Karrikins and strigolactones govern plant development and environmental responses through closely related signaling pathways.The transcriptional repressor proteins SUPPRESSOR OF MAX21(SMAX1),SMAX1-like2(SMXL2),and D53...Karrikins and strigolactones govern plant development and environmental responses through closely related signaling pathways.The transcriptional repressor proteins SUPPRESSOR OF MAX21(SMAX1),SMAX1-like2(SMXL2),and D53-like SMXLs mediate karrikin and strigolactone signaling by directly binding downstream genes or byinhibiting the activities of transcription factors.In this study,we characterized the non-transcriptional regulatory activities of SMXL proteins in Arabidopsis.We discovered that SMAX1 and SMXL2 with mutations in their ethylene-responsefactor-associated amphiphilic repression(EAR)motif had undetectable or weak transcriptional repression activities but still partially rescued the hypocotyl elongation defects and fully reversed the cotyledon epinasty defects of the smax1 smxl2 mutant.SMAX1 and SMXL2 directly interact with PHYTOCHROME INTERACTION FACTOR4(PIF4)and PIF5 to enhance their protein stability by interacting with phytochrome B(phyB)and suppressing the association of phyB with PIF4 and PIF5.The karrikin-responsive genes were then identified by treatment with GR24ent-ssa,GR24 analog showing karrikin activity.Interestingly,INDOLE-3-ACETIC ACID INDUCIBLE 29(IAA29)expression was repressed by GR24^(ent-5D)streatment in a PIF4-and PIF5-dependent and EARindependent manner,whereas KARRIKIN UPREGULATED F-BOX 1(KUF1)expression was induced in a PIF4-and PIF5-independent and EAR-dependent manner.Furthermore,the non-transcriptional regulatory activity of SMAX1,which is independent of the EAR motif,had a global effect on gene expression.Taken together,these results indicate that non-transcriptional regulatory activities of SMAX1 and SMXL2 mediate karrikin-regulated seedling response to red light.展开更多
Protein phosphorylation regulates a variety of important cellular and physiological processes in plants.In-depth profiling of plant phosphoproteomes has been more technically challenging than that of animal phosphopro...Protein phosphorylation regulates a variety of important cellular and physiological processes in plants.In-depth profiling of plant phosphoproteomes has been more technically challenging than that of animal phosphoproteomes.This is largely due to the need to improve protein extraction efficiency from plant cells,which have a dense cell wall,and to minimize sample loss resulting from the stringent sample clean-up steps required for the removal of a large amount of biomolecules interfering with phosphopeptide purification and mass spectrometry analysis.To this end,we developed a method with a streamlined workflow for highly efficient purification of phosphopeptides from tissues of various green organisms including Arabidopsis,rice,tomato,and Chlamydomonas reinhardtii,enabling in-depth identification with high quantitative reproducibility of about 11000 phosphosites,the greatest depth achieved so far with single liquid chromatography-mass spectrometry(LC-MS)runs operated in a data-dependent acquisition(DDA)mode.The mainstay features of the method are the minimal sample loss achieved through elimination of sample clean-up before protease digestion and of desalting before phosphopeptide enrichment and hence the dramatic increases of time-and cost-effectiveness.The method,named GreenPhos,combined with single-shot LC-MS,enabled in-depth quantitative identification of Arabidopsis phosphoproteins,including differentially phosphorylated spliceosomal proteins,at multiple time points during salt stress and a number of kinase substrate motifs.GreenPhos is expected to serve as a universal method for purification of plant phosphopeptides,which,if samples are further fractionated and analyzed by multiple LC-MS runs,could enable measurement of plant phosphoproteomes with an unprecedented depth using a given mass spectrometry technology.展开更多
Interorgan lipid transport is crucial for organism development and the maintenance of physiological function.Here,we demonstrate that Drosophila long-chain acyl-CoA synthetase(dAcsl),which catalyzes the conversion of ...Interorgan lipid transport is crucial for organism development and the maintenance of physiological function.Here,we demonstrate that Drosophila long-chain acyl-CoA synthetase(dAcsl),which catalyzes the conversion of fatty acids into acyl-coenzyme As(acyl-CoAs),plays a critical role in regulating systemic lipid homeostasis.dAcsl deficiency in the fat body led to the ectopic accumulation of neutral lipids in the gut,along with significantly reduced lipoprotein contents in both the fat body and hemolymph.The aberrant phenotypes were rescued by fat body-specific overexpression of apolipophorin.A multi-omics investigation comprising lipidomics,metabolomics,and proteomics in conjunction with genetic screening revealed that glycosylation processes were suppressed in dAcsl knockdown flies.Overexpression of CG9035,human ortholog of which is implicated in the congenital disorder of glycosylation,ame-liorated gut lipid accumulation in Drosophila.Aberrant lipoprotein glycosylation led to accelerated proteasome-related degradation and induced ER stress in dAcsl knockdown flies,impairing lipoprotein release into the circulation which compromised interorgan lipid transport between the fat body and the gut.Inhibition of ubiquitin-proteasome-dependent degradation alleviated the phenotype of gut ectopic fat accumulation in dAcsl knockdown flies.Finally,we verified that ACSL4,the human homolog of dAcsl,also regulated lipoprotein levels in HepG2 cells,indicating that the role of dAcsl in modulating lipoprotein secretion and systemic lipid homeostasis is possibly conserved in humans.展开更多
RNA polymerase transcriptional pausing represents a major checkpoint in transcription in bacteria and metazoans,but it is unknown whether this phenomenon occurs in plant organelles.Here,we report that transcriptional ...RNA polymerase transcriptional pausing represents a major checkpoint in transcription in bacteria and metazoans,but it is unknown whether this phenomenon occurs in plant organelles.Here,we report that transcriptional pausing occurs in chloroplasts.We found that mTERF5 specifically and positively regulates the transcription of chloroplast psbEFLJ in Arabidopsis thaliana that encodes four key subunits of photosystem II.We found that mTERF5 causes the plastid-encoded RNA polymerase(PEP)complex to pause at psbEFLJ by binding to the+30 to+51 region of double-stranded DNA.Moreover,we revealed that mTERF5 interacts with pTAC6,an essential subunit of the PEP complex,although pTAC6 is not involved in the transcriptional pausing at psbEFLJ.We showed that mTERF5 recruits additional pTAC6 to the transcriptionally paused region of psbEFLJ,and the recruited pTAC6 proteins could be assembled into the PEP complex to regulate psbEFLJ transcription.Taken together,our findings shed light on the role of transcriptional pausing in chloroplast transcription in plants.展开更多
The coordinated utilization of nitrogen(N)and phosphorus(P)is vital for plants to maintain nutrient balance and achieve optimal growth.Previously,we revealed a mechanism by which nitrate induces genes for phosphate ut...The coordinated utilization of nitrogen(N)and phosphorus(P)is vital for plants to maintain nutrient balance and achieve optimal growth.Previously,we revealed a mechanism by which nitrate induces genes for phosphate utilization;this mechanism depends on NRT1.1B-facilitated degradation of cytoplasmic SPX4,which in turn promotes cytoplasmic-nuclear shuttling of PHR2,the central transcription factor of phosphate signaling,and triggers the nitrate-induced phosphate response(NIPR)and N-P coordinated utilization in rice.In this study,we unveiled a fine-tuning mechanism of NIPR in the nucleus regulated by Highly Induced by Nitrate Gene 1(HINGE1,also known as RLI1),a MYB-transcription factor closely related to PHR2.RLI1/HINGE1,which is transcriptionally activated by PHR2 under nitrate induction,can directly activate the expression of phosphate starvation-induced genes.More importantly,RLI1/HINGE1 competes with PHR2 for binding to its repressor proteins in the nucleus(SPX proteins),and consequently releases PHR2 to further enhance phosphate response.Therefore,RLI1/HINGE1 amplifies the phosphate response in the nucleus downstream of the cytoplasmic SPX4-PHR2 cascade,thereby enabling fine-tuning of N-P balance when nitrate supply is sufficient.展开更多
Salt stress adversely affects plant growth,development,and crop yield.Rice(Oryza sativa L.)is one of the most salt-sensitive cereal crops,especially at the early seedling stage.Mitogen-activated protein kinase(MAPK/MP...Salt stress adversely affects plant growth,development,and crop yield.Rice(Oryza sativa L.)is one of the most salt-sensitive cereal crops,especially at the early seedling stage.Mitogen-activated protein kinase(MAPK/MPK)cascades have been shown to play critical roles in salt response in Arabidopsis.However,the roles of the MPK cascade signaling in rice salt response and substrates of Os MPK remain largely unknown.Here,we report that the salt-induced Os MPK4-Ideal Plant Architecture 1(IPA1)signaling pathway regulates the salt tolerance in rice.Under salt stress,Os MPK4 could interact with IPA1 and phosphorylate IPA1 at Thr180,leading to degradation of IPA1.Genetic evidence shows that IPA1 is a negative regulator of salt tolerance in rice,whereas Os MPK4 promotes salt response in an IPA1-dependent manner.Taken together,our results uncover an Os MPK4-IPA1 signal cascade that modulates the salt stress response in rice and sheds new light on the breeding of salt-tolerant rice varieties.展开更多
N-terminal acetylation is one of the most common protein modifications in eukaryotes,and approximately 40%of human and plant proteomes are acetylated by ribosome-associated N-terminal acetyltransferase A(NatA)in a co-...N-terminal acetylation is one of the most common protein modifications in eukaryotes,and approximately 40%of human and plant proteomes are acetylated by ribosome-associated N-terminal acetyltransferase A(NatA)in a co-translational manner.However,the in vivo regulatory mechanism of NatA and the global impact of NatA-mediated N-terminal acetylation on protein fate remain unclear.Here,we identify Huntingtin Yeast partner K(HYPK),an evolutionarily conserved chaperone-like protein,as a positive regulator of NatA activity in rice.We found that loss of OsHYPK function leads to developmental defects in rice plant architecture but increased resistance to abiotic stresses,attributable to perturbation of the N-terminal acetylome and accelerated global protein turnover.Furthermore,we demonstrated that OsHYPK is also a substrate of NatA and that N-terminal acetylation of OsHYPK promotes its own degradation,probably through the Ac/N-degron pathway,which could be induced by abiotic stresses.Taken together,our findings suggest that the OsHYPK-NatA complex plays a critical role in coordinating plant development and stress responses by dynamically regulating NatA-mediated N-terminal acetylation and global protein turnover,which are essential for maintaining adaptive phenotypic plasticity in rice.展开更多
Multisubunit SKP1/Cullin1/F-box(SCF)E3 ligases play essential roles in regulating the stability of crucial regulatory factors and controlling growth and development in eukaryotes.Detecting E3 ligase activity in vitro ...Multisubunit SKP1/Cullin1/F-box(SCF)E3 ligases play essential roles in regulating the stability of crucial regulatory factors and controlling growth and development in eukaryotes.Detecting E3 ligase activity in vitro is important forexploring the molecular mechanism of protein ubiquitination.However,in vitro ubiquitination assay systems for multisubunit E3 ligases remain difficult to achieve,especially in plants,mainly owing to difficulties in achieving active components of multisubunit E3 ligases with high purity and characterizing specific E2 and E3 pairs.In this study,we characterized components of the rice ScFDiwARF3(SCFDs)E3 ligase,screened the coordinated E2,and reconstituted active ScFD3 E3 ligase in vitro.We further engineered SCFD3 E3 ligase using a fused SKP1-Cullin1-RBX1(eSCR)protein and found that both the wild-type SCFD3 E3 ligase and the engineered SCFD3 E3 ligase catalyzed ubiquitination of the substrate D53,which is the key transcriptional repressor in strigolactone signaling.Finally,we replaced D3 with other F-box proteins from rice and humans and reconstituted active escF E3 ligases,including escFaID2,escFBxL1s,and escFcDC4 E3 ligases.Our work reconstitutes functional SCF E3 ligases in vitro and generates an engineered system with interchangeable F-box proteins,providing a powerful platform for studying the mechanisms of multisubunit SCF E3 ligases in eukaryotes.展开更多
Nonalcoholic fatty liver disease(NAFLD)encompasses a spectrum of pathologies,ranging from steatosis to nonalcoholic steatohepatitis(NASH).The factors promoting the progression of steatosis to NASH are still unclear.Re...Nonalcoholic fatty liver disease(NAFLD)encompasses a spectrum of pathologies,ranging from steatosis to nonalcoholic steatohepatitis(NASH).The factors promoting the progression of steatosis to NASH are still unclear.Recent studies suggest that mitochondrial lipid composition is critical in NASH develop-ment.Here,we showed that CDP-DAG synthase 2(Cds2)was downregulated in genetic or diet-induced NAFLD mouse models.Liver-specific deficiency of Cds2 provoked hepatic steatosis,inflammation and fibrosis in five-week-old mice.CDS2 is enriched in mitochondria-associated membranes(MAMs),and hepatic Cds2 deficiency impaired mitochondrial function and decreased mitochondrial PE levels.Overexpression of phosphatidylserine decarboxylase(PISD)alleviated the NASH-like phenotype in Cds2^(f/f);AlbCre mice and abnormal mitochondrial morphology and function caused by CDS2 deficiency in hepatocytes.Additionally,dietary supplementation with an agonist of peroxisome proliferator-activated receptor alpha(PPARa)attenuated mitochondrial defects and ameliorated the NASH-like phe-notype in Cds2^(f/f);AlbCre mice.Finally,Cds2 overexpression protected against high-fat diet-induced hepatic steatosis and obesity.Thus,Cds2 modulates mitochondrial function and NASH development.展开更多
Adventitious roots form from non-root tissues as part of normal development or in response to stress or wounding.The root primordia form in the source tissue,and during emergence the adventitious roots penetrate the i...Adventitious roots form from non-root tissues as part of normal development or in response to stress or wounding.The root primordia form in the source tissue,and during emergence the adventitious roots penetrate the inner cell layers and the epidermis;however,the mechanisms underlying this emergence remain largely unexplored.Here,we report that a regulatory module composed of the AP2/ERF transcription factor ABSCISIC ACID INSENSITIVE 4(ABI4),the MAP kinases MPK3 and MPK6,and the phosphatase PP2C12 plays an important role in the emergence of junction adventitious roots(J-ARs)from the root-hypocotyl junctions in Arabidopsis thaliana.ABI4 negatively regulates J-AR emergence,preventing the accumulation of reactive oxygen species and death of epidermal cells,which would otherwise facilitate J-AR emergence.Phosphorylation by MPK3/MPK6 activates ABI4 and dephosphorylation by PP2C12 inactivates ABI4.MPK3/MPK6 also directly phosphorylate and inactivate PP2C12 during J-AR emergence.We propose that this"double-check"mechanism increases the robustness of MAP kinase signaling and finely regulates the local programmed cell death required for J-AR emergence.展开更多
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 ...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.展开更多
Dear Editor,Rice dwarf virus(RDV),a member of the Phytoreoviurs genus,is transmitted to rice(Oryza sativa)plants by leaf-hopper(Nephotettix cincticeps)in a propagative manner.Infection by RDV results in severe stuntin...Dear Editor,Rice dwarf virus(RDV),a member of the Phytoreoviurs genus,is transmitted to rice(Oryza sativa)plants by leaf-hopper(Nephotettix cincticeps)in a propagative manner.Infection by RDV results in severe stunting growth pheno-types and a dramatic reduction in grain yield.The genome of RDV is composed of 12-segmented double-stranded RNAs(S1-S12 based on their migration rates in agarose gel electro-phoresis).The 12 segments encode seven structural proteins(P1,P2,P3,P5,P7,P8,and P9 as the products of Sl,S2,S3,S5,S7,S8,and S9,respectively)and five nonstructural proteins(Pns4,Pns6,Pnsl0,Pns11,and Pns12 as the products of S4,S6,S10,S11,and S12,respectively).The outer capsid protein P2 is essential for RDV infection of insects and thus influences transmission of RDV by the insect vector(Omura et al.,1998;Zhou et al.,2007).P2 also contributes to the dwarf phenotype of infected rice by interfering with gibberellic acid synthesis(Zhu et al.,2005).When RDV-infected rice plants were main-tained via vegetative propagation for several years without insect transmission,they regained normal growth height due to loss of RDV P2 and Pnsl0 proteins(Pu et al.,2011).展开更多
Cyanobacteria are a group of oxygenic photosynthetic bacteria with great potentials in biotechnological applications and advantages as models for photosynthesis research. The subcellular localizations of the majority ...Cyanobacteria are a group of oxygenic photosynthetic bacteria with great potentials in biotechnological applications and advantages as models for photosynthesis research. The subcellular localizations of the majority of proteins in any cyanobacteria remain undetermined, representing a major challenge in using cyanobacteria for both basic and industrial researches. Here, using label-free quantitative proteomics, we map 2027 proteins of Synechocystis sp. PCC6803, a model cyanobacterium, to different subcellular compartments and generate a proteome atlas with such information. The atlas leads to numerous unexpected but important findings, including the predominant localization of the histidine kinases Hik33 and Hik27 on the thylakoid but not the plasma membrane. Such information completely changes the concept regarding how the two kinases are activated. Together, the atlas provides subcellular localization information for nearly 60% proteome of a model cyanobacterium, and will serve as an important resource for the cyanobacterial research community.展开更多
Atherosclerotic cardiovascular disease resulting from dysregulated lipid metabolism is the leading cause of morbidity and mortality worldwide.Apolipoprotein E(ApoE)plays a critical role in cholesterol metabolism.Knock...Atherosclerotic cardiovascular disease resulting from dysregulated lipid metabolism is the leading cause of morbidity and mortality worldwide.Apolipoprotein E(ApoE)plays a critical role in cholesterol metabolism.Knockouts in lipid-metabolizing proteins including ApoE in multiple model organisms such as mice and rats exhibiting elevated levels of cholesterol have been widely used for dissecting the pathology of atherosclerosis,but few of these animal models exhibit advanced atherosclerotic plaques leading to ischemia-induced clinical symptoms,limiting their use for translational studies.Here we report hypercholesterolemia and severe atherosclerosis characterized by stenosis and occlusion of arteries,together with clinical manifestations of stroke and gangrene,in ApoE knockout dogs generated by CRISPR/Cas9 and cloned by somatic cell nuclear transfer technologies.Importantly,the hypercholesterolemia and atherosclerotic complications in F0 mutants are recapitulated in their offspring.As the ApoE-associated atherosclerosis and clinical manifestations in mutant dogs are more similar to that in human patients compared with those in other animal models,these mutant dogs will be invaluable in developing and evaluating new therapies,including endovascular procedures,against atherosclerosis and related disorders.展开更多
Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins.The current methods that applied in protein-protein interaction,such as co-immunoprecipit...Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins.The current methods that applied in protein-protein interaction,such as co-immunoprecipitation and pull down etc.,often cause plenty of working time due to the burdensome cloning and puri-fication procedures.Here we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within E.coli system.We modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease,two cleavage site F and two multiple cloning sites that flanking cleavage sites.The target proteins(for example:protein A and protein B)were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His6 tag.PPV NIa protease carried out intracellular cleavage along expression,then led to the separation of polyprotein components,therefore,the interaction between protein A-protein B can be detected through one-step purification and analysis.Negative control for protein B was brought into this system for monitoring interaction specificity.We successfully employed this system to prove two cases of reported protien-protein interaction:RHA2a/ANAC and FTA/FTB.In conclusion,a convenient and efficient system has been successfully developed for detecting protein-protein interaction.展开更多
基金the National Natural Science Foundation of China(32170320,32122012,and 32270327)the Hebei Natural Science Foundation(C2022503003)the Youth Innovation Promotion Association of the Chinese Academy of Sciences(Y2023025).
文摘Karrikins and strigolactones govern plant development and environmental responses through closely related signaling pathways.The transcriptional repressor proteins SUPPRESSOR OF MAX21(SMAX1),SMAX1-like2(SMXL2),and D53-like SMXLs mediate karrikin and strigolactone signaling by directly binding downstream genes or byinhibiting the activities of transcription factors.In this study,we characterized the non-transcriptional regulatory activities of SMXL proteins in Arabidopsis.We discovered that SMAX1 and SMXL2 with mutations in their ethylene-responsefactor-associated amphiphilic repression(EAR)motif had undetectable or weak transcriptional repression activities but still partially rescued the hypocotyl elongation defects and fully reversed the cotyledon epinasty defects of the smax1 smxl2 mutant.SMAX1 and SMXL2 directly interact with PHYTOCHROME INTERACTION FACTOR4(PIF4)and PIF5 to enhance their protein stability by interacting with phytochrome B(phyB)and suppressing the association of phyB with PIF4 and PIF5.The karrikin-responsive genes were then identified by treatment with GR24ent-ssa,GR24 analog showing karrikin activity.Interestingly,INDOLE-3-ACETIC ACID INDUCIBLE 29(IAA29)expression was repressed by GR24^(ent-5D)streatment in a PIF4-and PIF5-dependent and EARindependent manner,whereas KARRIKIN UPREGULATED F-BOX 1(KUF1)expression was induced in a PIF4-and PIF5-independent and EAR-dependent manner.Furthermore,the non-transcriptional regulatory activity of SMAX1,which is independent of the EAR motif,had a global effect on gene expression.Taken together,these results indicate that non-transcriptional regulatory activities of SMAX1 and SMXL2 mediate karrikin-regulated seedling response to red light.
基金support from the Ministry of Science and Technology of the People's Republic of China(2019YFA0707100,2019YFA0802203)Strategic Priority Research Program of Chinese Academy of Sciences(XDA24040202)National Key Research and Development Program of China(2022YFF1001704)。
文摘Protein phosphorylation regulates a variety of important cellular and physiological processes in plants.In-depth profiling of plant phosphoproteomes has been more technically challenging than that of animal phosphoproteomes.This is largely due to the need to improve protein extraction efficiency from plant cells,which have a dense cell wall,and to minimize sample loss resulting from the stringent sample clean-up steps required for the removal of a large amount of biomolecules interfering with phosphopeptide purification and mass spectrometry analysis.To this end,we developed a method with a streamlined workflow for highly efficient purification of phosphopeptides from tissues of various green organisms including Arabidopsis,rice,tomato,and Chlamydomonas reinhardtii,enabling in-depth identification with high quantitative reproducibility of about 11000 phosphosites,the greatest depth achieved so far with single liquid chromatography-mass spectrometry(LC-MS)runs operated in a data-dependent acquisition(DDA)mode.The mainstay features of the method are the minimal sample loss achieved through elimination of sample clean-up before protease digestion and of desalting before phosphopeptide enrichment and hence the dramatic increases of time-and cost-effectiveness.The method,named GreenPhos,combined with single-shot LC-MS,enabled in-depth quantitative identification of Arabidopsis phosphoproteins,including differentially phosphorylated spliceosomal proteins,at multiple time points during salt stress and a number of kinase substrate motifs.GreenPhos is expected to serve as a universal method for purification of plant phosphopeptides,which,if samples are further fractionated and analyzed by multiple LC-MS runs,could enable measurement of plant phosphoproteomes with an unprecedented depth using a given mass spectrometry technology.
基金supported by grants from the National Natural Science Foundation of China(92057202 and 31871194)the National Key R&D Program of China(2018YFA0506900 and 2018YFA0800901).
文摘Interorgan lipid transport is crucial for organism development and the maintenance of physiological function.Here,we demonstrate that Drosophila long-chain acyl-CoA synthetase(dAcsl),which catalyzes the conversion of fatty acids into acyl-coenzyme As(acyl-CoAs),plays a critical role in regulating systemic lipid homeostasis.dAcsl deficiency in the fat body led to the ectopic accumulation of neutral lipids in the gut,along with significantly reduced lipoprotein contents in both the fat body and hemolymph.The aberrant phenotypes were rescued by fat body-specific overexpression of apolipophorin.A multi-omics investigation comprising lipidomics,metabolomics,and proteomics in conjunction with genetic screening revealed that glycosylation processes were suppressed in dAcsl knockdown flies.Overexpression of CG9035,human ortholog of which is implicated in the congenital disorder of glycosylation,ame-liorated gut lipid accumulation in Drosophila.Aberrant lipoprotein glycosylation led to accelerated proteasome-related degradation and induced ER stress in dAcsl knockdown flies,impairing lipoprotein release into the circulation which compromised interorgan lipid transport between the fat body and the gut.Inhibition of ubiquitin-proteasome-dependent degradation alleviated the phenotype of gut ectopic fat accumulation in dAcsl knockdown flies.Finally,we verified that ACSL4,the human homolog of dAcsl,also regulated lipoprotein levels in HepG2 cells,indicating that the role of dAcsl in modulating lipoprotein secretion and systemic lipid homeostasis is possibly conserved in humans.
基金the National Natural Science Foundation of China(reference number 31730102)the State Key Basic Research and Development Plan of China(reference number 2015CB150105)+2 种基金the Key Research Plan of Frontier Sciences of the Chinese Academy of Sciences(reference number QYZDJ-SSW-SMC003)the Strategic Priority Research Program of the Chinese Academy of Sciences(reference number XDB17030100)the National Key Scientific Instrument and Equipment Development Project of China(grant no.2013YQ030595).
文摘RNA polymerase transcriptional pausing represents a major checkpoint in transcription in bacteria and metazoans,but it is unknown whether this phenomenon occurs in plant organelles.Here,we report that transcriptional pausing occurs in chloroplasts.We found that mTERF5 specifically and positively regulates the transcription of chloroplast psbEFLJ in Arabidopsis thaliana that encodes four key subunits of photosystem II.We found that mTERF5 causes the plastid-encoded RNA polymerase(PEP)complex to pause at psbEFLJ by binding to the+30 to+51 region of double-stranded DNA.Moreover,we revealed that mTERF5 interacts with pTAC6,an essential subunit of the PEP complex,although pTAC6 is not involved in the transcriptional pausing at psbEFLJ.We showed that mTERF5 recruits additional pTAC6 to the transcriptionally paused region of psbEFLJ,and the recruited pTAC6 proteins could be assembled into the PEP complex to regulate psbEFLJ transcription.Taken together,our findings shed light on the role of transcriptional pausing in chloroplast transcription in plants.
基金This work was supported by the National Key Research and Development Program of China(2016YFD0101801,2009CB118506)the National Natural Sciences Foundation of China(31771348,32002119)China Postdoctoral Science Foundation(2020M672569).
文摘The coordinated utilization of nitrogen(N)and phosphorus(P)is vital for plants to maintain nutrient balance and achieve optimal growth.Previously,we revealed a mechanism by which nitrate induces genes for phosphate utilization;this mechanism depends on NRT1.1B-facilitated degradation of cytoplasmic SPX4,which in turn promotes cytoplasmic-nuclear shuttling of PHR2,the central transcription factor of phosphate signaling,and triggers the nitrate-induced phosphate response(NIPR)and N-P coordinated utilization in rice.In this study,we unveiled a fine-tuning mechanism of NIPR in the nucleus regulated by Highly Induced by Nitrate Gene 1(HINGE1,also known as RLI1),a MYB-transcription factor closely related to PHR2.RLI1/HINGE1,which is transcriptionally activated by PHR2 under nitrate induction,can directly activate the expression of phosphate starvation-induced genes.More importantly,RLI1/HINGE1 competes with PHR2 for binding to its repressor proteins in the nucleus(SPX proteins),and consequently releases PHR2 to further enhance phosphate response.Therefore,RLI1/HINGE1 amplifies the phosphate response in the nucleus downstream of the cytoplasmic SPX4-PHR2 cascade,thereby enabling fine-tuning of N-P balance when nitrate supply is sufficient.
基金supported by the National Natural Science Foundation of China (31788103, 32122064, 32122012)the Chinese Academy of Sciences (XDA24030504, 2019099)+1 种基金China Agricultural Research System (CARS-01-4)the China Postdoctoral Science Foundation (2019M650885)
文摘Salt stress adversely affects plant growth,development,and crop yield.Rice(Oryza sativa L.)is one of the most salt-sensitive cereal crops,especially at the early seedling stage.Mitogen-activated protein kinase(MAPK/MPK)cascades have been shown to play critical roles in salt response in Arabidopsis.However,the roles of the MPK cascade signaling in rice salt response and substrates of Os MPK remain largely unknown.Here,we report that the salt-induced Os MPK4-Ideal Plant Architecture 1(IPA1)signaling pathway regulates the salt tolerance in rice.Under salt stress,Os MPK4 could interact with IPA1 and phosphorylate IPA1 at Thr180,leading to degradation of IPA1.Genetic evidence shows that IPA1 is a negative regulator of salt tolerance in rice,whereas Os MPK4 promotes salt response in an IPA1-dependent manner.Taken together,our results uncover an Os MPK4-IPA1 signal cascade that modulates the salt stress response in rice and sheds new light on the breeding of salt-tolerant rice varieties.
基金supported by grants from the National Natural Science Foundation of China(91935301,91635301,31601276)the Strategic Priority Research Program“Molecular Mechanism of Plant Growth and Development”of CAS(XDB27010100)+2 种基金the Top Talents Program“One Case One Discussion(Yishiyiyi)”of Shandong Province,ChinaThe Deutsche Forschungsgemeinschaft funded research at Heidelberg University via the Collaborative Research Center 1036(Project-ID:201348542-SFB 1036)individual research grants(WI 3560/4-1,Project-ID:353859218 and WI 3560/7-1,Project-ID:496871662).
文摘N-terminal acetylation is one of the most common protein modifications in eukaryotes,and approximately 40%of human and plant proteomes are acetylated by ribosome-associated N-terminal acetyltransferase A(NatA)in a co-translational manner.However,the in vivo regulatory mechanism of NatA and the global impact of NatA-mediated N-terminal acetylation on protein fate remain unclear.Here,we identify Huntingtin Yeast partner K(HYPK),an evolutionarily conserved chaperone-like protein,as a positive regulator of NatA activity in rice.We found that loss of OsHYPK function leads to developmental defects in rice plant architecture but increased resistance to abiotic stresses,attributable to perturbation of the N-terminal acetylome and accelerated global protein turnover.Furthermore,we demonstrated that OsHYPK is also a substrate of NatA and that N-terminal acetylation of OsHYPK promotes its own degradation,probably through the Ac/N-degron pathway,which could be induced by abiotic stresses.Taken together,our findings suggest that the OsHYPK-NatA complex plays a critical role in coordinating plant development and stress responses by dynamically regulating NatA-mediated N-terminal acetylation and global protein turnover,which are essential for maintaining adaptive phenotypic plasticity in rice.
基金The research was supported by grants from the National Natural Science Foundation of China(32133012,31788103,32100268,and 31900244)the Youth Innovation Promotion Association of the Chinese Academy of Sciences(2019099).
文摘Multisubunit SKP1/Cullin1/F-box(SCF)E3 ligases play essential roles in regulating the stability of crucial regulatory factors and controlling growth and development in eukaryotes.Detecting E3 ligase activity in vitro is important forexploring the molecular mechanism of protein ubiquitination.However,in vitro ubiquitination assay systems for multisubunit E3 ligases remain difficult to achieve,especially in plants,mainly owing to difficulties in achieving active components of multisubunit E3 ligases with high purity and characterizing specific E2 and E3 pairs.In this study,we characterized components of the rice ScFDiwARF3(SCFDs)E3 ligase,screened the coordinated E2,and reconstituted active ScFD3 E3 ligase in vitro.We further engineered SCFD3 E3 ligase using a fused SKP1-Cullin1-RBX1(eSCR)protein and found that both the wild-type SCFD3 E3 ligase and the engineered SCFD3 E3 ligase catalyzed ubiquitination of the substrate D53,which is the key transcriptional repressor in strigolactone signaling.Finally,we replaced D3 with other F-box proteins from rice and humans and reconstituted active escF E3 ligases,including escFaID2,escFBxL1s,and escFcDC4 E3 ligases.Our work reconstitutes functional SCF E3 ligases in vitro and generates an engineered system with interchangeable F-box proteins,providing a powerful platform for studying the mechanisms of multisubunit SCF E3 ligases in eukaryotes.
基金the Ministry of Science and Technology of China(2018YFA0506902,2016YFA0500100,and 2018YFA081104)the National Natural Science Foundation of China(9195420001,31771305,and 31630019)Chinese Academy of Sciences(XDPB17)。
文摘Nonalcoholic fatty liver disease(NAFLD)encompasses a spectrum of pathologies,ranging from steatosis to nonalcoholic steatohepatitis(NASH).The factors promoting the progression of steatosis to NASH are still unclear.Recent studies suggest that mitochondrial lipid composition is critical in NASH develop-ment.Here,we showed that CDP-DAG synthase 2(Cds2)was downregulated in genetic or diet-induced NAFLD mouse models.Liver-specific deficiency of Cds2 provoked hepatic steatosis,inflammation and fibrosis in five-week-old mice.CDS2 is enriched in mitochondria-associated membranes(MAMs),and hepatic Cds2 deficiency impaired mitochondrial function and decreased mitochondrial PE levels.Overexpression of phosphatidylserine decarboxylase(PISD)alleviated the NASH-like phenotype in Cds2^(f/f);AlbCre mice and abnormal mitochondrial morphology and function caused by CDS2 deficiency in hepatocytes.Additionally,dietary supplementation with an agonist of peroxisome proliferator-activated receptor alpha(PPARa)attenuated mitochondrial defects and ameliorated the NASH-like phe-notype in Cds2^(f/f);AlbCre mice.Finally,Cds2 overexpression protected against high-fat diet-induced hepatic steatosis and obesity.Thus,Cds2 modulates mitochondrial function and NASH development.
基金This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciencesthe Ministry of Agriculture of China(grant no.2016ZX08009003-005).
文摘Adventitious roots form from non-root tissues as part of normal development or in response to stress or wounding.The root primordia form in the source tissue,and during emergence the adventitious roots penetrate the inner cell layers and the epidermis;however,the mechanisms underlying this emergence remain largely unexplored.Here,we report that a regulatory module composed of the AP2/ERF transcription factor ABSCISIC ACID INSENSITIVE 4(ABI4),the MAP kinases MPK3 and MPK6,and the phosphatase PP2C12 plays an important role in the emergence of junction adventitious roots(J-ARs)from the root-hypocotyl junctions in Arabidopsis thaliana.ABI4 negatively regulates J-AR emergence,preventing the accumulation of reactive oxygen species and death of epidermal cells,which would otherwise facilitate J-AR emergence.Phosphorylation by MPK3/MPK6 activates ABI4 and dephosphorylation by PP2C12 inactivates ABI4.MPK3/MPK6 also directly phosphorylate and inactivate PP2C12 during J-AR emergence.We propose that this"double-check"mechanism increases the robustness of MAP kinase signaling and finely regulates the local programmed cell death required for J-AR emergence.
基金supported by grants from the Ministry of Agriculture and Rural Affairs of China(2016ZX08009003-0022016ZX08009003-005 and 2016ZX08009003-004)+2 种基金Chinese Academy of Sciences(XDA08010401-2)the Ministry of Science and Technology of the People’s Republic of China(2016YFD0100706)the State Key Laboratory of Plant Genomics。
文摘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.
文摘Dear Editor,Rice dwarf virus(RDV),a member of the Phytoreoviurs genus,is transmitted to rice(Oryza sativa)plants by leaf-hopper(Nephotettix cincticeps)in a propagative manner.Infection by RDV results in severe stunting growth pheno-types and a dramatic reduction in grain yield.The genome of RDV is composed of 12-segmented double-stranded RNAs(S1-S12 based on their migration rates in agarose gel electro-phoresis).The 12 segments encode seven structural proteins(P1,P2,P3,P5,P7,P8,and P9 as the products of Sl,S2,S3,S5,S7,S8,and S9,respectively)and five nonstructural proteins(Pns4,Pns6,Pnsl0,Pns11,and Pns12 as the products of S4,S6,S10,S11,and S12,respectively).The outer capsid protein P2 is essential for RDV infection of insects and thus influences transmission of RDV by the insect vector(Omura et al.,1998;Zhou et al.,2007).P2 also contributes to the dwarf phenotype of infected rice by interfering with gibberellic acid synthesis(Zhu et al.,2005).When RDV-infected rice plants were main-tained via vegetative propagation for several years without insect transmission,they regained normal growth height due to loss of RDV P2 and Pnsl0 proteins(Pu et al.,2011).
基金supported by a grant from National Natural Science Foundation of China (31670234 to YW)the Strategic Priority Research Program (XDB No. 17030300)the Ministry of Science and Technology of the People’s Republic of China (No. 2015CB150100)。
文摘Cyanobacteria are a group of oxygenic photosynthetic bacteria with great potentials in biotechnological applications and advantages as models for photosynthesis research. The subcellular localizations of the majority of proteins in any cyanobacteria remain undetermined, representing a major challenge in using cyanobacteria for both basic and industrial researches. Here, using label-free quantitative proteomics, we map 2027 proteins of Synechocystis sp. PCC6803, a model cyanobacterium, to different subcellular compartments and generate a proteome atlas with such information. The atlas leads to numerous unexpected but important findings, including the predominant localization of the histidine kinases Hik33 and Hik27 on the thylakoid but not the plasma membrane. Such information completely changes the concept regarding how the two kinases are activated. Together, the atlas provides subcellular localization information for nearly 60% proteome of a model cyanobacterium, and will serve as an important resource for the cyanobacterial research community.
基金supported by the National Natural Science Foundation of China(NSFC,81620108011)the Ministry of Science and Technology of China(MOST,2017YFC1308401)+3 种基金supported by NSFC(31830036 and 31921002)the Beijing Municipal Science&Technology Commission(Z181100001518001)MOST(2019YFA0707100)the Chinese Academy of Sciences(XDBS1020100)。
文摘Atherosclerotic cardiovascular disease resulting from dysregulated lipid metabolism is the leading cause of morbidity and mortality worldwide.Apolipoprotein E(ApoE)plays a critical role in cholesterol metabolism.Knockouts in lipid-metabolizing proteins including ApoE in multiple model organisms such as mice and rats exhibiting elevated levels of cholesterol have been widely used for dissecting the pathology of atherosclerosis,but few of these animal models exhibit advanced atherosclerotic plaques leading to ischemia-induced clinical symptoms,limiting their use for translational studies.Here we report hypercholesterolemia and severe atherosclerosis characterized by stenosis and occlusion of arteries,together with clinical manifestations of stroke and gangrene,in ApoE knockout dogs generated by CRISPR/Cas9 and cloned by somatic cell nuclear transfer technologies.Importantly,the hypercholesterolemia and atherosclerotic complications in F0 mutants are recapitulated in their offspring.As the ApoE-associated atherosclerosis and clinical manifestations in mutant dogs are more similar to that in human patients compared with those in other animal models,these mutant dogs will be invaluable in developing and evaluating new therapies,including endovascular procedures,against atherosclerosis and related disorders.
基金supported by National Natural Science Foundation of China(Grant No.31030047)the National Basic Research Program(973 Program)(No.2011CB915402).
文摘Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins.The current methods that applied in protein-protein interaction,such as co-immunoprecipitation and pull down etc.,often cause plenty of working time due to the burdensome cloning and puri-fication procedures.Here we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within E.coli system.We modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease,two cleavage site F and two multiple cloning sites that flanking cleavage sites.The target proteins(for example:protein A and protein B)were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His6 tag.PPV NIa protease carried out intracellular cleavage along expression,then led to the separation of polyprotein components,therefore,the interaction between protein A-protein B can be detected through one-step purification and analysis.Negative control for protein B was brought into this system for monitoring interaction specificity.We successfully employed this system to prove two cases of reported protien-protein interaction:RHA2a/ANAC and FTA/FTB.In conclusion,a convenient and efficient system has been successfully developed for detecting protein-protein interaction.
