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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., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

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., (© 2024 Institute of Botany, Chinese Academy of Sciences.)

Grain size is an important determinant of grain yield in rice. Although dozens of grain size genes have been reported, the molecular mechanisms that control grain size remain to be fully clarified. Here, we report the cloning and characterization of GR5 (GRAIN ROUND 5), which is allelic to SMOS1/SHB/RLA1/NGR5 and encodes an AP2 transcription factor. GR5 acts as a transcriptional activator and determines grain size by influencing cell proliferation and expansion. We demonstrated that GR5 physically interacts with five Gγ subunit proteins (RGG1, RGG2, DEP1, GS3, and GGC2) and acts downstream of the G protein complex. Four downstream target genes of GR5 in grain development (DEP2, DEP3, DRW1, and CyCD5;2) were revealed and their core T/CGCAC motif identified by yeast one-hybrid, EMSA, and ChIP-PCR experiments. Our results revealed that GR5 interacts with Gγ subunits and cooperatively determines grain size by regulating the expression of downstream target genes. These findings provide new insight into the genetic regulatory network of the G protein signaling pathway in the control of grain size and provide a potential target for high-yield rice breeding., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The elimination of seed shattering was a key step in rice (Oryza sativa) domestication. In this paper, we show that increasing the gibberellic acid (GA) content or response in the abscission region enhanced seed shattering in rice. We demonstrate that SLENDER RICE1 (SLR1), the key repressor of GA signaling, could physically interact with the rice seed shattering-related transcription factors quantitative trait locus of seed shattering on chromosome 1 (qSH1), O. sativa HOMEOBOX 15 (OSH15), and SUPERNUMERARY BRACT (SNB). Importantly, these physical interactions interfered with the direct binding of these three regulators to the lignin biosynthesis gene 4-COUMARATE: COENZYME A LIGASE 3 (4CL3), thereby derepressing its expression. Derepression of 4CL3 led to increased lignin deposition in the abscission region, causing reduced rice seed shattering. Importantly, we also show that modulating GA content could alter the degree of seed shattering to increase harvest efficiency. Our results reveal that the "Green Revolution" phytohormone GA is important for regulating rice seed shattering, and we provide an applicable breeding strategy for high-efficiency rice harvesting., Competing Interests: Conflict of interest statement. The authors declare no competing interests., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Drought stress is a major environmental factor that limits the growth, development, and yield of rice (Oryza sativa L.). Histone deacetylases (HDACs) are involved in the regulation of drought stress responses. HDA704 is an RPD3/HDA1 class HDAC that mediates the deacetylation of H4K8 (lysine 8 of histone H4) for drought tolerance in rice. In this study, we show that plants overexpressing HDA704 (HDA704-OE) are resistant to drought stress and sensitive to abscisic acid (ABA), whereas HDA704 knockout mutant (hda704) plants displayed decreased drought tolerance and ABA sensitivity. Transcriptome analysis revealed that HDA704 regulates the expression of ABA-related genes in response to drought stress. Moreover, HDA704 was recruited by a drought-resistant transcription factor, WAX SYNTHESIS REGULATORY 2 (OsWR2), and co-regulated the expression of the ABA biosynthesis genes NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3), NCED4, and NCED5 under drought stress. HDA704 also repressed the expression of ABA-INSENSITIVE 5 (OsABI5) and DWARF AND SMALL SEED 1 (OsDSS1) by regulating H4K8ac levels in the promoter regions in response to polyethylene glycol 6000 treatment. In agreement, the loss of OsABI5 function increased resistance to dehydration stress in rice. Our results demonstrate that HDA704 is a positive regulator of the drought stress response and offers avenues for improving drought resistance in rice., (© 2023 Institute of Botany, Chinese Academy of Sciences.)

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 (H 2 O 2 ) 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., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

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.

Lesion mutants can be valuable tools to reveal the interactions between genetic factors and environmental signals and to improve grain production. Here we identified a rice (Oryza sativa) mutant, lesion spotleaf1 (lsl1), which produces necrotic leaf lesions throughout its life cycle. LSL1 encodes a protein of unknown function and belongs to a grass-specific clade. The lesion phenotype of the lsl1 mutant was sharply induced by shading, and its detached leaves incubated in 6-benzylamino purine similarly formed lesions in the dark. In addition, the lsl1 mutant exhibited reactive oxygen species accumulation and cell death. The terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) and comet assays revealed that the lsl1 mutant contained severe DNA damage, resulting in reduced grain yield and quality. RNA sequencing, gene expression, and protein activity analyses indicate that LSL1 is required for chloroplast function. Furthermore, LSL1 interacts with PsaD and PAP10 to form a regulatory module that functions in chlorophyll synthesis and chloroplast development to maintain redox balance. Our results reveal that LSL1 maintains chloroplast structure, redox homeostasis, and DNA stability, and plays important roles in the interaction between genetic factors and environmental signals and in regulating grain size and quality., (© 2022. Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

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.

Panicle development is an important determinant of the grain number in rice. A thorough characterization of the molecular mechanism underlying panicle development will lead to improved breeding of high-yielding rice varieties. Frizzy Panicle (FZP), a critical gene for panicle development, is regulated by OsBZR1 and OsARFs at the transcriptional stage. However, the translational modulation of FZP has not been reported. We reveal that the CU-rich elements (CUREs) in the 3' UTR of the FZP mRNA are crucial for efficient FZP translation. The knockout of CUREs in the FZP 3' UTR or the over-expression of the FZP 3' UTR fragment containing CUREs resulted in an increase in FZP mRNA translation efficiency. Moreover, the number of secondary branches (NSB) and the grain number per panicle (GNP) decreased in the transformed rice plants. The CUREs in the 3' UTR of FZP mRNA were verified as the targets of the polypyrimidine tract-binding proteins OsPTB1 and OsPTB2 in rice. Both OsPTB1 and OsPTB2 were highly expressed in young panicles. The knockout of OsPTB1/2 resulted in an increase in the FZP translational efficiency and a decrease in the NSB and GNP. Furthermore, the over-expression of OsPTB1/2 decreased the translation of the reporter gene fused to FZP 3' UTR in vivo and in vitro. These results suggest that OsPTB1/2 can mediate FZP translational repression by interacting with CUREs in the 3' UTR of FZP mRNA, leading to changes in the NSB and GNP. Accordingly, in addition to transcriptional regulation, FZP expression is also fine-tuned at the translational stage during rice panicle development., (© 2022 Society for Experimental Biology and John Wiley & Sons Ltd.)

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., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

The riboflavin derivatives flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential cofactors for enzymes in multiple cellular processes. Characterizing mutants with impaired riboflavin metabolism can help clarify the role of riboflavin in plant development. Here, we characterized a rice (Oryza sativa) white and lesion-mimic (wll1) mutant, which displays a lesion-mimic phenotype with white leaves, chlorophyll loss, chloroplast defects, excess reactive oxygen species (ROS) accumulation, decreased photosystem protein levels, changes in expression of chloroplast development and photosynthesis genes, and cell death. Map-based cloning and complementation test revealed that WLL1 encodes lumazine synthase, which participates in riboflavin biosynthesis. Indeed, the wll1 mutant showed riboflavin deficiency, and application of FAD rescued the wll1 phenotype. In addition, transcriptome analysis showed that cytokinin metabolism was significantly affected in wll1 mutant, which had increased cytokinin and δ-aminolevulinic acid contents. Furthermore, WLL1 and riboflavin synthase (RS) formed a complex, and the rs mutant had a similar phenotype to the wll1 mutant. Taken together, our findings revealed that WLL1 and RS play pivotal roles in riboflavin biosynthesis, which is necessary for ROS balance and chloroplast development in rice., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Crown roots are essential for plants to obtain water and nutrients, perceive environmental changes, and synthesize plant hormones. In this study, we identified and characterized short crown root 8 ( scr8 ), which exhibited a defective phenotype of crown root and vegetative development. Temperature treatment showed that scr8 was sensitive to temperature and that the mutant phenotypes were rescued when grown under low temperature condition (20 °C). Histological and EdU staining analysis showed that the crown root formation was hampered and that the root meristem activity was decreased in scr8 . With map-based cloning strategy, the SCR8 gene was fine-mapped to an interval of 126.4 kb on chromosome 8. Sequencing analysis revealed that the sequence variations were only found in LOC_Os08g14850 , which encodes a CC-NBS-LRR protein. Expression and inoculation test analysis showed that the expression level of LOC_Os08g14850 was significantly decreased under low temperature (20 °C) and that the resistance to Xanthomonas oryzae pv. Oryzae ( Xoo ) was enhanced in scr8 . These results indicated that LOC_Os08g14850 may be the candidate of SCR8 and that its mutation activated the plant defense response, resulting in a crown root growth defect.

The genetic improvement of nitrogen use efficiency (NUE) of crops is vital for grain productivity and sustainable agriculture. However, the regulatory mechanism of NUE remains largely elusive. Here, we report that the rice Grain number, plant height, and heading date7 (Ghd7) gene genetically acts upstream of ABC1 REPRESSOR1 (ARE1), a negative regulator of NUE, to positively regulate nitrogen utilization. As a transcriptional repressor, Ghd7 directly binds to two Evening Element-like motifs in the promoter and intron 1 of ARE1, likely in a cooperative manner, to repress its expression. Ghd7 and ARE1 display diurnal expression patterns in an inverse oscillation manner, mirroring a regulatory scheme based on these two loci. Analysis of a panel of 2656 rice varieties suggests that the elite alleles of Ghd7 and ARE1 have undergone diversifying selection during breeding. Moreover, the allelic distribution of Ghd7 and ARE1 is associated with the soil nitrogen deposition rate in East Asia and South Asia. Remarkably, the combination of the Ghd7 and ARE1 elite alleles substantially improves NUE and yield performance under nitrogen-limiting conditions. Collectively, these results define a Ghd7-ARE1-based regulatory mechanism of nitrogen utilization, providing useful targets for genetic improvement of rice NUE., (Copyright © 2021 The Author. Published by Elsevier Inc. All rights reserved.)

Chloroplasts are closely associated with the growth and development of higher plants. Accumulating evidence has revealed that the multiple organellar RNA editing factors (MORF) family of proteins influences plastidic and mitochondrial development through post-transcriptional regulation. However, the role of MORFs in regulating the development of chloroplasts in rice is still unclear. The OsMORF9 gene belongs to a small family of 7 genes in rice and is highly expressed in young leaves. We used the CRISPR/Cas9 system to mutate OsMORF9. The resulting knockout lines osmorf9-1 and osmorf9-2 exhibited an albino seedling lethal phenotype. Besides, the expression of many plastid-encoded genes involved in photosynthesis, the biogenesis of plastidic ribosomes and the editing and splicing of specific plastidic RNA molecules were severely affected in these two OsMORF9 mutants. Furthermore, yeast two-hybrid analysis revealed that OsMORF9 could interact with OsSLA4 and DUA1 which are members of the pentatricopeptide repeat (PPR) family of proteins. Analysis of subcellular localization of OsMORF9 also suggested that it might function in chloroplasts. The findings from the present study demonstrated the critical role of OsMORF9 in the biogenesis of chloroplast ribosomes, chloroplast development and seedling survival. This therefore provides new insights on the function of MORF proteins in rice., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Lesion-mimic mutants (LMMs) provide a valuable tool to reveal the molecular mechanisms determining programmed cell death (PCD) in plants. Despite intensive research, the mechanisms behind PCD and the formation of lesions in various LMMs still remain to be elucidated. Here, we identified a rice (Oryza sativa) LMM, early lesion leaf 1 (ell1), cloned the causal gene by map-based cloning, and verified this by complementation. ELL1 encodes a cytochrome P450 monooxygenase, and the ELL1 protein was located in the endoplasmic reticulum. The ell1 mutant exhibited decreased chlorophyll contents, serious chloroplast degradation, upregulated expression of chloroplast degradation-related genes, and attenuated photosynthetic protein activity, indicating that ELL1 is involved in chloroplast development. RNA sequencing analysis showed that genes related to oxygen binding were differentially expressed in ell1 and wild-type plants; histochemistry and paraffin sectioning results indicated that hydrogen peroxide (H 2 O 2 ) and callose accumulated in the ell1 leaves, and the cell structure around the lesions was severely damaged, which indicated that reactive oxygen species (ROS) accumulated and cell death occurred in the mutant. TUNEL staining and comet experiments revealed that severe DNA degradation and abnormal PCD occurred in the ell1 mutants, which implied that excessive ROS accumulation may induce DNA damage and ROS-mediated cell death in the mutant. Additionally, lesion initiation in the ell1 mutant was light dependent and temperature sensitive. Our findings revealed that ELL1 affects chloroplast development or function, and that loss of ELL1 function induces ROS accumulation and lesion formation in rice., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

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., (© 2020 The Authors New Phytologist © 2020 New Phytologist Trust.)

Leaves are the primary structures responsible for photosynthesis, making leaf morphology one of the most important traits of rice plant architecture. Both plant architecture and nutrient utilization jointly affect rice yield, however, their molecular association is still poorly understood. We identified a rice mutant, leaf width 5 (lw5), that displayed small grains and wide leaves and possesses characteristics typical of a small "sink" and a large "source". Map-based cloning and CRISPR-Cas9 gene editing indicated that LW5 affects both the plant architecture and yield. It is an allele of D1, encoding the rice G protein α subunit. The loss of LW5 functioning leads to an increase in the rate of photosynthesis, vascular bundles, and chlorophyll content. However, the grain-straw ratio and the rate of grain filling decreased significantly. The detection results of 15 N-ammonium nitrate and an expression analysis of genes associated with nitrogen demonstrated that LW5 serves an important role in nitrate uptake and transport. LW5 affects plant architecture and grain size by regulating nitrogen transfer. These results provide a theoretical foundation for further research surrounding the molecular mechanism of "source-sink" balance in rice and suggest novel methods of molecular design for the cultivation of breeding super rice in ideal plant types., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

The juvenile-to-adult transition in plants involves changes in vegetative growth and plant architecture; the timing of this transition has important implications for agriculture. The microRNA miR156 regulates this transition and shoot maturation in plants. In Arabidopsis thaliana, deposition of histone H3 trimethylation on lysine 27 (H3K27me3, a repressive mark) at the MIR156A/C loci is regulated by Polycomb Repressive Complex 1 (PRC1) or PRC2, depending on the developmental stage. The levels of miR156 progressively decline during shoot maturation. The amount of H3K27me3 at MIR156A/C loci affects miR156 levels; however, whether this epigenetic regulation is conserved remains unclear. Here, we found that in rice (Oryza sativa), the putative PRC1 subunit LIKE HETEROCHROMATIN PROTEIN 1 (OsLHP1), with the miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) module, affects developmental phase transitions. Loss of OsLHP1 function results in ectopic expression of MIR156B/C/I/E, phenocopy of miR156 overexpression, and reduced H3k27me3 levels at MIR156B/C/I/E. This indicates that OsLHP1 has functionally diverged from Arabidopsis LHP1. Genetic and transcriptome analyses of wild-type, miR156b/c-overexpression, and Oslhp1-2 mutant plants suggest that OsLHP1 acts upstream of miR156 and SPL during the juvenile-to-adult transition. Therefore, modifying the OsLHP1-miR156-SPL pathway may enable alteration of the vegetative period and plant architecture., (© 2020 Institute of Botany, Chinese Academy of Sciences.)

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., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Seedling emergence in monocots depends mainly on mesocotyl elongation, requiring coordination between developmental signals and environmental stimuli. Strigolactones (SLs) and karrikins are butenolide compounds that regulate various developmental processes; both are able to negatively regulate rice ( Oryza sativa ) mesocotyl elongation in the dark. Here, we report that a karrikin signaling complex, DWARF14-LIKE (D14L)-DWARF3 (D3)- O. sativa SUPPRESSOR OF MAX2 1 (OsSMAX1) mediates the regulation of rice mesocotyl elongation in the dark. We demonstrate that D14L recognizes the karrikin signal and recruits the SCF D3 ubiquitin ligase for the ubiquitination and degradation of OsSMAX1, mirroring the SL-induced and D14- and D3-dependent ubiquitination and degradation of D53. Overexpression of OsSMAX1 promoted mesocotyl elongation in the dark, whereas knockout of OsSMAX1 suppressed the elongated-mesocotyl phenotypes of d14l and d3 OsSMAX1 localizes to the nucleus and interacts with TOPLESS-RELATED PROTEINs, regulating downstream gene expression. Moreover, we showed that the GR24 enantiomers GR24 5DS and GR24 ent- 5DS specifically inhibit mesocotyl elongation and regulate downstream gene expression in a D14- and D14L-dependent manner, respectively. Our work revealed that karrikin and SL signaling play parallel and additive roles in modulating downstream gene expression and negatively regulating mesocotyl elongation in the dark., (© 2020 American Society of Plant Biologists. All rights reserved.)

Rice ( Oryza sativa ) spikelets have a unique inflorescence structure, and the mechanisms regulating their development are not yet fully understood. Moreover, approaches to manipulate spikelet development have the potential to increase grain yield. In this study, we identified and characterized a recessive spikelet mutant, namely more floret1 ( mof1 ). The mof1 mutant has a delayed transition from the spikelet to the floral meristem, inducing the formation of extra lemma-like and palea-like organs. In addition, the main body of the palea was reduced, and the sterile lemma was enlarged and partially acquired hull (lemma and/or palea) identity. We used map-based cloning to identify the MOF1 locus and confirmed our identification by complementation and by generating new mof1 alleles using CRISPR-Cas9 gene editing. MOF1 encodes a MYB domain protein with the typical ethylene response factor-associated amphiphilic repression motifs, is expressed in all organs and tissues, and has a strong repression effect. MOF1 localizes to the nucleus and interacts with TOPLESS-RELATED PROTEINs to possibly repress the expression of downstream target genes. Taken together, our results reveal that MOF1 plays an important role in the regulation of organ identity and spikelet determinacy in rice., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Background: Chloroplasts play an important role in plant growth and development. The chloroplast genome contains approximately twenty group II introns that are spliced due to proteins encoded by nuclear genes. CAF2 is one of these splicing factors that has been shown to splice group IIB introns in maize and Arabidopsis thaliana. However, the research of the OsCAF2 gene in rice is very little, and the effects of OsCAF2 genes on chloroplasts development are not well characterized., Results: In this study, oscaf2 mutants were obtained by editing the OsCAF2 gene in the Nipponbare variety of rice. Phenotypic analysis showed that mutations to OsCAF2 led to albino leaves at the seeding stage that eventually caused plant death, and oscaf2 mutant plants had fewer chloroplasts and damaged chloroplast structure. We speculated that OsCAF2 might participate in the splicing of group IIA and IIB introns, which differs from its orthologs in A. thaliana and maize. Through yeast two-hybrid experiments, we found that the C-terminal region of OsCAF2 interacted with OsCRS2 and formed an OsCAF2-OsCRS2 complex. In addition, the N-terminal region of OsCAF2 interacted with itself to form homodimers., Conclusion: Taken together, this study improved our understanding of the OsCAF2 protein, and revealed additional information about the molecular mechanism of OsCAF2 in regulating of chloroplast development in rice.

Understanding the genetic basis of natural variation in grain size among diverse rice varieties can help breeders develop high-yielding rice cultivars. Here, we report the discovery of qTGW2, a new semidominant quantitative trait locus for grain width and weight. The corresponding gene, TGW2, encodes CELL NUMBER REGULATOR 1 (OsCNR1) localized to the plasma membrane. A single nucleotide polymorphism (SNP) variation 1818 bp upstream of TGW2 is responsible for its different expression, leading to alteration in grain width and weight by influencing cell proliferation and expansion in glumes. TGW2 interacts with KRP1, a regulator of cell cycle in plants, to negatively regulate grain width and weight. Genetic diversity analysis of TGW2 in 141 rice accessions revealed it as a breeding target in a selective sweep region. Our findings provide new insights into the genetic mechanism underlying grain morphology and grain weight, and uncover a promising gene for improving rice yield., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.)

Background: Copy number variations (CNVs) are an important type of structural variations in the genome that usually affect gene expression levels by gene dosage effect. Understanding CNVs as part of genome evolution may provide insights into the genetic basis of important agricultural traits and contribute to the crop breeding in the future. While available methods to detect CNVs utilizing next-generation sequencing technology have helped shed light on prevalence and effects of CNVs, the complexity of crop genomes poses a major challenge and requires development of additional tools., Results: Here, we generated genomic and transcriptomic data of 93 rice (Oryza sativa L.) accessions and developed a comprehensive pipeline to call CNVs in this large-scale dataset. We analyzed the correlation between CNVs and gene expression levels and found that approximately 13% of the identified genes showed a significant correlation between their expression levels and copy numbers. Further analysis showed that about 36% of duplicate pairs were involved in pseudogenetic events while only 5% of them showed functional differentiation. Moreover, the offspring copy mainly contributed to the expression levels and seemed more likely to become a pseudogene, whereas the parent copy tended to maintain the function of ancestral gene., Conclusion: We provide a high-accuracy CNV dataset that will contribute to functional genomics studies and molecular breeding in rice. We also showed that gene dosage effect of CNVs in rice is not exponential or linear. Our work demonstrates that the evolution of duplicated genes is asymmetric in both expression levels and gene fates, shedding a new insight into the evolution of duplicated genes.

Plant architecture is a complex agronomic trait and a major factor of crop yield, which is affected by several important hormones. Strigolactones (SLs) are identified as a new class hormoneinhibiting branching in many plant species and have been shown to be involved in various developmental processes. Genetical and chemical modulation of the SL pathway is recognized as a promising approach to modify plant architecture. However, whether and how the genes involved in the SL pathway could be utilized in breeding still remain elusive. Here, we demonstrate that a partial loss-of-function allele of the SL biosynthesis gene, HIGH TILLERING AND DWARF 1/DWARF17 (HTD1/D17), which encodes CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), increases tiller number and improves grain yield in rice. We found that the HTD1 gene had been widely utilized and co-selected with Semidwarf 1 (SD1), both contributing to the improvement of plant architecture in modern rice varieties since the Green Revolution in the 1960s. Understanding how phytohormone pathway genes regulate plant architecture and how they have been utilized and selected in breeding will lay the foundation for developing the rational approaches toward improving crop yield., (Copyright © 2020 The Author. Published by Elsevier Inc. All rights reserved.)

The chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins regulate the expression of chloroplast or mitochondrial genes that influence plant growth and development. Although 14 CRM domain proteins have previously been identified in rice, there are few studies of these gene expression patterns in various tissues and under abiotic stress. In our study, we found that 14 CRM domain-containing proteins have a conservative motif1. Under salt stress, the expression levels of 14 CRM genes were downregulated. However, under drought and cold stress, the expression level of some CRM genes was increased. The analysis of gene expression patterns showed that 14 CRM genes were expressed in all tissues but especially highly expressed in leaves. In addition, we analyzed the functions of OsCFM2 and found that this protein influences chloroplast development by regulating the splicing of a group I and five group II introns. Our study provides information for the function analysis of CRM domain-containing proteins in rice., Competing Interests: The authors declare no conflict of interest.

The palea and lemma are floral organ structures unique to grasses; these structures form the hull and directly affect grain size and quality. However, the molecular mechanisms controlling the development of the hull are not well understood. In this study, we characterized the rice (Oryza sativa) abnormal flower and grain1 (afg1) mutant, a new allele of OsMADS6. Similar to previously characterized osmads6 alleles, in the afg1 floret, the palea lost its marginal region and acquired the lemma identity. However, in contrast to other osmads6 alleles, the afg1 mutant showed altered grain size and grain quality, with decreased total starch and amylose contents, and increased protein and soluble sugar contents. The analysis of transcriptional activity suggested that AFG1 is a transcriptional activator and may affect grain size by regulating the expression levels of several genes related to cell expansion and proliferation in the afg1 mutant. These results revealed that AFG1 plays an important role in determining palea identity and affecting grain yield and quality in rice.

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, SNP 316 (C-T) was the only difference between the two major japonica haplotypes, and SNP 495 (C-G) and SNP 614 (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., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

A panel of ethane-methyl-sulfonate-mutagenized japonica rice lines was grown in the presence of salinity in order to identify genes required for the expression of salinity tolerance. A highly nontolerant selection proved to harbor a mutation in FLN2, a gene which encodes fructokinase-like protein2. Exposure of wild-type rice to salinity up-regulated FLN2, while a CRISPR/Cas9-generated FLN2 knockout line was hypersensitive to the stress. Both ribulose 1,5-bisphosphate carboxylase/oxygenase activity and the abundance of the transcript generated by a number of genes encoding components of sucrose synthesis were lower in the knockout line than in wild-type plants' leaves, while the sucrose contents of the leaf and root were, respectively, markedly increased and decreased. That sugar partitioning to the roots was impaired in FLN2 knockout plants was confirmed by the observation that several genes involved in carbon transport were down-regulated in both the leaf and in the leaf sheath. The levels of sucrose synthase, acid invertase, and neutral invertase activity were distinctly lower in the knockout plants' roots than in those of wild-type plants, particularly when the plants were exposed to salinity stress. The compromised salinity tolerance exhibited by the FLN2 knockout plants was likely a consequence of an inadequate supply of the assimilate required to support growth, a problem which was rectifiable by providing an exogenous supply of sucrose. The conclusion was that FLN2, on account of its influence over sugar metabolism, is important in the context of seedling growth and the rice plant's response to salinity stress., Competing Interests: The authors declare no conflict of interest.

Genetic erosion refers to the loss of genetic variation in a crop. In China, only a few original landraces of rice (Oryza sativa) were used in breeding and these became the primary genetic background of modern varieties. Expanding the genetic diversity among Chinese rice varieties and cultivating high-yielding and high-quality varieties with resistance to different biotic and abiotic stresses is critical. Here, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9(Cas9) genome editing system to edit Semi-Dwarf1 (SD1) in the elite landraces Kasalath and TeTePu (TTP), which contain many desired agronomic traits such as tolerance to low phosphorous and broad-spectrum resistance to several diseases and insects. Mutations of SD1 confer shorter plant height for better resistance to lodging. Field trials demonstrated that the yield of the new Kasalath and TTP mutant lines was better than that of the wild type under modern cultivation and that the lines maintained the same desirable agronomic characteristics as their wild-type progenitors. Our results showed that breeding using available landraces in combination with genomic data of different landraces and gene-editing techniques is an effective way to relieve genetic erosion in modern rice varieties.

The indica and japonica rice (Oryza sativa) subspecies differ in nitrate (NO 3 - ) assimilation capacity and nitrogen (N) use efficiency (NUE). Here, we show that a major component of this difference is conferred by allelic variation at OsNR2, a gene encoding a NADH/NADPH-dependent NO 3 - reductase (NR). Selection-driven allelic divergence has resulted in variant indica and japonica OsNR2 alleles encoding structurally distinct OsNR2 proteins, with indica OsNR2 exhibiting greater NR activity. Indica OsNR2 also promotes NO 3 - uptake via feed-forward interaction with OsNRT1.1B, a gene encoding a NO 3 - uptake transporter. These properties enable indica OsNR2 to confer increased effective tiller number, grain yield and NUE on japonica rice, effects enhanced by interaction with an additionally introgressed indica OsNRT1.1B allele. In consequence, indica OsNR2 provides an important breeding resource for the sustainable increases in japonica rice yields necessary for future global food security.

The palea and lemma (hull) are grass-specific organs, and determine grain size and quality. In the study, AH2 encodes a MYB domain protein, and functions in the development of hull and grain. Mutation of AH2 produces smaller grains and alters grain quality including decreased amylose content and gel consistency, and increased protein content. Meantime, part of the hull lost the outer silicified cells, and induces a transformation of the outer rough epidermis to inner smooth epidermis cells, and the body of the palea was reduced in the ah2 mutant. We confirmed the function of AH2 by complementation, CRISPR-Cas9, and cytological and molecular tests. Additionally, AH2, as a repressor, repress transcription of the downstream genes. Our results revealed that AH2 plays an important role in the determination of hull epidermis development, palea identity, and grain size., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

ζ-carotene desaturase (ZDS) is a key enzyme in carotenoid biosynthesis and plays an important role in plant photosynthesis. We characterized an albino leaf-color mutant obtained from ethyl methanesulfonate treatment: albino and seedling lethality 1 ( ale1 ). The material contains a chloroplast thylakoid defect where photosynthetic pigments declined and reactive oxygen species accumulated resulting in ale1 death within 3 weeks. Positional cloning and sequencing revealed that there was a single base substitution in ALE1 , which encoded a ZDS involved in carotenoid biosynthesis. RNAi and complementation tests confirmed the identity of ALE1 . Subcellular localization showed that the ALE1 protein is localized in the chloroplast. Expression analysis indicated that the genes involved in chlorophyll and carotenoid biosynthesis were downregulated. We conclude that ALE1 plays an important role in chloroplast and plant growth in rice.

Cadmium (Cd) is one of the most toxic heavy metals, and its accumulation in plants will seriously affect growth and yield. In this study, Cd-sensitive line D69 and Cd-tolerant line D28 were selected, which the Cd content of D28 was higher than D69 in both above and underground parts after Cd treatment. Using a combination of two-dimensional gel electrophoresis (2-DE) and MALDI-TOF-TOF MS/MS, the differential expression changes of phosphorylated proteins between D69 and D28 in leaves were classified and analyzed after Cd treatment. A total of 53 differentially expressed phosphoproteins were identified, which mainly involved in metabolism, signal transduction, gene expression regulation, material transport, and membrane fusion. The phosphorylated proteins of Cd-tolerant and Cd-sensitive lines were all analyzed, and found that some proteins associated with carbon metabolism, proteolytic enzymes, F-box containing transcription factors, RNA helicases, DNA replication/transcription/repair enzymes and ankyrins were detected in Cd-tolerant line D28, which might alleviate the abiotic stress caused by Cd treatment. These results will clarify the phosphorylated pathways in response and resistance to Cd stress in rice., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The multiple organellar RNA editing factors (MORF) gene family plays a key role in organelle RNA editing in flowering plants. MORF genes expressions are also affected by abiotic stress. Although seven OsMORF genes have been identified in rice, few reports have been published on their expression patterns in different tissues and under abiotic stress, and OsMORF-OsMORF interactions. In this study, we analyzed the gene structure of OsMORF family genes. The MORF family members were divided into six subgroups in different plants based on phylogenetic analysis. Seven OsMORF genes were highly expressed in leaves. Six and seven OsMORF genes expressions were affected by cold and salt stresses, respectively. OsMORF-OsMORF interaction analysis indicated that OsMORF1, OsMORF8a, and OsMORF8b could each interact with themselves to form homomers. Moreover, five OsMORF proteins were shown to be able to interact with each other, such as OsMORF8a and OsMORF8b interacting with OsMORF1 and OsMORF2b, respectively, to form heteromers. These results provide information for further study of OsMORF gene function.

ATP-citrate lyases (ACL) play critical roles in tumour cell propagation, foetal development and growth, and histone acetylation in human and animals. Here, we report a novel function of ACL in cell death-mediated pathogen defence responses in rice. Using ethyl methanesulphonate (EMS) mutagenesis and map-based cloning, we identified an Oryza sativa ACL-A2 mutant allele, termed spotted leaf 30-1 (spl30-1), in which an A-to-T transversion converts an Asn at position 343 to a Tyr (N343Y), causing a recessive mutation that led to a lesion mimic phenotype. Compared to wild-type plants, spl30-1 significantly reduces ACL enzymatic activity, accumulates high reactive oxygen species and increases degradation rate of nuclear deoxyribonucleic acids. CRISPR/Cas9-mediated insertion/deletion mutation analysis and complementation assay confirmed that the phenotype of spl30-1 resulted from the defective function of OsACL-A2 protein. We further biochemically identified that the N343Y mutation caused a significant degradation of SPL30 N343Y in a ubiquitin-26S proteasome system (UPS)-dependent manner without alteration in transcripts of OsACL-A2 in spl30-1. Transcriptome analysis identified a number of up-regulated genes associated with pathogen defence responses in recessive mutants of OsACL-A2, implying its role in innate immunity. Suppressor mutant screen suggested that OsSL, which encodes a P450 monooxygenase protein, acted as a downstream key regulator in spl30-1-mediated pathogen defence responses. Taken together, our study discovered a novel role of OsACL-A2 in negatively regulating innate immune responses in rice., (© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

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., (© 2019 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.)

High light and high temperature (HLHT) stress may become more frequent and severe as the climate changes, affecting crop growth and resulting in reduced production. However, the mechanism of the response to HLHT stress in rice is not yet fully understood. In the present study, we screened a rice mutant library using HLHT conditions and isolated an HLHT-sensitive mutant, local lesions 1 (ls1), which showed decreased pigment contents, defective stomata and chloroplasts, and a local lesions phenotype under HLHT. We characterized and cloned LS1 by map-based cloning and genetic complementation. LS1 encodes the A subunit of the RNase H2 complex (RNASEH2A). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and comet assays indicated that mutation of LS1 led to severe DNA damage under HLHT stress. Furthermore, we found excessive reactive oxygen species (ROS) accumulation in the ls1 mutant under HLHT stress. Exogenous antioxidants eased the local lesions phenotype of the ls1 mutant under HLHT. DNA damage caused by HLHT stress induces ROS accumulation, which causes the injury and apoptosis of leaf cells in the ls1 mutant. These results enhance our understanding of the regulatory mechanism in the response to HLHT stress in higher plants., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

In molecular breeding of super rice, it is essential to isolate the best quantitative trait loci (QTLs) and genes of leaf shape and explore yield potential using large germplasm collections and genetic populations. In this study, a recombinant inbred line (RIL) population was used, which was derived from a cross between the following parental lines: hybrid rice Chunyou84, that is, japonica maintainer line Chunjiang16B (CJ16); and indica restorer line Chunhui 84 (C84) with remarkable leaf morphological differences. QTLs mapping of leaf shape traits was analyzed at the heading stage under different environmental conditions in Hainan (HN) and Hangzhou (HZ). A major QTL qLL9 for leaf length was detected and its function was studied using a population derived from a single residual heterozygote (RH), which was identified in the original population. qLL9 was delimitated to a 16.17 kb region flanked by molecular markers C-1640 and C-1642, which contained three open reading frames (ORFs). We found that the candidate gene for qLL9 is allelic to DEP1 using quantitative real-time polymerase chain reaction (qRT-PCR), sequence comparison, and the clustered regularly interspaced short palindromic repeat-associated Cas9 nuclease (CRISPR/Cas9) genome editing techniques. To identify the effect of qLL9 on yield, leaf shape and grain traits were measured in near isogenic lines (NILs) NIL- qLL9 CJ16 and NIL- qLL9 C84 , as well as a chromosome segment substitution line (CSSL) CSSL- qLL9 KASA with a Kasalath introgressed segment covering qLL9 in the Wuyunjing (WYJ) 7 backgrounds. Our results showed that the flag leaf lengths of NIL- qLL9 C84 and CSSL- qLL9 KASA were significantly different from those of NIL- qLL9 CJ16 and WYJ 7, respectively. Compared with NIL- qLL9 CJ16 , the spike length, grain size, and thousand-grain weight of NIL- qLL9 C84 were significantly higher, resulting in a significant increase in yield of 15.08%. Exploring and pyramiding beneficial genes resembling qLL9 C84 for super rice breeding could increase both the source (e.g., leaf length and leaf area) and the sink (e.g., yield traits). This study provides a foundation for future investigation of the molecular mechanisms underlying the source⁻sink balance and high-yield potential of rice, benefiting high-yield molecular design breeding for global food security.

High light and high temperature (HLHT) stress induces the production of damaging reactive oxygen species (ROS) in many plants. Recently, we described a HLHT-sensitive rice ( Oryza sativa ) mutant, local lesions ( ls1 ), that exhibits local lesions under HLHT, due to DNA damage and excess ROS accumulation. Here, we determined that an HLHT treatment induced the local lesion phenotype in ls1 within 6 h. Corroborating this result, we found that transient HLHT treatment influenced the expression of many genes in the ls1 mutant, while affecting the growth and development of young leaves.

Improving a plant's level of tolerance to oxidative stress can frequently also enhance its tolerance to several other abiotic stresses. Here, a screen of a japonica type rice T-DNA insertion mutant library identified a highly oxidative stress-sensitive mutant. The line exhibited premature leaf senescence, starting at the three-leaf stage, and the symptoms were particularly severe from the five-leaf stage onwards. The leaves progressively lost chlorophyll, suffered protein degradation and were compromised with respect to their photosynthetic activity; their leaf mesophyll and bulliform cells became shrunken, and several senescence-associated genes ( SAG s), senescence-associated transcription factor genes ( SATF s) and autophagy-related genes ( ATG s) were progressively up-regulated. The product of the gene inactivated by the mutation, identified via positional cloning, was putatively a ubiquitin-conjugating enzyme. The gene was denoted here as RLS1 (reactive oxygen species-sensitive leaf senescence1). The phenotype of plants in which RLS1 was knocked down using RNA interference was comparable to that of the rls1 mutant. A comparative analysis of the knock-out line and the wild type leaves showed that the former accumulated more hydrogen peroxide and more malondialdehyde, expressed a heightened level of superoxide dismutase activity and a decreased level of catalase activity, and exhibited an altered transcriptional profile with respect to several SAG s, SATF s and ATG s, and that these effects were magnified when the plants were exposed to oxidative stress. The product of RLS1 is presumed to be a critical component of the rice oxidative stress response and is involved in ROS (reactive oxygen species)-mediated leaf senescence.

In grass, the spikelet is a unique inflorescence structure that directly determines grain yield. Despite a great deal of research, the molecular mechanisms behind spikelet development are not fully understood. In the study, FZP encodes an ERF domain protein, and functions in grain size and sterile lemma identity. Mutation of FZP causes smaller grains and degenerated sterile lemmas. The small fzp-12 grains were caused by a reduction in cell number and size in the hulls. Interestingly, the sterile lemma underwent a homeotic transformation into a rudimentary glume in the fzp-12 and fzp-13 mutants, whereas the sterile lemma underwent a homeotic transformation into a lemma in FZP over-expressing plants, suggesting that FZP specifically determines the sterile lemma identity. We confirmed the function of FZP by complementation, CRISPR-Cas9 gene editing, and cytological and molecular tests. Additionally, FZP interacts specifically with the GCC-box and DRE motifs, and may be involved in regulation of the downstream genes. Our results revealed that FZP plays a vital role in the regulation of grain size, and first provides clear evidence in support of the hypothesis that the lemma, rudimentary glume, and sterile lemma are homologous organs.

Plant growth and reproduction are both energy-requiring processes; the necessary energy is supplied by the products of photosynthesis. Both the vegetative growth and reproductive success of rice are compromised by the absence of a functional copy of the gene OsHAK1. Here, a comparison between wild type rice and OsHAK1 knockout mutants not only confirmed the known detrimental effect of the absence of OsHAK1 on root growth, pollen viability and fertility, but also showed that sucrose phosphate synthase activity was lowered, and the sucrose content of the leaves was markedly increased, due to a partial block on the up-loading of sucrose into the phloem. The impaired allocation of sugar to the roots and spikelets caused by the knocking out of OsHAK1 was accompanied by a down-regulation in the leaf sheaths and panicle axes of genes encoding sucrose transporters (SUT genes), which are active in the phloem, as well as in the roots and spikelets of those encoding monosaccharide transporters (MST genes), which transport hexose sugars across the plant plasma membrane. The activity of sucrose synthase, acid invertase and neutral invertase in the roots of mutant plants assayed at the tillering stage, and in their spikelets, assayed during grain-filling, was significantly lower than in the equivalent organs of wild type plants. As a result, the supply of total soluble sugar, glucose and fructose to sink organs was reduced, consistent with the effect of the mutation on root growth and panicle fertility. Compared to wild type plants, the mutants accumulated less potassium (K) throughout the plant. The conclusion was that the failure to fully supply the demand of the mutant's sink organs for assimilate was responsible for its compromised phenotype, and that the deficiency in K uptake induced by the loss of OsHAK1 functionality was responsible for the disruption of sugar metabolism., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In crops, nitrogen directly determines productivity and biomass. However, the improvement of nitrogen utilization efficiency (NUE) is still a major challenge in modern agriculture. Here, we report the characterization of are1, a genetic suppressor of a rice fd-gogat mutant defective in nitrogen assimilation. ARE1 is a highly conserved gene, encoding a chloroplast-localized protein. Loss-of-function mutations in ARE1 cause delayed senescence and result in 10-20% grain yield increases, hence enhance NUE under nitrogen-limiting conditions. Analysis of a panel of 2155 rice varieties reveals that 18% indica and 48% aus accessions carry small insertions in the ARE1 promoter, which result in a reduction in ARE1 expression and an increase in grain yield under nitrogen-limiting conditions. We propose that ARE1 is a key mediator of NUE and represents a promising target for breeding high-yield cultivars under nitrogen-limiting condition.

High temperature, a major abiotic stress, significantly affects the yield and quality of crops in many parts of the world. Components of the photosynthetic apparatus are highly susceptible to thermal damage. Although the responses to acute heat stress have been studied intensively, the mechanisms that regulate chloroplast development under heat stress remain obscure, especially in crop plants. Here, we cloned and characterized the gene responsible for the heat-sensitive albino1 (hsa1) mutation in rice (Oryza sativa). The hsa1 mutant harbors a recessive mutation in a gene encoding fructokinase-like protein2 (FLN2); the mutation causes a premature stop codon and results in a severe albino phenotype, with defects in early chloroplast development. The color of hsa1 mutant plants gradually changed from albino to green at later stages of development at various temperatures and chloroplast biogenesis was strongly delayed at high temperature (32 °C). HSA1 expression was strongly reduced in hsa1 plants compared to wild type (WT). HSA1 localizes to the chloroplast and regulates chloroplast development. An HSA1 deletion mutant induced by CRISPR/Cas9 was heat sensitive but had a faster greening phenotype than the original hsa1 allele at all temperatures. RNA and protein levels of plastid-encoded RNA polymerase-dependent plastid genes were markedly reduced in hsa1 plants compared to WT. These results demonstrated that HSA1 plays important roles in chloroplast development at early stages, and functions in protecting chloroplasts under heat stress at later stages in rice., (Copyright © 2017 Elsevier B.V. All rights reserved.)