A rice variation map derived from 10 548 rice accessions reveals the importance of rare variants.

Detailed knowledge of the genetic variations in diverse crop populations forms the basis for genetic crop improvement and gene functional studies. In the present study, we analyzed a large rice population with a total of 10 548 accessions to construct a rice super-population variation map (RSPVM), consisting of 54 378 986 single nucleotide polymorphisms, 11 119 947 insertion/deletion mutations and 184 736 presence/absence variations. Assessment of variation detection efficiency for different population sizes revealed a sharp increase of all types of variation as the population size increased and a gradual saturation of that after the population size reached 10 000. Variant frequency analysis indicated that ∼90% of the obtained variants were rare, and would therefore likely be difficult to detect in a relatively small population. Among the rare variants, only 2.7% were predicted to be deleterious. Population structure, genetic diversity and gene functional polymorphism of this large population were evaluated based on different subsets of RSPVM, demonstrating the great potential of RSPVM for use in downstream applications. Our study provides both a rich genetic basis for understanding natural rice variations and a powerful tool for exploiting great potential of rare variants in future rice research, including population genetics and functional genomics.

A centromere map based on super pan-genome highlights the structure and function of rice centromeres.

Rice (Oryza sativa) is a significant crop worldwide with a genome shaped by various evolutionary factors. Rice centromeres are crucial for chromosome segregation, and contain some unreported genes. Due to the diverse and complex centromere region, a comprehensive understanding of rice centromere structure and function at the population level is needed. We constructed a high-quality centromere map based on the rice super pan-genome consisting of a 251-accession panel comprising both cultivated and wild species of Asian and African rice. We showed that rice centromeres have diverse satellite repeat CentO, which vary across chromosomes and subpopulations, reflecting their distinct evolutionary patterns. We also revealed that long terminal repeats (LTRs), especially young Gypsy-type LTRs, are abundant in the peripheral CentO-enriched regions and drive rice centromere expansion and evolution. Furthermore, high-quality genome assembly and complete telomere-to-telomere (T2T) reference genome enable us to obtain more centromeric genome information despite mapping and cloning of centromere genes being challenging. We investigated the association between structural variations and gene expression in the rice centromere. A centromere gene, OsMAB, which positively regulates rice tiller number, was further confirmed by expression quantitative trait loci, haplotype analysis and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 methods. By revealing the new insights into the evolutionary patterns and biological roles of rice centromeres, our finding will facilitate future research on centromere biology and crop improvement.

Lateral transfers lead to the birth of momilactone biosynthetic gene clusters in grass.

Momilactone A, an important plant labdane-related diterpenoid, functions as a phytoalexin against pathogens and an allelochemical against neighboring plants. The genes involved in the biosynthesis of momilactone A are found in clusters, i.e., momilactone A biosynthetic gene clusters (MABGCs), in the rice and barnyardgrass genomes. In addition, we know little about the origin and evolution of MABGCs. Here, we integrated results from comprehensive phylogeny and comparative genomic analyses of the core genes of MABGC-like clusters and MABGCs in 40 monocot plant genomes, providing convincing evidence for the birth and evolution of MABGCs in grass species. The MABGCs found in the PACMAD clade of the core grass lineage (including Panicoideae and Chloridoideae) originated from a MABGC-like cluster in Triticeae (BOP clade) via lateral gene transfer (LGT) and followed by recruitment of MAS1/2 and CYP76L1 genes. The MABGCs in Oryzoideae originated from PACMAD through another LGT event and lost CYP76L1 afterwards. The Oryza MABGC and another Oryza diterpenoid cluster c2BGC are two distinct clusters, with the latter originating from gene duplication and relocation within Oryzoideae. Further comparison of the expression patterns of the MABGC genes between rice and barnyardgrass in response to pathogen infection and allelopathy provides novel insights into the functional innovation of MABGCs in plants. Our results demonstrate LGT-mediated origination of MABGCs in grass and shed lights into the evolutionary innovation and optimization of plant biosynthetic pathways.

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

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

WHITE AND LESION-MIMIC LEAF1, encoding a lumazine synthase, affects reactive oxygen species balance and chloroplast development in rice.

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.

Disruption of EARLY LESION LEAF 1, encoding a cytochrome P450 monooxygenase, induces ROS accumulation and cell death in rice.

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.

Recent origination of circular RNAs in plants.

Circular RNA (circRNA) is a kind of new regulatory RNA with diverse biological functions. Numerous circRNAs have been identified in many plant species; however, evolution of plant circRNAs remains largely unknown. In this study, we assembled full-length sequences of 6519 rice (Oryza sativa) circRNAs and analyzed their conservation in another 46 plant species based on comparison of sequences and expression patterns. We found that, at the genomic level, 8.7% of the 6519 circRNAs were conserved in dicotyledonous plants and 49.1% in Oryza genus. Meanwhile, 57.8% of parental protein-coding genes of the rice circRNAs originated recently after divergence of monocotyledonous plants, implying recent origin of the majority of rice circRNAs, a conclusion further supported by the results based on analysis of 4663 full-length circRNAs in Arabidopsis thaliana. Accordingly, we proposed three models to address the origination of different types of circRNAs. Taken together, the results obtained in this study provide new insights for the evolutionary dynamics of plant circRNAs and candidate circRNAs for further functional exploration.

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

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

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

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

Effects of Sample Size on Plant Single-Cell RNA Profiling.

Single-cell RNA (scRNA) profiling or scRNA-sequencing (scRNA-seq) makes it possible to parallelly investigate diverse molecular features of multiple types of cells in a given plant tissue and discover cell developmental processes. In this study, we evaluated the effects of sample size (i.e., cell number) on the outcome of single-cell transcriptome analysis by sampling different numbers of cells from a pool of ~57,000 Arabidopsis thaliana root cells integrated from five published studies. Our results indicated that the most significant principal components could be achieved when 20,000-30,000 cells were sampled, a relatively high reliability of cell clustering could be achieved by using ~20,000 cells with little further improvement by using more cells, 96% of the differentially expressed genes could be successfully identified with no more than 20,000 cells, and a relatively stable pseudotime could be estimated in the subsample with 5000 cells. Finally, our results provide a general guide for optimizing sample size to be used in plant scRNA-seq studies.

Loci and natural alleles for cadmium-mediated growth responses revealed by a genome wide association study and transcriptome analysis in rice.

BACKGROUND: Cadmium (Cd) is a toxic heavy metal that is harmful to the environment and human health. Cd pollution threatens the cultivation of rice (Oryza sativa L.) in many countries. Improving rice performance under Cd stress could potentially improve rice productivity. RESULTS: In this study, 9 growth traits of 188 different cultivated rice accessions under normal and Cd stress conditions were found to be highly variable during the seedling stage. Based on ~3.3 million single nucleotide polymorphisms (SNPs), 119 Cd-mediated growth response (CGR) quantitative trait loci (QTL) were identified by a genome-wide association study (GWAS), 55 of which have been validated by previously reported QTL and 64 were new CGR loci. Combined with the data from the GWAS, transcriptome analysis, gene annotations from the gene ontology (GO) Slim database, and annotations and functions of homologous genes, 148 CGR candidate genes were obtained. Additionally, several reported genes have been found to play certain roles in CGRs. Seven Cd-related cloned genes were found among the CGR genes. Natural elite haplotypes/alleles in these genes that increased Cd tolerance were identified by a haplotype analysis of a diverse mini core collection. More importantly, this study was the first to uncover the natural variations of 5 GST genes that play important roles in CGRs. CONCLUSION: The exploration of Cd-resistant rice germplasm resources and the identification of elite natural variations related to Cd-resistance will help improve the tolerance of current major rice varieties to Cd, as well as provide raw materials and new genes for breeding Cd-resistant varieties.

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

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

MORE FLORET1 Encodes a MYB Transcription Factor That Regulates Spikelet Development in Rice.

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.

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

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

Genome Resource of Sphingomonas carotinifaciens L9-754T, an Endophyte Isolated From Leaf Tissues of Jatropha curcas.

Sphingomonas carotinifaciens strain L9-754T (DSM 27347) is a gram-negative, chemoheterotrophic, and rod-shaped endophyte isolated from the stem tissues of Jatropha curcas L. This strain has putative in vitro antagonistic ability against the plant pathogenic fungus Magnaporthe grisea. A draft genome of L9-754T was obtained using the PacBio SMRT cell platform. By analyzing the genome of strain L9-754T, a gene cluster (GQR91_18700 - GQR91_18715) related to an antioxidant enzyme was identified in the obtained draft genome. The information obtained from the draft genome is expected to reveal the putative properties helpful in biocontrol applications.

Advances in Sensing, Response and Regulation Mechanism of Salt Tolerance in Rice.

Soil salinity is a serious menace in rice production threatening global food security. Rice responses to salt stress involve a series of biological processes, including antioxidation, osmoregulation or osmoprotection, and ion homeostasis, which are regulated by different genes. Understanding these adaptive mechanisms and the key genes involved are crucial in developing highly salt-tolerant cultivars. In this review, we discuss the molecular mechanisms of salt tolerance in rice-from sensing to transcriptional regulation of key genes-based on the current knowledge. Furthermore, we highlight the functionally validated salt-responsive genes in rice.

Identification and Characterization of Short Crown Root 8, a Temperature-Sensitive Mutant Associated with Crown Root 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.

AH2 encodes a MYB domain protein that determines hull fate and affects grain yield and quality in rice.

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.

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

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

Genome Sequence of Micromonospora terminaliae TMS7T, a New Endophytic Actinobacterium Isolated from the Medicinal Plant Terminalia mucronata.

Micromonospora terminaliae sp. nov., type strain TMS7T, is a gram-positive nonmotile aerobic actinobacterium that was recently isolated from a surface-sterilized stem of the medicinal plant Terminalia mucronata. This strain was described as a novel species in the Micromonospora genus. To elucidate the application potential of this species, its genome was completely sequenced, using the PacBio SMRT cell platform, and was compared with selected complete genome sequences of other Micromonospora species. Genomic analysis revealed that the genome of TMS7T consists of one circular DNA chromosome of 6,717,200 bp with a GC content of 73.35% and one plasmid of 24,912 bp with a GC content of 65.39%. The entire genome contains 6,311 predicted coding genes, 57 transfer RNAs, and nine ribosomal RNA genes. The genome contains a type III polyketide biosynthesis gene cluster, which encodes enzymes that catalyze the production of alkyl-O-dihydrogeranyl-methoxyhydroquinone. This information combined with the previous report that this strain can grow well on pH 10 medium with 4% NaCl (wt/vol) indicates that this strain may have potential biocontrol applications for economic plants cultivated on alkaline soil.