Wide grain 4,encoding an alpha-tubulin,regulates grain size by affecting cell expansion in rice

Rice is one of the three most important food crops in the world.Increasing rice yield is an effective way to ensure food security.Grain size is a key factor affecting rice yield;however,the genetic and molecular mechanisms regulating grain size have not been fully investigated.In this study,we identified a rice mutant,wide grain 4-D(wg4-D),that exhibited a significant increase in grain width and a decrease in grain length.Histological analysis demonstrated that WG4 affects cell expansion thereby regulating grain size.MutMap-based gene mapping and complementary transgenic experiments revealed that WG4 encodes an alpha-tubulin,OsTubA1.A SNP mutation in WG4 affected the arrangement of cortical microtubules and caused a wide-grain phenotype.WG4 is located in nuclei and cytoplasm and expressed in various tissues.Our results provide insights into the function of tubulin in rice and identifies novel targets the regulation of grain size in crop breeding.

qGW11a/OsCAT8,encoding an amino acid permease,negatively regulates grain size and weight in rice

Grain size is a key factor influencing grain weight in rice.In this study,a chromosome segment substitution line(CSSL9-17)was identified,that exhibits a significant reduction in both grain size and weight compared to its donor parent 93-11.Further investigation identified two quantitative trait loci(QTL)on chromosome 11,designated qGW11a and qGW11b,which contribute to 1000-grain weight with an additive effect.LOC_Os11g05690,encoding the amino acid permease OsCAT8,is the target gene of qGW11a.Overexpression of OsCAT8 resulted in decreased grain weight,while OsCAT8 knockout mutants exhibited increased grain weight.The 287-bp located within the OsCAT8 promoter region of 93-11 negatively regulates its activity,which is subsequently correlated with an increase in grain size and weight.These results suggest that OsCAT8 functions as a negative regulator of grain size and grain weight in rice.

MicroRNA396-mediated alteration in plant development and salinity stress response in creeping bentgrass

The conserved microRNA396(miR396)is involved in plant growth,development,and abiotic stress response in multiple plant species through regulating its targets,Growth Regulating Factor(GRF)transcription factor genes.However,the role of miR396 has not yet been characterized in perennial monocot species.In addition,the molecular mechanism of miR396-mediated abiotic stress response remains unclear.To elucidate the role of miR396 in perennial monocot species,we generated transgenic creeping bentgrass(Agrostis stolonifera)overexpressing Osa-miR396c,a rice miRNA396 gene.Transgenic plants exhibited altered development,including less shoot and root biomass,shorter internodes,smaller leaf area,fewer leaf veins,and epidermis cells per unit area than those of WT controls.In addition,transgenics showed enhanced salt tolerance associated with improved water retention,increased chlorophyll content,cell membrane integrity,and Na^(+)exclusion during high salinity exposure.Four potential targets of miR396 were identified in creeping bentgrass and up-regulated in response to salt stress.RNA-seq analysis indicates that miR396-mediated salt stress tolerance requires the coordination of stress-related functional proteins(antioxidant enzymes and Na+/H+antiporter)and regulatory proteins(transcription factors and protein kinases).This study establishes a miR396-associated molecular pathway to connect the upstream regulatory and downstream functional elements,and provides insight into the miRNA-mediated regulatory networks.

A natural allele of TAW1 contributes to high grain number and grain yield in rice

Grain number per panicle (GNP) is a complex trait controlled by quantitative trait loci (QTL),directly determining grain yield in rice.Identifying GNP-associated QTL is desirable for increasing rice yield.A rice chromosome segment substitution line (CSSL),F771,which showed increased panicle length and GNP,was identified in a set of CSSLs derived from a cross between two indica cultivars,R498 (recipient) and WY11327 (donor).Genetic analysis showed that the panicle traits in F771 were semidominant and controlled by multiple QTL.Six QTL were consistently identified by QTL-seq analysis.Among them,the major QTL q PLN10 for panicle length and GNP was localized to a 121-kb interval between markers N802 and N909 on chromosome 10.Based on quantitative real-time PCR and sequence analysis,TAWAWA1(TAW1),a known regulator of rice inflorescence architecture,was identified as the candidate gene for q PLN10.A near-isogenic line,NIL-TAW1,was developed to evaluate its effects.In comparison with the recurrent parent R498,NIL-TAW1 showed increased panicle length (14.0%),number of secondary branches (20.9%) and GNP (22.0%),and the final grain yield per plant of NIL-TAW1 was increased by18.6%.Transgenic experiments showed that an appropriate expression level of TAW1 was necessary for panicle development.Haplotype analysis suggested that the favorable F771-type (Hap 13) of TAW1was introduced from aus accessions and had great potential value in high-yield breeding both in indica and japonica varieties.Our results provide a promising genetic resource for rice grain yield improvement.

Identification and characterization of rice blast resistance gene Pid4 by a combination of transcriptomic profiling and genome analysis

Map-based cloning of plant disease resistance (R) genes is time-consuming. Here, we reported the isolation of blast R gene Pid4 using comparative transcriptomic profiling and genome-wide sequence analysis. Pid4 encodes a coiled-coil nucleotide-binding site leucine-rich repeat(CC-NBS-LRR) protein and is constitutively expressed at diverse developmental stages in the rice variety Digu. The Pid4 protein is localized in both the nucleus and cytoplasm. Introduction of Pid4 into susceptible rice cultivars confers race-specific resistance to leaf and neck blast. Amino acid sequence comparison and blast resistance spectrum tests showed that Pid4 is a novel R gene, different from the previously reported R genes located in the same gene cluster. A Pid4 Indel marker was developed to facilitate the identification of Pid4 in different rice varieties. We demonstrated that a plant R gene can be quickly isolated using transcriptomic profiling coupled with genome-wide sequence analysis.

Deletion of a Dna K protein gene causes seedling green-revertible albino by retarding chloroplast development in rice

Green-revertible albino mutants are important sources for studying chloroplast structure, chloroplast development, chlorophyll biosynthesis, and plant photo- synthesis. In the present study, we characterized a green- revertible albino mutant gra（k）, which was obtained from the tissue-cultured rice Kitaake. The mutant gra（k） exhib- ited albino on its first three leaves. The leaf color started to turn green at the four-leaf stage. The chlorophyll contents were deeply reduced at the seedling stage, and the chloroplast development was delayed in gra（k）. The green- revertible albino （gra） phenotype of the mutant gra（k） was temperature dependent. The main agronomic traits, including plant height, tilling number per plant, seed set- ting rate, and thousand-grain weight, slightly decreased in gra（k） comparing to those in the wild-type Kitaake. Genetic analysis showed that the gra phenotype was con- trolled by a single recessive nucleic gene. By using 5,168 recessive F2 individuals derived from the cross of gra（k） × Jodan, the locus of the gene Gra（k） was delimited in a DNA region of 200 kb between the makers B-31 and P11 on chromosome 5. Sequencing analysis indicated that the three functionally annotated genes, LOC_Os05g23700, LOC_Os05g23720, and LOC_Os05g23740, were all deleted in the 200 kb region in the mutant gra（k）. Trans- genic test revealed that the gra（k） plants over-expressing LOC_Os05g23740CDS were restored to normal green as the wild-type Kitaake. Our results proved that the deletion of the DnaK protein gene LOC_Os05g23740 （encoding the chaperon protein OsHsp70CP1） led to the gra phenotype in the mutant gra（k）.

OsELF3-2, an Ortholog of Arabidopsis ELF3, Interacts with the E3 Ligase APIP6 and Negatively Regulates Immunity against Magnaporthe oryzae in Rice

Dear Editor, The ubiquitin proteasome system （UPS） is a highly regulated mechanism of intracellular protein degradation in eukaryotes. The UPS comprises three key enzymes： ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase enzyme E3. There are nearly 1500 E3 ligase genes in rice and Arabidopsis genomes that target substrates for many biological processes for plant growth and tolerance to abiotic and biotic stresses.

Gradual daylength sensing coupled with optimum cropping modes enhances multi-latitude adaptation of rice and maize

To expand crop planting areas,reestablishment of crop latitude adaptation based on genetic variation in photoperiodic genes can be performed,but it is quite time consuming.By contrast,a crop variety that already exhibits multi-latitude adaptation has the potential to increase its planting areas to be more widely and quickly available.However,the importance and potential of multi-latitude adaptation of crop varieties have not been systematically described.Here,combining daylength-sensing data with the cropping system of elite rice and maize varieties,we found that varieties with gradual daylength sensing coupled with optimum cropping modes have an enhanced capacity for multi-latitude adaptation in China.Furthermore,this multi-latitude adaptation expanded their planting areas and indirectly improved China’s nationwide rice and maize unit yield.Thus,coupling the daylength-sensing process with optimum cropping modes to enhance latitude adaptability of excellent varieties represents an exciting approach for deploying crop varieties with the potential to expand their planting areas and quickly improve nationwide crop unit yield in developing countries.

The DTH8-Hd1 Module Mediates Day-Length-Dependent Regulation of Rice Flowering

Photoperiodic flowering is one of the most important pathways to govern flowering in rice （Oryza sativa）, in which Heading date 1 （Hdl）, an ortholog of the Arabidopsis CONSTANS gene, encodes a pivotal regulator. Hdl promotes flowering under short-day conditions （SD） but represses flowering under long-day conditions （LD） by regulating the expression of Heading date 3a （Hd3a）, the FLOWERING LOCUS T （FT） ortholog in rice. However, the molecular mechanism of how Hdl changes its regulatory activity in response to day length remains largely unknown. In this study, we demonstrated that the repression of flowering in LD by Hdl is dependent on the transcription factor DAYS TO HEADING 8 （DTHS）. Loss of DTH8 function results in the activation of Hd3a by Hdl, leading to early flowering. We found that Hdl directly interacts with DTH8 and that the formation of the DTH8-Hd1 complex is necessary for the transcriptional repression of Hd3a by Hdl in LD, implicating that the switch of Hdl function is mediated by DTH8 in LD rather than in SD. Furthermore, we revealed that DTH8 associates with the Hd3a promoter to modulate the level of H3K27 trimethylation （H3K27me3） at the Hd3a locus. In the presence of the DTH8-Hdl complex, the H3K27me3 level was increased at Hd3a, whereas loss of DTH8 function resulted in decreased H3K27me3 level at Hd3a. Taken together, our findings indicate that, in response to day length, DTH8 plays a critical role in mediating the transcriptional regulation of Hd3a by Hdl through the DTH8-Hd1 module to shape epigenetic modifications in photoperiodic flowering.

OsSPL18 controls grain weight and grain number in rice

Grain weight and grain number are two important traits directly determining grain yield in rice. To date,a lot of genes related to grain weight and grain number have been identified; however, the regulatory mechanism underlying these genes remains largely unknown. In this study, we studied the biological function of OsSPL18 during grain and panicle development in rice. Knockout (KO) mutants of OsSPL18exhibited reduced grain width and thickness, panicle length and grain number, but increased tiller number. Cytological analysis showed that OsSPL18 regulates the development of spikelet hulls by affecting cell proliferation. qRT-PCR and GUS staining analyses showed that OsSPL18 was highly expressed in developing young panicles and young spikelet hulls, in agreement with its function in regulating grain and panicle development. Transcriptional activation experiments indicated that OsSPL18is a functional transcription factor with activation domains in both the N-terminus and C-terminus, and both activation domains are indispensable for its biological functions. Quantitative expression analysis showed that DEP1, a major grain number regulator, was significantly down-regulated in OsSPL18 KO lines.Both yeast one-hybrid and dual-luciferase (LUC) assays showed that OsSPL18 could bind to the DEP1promoter, suggesting that OsSPL18 regulates panicle development by positively regulating the expression of DEP1. Sequence analysis showed that OsSPL18 contains the OsmiR156k complementary sequence in the third exon; 5?RLM-RACE experiments indicated that OsSPL18 could be cleaved by OsmiR156k. Taken together, our results uncovered a new OsmiR156k-OsSPL18-DEP1 pathway regulating grain number in rice.

Analysis of genetic architecture and favorable allele usage of agronomic traits in a large collection of Chinese rice accessions

Genotyping and phenotyping large natural populations provide opportunities for population genomic analysis and genome-wide association studies(GWAS). Several rice populations have been re-sequenced in the past decade;however, many major Chinese rice cultivars were not included in these studies. Here, we report large-scale genomic and phenotypic datasets for a collection mainly comprised of 1,275 rice accessions of widely planted cultivars and parental hybrid rice lines from China. The population was divided into three indica/Xian and three japonica/Geng phylogenetic subgroups that correlate strongly with their geographic or breeding origins. We acquired a total of 146 phenotypic datasets for 29 agronomic traits under multi-environments for different subpopulations. With GWAS, we identified a total of 143 significant association loci, including three newly identified candidate genes or alleles that control heading date or amylose content. Our genotypic analysis of agronomically important genes in the population revealed that many favorable alleles are underused in elite accessions, suggesting they may be used to provide improvements in future breeding efforts. Our study provides useful resources for rice genetics research and breeding.

From Green Super Rice to green agriculture:Reaping the promise of functional genomics research

Producing sufficient food with finite resources to feed the growing global population while having a smaller impact on the environment has always been a great challenge.Here,we review the concept and practices of Green Super Rice(GSR)that have led to a paradigm shift in goals for crop genetic improvement and models of food production for promoting sustainable agriculture.The momentous achievements and global deliveries of GSR have been fueled by the integration of abundant genetic resources,functional gene discoveries,and innovative breeding techniques with precise gene and whole-genome selection and efficient agronomic management to promote resource-saving,environmentally friendly crop production systems.We also provide perspectives on new horizons in genomic breeding technologies geared toward delivering green and nutritious crop varieties to further enhance the development of green agricul-ture and better nourish the world population.

Loss of Gn1a/OsCKX2 confers heavy-panicle rice with excellent lodging resistance

Significant achievements have been made in breeding programs for the heavy-panicle-type(HPT)rice(Oryza sativa) in Southwest China. The HPT varieties now exhibit excellent lodging resistance,allowing them to overcome the greater pressures caused by heavy panicles. However, the genetic mechanism of this lodging resistance remains elusive. Here, we isolated a major quantitative trait locus, Panicle Neck Diameter 1(PND1), andidentified the causal gene as GRAIN NUMBER 1 A/CYTOKININ OXIDASE 2(Gn1 A/Os CKX2). The null gn1 a allele from rice line R498(gn1 aR498) improved lodging resistance through increasing the culm diameter and promoting crown root development.Loss-of-function of Gn1 a/Os CKX2 led to cytokinin accumulation in the crown root tip and accelerated the development of adventitious roots. Gene pyramiding between the null gn1 aR498 allele with two gain-of-function alleles, STRONG CULM 2(SCM2)and SCM3, further improved lodging resistance.Moreover, Gn1 a/Os CKX2 had minimal influence on overall rice quality. Our research thus highlights the distinct genetic components of lodging resistance of HPT varieties and provides a strategy for tailormade crop improvement of both yield and lodging resistance in rice.

Overproduction of OsRACK1A,an effector-targeted scaffold protein promoting OsRBOHB-mediated ROS production,confers rice floral resistance to false smut disease without yield penalty

Grain formation is fundamental for crop yield but is vulnerable to abiotic and biotic stresses.Rice grain production is threatened by the false smut fungus Ustilaginoidea virens,which specifically infects rice floral organs,disrupting fertilization and seed formation.However,little is known about the molecular mechanisms of the U.virens-rice interaction and the genetic basis of floral resistance.Here,we report that U.virens secretes a cytoplasmic effector,UvCBP1,to facilitate infection of rice flowers.Mechanistically,UvCBP1 interacts with the rice scaffold protein OsRACK1A and competes its interaction with the reduced nicotinamide adenine dinucleotide phosphate oxidase OsRBOHB,leading to inhibition of reactive oxygen species(ROS)production.Although the analysis of natural variation revealed no OsRACK1A variants that could avoid being targeted by UvCBP1,expression levels of OsRACK1A are correlated with field resistance against U.virens in rice germplasm.Overproduction of OsRACK1A restores the OsRACK1A-OsRBOHB association and promotes OsRBOHB phosphorylation to enhance ROS production,conferring rice floral resistance to U.virens without yield penalty.Taken together,our findings reveal a new pathogenic mechanism mediated by an essential effector from a flower-specific pathogen and provide a valuable genetic resource for balancing disease resistance and crop yield.

NaCl胁迫下哈茨木霉ACCC32524的转录组和代谢组分析

【目的】通过分析NaCl胁迫下哈茨木霉(Trichoderma harzianum)ACCC32524转录组和代谢组数据,研究差异表达基因及次级代谢产物的变化情况,初步探索响应NaCl胁迫的分子机制。【方法】利用Illumina HiSeq XTen高通量测序平台完成0、0.4、0.6 mol/L NaCl浓度胁迫培养下哈茨木霉ACCC32524的转录组测序,GC-TOF-MS技术完成对0mol/L和0.6mol/LNaCl胁迫培养下的差异次级代谢产物检测,利用相关软件及数据库对差异表达基因(DEGs)和次级代谢产物的注释、筛选和分类,并进行RT-qPCR验证。【结果】本研究分别得到0.4 mol/L和0.6 mol/L NaCl胁迫下417和733条差异表达基因;GO富集分析显示,分别有318和582条差异表达基因注释到生物学过程、分子功能和细胞组分3个一级分类和40个二级分类;COG分类结果表明分别有232和414条转录本为20个类别,涉及差异表达基因最多的分别为氨基酸的转运和代谢、一般功能预测、碳水化合物的转运和代谢;KEGG代谢途径分析结果表明,分别有75和96条基因归到25个代谢通路中(P≤0.05),其中涉及差异基因最多的是氨基酸的生物合成和2-氧代羧酸代谢通路。从转录组数据中共筛选出与渗透调节、离子转运、活性氧清除等22个耐盐相关基因。0 mol/L和0.6 mol/L NaCl胁迫下的代谢组数据中共筛选出101个差异次级代谢产物,包括8种积累量上调和93种下调物质,其中36个得到定性,分属于糖类、有机酸和氨基酸等9个分类中。RT-qPCR验证挑选的差异表达基因的表达量变化,均与RNA-seq分析结果一致。【结论】NaCl胁迫下引起哈茨木霉ACCC32524基因及次级代谢产物发生明显变化,细胞代谢途径发生明显偏移,这些进程共同作用减少NaCl对细胞的毒害作用,为木霉菌的耐盐机理研究提供重要信息。

A single nucleotide substitution at 5'-UTR of GSN1 represses its translation and leads to an increase of grain length in rice

Rice grain size is an important trait that affects rice yield and quality, and thus the identification of genes related to grain size is of great significance for improving rice yield and quality. Many genes related to grain size, such as DEP1(Huang et al., 2009),GW5(Liu et al., 2017).

Characterization and Fine Mapping of GLABROUS RICE 2 in Rice

Glabrous rice exhibits glabrous leaves and hulls because neither of these structures have trichomes on their surfaces. Glabrous rice varieties have become an important germplasm resource in the rice industry because they have considerable packaging efficiency and can reduce skin itching and dust during harvesting, drying, and packing （Shim et al., 2012; Zhang et al., 2012）.

Improving the efficiency of hybrid combination preparation in rice breeding by a modified flowering stimulant

The utilization of hybrid vigor is an important breakthrough in the history of rice breeding.To select the best hybrid combinations,breeders manually perform extensive testing of hybridizations between restorer and sterile lines,which is a laborious and time-consuming process.Here,we report that a modified flowering stimulant containing methyl jasmonate(MeJA),6-benzylamine adenine and kinetin effectively promotes the flowering and seed set of male-sterile rice lines.Different concentrations of the ingredients were tested to identify an optimal formulation.Seed quality evaluation indicated that hybrid seeds from plants sprayed with the flowering stimulant had a higher germination rate than seeds from plants prepared by glume-cutting.In summary,the modified flowering stimulant described in this study may help reduce the labor requirement associated with hybrid rice breeding and improve yield and efficiency.

Characterization and Fine Mapping of a Novel Vegetative Senescence Lethal Mutant Locus in Rice

Mutants showing spontaneous cell death in the absence of pathogen attack are called lesion mimic mutants （lmm） （Lorrain et al., 2003）. These mutants usually exhibit typical hypersensitive responses （HRs） within or around the lesion spots, which are frequently observed in plants challenged with avirulent pathogens （Lorrain et al., 2003）. A number of these mutants have been characterized in rice （Zeng et al., 2004）, Arabidopsis （Guo et al., 2013）, maize （Wang et al., 2013） and barley （Persson et al., 2008）. Most lmm show enhanced resistance to various pathogens （Huang et al., 2011）, because HR is usually accompanied with enhanced defense responses, such as reactive oxygen species （ROS） activation （Qiao et al., 2010） and increased expression of pathogenesis-related genes （Lorrain et al., 2003）. Additionally, most lmm exhibit defects in growth and development due to the disordered physiolog- ical and molecular processes caused by the lesion spots. Thus, lmm are powerful tools for the study of the molecular mech- anisms of cell death, plant development and disease resistance （Lorrain et al., 2003; Babu et al., 2011）.