C-TERMINAL DOMAIN PHOSPHATASE-LIKE 3 contributes to GA-mediated growth and flowering by interaction with DELLA proteins.

Gibberellic acid (GA) plays a central role in many plant developmental processes and is crucial for crop improvement. DELLA proteins, the core suppressors in the GA signaling pathway, are degraded by GA via the 26S proteasomal pathway to release the GA response. However, little is known about the phosphorylation-mediated regulation of DELLA proteins. In this study, we combined GA response assays with protein-protein interaction analysis to infer the connection between Arabidopsis thaliana DELLAs and the C-TERMINAL DOMAIN PHOSPHATASE-LIKE 3 (CPL3), a phosphatase involved in the dephosphorylation of RNA polymerase II. We show that CPL3 directly interacts with DELLA proteins and promotes DELLA protein stability by inhibiting its degradation by the 26S proteasome. Consequently, CPL3 negatively modulates multiple GA-mediated processes of plant development, including hypocotyl elongation, flowering time, and anthocyanin accumulation. Taken together, our findings demonstrate that CPL3 serves as a novel regulator that could improve DELLA stability and thereby participate in GA signaling transduction.

Fine mapping of the grain chalkiness quantitative trait locus qCGP6 reveals the involvement of Wx in grain chalkiness formation.

Grain chalkiness is an important index of rice appearance quality and is negatively associated with rice processing and eating quality. However, the genetic mechanism underlying chalkiness formation is largely unknown. To identify the genetic basis of chalkiness, 410 recombinant inbred lines (RILs) derived from two representative indica rice varieties, Shuhui498 (R498) and Yihui3551 (R3551), were used to discover quantitative trait loci (QTLs). The two parental lines and RILs were grown in three locations in China under three controlled fertilizer application levels. Analyses indicated that chalkiness was significantly affected by genotype, the environment, and the interaction between the two, and that heritability was high. Several QTLs were isolated, including the two stable QTLs qCGP6 and qCGP8. Fine mapping and candidate gene verification of qCGP6 showed that Wx may play a key role in chalkiness formation. Chromosomal segment substitution lines (CSSLs) and near-isogenic lines (NILs) carrying the Wxa or Wxin allele produced more chalky grain than the R498 parent. A similar result was also observed in the 3611 background. Notably, the effect of the Wx genotype on rice chalkiness was shown to be dependent on environmental conditions, and Wx alleles exhibited different sensitivities to shading treatment. Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), the Wxa promoter region was successfully edited; down-regulating Wx alleviates chalkiness formation in NILR498-Wxa. This study developed a new strategy for synergistic improvement of eating and appearance qualities in rice, and created a novel Wx allele with great potential in breeding applications.

Wide Grain 3, a GRAS Protein, Interacts with DLT to Regulate Grain Size and Brassinosteroid Signaling in Rice.

BACKGROUND: Grain size is a direct determinant of grain weight and yield in rice; however, the genetic and molecular mechanisms determining grain size remain largely unknown. FINDINGS: We identified a mutant, wide grain 3 (wg3), which exhibited significantly increased grain width and 1000-grain weight. Cytological analysis showed that WG3 regulates grain size by affecting cell proliferation. MutMap-based gene cloning and a transgenic experiment demonstrated that WG3 encodes a GRAS protein. Moreover, we found that WG3 directly interacts with DWARF AND LOW-TILLERING (DLT), a previously reported GRAS protein, and a genetic experiment demonstrated that WG3 and DLT function in a common pathway to regulate grain size. Additionally, a brassinosteroid (BR) sensitivity test suggested that WG3 has a positive role in BR signaling in rice. Collectively, our results reveal a new genetic and molecular mechanism for the regulation of grain size in rice by the WG3-DLT complex, and highlight the important functions of the GRAS protein complex in plants. CONCLUSION: WG3 functions directly in regulating grain size and BR signaling in rice.

Short grain 5 controls grain length in rice by regulating cell expansion.

Grain shape is a crucial determinant of grain weight and quality and plays a vital role in rice breeding. Although many grain shape-related genes have been reported, the regulatory relationship between them has not been well characterized in rice. In this study, we report the isolation of a short-grain-length mutant called sg5 from the heavy-panicle-type hybrid rice elite restorer line 'ShuhuiR498' (R498) after ethyl methanesulfonate (EMS) treatment. MutMap cloning revealed that SG5 encodes a Myb-like transcription factor. A missense mutation in the first exon of SG5 was found to cause an amino acid change from leucine to proline at position 197 in the mutant SG5 protein. Gene knockout and genetic complementation experiments confirmed that the point mutation in SG5 was responsible for the sg5 mutant phenotype. SG5 is mainly expressed in young panicles and hulls. In addition, the SG5 protein is found in the nucleus and does not affect subcellular localization. Histochemical observation and gene expression analysis indicated that SG5 regulates spikelet hull development by mediating cell expansion. Moreover, the expression levels of BG1, GS2, and DEP1 were reduced in sg5 plants, and dual-luciferase (LUC) assays showed that SG5 can bind to the BG1 gene promoter. The effect of pyramiding sg5 and GS3 suggests that sg5 and GS3 regulate grain length independently. The results of our study show that the missense mutation in sg5 is essential for the molecular function of SG5 and SG5 is involved in regulating cell expansion and expression of grain-shape-related genes to regulate grain length. This work provides new data to help study and understand the molecular function of SG5.

OsBSK2, a putative brassinosteroid-signalling kinase, positively controls grain size in rice.

Grain size is an important trait that directly affects grain yield in rice; however, the genetic and molecular mechanisms regulating grain size remain unclear. In this study, we identified a mutant, grain length and grain weight 10 (glw10), which exhibited significantly reduced grain length and grain weight. Histological analysis demonstrated that GLW10 affects cell expansion, which regulates grain size. MutMap-based gene mapping and transgenic experiments demonstrated that GLW10 encodes a putative brassinosteroid (BR) signalling kinase, OsBSK2. OsBSK2 is a plasma membrane protein, and an N-myristoylation site is needed for both membrane localization and function. OsBSK2 directly interacts with the BR receptor kinase OsBRI1; however, genetic experiments have demonstrated that OsBSK2 may regulate grain size independent of the BR signalling pathway. OsBSK2 can form a homodimer or heterodimer with OsBSK3 and OsBSK4, and silencing OsBSK2, OsBSK3, and OsBSK4 reduce grain size. This indicates that OsBSKs seem to function as homodimers or heterodimers to positively regulate grain size in rice. OsBSK2/3/4 are all highly expressed in young panicles and spikelet hulls, suggesting that they control grain size. In summary, our results provide novel insights into the function of BSKs in rice, and identify novel targets for improving grain size during crop breeding.

Fine mapping and candidate gene analysis of qGSN5, a novel quantitative trait locus coordinating grain size and grain number in rice.

KEY MESSAGE: qGSN5, a novel quantitative trait locus coordinating grain size and grain number in rice, was fine-mapped to an 85.60-kb region. GS3 may be a suppressor of qGSN5. Grain size and grain number are two factors that directly determine rice grain yield; however, the underlying genetic mechanisms are complicated and remain largely unclear. In this study, a chromosome segment substitution line (CSSL), CSSL28, which showed increased grain size and decreased grain number per panicle, was identified in a set of CSSLs derived from a cross between 93-11 (recipient) and Nipponbare (donor). Four substitution segments were identified in CSSL28, and the substitution segment located on chromosome 5 was responsible for the phenotypes of CSSL28. Thus, we defined this quantitative trait locus (QTL) as grain size and grain number 5 (qGSN5). Cytological and quantitative PCR analysis showed that qGSN5 regulates the development of the spikelet hull by affecting cell proliferation. Genetic analysis showed that qGSN5 is a semi-dominant locus regulating grain size and grain number. Through map-based cloning and overlapping substitution segment analysis, qGSN5 was finally delimited to an 85.60-kb region. Based on sequence and quantitative PCR analysis, Os05g47510, which encodes a P-type pentatricopeptide repeat protein, is the most likely candidate gene for qGSN5. Pyramiding analysis showed that the effect of qGSN5 was significantly lower in the presence of a functional GS3 gene, indicating that GS3 may be a suppressor of qGSN5. In addition, we found that qGSN5 could improve the grain shape of hybrid rice. Together, our results lay the foundation for cloning a novel QTL coordinating grain size and grain number in rice and provide a good genetic material for long-grain hybrid rice breeding.

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), and identified the causal gene as GRAIN NUMBER 1A/CYTOKININ OXIDASE 2 (Gn1A/OsCKX2). The null gn1a allele from rice line R498 (gn1aR498 ) improved lodging resistance through increasing the culm diameter and promoting crown root development. Loss-of-function of Gn1a/OsCKX2 led to cytokinin accumulation in the crown root tip and accelerated the development of adventitious roots. Gene pyramiding between the null gn1aR498 allele with two gain-of-function alleles, STRONG CULM 2 (SCM2) and SCM3, further improved lodging resistance. Moreover, Gn1a/OsCKX2 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 tailor-made crop improvement of both yield and lodging resistance in rice.

OsSPL9 Regulates Grain Number and Grain Yield in Rice.

Rice grain yield consists of several key components, including tiller number, grain number per panicle (GNP), and grain weight. Among them, GNP is mainly determined by panicle branches and spikelet formation. In this study, we identified a gene affecting GNP and grain yield, OsSPL9, which encodes SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) family proteins. The mutation of OsSPL9 significantly reduced secondary branches and GNP. OsSPL9 was highly expressed in the early developing young panicles, consistent with its function of regulating panicle development. By combining expression analysis and dual-luciferase assays, we further confirmed that OsSPL9 directly activates the expression of RCN1 (rice TERMINAL FLOWER 1/CENTRORADIALIS homolog) in the early developing young panicle to regulate the panicle branches and GNP. Haplotype analysis showed that Hap3 and Hap4 of OsSPL9 might be favorable haplotypes contributing to high GNP in rice. These results provide new insights on high grain number breeding in rice.

Pan-genome analysis of 33 genetically diverse rice accessions reveals hidden genomic variations.

Structural variations (SVs) and gene copy number variations (gCNVs) have contributed to crop evolution, domestication, and improvement. Here, we assembled 31 high-quality genomes of genetically diverse rice accessions. Coupling with two existing assemblies, we developed pan-genome-scale genomic resources including a graph-based genome, providing access to rice genomic variations. Specifically, we discovered 171,072 SVs and 25,549 gCNVs and used an Oryza glaberrima assembly to infer the derived states of SVs in the Oryza sativa population. Our analyses of SV formation mechanisms, impacts on gene expression, and distributions among subpopulations illustrate the utility of these resources for understanding how SVs and gCNVs shaped rice environmental adaptation and domestication. Our graph-based genome enabled genome-wide association study (GWAS)-based identification of phenotype-associated genetic variations undetectable when using only SNPs and a single reference assembly. Our work provides rich population-scale resources paired with easy-to-access tools to facilitate rice breeding as well as plant functional genomics and evolutionary biology research.

Leaf-derived ABA regulates rice seed development via a transporter-mediated and temperature-sensitive mechanism.

Long-distance transport of the phytohormone abscisic acid (ABA) has been studied for ~50 years, yet its mechanistic basis and biological significance remain very poorly understood. Here, we show that leaf-derived ABA controls rice seed development in a temperature-dependent manner and is regulated by defective grain-filling 1 (DG1), a multidrug and toxic compound extrusion transporter that effluxes ABA at nodes and rachilla. Specifically, ABA is biosynthesized in both WT and dg1 leaves, but only WT caryopses accumulate leaf-derived ABA. Our demonstration that leaf-derived ABA activates starch synthesis genes explains the incompletely filled and floury seed phenotypes in dg1 Both the DG1-mediated long-distance ABA transport efficiency and grain-filling phenotypes are temperature sensitive. Moreover, we extended these mechanistic insights to other cereals by observing similar grain-filling defects in a maize DG1 ortholog mutant. Our study demonstrates that rice uses a leaf-to-caryopsis ABA transport-based mechanism to ensure normal seed development in response to variable temperatures.

Characterization of a novel allele of bc12/gdd1 indicates a differential leaf color function for BC12/GDD1 in Indica and Japonica backgrounds.

Leaf color is directly associated with plant photosynthesis. Here, we have isolated and identified a spontaneous rice mutant named yd1 that has yellowish leaves and dwarf stature. Map-based cloning reveals that YD1 encodes a previously reported kinesin protein from the kinesin-4 subfamily, BC12/GDD1. Arginine-328 is replaced by leucine in yd1, BC12328Leu. YD1 is mainly expressed in leaves and is involved in chlorophyll (Chl) synthesis. The yd1 mutant had less Chl and a reduced and disordered thylakoid ultrastructure. In yd1 plants, Chl biosynthesis and photosynthesis associated gene expression was decreased and Chl degradation gene expression was increased, thereby leading to a reduced photosynthesis rate and grain yield. In this study we reveal that the novel BC12328Leu allele of BC12 modulated plant leaf color in yd1 plants, which has not been previously reported in studies of BC12/GDD1/MTD1/SRG1. Gene knockout results indicated that YD1 regulates leaf color in the indica rice background, but not in the japonica rice background. Our study provides new insights into molecular regulation of rice growth by BC12/GDD1 in different genetic backgrounds.

GWC1 is essential for high grain quality in rice.

Appearance quality is an important determinant of rice quality. Many genes that affect grain appearance quality have been identified, but the regulatory mechanisms that contribute to this trait remain unclear. Here, two grains with chalkiness (gwc1) mutants, gwc1-1 and gwc1-2, were identified from an EMS-mutagenized population of indica rice cultivar Shuhui498 (R498). The gwc1 mutants had poor grain appearance quality consistent with the measured values for the percentage of grains with chalkiness, square of chalky endosperm, the total starch, amylose and sucrose contents. Milling quality and grain size were also affected in the gwc1 mutants. The gwc1-1 and gwc1-2 were found to be loss-of-function allelic mutants. GWC1 was mapped to the long arm of rice chromosome 8 using the MutMap strategy and incorrectly annotated in the reference genome for Nipponbare (MSU). The GWC1 gene corresponds to the WTG1/OsOTUB1 gene, which encodes an otubain-like protease with deubiquitinating activity that is homologous to human OTUB1. GWC1 transcripts accumulated to high levels in early endosperm after fertilization and developing inflorescences, and GWC1-green fluorescent protein (GFP) signal was detected in the nucleus and cytoplasm. GWC1 is likely to regulate grain appearance quality through genes involved in sucrose metabolism and starch biosynthesis. Overall, the present findings reveal that GWC1 is important for grain quality and yield due to its effects on grain chalkiness and size.

[Symbiotic bacteria facilitate algal growth and oil biosynthesis in Scenedesmus obliquus]. / å ±ç”ŸèŒä¿ƒè¿›æ–œç”Ÿæ 藻生长和油脂合成.

We isolated bacteria from the phycosphere of Scenedesmus obliques and sequenced 16S rDNAs to establish algae-bacterial co-culture systems. Further, we examined effects of the bacteria on algal growth, and parameters associated with physio-biochemical and oil-producing characteristics of S. obliquus. Seven bacterial strains were isolated, including Micrococcus (strains 1-1, 1-2 and 1-3), Pseudomonas sp. (strains 2-1 and 2-2), Exiguobacterium (strain-3) and Staphylococcus sp. (strain-4). Among them, two bacteria (strain 1-2 and strain 2-1) were characterized as the dominant growth-promoting bacterial strains, which significantly increased algal growth, pigment production, and oil enrichment. After eight days cultivation under microalgal-bacterial (strain 1-2) symbiotic systemat at an initial ratio of 1:10, biomass of S. obliquus was 4.27 g·L-1, about 46.0% higher than that of the control. The contents of chlorophyll a, chlorophyll b and carotenoids were increased by 12.1%, 16.7% and 25.0%, respectively. Oil content was increased by 14.0% and reached to 25.7%, and the oleic acid content was significantly higher than that of the control and up to 16.4%. When co-cultured with Pseudomonas sp. (strain 2-1) for eight days at an initial ratio of 1:5, algal biomass, chlorophyll a, chlorophyll b and carotenoids contents were higher than that of the control by 47.9%, 16.0%, 17.5% and 19.9%, respectively. The total oil (27.1%) and oleic acid (18.2%) contents were increased by 20.4% and 64.0%, respectively. We concluded that Micrococcus (strain 1-2) and Pseudomonas sp. (strain 2-1) could significantly promote algal growth and increase oil production by their beneficial interaction with S. obliques, which could be potentially used in commercial production of S. obliques.

Mitochondria-Associated Pyruvate Kinase Complexes Regulate Grain Filling in Rice.

Grain filling is a complex agronomic trait that directly determines grain weight and quality in rice (Oryza sativa). Nevertheless, key factors affecting grain filling remain poorly understood. Here, we identified a grain filling gene, OsPK3, encoding a pyruvate kinase (PK). The loss of function of OsPK3 caused reduced PK activity and Suc translocation defects from source to sink in rice, which led to compromised grain filling. OsPK3 was constitutively expressed but had relatively higher expression levels in leaf and developing caryopsis and specific expression signals in tissues involved in Suc transport and unloading, supporting its biological function in regulation of grain filling by affecting Suc translocation. Subcellular localization analysis of OsPK3 revealed its association with mitochondria, and OsPK3 physically interacted and formed heterodimers in vivo with two other PK isozymes, OsPK1 and OsPK4. Both OsPK1 and OsPK4 localized to the mitochondria and cytosol and were recruited to the mitochondria by OsPK3. Despite their high sequence similarity, OsPK1 and OsPK4 had distinct expression patterns. As observed for ospk3, disruption of OsPK1 caused pleiotropic defects, while OsPK4 loss of function led to severely chalky grains without other obvious defects. Collectively, we revealed that two mitochondria-associated pyruvate kinase complexes, OsPK3-OsPK1/OsPK4, are involved in regulation of grain filling by stage-specific fine-tuning of Suc translocation.

Membrane-associated xylanase-like protein OsXYN1 is required for normal cell wall deposition and plant development in rice.

The rice (Oryza sativa) genome encodes 37 putative ß-1,4-xylanase proteins, but none of them has been characterized at the genetic level. In this work, we report the isolation of slim stem (ss) mutants with pleiotropic defects, including dwarfism, leaf tip necrosis, and withered and rolled leaves under strong sunlight. Map-based cloning of the ss1 mutant identified the candidate gene as OsXyn1 (LOC_03g47010), which encodes a xylanase-like protein belonging to the glycoside hydrolase 10 (GH10) family. OsXyn1 was found to be widely expressed, especially in young tissues. Subcellular localization analysis showed that OsXyn1 encodes a membrane-associated protein. Physiological analysis of ss1 and the allelic ss2 mutant revealed that water uptake was partially compromised in these mutants. Consistently, the plant cell wall of the mutants exhibited middle lamella abnormalities or deficiencies. Immunogold assays revealed an unconfined distribution of xylan in the mutant cell walls, which may have contributed to a slower rate of plant cell wall biosynthesis and delayed plant growth. Additionally, water deficiency caused abscisic acid accumulation and triggered drought responses in the mutants. The findings that OsXyn1 is involved in plant cell wall deposition and the regulation of plant growth and development help to shed light on the functions of the rice GH10 family.

Sclerenchyma cell thickening through enhanced lignification induced by OsMYB30 prevents fungal penetration of rice leaves.

Broad-spectrum resistance is highly preferred in crop breeding programmes. Previously, we have reported the identification of the broad-spectrum resistance-Digu 1 (bsr-d1) allele from rice Digu. The bsr-d1 allele prevents activation of Bsr-d1 expression by Magnaporthe oryzae infection and degradation of H2 O2 by peroxidases, leading to resistance to M. oryzae. However, it remains unknown whether defence pathways other than H2 O2 burst and peroxidases contribute to the bsr-d1-mediated immunity. Blast resistance was determined in rice leaves by spray and punch inoculations. Target genes of OsMYB30 were identified by one-hybrid assays in yeast and electrophoretic mobility shift assay. Lignin content was measured by phloroglucinol-HCl staining, and acetyl bromide and thioacidolysis methods. Here, we report the involvement of the OsMYB30 gene in bsr-d1-mediated blast resistance. Expression of OsMYB30 was induced during M. oryzae infection or when Bsr-d1 was knocked out or downregulated, as occurs in bsr-d1 plants upon infection. We further found that OsMYB30 bound to and activated the promoters of 4-coumarate:coenzyme A ligase genes (Os4CL3 and Os4CL5) resulting in accumulation of lignin subunits G and S. This action led to obvious thickening of sclerenchyma cells near the epidermis, inhibiting M. oryzae penetration at the early stage of infection. Our study revealed novel components required for bsr-d1-mediated resistance and penetration-dependent immunity, and advanced our understanding of broad-spectrum disease resistance.

Retraction: Facile preparation of bithiazole-based material for inkjet printed light-emitting electrochemical cell.

[This retracts the article DOI: 10.1039/C9RA00093C.].

A minireview of hydroamination catalysis: alkene and alkyne substrate selective, metal complex design.

Organic compounds that contain nitrogen are very important intermediates in pharmaceutical and chemical industry. Hydroamination is the reaction that can form C-N bond with high atom economy. The research progress in metals catalyzed hydroamination of alkenes and alkynes from the perspective of reaction mechanism is categorized and summarized.

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 OsSPL18 exhibited 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 OsSPL18 is 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 DEP1 promoter, 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.