OsmiR396/growth regulating factor modulate rice grain size through direct regulation of embryo-specific miR408

microRNAs (miRNAs) are promising targets for crop improvement of complex agricultural traits. Coordinated activity between/among different miRNAs may fine-tune specific developmental processes in diverse organisms. Grain size is a main factor determining rice (Oryza sativa L.) crop yield, but the network of miRNAs influencing this trait remains uncharacterized. Here we show that sequestering OsmiR396 through target mimicry (MIM396) can substantially increase grain size in several japonica and indica rice subspecies and in plants with excessive tillers and a high panicle density. Thus, OsmiR396 has a major role related to the regulation of rice grain size. The grain shape of Growth Regulating Factor8 (OsGRF8)-overexpressing transgenic plants was most similar to that of MIM396 plants, suggesting OsGRF8 is a major mediator of OsmiR396 in grain size regulation. A miRNA microarray analysis revealed changes to the expression of many miRNAs, including OsmiR408, in the MIM396 plants. Analyses of gene expression patterns and functions indicated OsmiR408 is an embryo-specific miRNA that positively regulates grain size. Silencing OsmiR408 expression (miR408KO) using CRISPR technology resulted in small grains. Moreover, we revealed the direct regulatory effects of OsGRF8 on OsMIR408 expression. A genetic analysis further showed that the large-grain phenotype of MIM396 plants could be complemented by miR408KO. Also, several hormone signaling pathways might be involved in the OsmiR396/GRF-meditated grain size regulation. Our findings suggest that genetic regulatory networks comprising various miRNAs, such as OsmiR396 and OsmiR408, may be crucial for controlling rice grain size. Furthermore, the OsmiR396/GRF module may be important for breeding new high-yielding rice varieties.

The OsmiR396/Growth Regulating Factor module plays a pivotal role in rice grain size regulation and genetically regulates OsmiR408, which acts as an embryo-specific grain size regulator.     

Regulation of OsmiR156h through Alternative Polyadenylation Improves Grain Yield in Rice

Substantial increases in grain yield of cereal crops are required to feed a growing human population. Here we show that a natural variant of SEMIDWARF AND HIGH-TILLERING (SDT) increases harvest index and grain productivity in rice. Gain-of-function sdt mutation has a shortened polyadenylation tail on the OsmiR156h microRNA precursor, which cause the up-regulation of OsmiR156h. The plants carrying the semidominant sdt allele exhibit reduced plant height, enhanced lodging resistance, increased tiller numbers per plant, and resulting in an increased grain yield. We also show that combining the sdt allele with the OsSPL14^WFP allele can be effective in simultaneously improving tillering capacity and panicle branching, thereby leading to higher harvest index and grain yield. Most importantly, pyramiding of the sdt allele and the green revolution gene sd1 enhances grain yield by about 20% in hybrid rice breeding. Our results suggest that the manipulation of the polyadenylation status of OsmiR156 represents a novel strategy for improving the yield potential of rice over what is currently achievable.     

A putative AGO protein,OsAGO17, positively regulates grain size and grain weight through OsmiR397b in rice

Argonaute (AGO) proteins and small RNAs (sRNAs) are core components of the RNA‐induced silencing complex (RISC). It has been reported that miRNAs regulate plant height and grain size in rice, but which AGO is involved in grain size regulation remains unclear. Here, we report that enhanced expression of OsAGO17, a putative AGO protein, could improve grain size and weight and promote stem development in rice. Cytological evidence showed that these effects are mainly caused by alteration of cell elongation. Expression analyses showed that OsAGO17 was highly expressed in young panicles and nodes, which was consistent with the expression pattern of OsmiR397b. SRNA sequencing, stem‐loop RT‐PCR and sRNA blotting showed that the expression of OsmiR397b was reduced in ago17 and enhanced in the OsAGO17 OE lines. Four OsmiR397b target laccase (LAC) genes showed complementary expression patterns with OsAGO17 and OsmiR397b. Combined with the results of immunoprecipitation (IP) analysis, we suggested that OsAGO17 formed an RISC with OsmiR397b and affected rice development by suppression of LAC expression. In conclusion, OsAGO17 might be a critical protein in the sRNA pathway and positively regulates grain size and weight in rice.     

The alteration in the architecture of a T‐DNA insertion rice mutant osmtd1 is caused by up‐regulation of MicroRNA156f

Plant architecture is an important factor for crop production. Some members of microRNA156 (miR156) and their target genes SQUAMOSA Promoter‐Binding Protein‐Like (SPL) were identified to play essential roles in the establishment of plant architecture. However, the roles and regulation of miR156 is not well understood yet. Here, we identified a T‐DNA insertion mutant Osmtd1 (Oryza sativa multi‐tillering and dwarf mutant). Osmtd1 produced more tillers and displayed short stature phenotype. We determined that the dramatic morphological changes were caused by a single T‐DNA insertion in Osmtd1. Further analysis revealed that the T‐DNA insertion was located in the gene Os08g34258 encoding a putative inhibitor I family protein. Os08g34258 was knocked out and OsmiR156f was significantly upregulated in Osmtd1. Overexpression of Os08g34258 in Osmtd1 complemented the defects of the mutant architecture, while overexpression of OsmiR156f in wild‐type rice phenocopied Osmtd1. We showed that the expression of OsSPL3, OsSPL12, and OsSPL14 were significantly downregulated in Osmtd1 or OsmiR156f overexpressed lines, indicating that OsSPL3, OsSPL12, and OsSPL14 were possibly direct target genes of OsmiR156f. Our results suggested that OsmiR156f controlled plant architecture by mediating plant stature and tiller outgrowth and may be regulated by an unknown protease inhibitor I family protein.     

Molecular insights into the regulation of rice kernel elongation

Abstract

A large number of rice agronomic traits are complex, multi factorial and polygenic. As the mechanisms and genes determining grain size and yield are largely unknown, the identification of regulatory genes related to grain development remains a preeminent approach in rice genetic studies and breeding programs. Genes regulating cell proliferation and expansion in spikelet hulls and participating in endosperm development are the main controllers of rice kernel elongation and grain size. We review here and discuss recent findings on genes controlling rice grain size and the mechanisms, epialleles, epigenomic variation, and assessment of controlling genes using genome-editing tools relating to kernel elongation.     

不同生育阶段夜温升高对双季水稻产量的影响

利用两间玻璃温室内夜间不同的温度条件,研究了水稻不同生育阶段的夜温升高对双季早、晚稻产量的影响.结果表明：播种-幼穗分化（一次枝梗分化期）期间夜温升高,有利于双季水稻的分蘖,植株有效穗数显著增加,平均夜间最低温度每升高1 ℃,双季早、晚稻的产量分别提高10.02%～13.18%和6.52%～7.78%（P＜0.01）;幼穗分化-抽穗（10%稻穗抽出剑叶鞘）期间夜温升高,导致颖花退化,使每穗发育颖花数减少,平均夜间最低温度每升高1 ℃,双季早、晚稻的产量分别下降3.76%～6.67%和3.66%～6.94%（P＜0.01）;灌浆结实期（抽穗-成熟）夜温升高,双季早稻的结实率和产量显著下降,而双季晚稻的结实率和产量显著提高,平均夜间最低温度每升高1 ℃,双季早稻的产量下降2.07%～5.61%（P＜0.05）,双季晚稻的产量提高1.63%～2.28%（P＜0.05）.表明不同生育阶段的夜温升高对双季水稻产量的影响存在明显差异.     

Apoplasmic assimilates and grain growth of contrasting rice cultivars differing in grain dry mass and size

Apical dominance in assimilate filling impacts grain growth in basal spikelets of rice panicle. In this study, organic materials of the pericarp, apoplasmic space and endosperm of the apical and basal caryopses, and photosynthesis of the flag leaf were measured during early part of grain development in three types of rice cultivars with similar phenology, but difference in grain weight and size in the dry and wet seasons of 2006 and 2007, respectively. Photosynthetic activity of the flag leaf was consistently low in small-seeded cultivars. Rates of grain filling and cell division of endosperm and concentration of assimilates, starch, proteins and chlorophylls of the caryopsis were lower, but spikelet ethylene production and peroxidase activity were higher in a small-seeded cultivar compared to a big-seeded cultivar. Similar disparities in grain filling and other attributes were noticed for the inferior basal spikelets of the panicle compared to the superior apical spikelets, except the assimilate concentration of the pericarp and endosperm. Temporal fluctuation in assimilate concentration of the organs were similar between the cultivars. Concentration of apoplasmic assimilates mostly exhibited negative correlation with that of pericarp and endosperm. Compared to the apical spikelets, correlation was more negative for the basal spikelets. Conversely, correlation was positive between the concentration of apoplasmic assimilates and endosperm cell number and grain weight of the cultivars. Ethylene released from the spikelets at anthesis affected growth and cell division rates of endosperm and enhanced protein and chlorophyll degradation and peroxidase activity of the caryopsis. It was concluded that variation in spikelet ethylene production may be responsible for differences in size or weight of grains among rice cultivars and spikelets at different locations of the panicle. The concentration of apoplasmic assimilates could be an indicator for grain filling capacity, and ethylene regulated the concentration by affecting pericarp activity for assimilate unloading.     

高浓度CO2对稻穗不同位置籽粒结实和米质性状的影响

不断升高的大气CO₂浓度影响水稻颖花发育、灌浆结实和品质形成,但这种影响是否与籽粒在稻穗上的着生部位有关尚不清楚.利用稻田FACE (Free-Air CO₂ Enrichment)平台,以优质丰产粳稻‘武运粳23’为材料,CO₂处理设背景CO₂浓度(Ambient)和高CO₂浓度(增200 μmol·mol^-1, FACE)两个水平,研究开放大田条件下高浓度CO₂对水稻颖花密度、籽粒结实能力、稻米外观和食味品质的影响及其与稻穗不同着生位置的关系.结果表明:FACE处理使武运粳23籽粒产量平均增加18.3%,从产量构成因素看,穗数和饱粒重分别增加21.4%、9.4%,每穗颖花数、饱粒率平均减少9.0%、2.2%.FACE水稻饱粒率下降主要与稻穗不同部位空粒率大幅增加有关.FACE水稻每穗颖花数减少主要与稻穗上部、中部二次枝梗现存颖花大幅减少有关,而其他位置颖花数均无显著变化;稻穗不同位置饱粒重和饱粒率对FACE的响应无显著差异.FACE处理使绿粒率下降,但糙米长度和宽度均增加,稻穗不同部位趋势一致.FACE使垩白粒率(增幅59%)、垩白度(增幅55%)均极显著增加,增幅表现为稻穗一次枝梗>二次枝梗、上部>中部>下部.FACE使稻穗不同位置稻米直链淀粉含量略增,使最高粘度、热浆粘度、崩解值、最终粘度和消减值略降,但多未达显著水平.FACE使稻米糊化温度显著下降,弱势粒的降幅大于强势粒.综上,高浓度CO₂环境下武运粳23产量增加主要与穗数增多和籽粒增重有关,而稻穗明显变小;高浓度CO₂使稻米绿粒率减少,垩白增多,而对蒸煮食味品质影响较少;颖花着生位置对高浓度CO₂环境下水稻颖花发育、结实和品质的影响因不同测定指标而异.     

Functional characterization of the stunt lemma palea 1 mutant allele in rice

The rice stunted lemma/palea 1 (slp1) mutant displays a dwarf, shortened panicle length, degenerated lemma and palea, and sterility. A previous study suggested that a missense mutation at the sixth amino acid of the OsSPL16 protein was likely to be responsible for the slp1 mutant phenotype. The current study shows that the overexpression of the wild-type OsSPL16 allele in slp1/slp1 and Slp1/slp1 mutants was unable to convert the slp1 mutant phenotype to normal. However, the introduction of the mutant OsSPL16 allele into a normal rice cultivar led to the slp1 mutant phenotype in transgenic plants. These results indicated that the missense mutation in OsSPL16 creates a neomorphic allele, which affects plant height and the development of the inflorescence and spikelet.     

Comparative Proteomic Analysis Reveals Nitrogen Fertilizer Increases Spikelet Number per Panicle in Rice by Repressing Protein Degradation and 14-3-3 Proteins

The spikelet number per panicle is established in the early stages of panicle development. Nitrogen fertilizer application before panicle initiation is known to increase spikelet number, which is one of the most important traits in rice productivity determination. However, the basic proteomic mechanism remains poorly understood. The present study shows that nitrogen fertilizer significantly increased spikelet number and grain yield in rice. Proteomic variations were further analyzed in young panicles at the secondary panicle branch initiation and spikelet meristem initiation under nitrogen fertilizer treatment. Proteomic analysis identified 63 proteins with significant differential accumulation in young panicles under nitrogen fertilizer treatment. Proteolysis represents the largest functional category, which suggests that protein degradation is an important pathway in the response to nitrogen fertilizer. Importantly, nitrogen fertilizer significantly reduced 14-3-3 proteins, which interact with key enzymes associated with carbon and nitrogen metabolism, and the rice FT homologue Hd3a. Real-time PCR revealed that Hd3a signaling is also repressed by nitrogen fertilizer in leaves. This study contributes to a better understanding of the regulation of nitrogen fertilizers in the flowering pathway leading to panicle development. The identification of novel genes provides new insight into the profound impacts of nitrogen fertilizer on panicle development in rice.     

A Pyramid Breeding of Eight Grain-yield Related Quantitative Trait Loci Based on Marker-assistant and Phenotype Selection in Rice （Oryza sativa L.）

1000-Grain weight and spikelet number per panicle are two important components for rice grain yield.In our previous study,eight quantitative trait loci(QTLs)conferring spikelet number per panicle and 1000-grain weight were mapped through sequencing-based genotyping of 150 rice recombinant inbred lines(RILs).In this study,we validated the effects of four QTLs from Nipponbare using chromosome segment substitution lines(CSSLs),and pyramided eight grain yield related QTLs.The new lines containing the eight QTLs with positive effects showed increased panicle and spikelet size as compared with the parent variety 93-11.We further proposed a novel pyramid breeding scheme based on marker-assistant and phenotype selection(MAPS).This scheme allowed pyramiding of as many as 24 QTLs at a single hybridization without massive cross work.This study provided insights into the molecular basis of rice grain yield for direct wealth for high-yielding rice breeding.     

MATE transporter GFD1 cooperates with sugar transporters,mediates carbohydrate partitioning and controls grain-filling duration,grain size and number in rice

More than half of the world's food is provided by cereals, as humans obtain >60% of daily calories from grains. Producing more carbohydrates is always the final target of crop cultivation. The carbohydrate partitioning pathway directly affects grain yield, but the molecular mechanisms and biological functions are poorly understood, including rice (Oryza sativa L.), one of the most important food sources. Here, we reported a prolonged grain filling duration mutant 1 (gfd1), exhibiting a long grain-filling duration, less grain number per panicle and bigger grain size without changing grain weight. Map-based cloning and molecular biological analyses revealed that GFD1 encoded a MATE transporter and expressed high in vascular tissues of the stem, spikelet hulls and rachilla, but low in the leaf, controlling carbohydrate partitioning in the stem and grain but not in the leaf. GFD1 protein was partially localized on the plasma membrane and in the Golgi apparatus, and was finally verified to interact with two sugar transporters, OsSWEET4 and OsSUT2. Genetic analyses showed that GFD1 might control grain-filling duration through OsSWEET4, adjust grain size with OsSUT2 and synergistically modulate grain number per panicle with both OsSUT2 and OsSWEET4. Together, our work proved that the three transporters, which are all initially classified in the major facilitator superfamily family, could control starch storage in both the primary sink (grain) and temporary sink (stem), and affect carbohydrate partitioning in the whole plant through physical interaction, giving a new vision of sugar transporter interactome and providing a tool for rice yield improvement.     

云南不同类型一季稻产量形成及其与气象因子的关系

利用云南省14个不同海拔农业气象观测站1994-2010年水稻大田发育期和产量观测资料,研究水稻产量形成及气象因子对低产水稻产量构成因素的影响;根据14个站点水稻产量构成因素的系统聚类分析结果和水稻类型,将水稻分为低产粳型、低产籼型、高产粳型和高产籼型4个类型.对这4类水稻产量构成因素的分析结果表明,单位面积颖花量与产量呈极显著正相关,低产粳型水稻产量主要受结实率和颖花量影响,其他3类颖花量对产量影响较大.低产粳型水稻主要受低温的影响,孕穗期低温降低颖花量和有效穗数,孕穗期和抽穗开花期低温增加空壳率,以平均气温、平均最高气温和冷积温的影响较大;乳熟前期较低的平均气温增加秕谷率,同时降低千粒重.低产籼型水稻产量构成因素受多种气象因子的综合影响明显;分蘖期和拔节期一定程度的增温不利于增加低产籼型水稻有效穗数,而分蘖期较多的日照时数和较大的平均气温日较差有利于有效穗数的增加,分蘖期和拔节期气温和日照时数与每穗粒数的关系呈“抛物线型”;低产籼型水稻空壳率一定程度上受抽穗开花期低温的影响,而乳熟前期高温少雨不仅增加秕谷率,还导致千粒重明显下降.     

Studies on the nature of relationships between grain size,spikelet number,grain yield and spikelet filling in late duration varieties of rice (Oryza sativa L.)

Summary Studies on the nature of relationship between grain size (in terms of thousand grain weight), grain number, rate of filling and yield have been worked out with late duration (140–150 days) rice varieties. Grain number per m², grains per panicle and yield showed a negative relationship with grain size. There was no compensatory balance between grain size and grain number within a range of thousand grain weight of 15 to 30 g. Based on the peaks in grain filling activity, the varieties were classified into four groups. There was no relationship between grain size and rate of grain filling thereby suggesting that grain filling is determined by inherent endogenous factors.     

The contribution of intersubspecific hybridization to?the breeding of?super-high-yielding japonica rice in northeast China

Hybridization between indica and japonica rice combined with utilization of ideal plant type has led to the development of high-yielding japonica rice in northern China. However, the contribution at the genomic level of intersubspecific hybridization to the increased yield of northern Chinese japonica rice is uncertain. In this study, we analyzed the genomic pedigree of descendants of hybridization between indica and japonica rice grown in northeastern China between 1963 and 2008. Simple sequence repeat markers indicated that since 1990 the genetic diversity among northern japonica cultivars was enriched. Genome-wide analysis with subspecies-specific indel and intron length polymorphism markers showed indica-allele frequencies were significantly increased in cultivars bred after 1990, and were significantly positively correlated with spikelet number per panicle and significantly negatively correlated with panicle number per plant. Among eight genes controlling agronomic traits, GN1a and GS3 were partially fixed in the genome of northern japonica cultivars. In contrast, Waxy and qSH1 were eliminated, whereas DEP1 and qSW5 were retained. Indica germplasm is an important contributor to the increased yield of northern japonica rice. Breeding for high yield and grain quality in combination is a complicated process and difficult to achieve when relying on only one or several functional genes, thus the selection expertise of the breeder remains critical.     

Polyamines and ethylene in rice young panicles in response to soil drought during panicle differentiation

This study tested the hypothesis that polyamines (PA) and ethylene (ETH) mediate the effects of soil drought on spikelet development in rice (Oryza sativa L.). Two rice cultivars, Yong You-2640 and Yang Dao-6, with vastly different panicle sizes were grown in pots under three soil moisture treatments: well-watered (WW), moderate soil drought (MD) and severe soil drought (SD), from the onset of panicle initiation to the pollen completion stage. MD treatment significantly increased spikelet differentiation, spikelet number per panicle, fully filled grain percentage and grain yield, decreasing the percentage of degenerated spikelets, sterile spikelets and partially filled grains compared to WW treatment. In contrast, SD treatment showed opposite effects. MD also increased the contents of free spermidine (Spd), free spermine (Spm) and the ratios of free putrescine, free-Spd and free-Spm to 1-aminocylopropane-1-carboxylic acid (ACC), decreasing the ETH evolution rate and ACC content in young panicles. In contrast, SD treatment showed opposite effects. Furthermore, free-Spd and free-Spm contents increased significantly, while ETH and ACC levels, and the percentage of degenerated and sterile spikelets decreased significantly under application of Spd or an inhibitor of ETH synthesis. The results were reversed when ACC or an inhibitor of Spd and Spm synthesis was applied. These findings suggest antagonistic interactions between free-PA (Spd and Spm) and ETH in response to soil drought, mediating spikelet development in rice.