2-ZnF domain result in increased plant height and larger grain size, whereas frameshift mutations in both C2H2-ZnF and VEFS domains lead to dwarf and malformed spikelet. These observations indicated the dual functions of GW9 in regulating grain size and floral organ identity through the C2H2-ZnF and VEFS domains, respectively. Further investigation revealed the interaction between GW9 and the E3 ubiquitin ligase protein GW2, with GW9 being the target of ubiquitination by GW2. Genetic analyses suggest that GW9 and GW2 function in a coordinated pathway controlling grain size and weight. Our findings provide a novel insight into the functional role of GW9 in the regulation of grain size and weight, offering potential molecular strategies for improving rice yield.

Formin protein DRT1 affects gross morphology and chloroplast relocation in rice.

Plant height and tiller number are two major factors determining plant architecture and yield. However, in rice (Oryza sativa), the regulatory mechanism of plant architecture remains to be elucidated. Here, we reported a recessive rice mutant presenting dwarf and reduced tillering phenotypes (drt1). Map-based cloning revealed that the phenotypes are caused by a single point mutation in DRT1, which encodes the Class I formin protein O. sativa formin homolog 13 (OsFH13), binds with F-actin, and promotes actin polymerization for microfilament organization. DRT1 protein localized on the plasma membrane (PM) and chloroplast (CP) outer envelope. DRT1 interacted with rice phototropin 2 (OsPHOT2), and the interaction was interrupted in drt1. Upon blue light stimulus, PM localized DRT1 and OsPHOT2 were translocated onto the CP membrane. Moreover, deficiency of DRT1 reduced OsPHOT2 internalization and OsPHOT2-mediated CP relocation. Our study suggests that rice formin protein DRT1/OsFH13 is necessary for plant morphology and CP relocation by modulating the actin-associated cytoskeleton network.

The LARGE2-APO1/APO2 regulatory module controls panicle size and grain number in rice.

Panicle size and grain number are important agronomic traits and influence grain yield in rice (Oryza sativa), but the molecular and genetic mechanisms underlying panicle size and grain number control remain largely unknown in crops. Here we report that LARGE2 encodes a HECT-domain E3 ubiquitin ligase OsUPL2 and regulates panicle size and grain number in rice. The loss of function large2 mutants produce large panicles with increased grain number, wide grains and leaves, and thick culms. LARGE2 regulates panicle size and grain number by repressing meristematic activity. LARGE2 is highly expressed in young panicles and grains. Biochemical analyses show that LARGE2 physically associates with ABERRANT PANICLE ORGANIZATION1 (APO1) and APO2, two positive regulators of panicle size and grain number, and modulates their stabilities. Genetic analyses support that LARGE2 functions with APO1 and APO2 in a common pathway to regulate panicle size and grain number. These findings reveal a novel genetic and molecular mechanism of the LARGE2-APO1/APO2 module-mediated control of panicle size and grain number in rice, suggesting that this module is a promising target for improving panicle size and grain number in crops.

Carotenoid isomerase regulates rice tillering and grain productivity by its biosynthesis pathway.

Carotenoid isomerase activity and carotenoid content maintain the appropriate tiller number, photosynthesis, and grain yield. Interactions between the strigolactone and abscisic acid pathways regulates tiller formation.

NLG1, encoding a mitochondrial membrane protein, controls leaf and grain development in rice.

BACKGROUND: Mitochondrion is the key respiratory organ and participate in multiple anabolism and catabolism pathways in eukaryote. However, the underlying mechanism of how mitochondrial membrane proteins regulate leaf and grain development remains to be further elucidated. RESULTS: Here, a mitochondria-defective mutant narrow leaf and slender grain 1 (nlg1) was identified from an EMS-treated mutant population, which exhibits narrow leaves and slender grains. Moreover, nlg1 also presents abnormal mitochondria structure and was sensitive to the inhibitors of mitochondrial electron transport chain. Map-based cloning and transgenic functional confirmation revealed that NLG1 encodes a mitochondrial import inner membrane translocase containing a subunit Tim21. GUS staining assay and RT-qPCR suggested that NLG1 was mainly expressed in leaves and panicles. The expression level of respiratory function and auxin response related genes were significantly down-regulated in nlg1, which may be responsible for the declination of ATP production and auxin content. CONCLUSIONS: These results suggested that NLG1 plays an important role in the regulation of leaf and grain size development by maintaining mitochondrial homeostasis. Our finding provides a novel insight into the effects of mitochondria development on leaf and grain morphogenesis in rice.

SEMI-ROLLED LEAF 10 stabilizes catalase isozyme B to regulate leaf morphology and thermotolerance in rice (Oryza sativa L.).

Plant architecture and stress tolerance play important roles in rice breeding. Specific leaf morphologies and ideal plant architecture can effectively improve both abiotic stress resistance and rice grain yield. However, the mechanism by which plants simultaneously regulate leaf morphogenesis and stress resistance remains elusive. Here, we report that SRL10, which encodes a double-stranded RNA-binding protein, regulates leaf morphology and thermotolerance in rice through alteration of microRNA biogenesis. The srl10 mutant had a semi-rolled leaf phenotype and elevated sensitivity to high temperature. SRL10 directly interacted with catalase isozyme B (CATB), and the two proteins mutually increased one other's stability to enhance hydrogen peroxide (H2 O2 ) scavenging, thereby contributing to thermotolerance. The natural Hap3 (AGC) type of SRL10 allele was found to be present in the majority of aus rice accessions, and was identified as a thermotolerant allele under high temperature stress in both the field and the growth chamber. Moreover, the seed-setting rate was 3.19 times higher and grain yield per plant was 1.68 times higher in near-isogenic line (NIL) carrying Hap3 allele compared to plants carrying Hap1 allele under heat stress. Collectively, these results reveal a new locus of interest and define a novel SRL10-CATB based regulatory mechanism for developing cultivars with high temperature tolerance and stable yield. Furthermore, our findings provide a theoretical basis for simultaneous breeding for plant architecture and stress resistance.

Control of Grain Size and Weight by the GSK2-LARGE1/OML4 Pathway in Rice.

Regulation of grain size is crucial for improving crop yield and is also a basic aspect in developmental biology. However, the genetic and molecular mechanisms underlying grain size control in crops remain largely unknown despite their central importance. Here, we report that the MEI2-LIKE PROTEIN4 (OML4) encoded by the LARGE1 gene is phosphorylated by GLYCOGEN SYNTHASE KINASE2 (GSK2) and negatively controls grain size and weight in rice (Oryza sativa). Loss of function of OML4 leads to large and heavy grains, while overexpression of OML4 causes small and light grains. OML4 regulates grain size by restricting cell expansion in the spikelet hull. OML4 is expressed in developing panicles and grains, and the GFP-OML4 fusion protein is localized in the nuclei. Biochemical analyses show that the GSK2 physically interacts with OML4 and phosphorylates it, thereby possibly influencing the stability of OML4. Genetic analyses support that GSK2 and OML4 act, at least in part, in a common pathway to control grain size in rice. These results reveal the genetic and molecular mechanism of a GSK2-OML4 regulatory module in grain size control, suggesting that this pathway is a suitable target for improving seed size and weight in crops.

UDP-N-acetylglucosamine pyrophosphorylase enhances rice survival at high temperature.

High-temperature stress inhibits normal cellular processes and results in abnormal growth and development in plants. However, the mechanisms by which rice (Oryza sativa) copes with high temperature are not yet fully understood. In this study, we identified a rice high temperature enhanced lesion spots 1 (hes1) mutant, which displayed larger and more dense necrotic spots under high temperature conditions. HES1 encoded a UDP-N-acetylglucosamine pyrophosphorylase, which had UGPase enzymatic activity. RNA sequencing analysis showed that photosystem-related genes were differentially expressed in the hes1 mutant at different temperatures, indicating that HES1 plays essential roles in maintaining chloroplast function. HES1 expression was induced under high temperature conditions. Furthermore, loss-of-function of HES1 affected heat shock factor expression and its mutation exhibited greater vulnerability to high temperature. Several experiments revealed that higher accumulation of reactive oxygen species occurred in the hes1 mutant at high temperature. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and comet experiments indicated that the hes1 underwent more severe DNA damage at high temperature. The determination of chlorophyll content and chloroplast ultrastructure showed that more severe photosystem defects occurred in the hes1 mutant under high temperature conditions. This study reveals that HES1 plays a key role in adaptation to high-temperature stress in rice.

The kinesin-13 protein BR HYPERSENSITIVE 1 is a negative brassinosteroid signaling component regulating rice growth and development.

Phytohormones performed critical roles in regulating plant architecture and thus determine grain yield in rice. However, the roles of brassinosteroids (BRs) compared to other phytohormones in shaping rice architecture are less studied. In this study, we report that BR hypersensitive1 (BHS1) plays a negative role in BR signaling and regulate rice architecture. BHS1 encodes the kinesin-13a protein and regulates grain length. We found that bhs1 was hypersensitive to BR, while BHS1-overexpression was less sensitive to BR compare to WT. BHS1 was down-regulated at RNA and protein level upon exogenous BR treatment, and proteasome inhibitor MG132 delayed the BHS1 degradation, indicating that both the transcriptional and posttranscriptional regulation machineries are involved in BHS1-mediated regulation of plant growth and development. Furthermore, we found that the BR-induced degradation of BHS1 was attenuated in Osbri1 and Osbak1 mutants, but not in Osbzr1 and Oslic mutants. Together, these results suggest that BHS1 is a novel component which is involved in negative regulation of the BR signaling downstream player of BRI1.

Molecular Events of Rice AP2/ERF Transcription Factors.

APETALA2/ethylene response factor (AP2/ERF) is widely found in the plant kingdom and plays crucial roles in transcriptional regulation and defense response of plant growth and development. Based on the research progress related to AP2/ERF genes, this paper focuses on the classification and structural features of AP2/ERF transcription factors, reviews the roles of rice AP2/ERF genes in the regulation of growth, development and stress responses, and discusses rice breeding potential and challenges. Taken together; studies of rice AP2/ERF genes may help to elucidate and enrich the multiple molecular mechanisms of how AP2/ERF genes regulate spikelet determinacy and floral organ development, flowering time, grain size and quality, embryogenesis, root development, hormone balance, nutrient use efficiency, and biotic and abiotic response processes. This will contribute to breeding excellent rice varieties with high yield and high resistance in a green, organic manner.

Genome-Wide Association Study Reveals Novel QTLs and Candidate Genes for Grain Number in Rice.

Grain number per panicle (GNPP), determined mainly by panicle branching, is vital for rice yield. The dissection of the genetic basis underlying GNPP could help to improve rice yield. However, genetic resources, including quantitative trait loci (QTL) or genes for breeders to enhance rice GNPP, are still limited. Here, we conducted the genome-wide association study (GWAS) on the GNPP, primary branch number (PBN), and secondary branch number (SBN) of 468 rice accessions. We detected a total of 18 QTLs, including six for GNPP, six for PBN, and six for SBN, in the whole panel and the indica and japonica subpanels of 468 accessions. More importantly, qPSG1 was a common QTL for GNPP, PBN, and SBN and was demonstrated by chromosome segment substitution lines (CSSLs). Considering gene annotation, expression, and haplotype analysis, seven novel and strong GNPP-related candidate genes were mined from qPSG1. Our results provide clues to elucidate the molecular regulatory network of GNPP. The identified QTLs and candidate genes will contribute to the improvement of GNPP and rice yield via molecular marker-assisted selection (MAS) breeding and genetic engineering techniques.

LEAF TIP RUMPLED 1 Regulates Leaf Morphology and Salt Tolerance in Rice.

Leaf morphology is one of the important traits related to ideal plant architecture and is an important factor determining rice stress resistance, which directly affects yield. Wax layers form a barrier to protect plants from different environmental stresses. However, the regulatory effect of wax synthesis genes on leaf morphology and salt tolerance is not well-understood. In this study, we identified a rice mutant, leaf tip rumpled 1 (ltr1), in a mutant library of the classic japonica variety Nipponbare. Phenotypic investigation of NPB and ltr1 suggested that ltr1 showed rumpled leaf with uneven distribution of bulliform cells and sclerenchyma cells, and disordered vascular bundles. A decrease in seed-setting rate in ltr1 led to decreased per-plant grain yield. Moreover, ltr1 was sensitive to salt stress, and LTR1 was strongly induced by salt stress. Map-based cloning of LTR1 showed that there was a 2-bp deletion in the eighth exon of LOC_Os02g40784 in ltr1, resulting in a frameshift mutation and early termination of transcription. Subsequently, the candidate gene was confirmed using complementation, overexpression, and knockout analysis of LOC_Os02g40784. Functional analysis of LTR1 showed that it was a wax synthesis gene and constitutively expressed in entire tissues with higher relative expression level in leaves and panicles. Moreover, overexpression of LTR1 enhanced yield in rice and LTR1 positively regulates salt stress by affecting water and ion homeostasis. These results lay a theoretical foundation for exploring the molecular mechanism of leaf morphogenesis and stress response, providing a new potential strategy for stress-tolerance breeding.

Anchoring Black Phosphorus Quantum Dots on Fe-Doped W18 O49 Nanowires for Efficient Photocatalytic Nitrogen Fixation.

Herein, we demonstrate that the surface anchoring of black phosphorus quantum dots (BPQDs) and bulk iron-doping in W18 O49 nanowires significantly promotes the photocatalytic activity toward N2 fixation into NH3 . More specifically, a NH3 production rate of up to 187.6 µmol g-1 h-1 could be achieved, nearly one order of magnitude higher than that of pristine W18 O49 (18.9 µmol g-1 h-1 ). Comprehensive experiments and density-functional theory calculations reveal that Fe-doping could enhance the reducing ability of photo-generated electrons by decreasing the work function and elevating the defect band (d-band) centers. Additionally, the surface BPQDs anchoring could facilitate the N2 adsorption/activation owing to the increased adsorption energy and advantaged W-P dimer bonding-mode. Therefore, synergizing the surface BPQD anchoring and bulk Fe-doping remarkably enhanced the photocatalytic activity of W18 O49 nanowires for NH3 production.

Primary leaf-type ferredoxin 1 participates in photosynthetic electron transport and carbon assimilation in rice.

Ferredoxins (Fds) play a crucial role in photosynthesis by regulating the distribution of electrons to downstream enzymes. Multiple Fd genes have been annotated in the Oryza sativa L. (rice) genome; however, their specific functions are not well understood. Here, we report the functional characterization of rice Fd1. Sequence alignment, phylogenetic analysis of seven rice Fd proteins and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that rice Fd1 is a primary leaf-type Fd. Electron transfer assays involving NADP+ and cytochrome c indicated that Fd1 can donate electrons from photosystem I (PSI) to ferredoxin-NADP+ reductase. Loss-of-function fd1 mutants showed chlorosis and seedling lethality at the three-leaf stage. The deficiency of Fd1 impaired photosynthetic electron transport, which affected carbon assimilation. Exogenous glucose treatment partially restored the mutant phenotype, suggesting that Fd1 plays an important role in photosynthetic electron transport in rice. In addition, the transcript levels of Fd-dependent genes were affected in fd1 mutants, and the trend was similar to that observed in fdc2 plants. Together, these results suggest that OsFd1 is the primary Fd in photosynthetic electron transport and carbon assimilation in rice.

iTRAQ-based proteomic analysis provides insights into the molecular mechanisms of rice formyl tetrahydrofolate deformylase in salt response.

MAIN CONCLUSION: A new molecular mechanism of tetrahydrofolate deformylase involved in the salt response presumably affects mitochondrial and chloroplast function by regulating energy metabolism and accumulation of reactive oxygen species. High salinity severely restrains plant growth and development, consequently leading to a reduction in grain yield. It is therefore critical to identify the components involved in plant salt resistance. In our previous study, we identified a rice leaf early-senescence mutant hpa1, which encodes a formyl tetrahydrofolate deformylase (Xiong et al. in Sci China Life Sci 64(5):720-738, 2021). Here, we report that HPA1 also plays a role in the salt response. To explore the molecular mechanism of HPA1 in salt resistance, we attempted to identify the differentially expressed proteins between wild type and hpa1 mutant for salinity treatment using an iTRAQ-based comparative protein quantification approach. A total of 4598 proteins were identified, of which 279 were significantly altered, including 177 up- and 102 down-regulated proteins. A functional analysis suggested that the 279 differentially expressed proteins are involved mainly in the regulation of oxidative phosphorylation, phenylpropanoid biosynthesis, photosynthesis, posttranslational modifications, protein turnover and energy metabolism. Moreover, a deficiency in HPA1 impaired chlorophyll metabolism and photosynthesis in chloroplasts and affected the electron flow of the electron transport chain in mitochondria. These changes led to abnormal energy metabolism and accumulation of reactive oxygen species, which may affect the permeability and integrity of cell membranes, leading to cell death. In addition, the results were verified by transcriptional or physiological experiments. Our results provide an insight into a new molecular mechanism of the tetrahydrofolate cycle protein formyl tetrahydrofolate deformylase, which is involved in the salt response, presumably by affecting mitochondrial and chloroplast function regulating energy metabolism and accumulation of reactive oxygen species under salt stress.

PHOTO-SENSITIVE LEAF ROLLING 1 encodes a polygalacturonase that modifies cell wall structure and drought tolerance in rice.

The biosynthesis and modification of cell wall composition and structure are controlled by hundreds of enzymes and have a direct consequence on plant growth and development. However, the majority of these enzymes has not been functionally characterised. Rice mutants with leaf-rolling phenotypes were screened in a field. Phenotypic analysis under controlled conditions was performed for the selected mutant and the relevant gene was identified by map-based cloning. Cell wall composition was analysed by glycome profiling assay. We identified a photo-sensitive leaf rolling 1 (psl1) mutant with 'napping' (midday depression of photosynthesis) phenotype and reduced growth. The PSL1 gene encodes a cell wall-localised polygalacturonase (PG), a pectin-degrading enzyme. psl1 with a 260-bp deletion in its gene displayed leaf rolling in response to high light intensity and/or low humidity. Biochemical assays revealed PG activity of recombinant PSL1 protein. Significant modifications to cell wall composition in the psl1 mutant compared with the wild-type plants were identified. Such modifications enhanced drought tolerance of the mutant plants by reducing water loss under osmotic stress and drought conditions. Taken together, PSL1 functions as a PG that modifies cell wall biosynthesis, plant development and drought tolerance in rice.

A ß-ketoacyl carrier protein reductase confers heat tolerance via the regulation of fatty acid biosynthesis and stress signaling in rice.

Heat stress is a major environmental threat affecting crop growth and productivity. However, the molecular mechanisms associated with plant responses to heat stress are poorly understood. Here, we identified a heat stress-sensitive mutant, hts1, in rice. HTS1 encodes a thylakoid membrane-localized ß-ketoacyl carrier protein reductase (KAR) involved in de novo fatty acid biosynthesis. Phylogenetic and bioinformatic analysis showed that HTS1 probably originated from streptophyte algae and is evolutionarily conserved in land plants. Thermostable HTS1 is predominantly expressed in green tissues and strongly induced by heat stress, but is less responsive to salinity, cold and drought treatments. An amino acid substitution at A254T in HTS1 causes a significant decrease in KAR enzymatic activity and, consequently, impairs fatty acid synthesis and lipid metabolism in the hts1 mutant, especially under heat stress. Compared to the wild-type, the hts1 mutant exhibited heat-induced higher H2 O2 accumulation, a larger Ca2+ influx to mesophyll cells, and more damage to membranes and chloroplasts. Also, disrupted heat stress signaling in the hts1 mutant depresses the transcriptional activation of HsfA2s and the downstream target genes. We suggest that HTS1 is critical for underpinning membrane stability, chloroplast integrity and stress signaling for heat tolerance in rice.

The rice LRR-like1 protein YELLOW AND PREMATURE DWARF 1 is involved in leaf senescence induced by high light.

Senescence in plants is induced by endogenous physiological changes and exogenous stresses. In this study, we isolated two alleles of a novel rice (Oryza sativa) mutant, yellow and premature dwarf 1 (ypd1). The ypd1 mutants exhibited a yellow and dwarf phenotype from germination, and premature senescence starting at tillering. Moreover, the ypd1 mutants were sensitive to high light, which accelerated cell death and senescence. Consistent with their yellow phenotype, the ypd1 mutants had abnormal chloroplasts and lower levels of photosynthetic pigments. TUNEL assays together with histochemical staining demonstrated that ypd1 mutants showed cell death and that they accumulated reactive oxygen species. The ypd1 mutants also showed increased expression of genes associated with senescence. Map-based cloning revealed a G→A substitution in exon 6 (ypd1-1) and exon 13 (ypd1-2) of LOC_Os06g13050 that affected splicing and caused premature termination of the encoded protein. YPD1 was found to be preferentially expressed in the leaf and it encodes a LRR-like1 protein. Complementation, overexpression, and targeted deletion confirmed that the mutations in YPD1 caused the ypd1 phenotype. YPD1 was localized on the chloroplast membrane. Our results thus demonstrate that the novel rice LRR-like1 protein YPD1 affects chloroplast development and leaf senescence.

A Nck-associated protein 1-like protein affects drought sensitivity by its involvement in leaf epidermal development and stomatal closure in rice.

Water deficit is a major environmental threat affecting crop yields worldwide. In this study, a drought stress-sensitive mutant drought sensitive 8 (ds8) was identified in rice (Oryza sativa L.). The DS8 gene was cloned using a map-based approach. Further analysis revealed that DS8 encoded a Nck-associated protein 1 (NAP1)-like protein, a component of the SCAR/WAVE complex, which played a vital role in actin filament nucleation activity. The mutant exhibited changes in leaf cuticle development. Functional analysis revealed that the mutation of DS8 increased stomatal density and impaired stomatal closure activity. The distorted actin filaments in the mutant led to a defect in abscisic acid (ABA)-mediated stomatal closure and increased ABA accumulation. All these resulted in excessive water loss in ds8 leaves. Notably, antisense transgenic lines also exhibited increased drought sensitivity, along with impaired stomatal closure and elevated ABA levels. These findings suggest that DS8 affects drought sensitivity by influencing actin filament activity.

Using Heading date 1 preponderant alleles from indica cultivars to breed high-yield, high-quality japonica rice varieties for cultivation in south China.

Heading date 1 (Hd1) is an important gene for the regulation of flowering in rice, but its variation in major cultivated rice varieties, and the effect of this variation on yield and quality, remains unknown. In this study, we selected 123 major rice varieties cultivated in China from 1936 to 2009 to analyse the relationship between the Hd1 alleles and yield-related traits. Among these varieties, 19 haplotypes were detected in Hd1, including two major haplotypes (H8 and H13) in the japonica group and three major haplotypes (H14, H15 and H16) in the indica group. Analysis of allele frequencies showed that the secondary branch number was the major aimed for Chinese indica breeding. In the five major haplotypes, SNP316 (C-T) was the only difference between the two major japonica haplotypes, and SNP495 (C-G) and SNP614 (G-A) are the major SNPs in the three indica haplotypes. Association analysis showed that H16 is the most preponderant allele in modern cultivated Chinese indica varieties. Backcrossing this allele into the japonica variety Chunjiang06 improved yield without decreasing grain quality. Therefore, our analysis offers a new strategy for utilizing these preponderant alleles to improve yield and quality of japonica varieties for cultivation in the southern areas of China.