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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.

Xanthine dehydrogenase, a member of the molybdenum enzyme family, participates in purine metabolism and catalyzes the generation of ureides from xanthine and hypoxanthine. However, the mechanisms by which xanthine dehydrogenase affects rice growth and development are poorly understood. In the present study, we identified a mutant with early leaf senescence and reduced tillering that we named early senescence and less-tillering 1 (esl1). Map-based cloning revealed that ESL1 encodes a xanthine dehydrogenase, and it was expressed in all tissues. Chlorophyll content was reduced and chloroplast maldevelopment was severe in the esl1 mutant. Mutation of ESL1 led to decreases in allantoin, allantoate, and ABA contents. Further analysis revealed that the accumulation of reactive oxygen species in esl1 resulted in decreased photosynthesis and impaired chloroplast development, along with increased sensitivity to abscisic acid and abiotic stresses. Ttranscriptome analysis showed that the ESL1 mutation altered the expression of genes involved in the photosynthesis process and reactive oxygen species metabolism. Our results suggest that ESL1 is involved in purine metabolism and the induction of leaf senescence. These findings reveal novel molecular mechanisms of ESL1 gene-mediated plant growth and leaf senescence.

Background Mitochondrion is the key respiratory organ and participates in various anabolic and catabolic metabolic 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. Map-based cloning and transgenic functional confirmation revealed that NLG1encodes a mitochondrial import inner membrane translocase containing a subunit Tim21 domain. NLG1 was identified as a mitochondria-localized translocase protein, which is mainly transcribed in leaves and panicles. Further analysis showed that the expression level of respiratory function and auxin response related genes were significantly down-regulated, resulting in ATP production and auxin content declined remarkably in nlg1. Moreover, nlg1 also exhibited abnormal mitochondria structure and was sensitive to the inhibitors of mitochondrial electron transport chain. 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 for exploring the relationship between mitochondria development and plant growth in rice.

OsPPR11 belongs to the P-type PPR protein family and can interact with OsCAF2 to regulate Group II intron splicing and affect chloroplast development in rice. Pentatricopeptide repeat (PPR) proteins participate in chloroplasts or mitochondria group II introns splicing in plants. The PPR protein family contains 491 members in rice, but most of their functions are unknown. In this study, we identified a nuclear gene encoding the P-type PPR protein OsPPR11 in chloroplasts. The qRT-PCR analysis demonstrated that OsPPR11 was expressed in all plant tissues, but leaves had the highest expression. The osppr11 mutants had yellowing leaves and a lethal phenotype that inhibited chloroplast development and photosynthesis-related gene expression and reduced photosynthesis-related protein accumulation in seedlings. Moreover, photosynthetic complex accumulation decreased significantly in osppr11 mutants. The OsPPR11 is required for ndhA, and ycf3-1 introns splicing and interact with CRM family protein OsCAF2, suggesting that these two proteins may form splicing complexes to regulate group II introns splicing. Further analysis revealed that OsCAF2 interacts with OsPPR11 through the N-terminus. These results indicate that OsPPR11 is essential for chloroplast development and function by affecting group II intron splicing in rice.

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

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.

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 (H2 O2 ) 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.

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 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.

Chloroplasts are identified as important organelles for photosynthesis, biosynthesis, and storage of metabolites. So far, the molecular regulatory mechanism of chloroplast development remains to be unclear. The chloroplast RNA splicing 2 (CRS2) protein was determined to play a key role in chloroplast development as they affect the splicing of chloroplast gene introns in maize. However, the functions of OsCRS2 in rice chloroplast development have rarely been studied. In this study, oscrs2-1 and oscrs2-2 mutants were obtained to gene function research using the CRISPR/Cas9 gene editing technology. Both of the mutants displayed an albino seedling phenotype. The OsCRS2 is located in the chloroplast, and is highly expressed in green tissues. OsCRS2 could affect group I and seven group II introns splicing and participate in chloroplast rRNA biogenesis. RNA sequencing analysis indicated that both nuclear-encoded photosynthesis and chloroplast development-related genes expression were significantly reduced in the oscrs2-1 mutant. To conclude, these results suggested that OsCRS2 is necessary for early chloroplast development 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.

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.

Rice (Oryza sativa) spikelets are a unique inflorescence structure and their development directly determines grain size and yield. Although many genes related to spikelet development have been reported, the molecular mechanisms underlying this process have not been fully elucidated. In this study, we identified a new recessive rice mutant, lacking rudimentary glume 1 (lrg1). The lrg1 spikelets only formed one rudimentary glume, which, along with the sterile lemmas, was homeotically transformed into lemma-like organs and acquired lemma identity. The transition from the spikelet to the floral meristem was delayed in the lrg1 mutant, resulting in the formation of an ectopic lemma-like organ between the sterile lemma and the terminal floret. In addition, we found that the abnormal lrg1 grain phenotype resulted from the alteration of cell numbers and the hull size. LRG1 encodes a ZOS4-06-C2H2 zinc-finger protein with the typical EAR motifs, and is expressed in all organs and tissues. LRG1 localizes to the nucleus and can interact with the TOPLESS-RELATED PROTEINs (TPRs) to repress the expressions of their downstream target genes. Taken together, our results reveal that LRG1 plays an important role in the regulation of spikelet organ identity and grain size.

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.

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.

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.

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.

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.

Rice source- and sink-associated traits are important for grain yield and are sensitive to environmental conditions. The continuing increase of CO2 concentrations in the atmosphere will become a major challenge for rice growth and development in the future due to changes in our climate such as extremes in temperature. To guarantee food safety, novel genetic loci need to be identified for source- and sink-associated traits that are specifically expressed under elevated CO2 conditions. Eighty chromosome segment substitution lines carrying japonica (Nipponbare) chromosome segments in the indica (9311) background were used in this study. QTL analysis was conducted for source- and sink-related traits, including flag leaf length, flag leaf width, flag leaf fresh weight, flag leaf dry weight, primary branch number, secondary branch number, grain number per panicle, panicle weight per plant, chlorophyll a, chlorophyll b, and carotenoid contents, under ambient CO2 concentrations and free-air CO2 enrichment. A total of 49 QTLs for these traits were detected on chromosomes 1, 3, 5, 6, 7, 9, and 12 under the two conditions; the variance explained by these QTLs varied from 6.22 to 38.15%. Among these QTLs, 19 of them were detected under the natural field conditions and 30 were detected in the elevated CO2 conditions. In addition, 2 and 13 QTLs were specifically expressed in the natural and CO2-enriched conditions, respectively. Our findings have important implications on the utilization of germplasm resources for ensuring food security under elevated CO2 levels, especially for QTLs that were specifically detected under the elevated CO2 condition.

The typical rice (Oryza sativa) spikelet contains a single fertile floret and produces only one grain; by contrast, Brachypodium distachyon spikelets contain multiple fertile florets and produce several grains. To increase yield, rice breeders have traditionally focused on panicle morphology (branch number and length, spikelet density), but have not considered the number of florets in each spikelet. Production of rice spikelets with more florets could further increase the number of grains per panicle. Here, we describe two novel approaches - altering meristem determinacy and restoring lateral floret formation - for breeding rice cultivars with a multifloret spikelet, thereby increasing the number of grains per panicle and potentially improving yield.

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.

Summary 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., Significance Statement DS8 encodes a NAP1‐like protein that affects drought sensitivity by its involvement in leaf epidermal development and stomatal closure in rice, revealing a molecular mechanism concerning the SCAR/WAVE‐Arp2/3 complex in the drought stress response.

Chloroplast transit peptides (CTPs) can be used to transport non-chloroplastic proteins into the chloroplasts. Here, we studied the CTPs of three rice (Oryza sativa L.) chloroplast-localized proteins and found that their CTPs could be used to transport non-chloroplast-localized proteins into the chloroplasts. Fusion proteins lacking the CTP remained located in the cytoplasm. Furthermore, we constructed green fluorescent protein fusion vectors with the three CTPs and three non-chloroplast-localized proteins, Ghd10, MULTI-FLORET SPIKELET1 (MFS1), and SHORTENED UPPERMOST INTERNODE 1 (SUI1). After transforming these constructs into rice protoplasts, the fusion proteins all localized in the chloroplasts. Collectively, our results showed that these CTPs can transport non-chloroplast-localized proteins into the chloroplasts, and more importantly, these CTPs can be applied to engineer chloroplast metabolism. Keywords: rice, chloroplast transit peptide, translocated function, subcellular location