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Targeted mutagenesis of POLYAMINE OXIDASE 5 that negatively regulates mesocotyl elongation enables the generation of direct-seeding rice with improved grain yield

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Molecular Plant 2021年第2期14卷 344-351页

作者： Yusong Lv gaoneng shao Guiai Jiao Zhonghua Sheng Lihong Xie Shikai Hu shaoqing Tang Xiangjin Wei Peisong Hu State Key Laboratory of Rice Biology China National Center for Rice ImprovementChina National Rice Research InstituteHangzhou 310006China National Key Laboratory of Crop Genetic Improvement Huazhong Agricultural UniversityWuhan 430070China

Under conditions of labor or resource scarcity,direct seeding,rather than transplantation,is the preferred mode of rice(Oryza sativa)cultivation.This approach requires varieties that exhibit uniform seedling emergence.Mesocotyl elongation(ME),the main driver of rapid emergence of rice seedlings from soil,is enhanced by darkness and inhibited by light.Plant polyamine oxidases(PAOs)oxidize polyamines(PAs)and release H2O2,Here,we established that OsPAO5 expression in rice seedlings is increased in the presence of light and inhibited by darkness.To determine its role in ME,we created OsPAO5 mutants using CRISPR/Cas9.Compared with the wild type,pao5 mutants had longer mesocotyls,released less H2O2,and synthesized more ethylene.The mutant seedlings emerged at a higher and more uniform rate,indicating their potential for use in direct seeding.Nucleotide polymorphism analysis revealed that an SNP(PAO5-578G/A)located 578 bp upstream of the OsPAO5 start codon alters its expression,and was selected during rice mesocotyl domestication.The PAO5-578G genotype conferring a long mesocotyl mainly exists in wild rice,most Aus accessions,and some Geng(Japonica)accessions.Intriguingly,knocking out OsPAO5 can remarkably increase the grain weight,grain number,and yield potential.In summary,we developed a novel strategy to obtain elite rice with higher emergence vigor and yield potential,which can be conveniently and widely used to breed varieties of direct-seeding rice.

关键词： Oryza sativa direct seeding mesocotyl elongation grain yield polyamine oxidase 5

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GRAIN INCOMPLETE FILLING 2 regulates grain filling and starch synthesis during rice caryopsis development

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Journal of Integrative Plant Biology 2017年第2期59卷 134-153页

作者： Xiangjin Wei Guiai Jiao Haiyan Lin Zhonghua Sheng gaoneng shao Lihong Xie shaoqing Tang Qingguo Xu Peisong Hu State Key Laboratory of Rice Biology Key Laboratory of Rice Biology and Breeding of Ministry of Agriculture China National Rice Research Institute Hangzhou 310006 China . College of Agronomy Hunan Agricultural University Changsha 410128 China Agricultural Genomes Institute at Shenzhen Chinese Academy of Agricultural Sciences Shenzhen 518120 China

Rice grain filling determines grain weight, final yield and grain quality. Here, a rice defective grain filling mutant, gif2, was identified. Grains ofgif2 showed a slower filling rate and a significant lower final grain weight and yield compared to wild-type. The starch content in gilt2 was noticeably decreased and its physicochemical properties were also altered. Moreover, gif2 endosperm cells showed obvious defects in compound granule formation. Posi- tional cloning identified GIF2 to encode an ADP-glucose pyrophosphorylase （AGP） large subunit, AGPL2; consequently, AGP enzyme activity in gif2 endosperms was remarkably decreased. GIF2 is mainly expressed in developing grains and the coded protein localizes in the cytosol. Yeast two hybrid assay showed that GIF2 interacted with AGP small subunits OsAGPS% OsAGPS2a and OsAGPS2b. Transcript levels for granule-bound starch synthase, starch synthase, starch branching enzyme and starch debranching enzyme were distinctly elevated in gif2 grains. In addition, the level of nucleotide diversity of the GIF2 locus was extremely low in both cultivated and wild rice. All of these results suggest that GIF2 plays important roles in the regulation of grain filling and starch biosynthesis during caryopsis development, and that it has been preserved during selection throughout domestication of modern rice.

关键词： GIF GRAIN INCOMPLETE FILLING 2 regulates grain filling and starch synthesis during rice caryopsis development Figure AGPS RNAi

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CDE4 encodes a pentatricopeptide repeat protein involved in chloroplast RNA splicing and affects chloroplast development under low-temperature conditions in rice

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Journal of Integrative Plant Biology 2021年第10期63卷 1724-1739页

作者： Xinyong Liu Xichun Zhang Ruijie Cao Guiai Jiao Shikai Hu gaoneng shao Zhonghua Sheng Lihong Xie shaoqing Tang Xiangjin Wei Peisong Hu State Key Laboratory of Rice Biology China National Center for Rice ImprovementChina National Rice Research InstituteHangzhou 310006China Guizhou Rice Research Institute Guizhou Academy of Agricultural SciencesGuiyang 550006China

Pentatricopeptide repeat(PPR)proteins play important roles in the post-transcriptional modification of organellar RNAs in plants.However,the function of most PPR proteins remains unknown.Here,we characterized the rice(Oryza sativa L.)chlorophyll deficient 4(cde4)mutant which exhibits an albino phenotype during early leaf development,with decreased chlorophyll contents and abnormal chloroplasts at low-temperature(20℃).Positional cloning revealed that CDE4 encodes a P-type PPR protein localized in chloroplasts.In the cde4 mutant,plastid-encoded polymerase(PEP)-dependent transcript levels were significantly reduced,but transcript levels of nuclear-encoded genes were increased compared to wild-type plants at 20℃.CDE4 directly binds to the transcripts of the chloroplast genes rpl2,ndhA,and ndhB.Intron splicing of these transcripts was defective in the cde4 mutant at 20℃,but was normal at 32℃.Moreover,CDE4 interacts with the guanylate kinase VIRESCENT 2(V2);overexpression of V2 enhanced CDE4 protein stability,thereby rescuing the cde4 phenotype at 20℃.Our results suggest that CDE4 participates in plastid RNA splicing and plays an important role in rice chloroplast development under lowtemperature conditions.

关键词： CDE4 chloroplast development pentatricopeptide repeat(PPR)protein RNA splicing low-temperature Oryza sativa L.

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MONOCULM 3,an Ortholog of WUSCHEL in Rice,Is Required for Tiller Bud Formation

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Journal of Genetics and Genomics 2015年第2期42卷 71-78页

作者： Zefu Lu gaoneng shao Jinsong Xiong Yongqing Jiao Jing Wang Guifu Liu Xiangbing Meng Yan Liang Guosheng Xiong Yonghong Wang Jiayang Li State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing) Institute of Genetics and Developmental BiologyChinese Academy of Sciences College of Horticulture Nanjing Agricultural University Oil Crops Research Institute China Academy of Agricultural Science Rice Research Institute Sichuan Agricultural University Agricultural Genome Institute China Academy of Agricultural Science

WUSCHEL （WUS） plays an essential role for the maintenance of meristem activity in dicots, but its function is still elusive in monocots. We isolated a new monoculm mutant, monoculm 3 （moc3）, in which a point mutation causes the premature termination of rice O. sativa WUS （OsWUS）. Morphological observation revealed that the formation of tiller buds was disrupted in moc3. MOC3 was localized in the nuclear and could interact with TOPLESS-RELATED PROTEINS （TPRs）. The expression of MOC3 was induced by cytokinins and defection of MOC3 affected the expression of several two-component cytokinin response regulators, OsRRs and ORRs. Our results suggest that MOC3 is required for the formation of axillary buds and has a complex relationship with cytokinins.

关键词： MOC3 WUSCHEL Tiller bud formation Cytokinins Oryza sativa L

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Tiller Bud Formation Regulators MOC1 and MOC3 Cooperatively Promote Tiller Bud Outgrowth by Activating F0N1 Expression in Rice

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Molecular Plant 2019年第8期12卷 1090-1102页

作者： gaoneng shao Zefu Lu Jinsong Xiong Bing Wang Yanhui Jing Xiangbing Meng Guifu Liu Haiyan Ma Yan Liang Fan Chen Yonghong Wang Jiayang Li Hong Yu State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing) Institute of Genetics and Developmental BiologyThe Innovative Academy of Seed DesignChinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100049China State Key Laboratory of Rice Biology China National Rice Research InstituteHangzhou 310006China State Key Laboratory of Molecular Developmental Biology Institute of Genetics and Developmental BiologyThe Innovative Academy of Seed DesignChinese Academy of SciencesBeijing 100101China Present address: Department of Genetics University of GeorgiaAthensGA 30605USA Present address: College of Horticulture Nanjing Agricultural UniversityNanjingJiangsu 210095China

Tillering in rice is one of the most important agronomic traits.Rice tiller development can be divided into two main processes: the formation of the axillary bud and its subsequent outgrowth.Several genes critical for bud formation in rice have been identified by genetic studies;however,their molecular functions and relationships are still largely unknown.Here,we report that MONOCULM 1 (MOC1) and MONOCULM 3/ TILLERS ABSENT 1/STERILE AND REDUCED TILLERING 1 (MOC3/TAB1/SRT1),two vital regulators for tiller formation in rice,physically interact to regulate tiller bud outgrowth through upregulating the expression of FLORAL ORGAN NUMBER 1 (FON1),the homolog of CLAVATA1 in rice.We found that M0C3 is able to directly bind the promoter ofFONI and subsequently activate FON1 expression.MOC1 functions as a coactivator of MOC3,whereas it could not directly bind the FON1 promoter,and further activated FON1 expression in the presence of MOC3.Accordingly,FON1 is highly expressed at axillary meristems and shows remarkably decreased expression levels in mod and moc3 mutants.Loss-of-function mutants of FON1 exhibit normal bud formation but defective bud outgrowth and reduced tiller number.Collectively,these results shed light on a joint transcriptional regulatory mechanim by MOC1 and MOC3,and establish a new framework for the control of tiller bud formation and outgrowth.

关键词： rice tiller formation MOC3 M0C1 FON1

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OsVP1 activates Sdr4 expression to control rice seed dormancy via the ABA signaling pathway

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The Crop Journal 2021年第1期9卷 68-78页

作者： Wenqiang Chen Wei Wang Yusong Lyu Yawen Wu Pingliang Huang Shikai Hu Xiangjin Wei Guiai Jiao Zhonghua Sheng shaoqing Tang gaoneng shao Ju Luo State Key Laboratory of Rice Biology China National Rice Research InstituteHangzhou 310006ZhejiangChina Institute of Crop Science and Nuclear Technology Utilization Zhejiang Academy of Agricultural SciencesHangzhou 310021ZhejiangChina

Pre-harvest sprouting(PHS)is a disadvantageous trait in cereal production worldwide,causing large economic losses each year.Its regulation mechanism is still unclear.We generated the Oryza sativa Viviparous1(OsVP1)mutant using gene editing technique,which shows increased PHS compared with that of the wild type Nipponbare.OsVP1 is localized mainly in the nucleus and expressed in various tissues and organs.Expression of Seed dormancy 4(Sdr4),a key gene controlling PHS,was sharply reduced in OsVP1 mutants.OsVP1 bound to the specific motif CACCTG in the promoter of Sdr4 and activated its expression in rice protoplasts.Overexpression of Sdr4 reduced the high seed germination rate of OsVP1 mutant cr-osvp1-1,showing that Sdr4 acts as a downstream target of OsVP1.Both OsVP1 and Sdr4 loss-of-function mutants were insensitive to exogenous ABA and employed the ABA signaling pathway in regulating seed dormancy.These findings shed light on the control of seed dormancy aimed at preventing PHS in rice.

关键词： Rice Pre-harvest sprouting Seed dormancy OsVP1 Sdr4 ABA

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Integrated transcriptome, small RNA, and degradome analysis to elucidate the regulation of rice seedling mesocotyl development during the passage from darkness to light

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The Crop Journal 2020年第6期8卷 918-928页

作者： Yusong Lyu Xiangjin Wei Min Zhong Shipeng Niu Shakeel Ahmad gaoneng shao Guiai Jiao Zhonghua Sheng Lihong Xie Shikai Hu Yawen Wu shaoqing Tang Peisong Hu State Key Laboratory of Rice Biology China National Center for Rice ImprovementChina National Rice Research InstituteHangzhou 310006ZhejiangChina National Key Laboratory of Crop Genetic Improvement Huazhong Agricultural UniversityWuhan 430070HubeiChina

The mesocotyl,a structure located between the basal part of the seminal root and the coleoptile node of seedlings,contributes to pushing the shoot tip through the soil surface,a function that is essential for the uniform emergence of direct-seeded rice.Its elongation is inhibited by light and induced in darkness.This investigation of an indica rice(P25)with vigorous mesocotyl elongation was aimed at identifying the"omics"basis of its lightinduced growth inhibition.A transcriptomic comparison between mesocotyl tissues that had developed in the dark and then been exposed to light identified many differentially expressed genes(DEGs)and differentially abundant micro RNAs(mi RNAs).Degradome sequencing analysis revealed 27 negative mi RNA-target pairs.A co-expression regulatory network was constructed based on the mi RNAs,their corresponding targets,and DEGs with a common Gene Ontology term.It suggested that auxin and light,probably antagonistically,affect mesocotyl elongation by regulating polyamine oxidase activity.

关键词： Oryza sativa L. Mesocotyl Transcriptome MicroRNAome Degradome

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OPAQUE3, encoding a transmembrane bZIP transcription factor, regulates endosperm storage protein and starch biosynthesis in rice

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Plant Communications 2022年第6期3卷 299-313页

作者： Ruijie Cao shaolu Zhao Guiai Jiao Yingqing Duan Liuyang Ma Nannan Dong Feifei Lu Mingdong Zhu gaoneng shao Shikai Hu Zhonghua Sheng Jian Zhang shaoqing Tang Xiangjin Wei Peisong Hu State Key Laboratory of Rice Biology China National Center for Rice ImprovementChina National Rice Research InstituteHangzhou 310006China Institute of Agricultural Science in Jiangsu Coastal Areas Yancheng 224002China

Starch and storage proteins are the main components of rice(Oryza sativa L.)grains.Despite their importance,the molecular regulatory mechanisms of storage protein and starch biosynthesis remain largely elusive.Here,we identified a rice opaque endosperm mutant,opaque3(o3),that overaccumulates 57-kDa proglutelins and has significantly lower protein and starch contents than the wild type.The o3 mutant also has abnormal protein body structures and compound starch grains in its endosperm cells.OPAQUE3(O3)encodes a transmembrane basic leucine zipper(bZIP)transcription factor(OsbZIP60)and is localized in the endoplasmic reticulum(ER)and the nucleus,but it is localized mostly in the nucleus under ER stress.We demonstrated that O3 could activate the expression of several starch synthesis-related genes(GBSSI,AGPL2,SBEI,and ISA2)and storage protein synthesis-related genes(OsGluA2,Prol14,and Glb1).O3 also plays an important role in protein processing and export in the ER by directly binding to the promoters and activating the expression of OsBIP1 and PDIL1-1,two major chaperones that assist with folding of immature secretory proteins in the ER of rice endosperm cells.High-temperature conditions aggravate ER stress and result in more abnormal grain development in o3 mutants.We also revealed that OsbZIP50 can assist O3 in response to ER stress,especially under high-temperature conditions.We thus demonstrate that O3 plays a central role in rice grain development by participating simultaneously in the regulation of storage protein and starch biosynthesis and the maintenance of ER homeostasis in endosperm cells.

关键词： grain development ER stress OPAQUE3 high temperature rice

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外源氨基酸对层出镰刀菌菌丝生长和伏马毒素合成的影响

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中国稻米 2024年第1期30卷 47-52页

作者：梁文豪胡时开圣忠华魏祥进焦桂爱邵高能谢黎虹王玲唐绍清胡培松中国水稻研究所/水稻生物育种全国重点实验室杭州310006

由层出镰刀菌引起的水稻穗腐病,不仅造成水稻产量损失,而且因产生真菌毒素而导致稻米品质下降。本研究通过在生长培养基中添加外源氨基酸,评价其对层出镰刀菌菌丝生长和伏马毒素合成的影响。结果表明,不同氨基酸和不同添加量对菌丝生长... 详细信息

由层出镰刀菌引起的水稻穗腐病,不仅造成水稻产量损失,而且因产生真菌毒素而导致稻米品质下降。本研究通过在生长培养基中添加外源氨基酸,评价其对层出镰刀菌菌丝生长和伏马毒素合成的影响。结果表明,不同氨基酸和不同添加量对菌丝生长均有影响。与未添加氨基酸的对照相比,在MM培养基上添加L-精氨酸、L-谷氨酸、L-脯氨酸和L-瓜氨酸,在PDA培养基上添加L-谷氨酸,均抑制菌丝生长。添加氨基酸显著提高了伏马毒素含量,且随着氨基酸浓度升高,在PDA中产毒量减少,在MM中则呈增加趋势。外源氨基酸明显改变了伏马毒素生物合成基因在转录水平上的表达,但基因表达模式与毒素含量并不一致。综合分析表明,外源氨基酸会影响层出镰刀菌菌丝生长和伏马毒素含量,且不同氨基酸组分与菌丝生长和伏马毒素合成的关联性不同。

关键词：水稻穗腐病层出镰刀菌外源氨基酸菌丝生长伏马毒素

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NRL3 Interacts with OsK4 to Regulate Heading Date in Rice

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Rice science 2022年第3期29卷 237-246页

作者： CHEN Wei CAI Yicong Shakeel AHMAD WANG Yakun AN Ruihu TANG Shengjia GUO Naihui WEI Xiangjin TANG shaoqing shao gaoneng JIAO Guiai XIE Lihong HU Shikai SHENG Zhonghua HU Peisong Key Laboratory of Crop Physiology Ecology and Genetic BreedingMinistry of Education/Collaboration Center for Double-Season Rice Modernization ProductionJiangxi Agricultural University/Research Center of Super Rice Engineering and TechnologyJiangxi ProvinceNanchang 330045China State Key Laboratory of Rice Biology/Key Laboratory of Rice Biology and Breeding Ministry of Agriculture and Rural Affairs/China National Rice Improvement Center/China National Rice Research InstituteHangzhou 310006China

NRL3 is essential for the growth and development of rice leaves.In this study,we found that the loss function of NRL3 also delayed heading date under natural long daylight and short daylight conditions.The yeast two-hybrid and the bimolecular fluorescence complementation proved that NRL3interacts with OsK4,a Snf1-related kinase.OsK4 localized to the nucleus and expressed in various rice tissues.The rhythmic expression pattern of Os K4 was similar to NRL3 under long daylight and short daylight conditions.Knock-out mutants of Os K4 exhibited early heading under long daylight conditions,indicating that it acts as a negative regulator of heading date in rice.Interestingly,the OsK4 mutant under the nrl3 mutant background rescued the late heading phenotype of nrl3 under long daylight conditions,suggesting that Os K4 functions downstream of NRL3.Moreover,both NRL3 and Os K4 controlled heading date through regulating the expression of Hd3a and RFT1 genes.These findings shed light on the heading date regulation in rice and provide a sound theoretical base to improve regional adaptability of rice.

关键词： Oryza sativa heading date NRL3 gene OsK4 gene negative regulator loss function

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