Rice AGL1 determines grain size and sterile lemma identity

The grass spikelet is a unique inflorescence structure that determines grain size.Although many genetic factors have been well characterized for grain size and glume development,the underlying molecular mechanisms in rice are far from established.Here,we isolated rice gene,AGL1 that controlled grain size and determines the fate of the sterile lemma.Loss of function of AGL1 produced larger grains and reduced the size of the sterile lemma.Larger grains in the agl1 mutant were caused by a larger number of cells that were longer and wider than in the wild type.The sterile lemma in the mutant spikelet was converted to a rudimentary glume-like organ.Our findings showed that the AGL1(also named LAX1)protein positively regulated G1 expression,and negatively regulated NSG1 expression,thereby affecting the fate of the sterile lemma.Taken together,our results revealed that AGL1 played a key role in negative regulation of grain size by controlling cell proliferation and expansion,and supported the opinion that rudimentary glume and sterile lemma in rice are homologous organs.

Genome-wide association study and transcriptome analysis reveal new QTL and candidate genes for nitrogen-deficiency tolerance in rice

The development of rice cultivars with improved nitrogen use efficiency(NUE)is desirable for sustainable agriculture.Achieving this goal depends in part on understanding how rice responds to low soil nitrogen(N)and identifying causative genes underlying this trait.To identify quantitative trait loci(QTL)or genes associated with low N response,we conducted a genome-wide association study(GWAS)using a diverse panel of 230 rice accessions and performed a transcriptomic investigation of rice accessions with differential responses to low N stress at two N levels.We detected 411 GWAS-associated genes in 5 QTL and 2722 differentially expressed genes in response to low N,of which 24 were identified by both methods and ranked according to gene annotations,literature queries,gene expression,and genetic diversity analysis.The large-scale datasets obtained from this study reveal low N-responsive characteristics and provide insights towards understanding the regulatory mechanisms of N-deficiency tolerance in rice,and the candidate genes or QTL would be valuable resources for increasing rice NUE via molecular biotechnology.

Temporal dynamic changes of connexin 43 expression in C6 cells following lipopolysaccharide stimulation

Connexin 43, a gap junction protein, is expressed mainly in glia in the central nervous system. Neuroinflammation plays an important role in central nervous system injury. Changes to glial connexin 43 levels and neuroinflammation may trigger brain injury and neurodegenerative diseases To illustrate the relationship between connexin 43 and neuroinflammation, this study investigated how connexin 43 expression levels change in lipopolysaccharide-stimulated rat C6 glioma cells. C6 cells were treated with 0.05, 0.25, 0.5, 1,2.5 and 5 IJg/mL lipopolysaccharide for 24 hours. The nitrite estimation-detected nitric oxide release level was elevated substantially after lipopolysaccharide stimulation. To test the transcriptional level changes of inducible nitric oxide synthase, tumor necrosis factor-a and connexin 43 mRNA, C6 cells were treated with 5 pg/mL lipopolysaccharide for 3 48 hours. Reverse transcription-PCR showed that the expression of inducible nitric oxide synthase and tumor necrosis factor-a mRNA increased over time, but connexin 43 mRNA levels increased in lipopolysaccharide-stimulated C6 cells at 3 and 6 hours, and then decreased from 12 to 48 hours. Connexin 43 protein expression was detected by immunofluorescence staining, and the protein levels matched the mRNA expression levels. These results suggest that connexin 43 expression is biphasic in lipopo^ysacchadde-induced neuroinflammation in C6 cells, which may be correlated with the connexin 43 compensatory mechanism.

Carotenoid isomerase regulates rice tillering and grain productivity by its biosynthesis pathway

Tillers are unique inflorescence-like branches in grasses,and their number determines the panicle number,plant architecture,and yield(Shang et al.,2021).Tiller formation mainly undergoes axillary meristem(AM)initiation and tiller bud outgrowth(Wang et al.,2018;Yan et al.,2023).The rice(Oryza sativa)KNOX gene OSH1 is expressed in AMs,and an osh1 mutant produces fewer tillers(Tanaka et al.,2015).

Effect of functional groups on tribological properties of lubricants and mechanism investigation

Nine organic compounds were utilized as model lubricants to investigate the impact of functional groups on tribological performances.Nonanoic Acid with carboxyl showed the best lubrication properties,and fluid film and tribofilm were coexistent in its friction test,bringing a low friction coefficient and wear rate.In addition,the lubricant with low friction coefficient corresponded to high adsorption energy in density functional theory(DFT)calculations.And the lubricant forming adsorption film with large surface energy displayed small wear rate in friction test.Moreover,adsorption energies positively correlated surface energies.Based on the experimental results,the action mechanism of functional groups on tribological properties of lubricants was proposed.Various functional groups make lubricant molecules show different adsorption energies and surface energies.Lubricant molecules with high adsorption energy are more likely to adsorb on substrates and form a vertical monolayer,which can maintain a regular molecular brush structure during friction and bring a low friction coefficient.And lubricant molecules with high surface energy may be more prone having tribochemical reactions during friction and forming protective tribofilm,which leads to a low wear rate.

A Rare Allele of GS2 Enhances Grain Size anc Grain Yield in Rice

Grain size determines grain weight and affects grain quality. Several major quantitative trait loci （QTLs） regulating grain size have been cloned; however, our understanding of the underlying mechanism that regulates the size of rice grains remains fragmentary. Here, we report the cloning and characterization of a dominant QTL, GRAIN SIZE ON CHROMOSOME 2 （GS2）, which encodes Growth-Regulating Factor 4 （OsGRF4）, a transcriptional regulator. GS2 localizes to the nucleus and may act as a transcription activator. A rare mutation of GS2 affecting the binding site of a microRNA, OsmiR396c, causes elevated expression of GS2/OsGRF4. The increase in GS2 expression leads to larger cells and increased numbers of cells, which thus enhances grain weight and yield. The introduction of this rare allele of GS2/OsGRF4 into rice cultivars could significantly enhance grain weight and increase grain yield, with possible applications in breeding high-yield rice varieties.

The GW2-WG1-OsbZIP47 pathway controls grain size and weight in rice

Regulation of seed size is a key strategy for improving crop yield and is also a basic biological question.However,the molecular mechanisms by which plants determine their seed size remain elusive.Here,we report that the GW2-WG1-OsbZIP47 regulatory module controls grain width and weight in rice.WG1,which encodes a glutaredoxin protein,promotes grain growth by increasing cell proliferation.Interestingly,WG1 interacts with the transcription factor OsbZIP47 and represses its transcriptional activity by associating with the transcriptional co-repressor ASP1,indicating that WG1 may act as an adaptor protein to recruit the transcriptional co-repressor.In contrary,OsbZIP47 restricts grain growth by decreasing cell proliferation.Further studies reveal that the E3 ubiquitin ligase GW2 ubiquitinates WG1 and targets it for degradation.Genetic analyses confirm that GW2,WG1,and OsbZIP47 function in a comm on pathway to control grain growth.Taken together,ourfindi ngs reveal a genetic and molecular framework for the control of grain size and weight by the GW2-WG1-OsbZIP47 regulatory module,providing new targets for improving seed size and weight in crops.

Rice Ferredoxin-Dependent Glutamate Synthase Regulates Nitrogen-Carbon Metabolomes and Is Genetically Differentiated between japonica and indica Subspecies

Plants assimilate inorganic nitrogen absorbed from soil into organic forms as Gin and Glu through the glutamine synthetase/glutamine：2-oxoglutarate amidotransferase （GS/GOGAT） cycle. Whereas GS cata- lyzes the formation of Gin from Glu and ammonia, GOGAT catalyzes the transfer of an amide group from Gin to 2-oxoglutarate to produce two molecules of Glu. However, the regulatory role of the GS/GOGAT cycle in the carbon-nitrogen balance is not well understood. Here, we report the functional characterization of rice ABNORMAL CYTOKININ RESPONSE 1 （ABC1） gene that encodes a ferredoxin-dependent （Fd）- GOGAT. The weak mutant allele abcl-1 mutant shows a typical nitrogen-deficient syndrome, whereas the T-DNA insertional mutant abcl-2 is seedling lethal. Metabolomics analysis revealed the accumulation of an excessive amount of amino acids with high N/C ratio （Gin and Asn） and several intermediates in the tricarboxylic acid cycle in abcl-1, suggesting that ABC1 plays a critical role in nitrogen assimilation and carbon-nitrogen balance. Five non-synonymous single-nucleotide polymorphisms were identified in the ABC1 coding region and characterized as three distinct haplotypes, which have been highly and specifically differentiated between japonica and indica subspecies. Collectively, these results suggest that ABC1/ OsFd-GOGAT is essential for plant growth and development by modulating nitrogen assimilation and the carbon-nitrogen balance.

ALK,the Key Gene for Gelatinization Temperature,is a Modifier Gene for Gel Consistency in Rice

Gelatinization temperature（GT） is an important parameter in evaluating the cooking and eating quality of rice.Indeed,the phenotype,biochemistry and inheritance of GT have been widely studied in recent times.Previous map-based cloning revealed that GT was controlled by ALK gene,which encodes a putative soluble starch synthase II-3.Complementation vector and RNAi vector were constructed and transformed into Nipponbare mediated by Agrobacterium.Phenotypic and molecular analyses of transgenic lines provided direct evidence for ALK as a key gene for GT.Meanwhile,amylose content,gel consistency and pasting properties were also affected in transgenic lines.Two of four nonsynonymous single nucleotide polymorphisms in coding sequence of ALK were identified as essential for GT.Based on the single nucleotide polymorphisms（SNPs）,two new sets of SNP markers combined with one cleaved amplified polymorphic sequence marker were developed for application in rice quality breeding.

Xiaowei, a New Rice Germplasm for Large-Scale Indoor Research

Dear Editor,Rice (Oryza sativa)is a model monocot plant for biological studies due to its relatively small genome,rich germplasm resources,and high-efficiency transformation methods.Although significant progress has been made in rice genomics and functional genomics research (Li et al.,2018),large-scale indoor research toward better understanding of rice biology is hampered by its long growth period and extreme dependence on the natural environment for cultivation.There are some rice germplasms that have a short growth period and short plant height,such as Kitaake.

Quantitative Trait Loci Mapping of Flag-leaf Ligule Length in Rice and Alignment with ZmLG1 Gene

A doubled haploid （DH） population, which consists of 120 lines derived from anther culture of a typical indica and japonica hybrid‘CJ06＇/‘TNI＇, was used in this study. Ligule lengths of flag leaf were investigated for quantitative trait loci （QTL） mapping using the DH population. Five QTLs （qLL-2, qLL.4, qLL-6, qLL-IO and qLL-12） controlling the ligule length （LL） were detected on chromosomes 2, 4, 6, 10 and 12, with the variances explained 11.4%, 13.6%, 27.8%, 22.1% and 11.0%, respectively. Using four known genes of ZmGL1, ZmGL2, ZmGL3 and ZmGL4 in maize from the MaizeGDB, their homologs in rice were aligned and integrated into the existing simple sequence repeats linkage map by in silico mapping. A ZmLG1 homolog gene, OsLG1 encoding a squamosa promoter binding protein, was located between the markers RM255 and RM280, which is just identical to the interval of qLL.4 on the long arm of chromosome 4. The results are beneficial to dissection of the ligule molecular mechanism and the study of cereal evolution.

The Ghd7 transcription factor represses ARE1 expression to enhance nitrogen utilization and grain yield in rice

The genetic improvement of nitrogen use efficiency(NUE)of crops is vital for grain productivity and sustainable agriculture.However,the regulatory mechanism of NUE remains largely elusive.Here,we report that the rice Grain number,plant height,and heading date7(Ghd7)gene genetically acts upstream of ABC1 REPRESSOR1(ARE1),a negative regulator of NUE,to positively regulate nitrogen utilization.As a transcriptional repressor,Ghd7 directly binds to two Evening Element-like motifs in the promoter and intron 1 of ARE1,likely in a cooperative manner,to repress its expression.Ghd7 and ARE1 display diurnal expression patterns in an inverse oscillation manner,mirroring a regulatory scheme based on these two loci.Analysis of a panel of 2656 rice varieties suggests that the elite alleles of Ghd7 and ARE1 have undergone diversifying selection during breeding.Moreover,the allelic distribution of Ghd7 and ARE1 is associated with the soil nitrogen deposition rate in East Asia and South Asia.Remarkably,the combination of the Ghd7 and ARE1 elite alleles substantially improves NUE and yield performance under nitrogen-limiting conditions.Collectively,these results define a Ghd7–ARE1-based regulatory mechanism of nitrogen utilization,providing useful targets for genetic improvement of rice NUE.

Genetic analysis of leaffolder resistance in rice

A double haploid （DH） population, which consists of 120 lines derived from anther culture of a typical indica and japonica hybrid ‘CJ06＇/‘TNI＇, was used to investigate the genetic basis for rice leaffolder resistance. Using a constructed molecular linkage map, five QTLs for rolled leaves were detected on chromosomes 1, 2, 3, 4, and 8. The positive alleles from C J06 on chromosomes 3, 4, and 8 increased the resistance to rice leaffolder, and the alleles from TN1 on chromosomes 1 and 2 also enhanced resistance to leaffolder. The interactions between QTLs were identified and tested, and four conditional interactions were acquired for resistance to rice leaffolder. These loci were located on chromosomes 2, 9, 10, and 11, respectively. QTL pyramiding indicated that the positive alleles affect resistance to leaffolder. The prospective application of this data in rice breeding was also discussed.

Development of nutritious rice with high zinc/selenium and low cadmium in grains through QTL pyramiding

Enriching zinc(Zn) and selenium(Se) levels,while reducing cadmium(Cd) concentration in rice grains is of great benefit for human diet and health.Large natural variations in grain Zn, Se, and Cd concentrations in different rice accessions enable Zn/Sebiofortification and Cd-minimization through molecular breeding. Here, we report the development of new elite varieties by pyramiding major quantitative trait loci(QTLs) that significantly contribute to high Zn/Se and low Cd accumulation in grains. A chromosome segment substitution line CSSLGCC7 with the PA64s-derived GCC7 allele in the 93-11 background, exhibited steadily higher Mn and lower Cd concentrations in grains than those of 93-11. This elite chromosome segment substitution line(CSSL) was used as the core breeding material to cross with CSSLs harboring other major QTLs for essential mineral elements, especially CSSLGZC6 for grain Zn concentration and CSSLGSC5 for grain Se concentration. The CSSLGCC7+GZC6 and CSSLGCC7+GSC5 exhibited lower Cd concentration with higher Zn and Se concentrations in grains, respectively. Our study thus provides elite materials for rice breeding targeting high Zn/Se and low Cd concentrations in grains.

Identification and characterization of Mini1, a gene regulating rice shoot development

The aerial parts of higher plants are generated from the shoot apical meristem （SAM）. In this study, we isolated a small rice （Oryza sativa L.） mutant that showed premature termination of shoot development and was named mini rice 1 （mini1）. The mutant was first isolated from a japonica cultivar Zhonghua11 （ZH11） subjected to ethyl methanesulfonate （EMS） treatment. With bulked segregant analysis （BSA） and mapbased cloning method, Mini1 gene was finally fine-mapped to an interval of 48.6 kb on chromosome 9. Sequence analyses revealed a single base substitution from G to A was found in the region, which resulted in an amino acid change from Gly to Asp. The candidate gene Os09go363900 was predicted to encode a putative adhesion of calyx edges protein ACE （putative HOTHEAD precursor） and genetic complementation experiment confirmed the identity of Minil. Os09go36：3900 contains glucose-methanol-choline （GMC） oxidoreductase and NAD（P）-binding Rossmann-like domain, and exhibits high similarity to Arabidopsis HOTHEAD （HTH）. Expression analysis indicated Minil was highly expressed in young shoots but lowly in roots and the expression level of most genes involved in auxin biosynthesis and signal transduction were reduced in mutant. We conclude that Mini1 plays an important role in maintaining SAM activity and promoting shoot development in rice.

Formyl tetrahydrofolate deformylase affects hydrogen peroxide accumulation and leaf senescence by regulating the folate status and redox homeostasis in rice

It is well established that an abnormal tetrahydrofolate(THF)cycle causes the accumulation of hydrogen peroxide(H_(2)O_(2))and leaf senescence,however,the molecular mechanism underlying this relationship remains largely unknown.Here,we reported a novel rice tetrahydrofolate cycle mutant,which exhibited H_(2)O_(2)accumulation and early leaf senescence phenotypes.Map-based cloning revealed that HPA1 encodes a tetrahydrofolate deformylase,and its deficiency led to the accumulation of tetrahydrofolate,5-formyl tetrahydrofolate and 10-formyl tetrahydrofolate,in contrast,a decrease in 5,10-methenyl-tetrahydrofolate.The expression of tetrahydrofolate cycle-associated genes encoding serine hydroxymethyl transferase,glycine decarboxylase and 5-formyl tetrahydrofolate cycloligase was significantly down-regulated.In addition,the accumulation of H_(2)O_(2)in hpa1 was not caused by elevated glycolate oxidation.Proteomics and enzyme activity analyses further revealed that mitochondria oxidative phosphorylation complex I and complex V were differentially expressed in hpa1,which was consistent with the H_(2)O_(2)accumulation in hpa1.In a further feeding assay with exogenous glutathione(GSH),a non-enzymatic antioxidant that consumes H_(2)O_(2),the H_(2)O_(2)accumulation and leaf senescence phenotypes of hpa1 were obviously compensated.Taken together,our findings suggest that the accumulation of H_(2)O_(2)in hpa1 may be mediated by an altered folate status and redox homeostasis,subsequently triggering leaf senescence.

Involvement of a Putative Bipartite Transit Peptide in Targeting Rice Pheophorbide a Oxygenase into Chloroplasts for Chlorophyll Degradation during Leaf Senescence

Leaf senescence is one of the major factors contributing to the productivity and the grain quality in crops. The regulatory mechanism of leaf senescence remains largely unknown. Here, we report the identification and characterization of a rice e_aarly senescence 1 （easl） mutant, which displayed an early leaf senescence phenotype, accompanying by dwarfism and reduced tiller number, eventually leading to the reduction of grain yield. Map-based cloning revealed that the nuclear gene EAS1 encodes a pheophorbide a oxygenase （PaO）, a key enzyme for chlorophyll breakdown. A highly conserved Thr residue of PaO was mutated into Ile in the easl mutant. Phylogenetic analysis indicates that PaO is an evolutionarily conserved protein, and EAS1 is 68% identical to the Arabidopsis ACCERLERATED CELL DEATH （ACD1） protein. Unlike ACD1 that contains a single transit peptide, EAS1 contains two putative transit peptides at its N-ter- minus, which are essential for its functionality, suggesting that targeting of EAS1 to the chloroplast is likely mediated by a putative bipartite transit peptide. Consistently, only a short version of EAS1 lacking the first putative transit peptide, but not the full-length EAS1, was capable of rescuing the Arabidopsis acdl mutant phenotype. These results suggest that rice EASI represents a functional PaO, which is involved in chlorophyl/degradation and may utilize a unique mechanism for its import into the chloroplast.

Regulation of nitrogen starvation responses by the alarmone(p)ppGpp in rice

Nitrogen is an essential macronutrient for all living organisms and is critical for crop productivity and quality.In higher plants,inorganic nitrogen is absorbed through roots and then assimilated into amino acids by the highly conserved glutamine synthetase/glutamine:2-oxoglutarate aminotransferase(GS/GOGAT)cycle.How nitrogen metabolism and nitrogen starvation responses of plants are regulated remains largely unknown.Previous studies revealed that mutations in the rice ABNORMAL CYTOKININ RESPONSE1(ABC1)gene encoding Fd-GOGAT cause a typical nitrogen deficiency syndrome.Here,we show that ARE2(for ABC1 REPRESSOR2)is a key regulator of nitrogen starvation responses in rice.The are2 mutations partially rescue the nitrogen-deficient phenotype of abc1 and the are2 mutants show enhanced tolerance to nitrogen deficiency,suggesting that ARE2 genetically interacts with ABC1/Fd-GOGAT.ARE2 encodes a chloroplast-localized Rel A/Spo T homolog protein that catalyzes the hydrolysis of guanosine pentaphosphate or tetraphosphate(p)pp Gpp,an alarmone regulating the stringent response in bacteria under nutritional stress conditions.The are2 mutants accumulate excessive amounts of(p)pp Gpp,which correlate with lower levels of photosynthetic proteins and higher amino acid levels.Collectively,these observations suggest that the alarmone(p)pp Gpp mediates nitrogen stress responses and may constitute a highly conserved mechanism from bacteria to plants.

The pleiotropic ABNORMAL FLOWER AND DWARF1 affects plant height, floral development and grain yield in rice

Moderate plant height and successful establishment of reproductive organs play pivotal roles in rice grain production. The molecular mechanism that controls the two aspects remains unclear in rice. In the present study,we characterized a rice gene, ABNORMAL FLOWER AND DWARF1（AFD1） that determined plant height, floral development and grain yield. The afd1 mutant showed variable defects including the dwarfism, long panicle, low seed setting and reduced grain yield. In addition, abnormal floral organs were also observed in the afd1 mutant including slender and thick hulls, and hull-like lodicules.AFD1 encoded a DUF640 domain protein and was expressed in all tested tissues and organs. Subcellular localization showed AFD1-green fluorescent fusion protein（GFP） was localized in the nucleus. Meantime, our results suggested that AFD1 regulated the expression of cell division and expansion related genes.

A New-Nipponbare Rice Germplasm with High Seed-Setting Rate

Rice （Oryza sativa L.） is an important crop providing staple food for more than half the world＇s population and also considered as a model plant for molecular biological study of the cereals. In 1998, the large-scale sequencing of japonica rice cultivar Nipponbare （bred at Aichi Agricultural Center in Japan and released in 1963） was initiated by International Rice Genome Sequencing Project （IRGSP） and the high-quality draft of genome was announced in 2002 （Goff et al,, 2002）. Owing to its easy genetic transformation and released whole genome sequencing data, Nipponbare is widely used in functional genomic research （Piffanelli et al., 2007; Luan et al., 2008; Hu et al., 2010; Thang et al., 2010; Tabuchi et al., 2011; Zhang et al., 2011; Jiang et al., 2012; Lu et al.. 2013）.