The evolutionarily conserved mitogen-activated protein kinase(MAPK)cascades relay extracellular signals into cells,triggering a variety of cellular responses.We previously revealed NtMPK4 as a positive regulator of ni...The evolutionarily conserved mitogen-activated protein kinase(MAPK)cascades relay extracellular signals into cells,triggering a variety of cellular responses.We previously revealed NtMPK4 as a positive regulator of nicotine biosynthesis;however,its upstream regulation remains unclear.Here,we characterized a MAPK cascade,comprising NtMEKK1b,NtMPKK2a,and NtMPK4,that promotes nicotine biosynthesis.This signaling module transduces external cues,including jasmonate and pathogen elicitors such as flg22,into post-translational modifications that enhance transcriptional activity and pathway gene expression.NtMPKK2a physically interacts with and phosphorylates NtMPK4 in vivo,confirming its role as an upstream kinase.RNAi-mediated silencing of NtMPKK2a significantly reduced the expression of nicotine pathway genes and decreased nicotine accumulation,whereas induced-overexpression of NtMPKK2a upregulated nicotine pathway genes and increased nicotine contents in tobacco hairy roots.Overexpression of NtMPKK2a in tobacco cells enhanced the transactivation activity of a NIC2-locus Ethylene Response Factor NtERF221 on Putrescine N-methyltransferase(NtPMT)promotor,further supporting its role in promoting nicotine biosynthesis.Furthermore,we identified NtMEKK1b,a tobacco MEKK that interacts with NtMAPKK2a in yeast cells.Knock-down of NtMEKK1b in transgenic tobacco plants attenuated the expression of nicotine pathway genes and reduced nicotine contents,whereas induced-overexpression of NtMEKK1b upregulated gene expression and nicotine accumulation.Our findings uncover a previously uncharacterized MAPK cascade module,NtMEKK1b-NtMPKK2a-NtMPK4,that regulates nicotine biosynthesis,highlighting the importance of posttranslational regulation in nicotine biosynthesis.展开更多
The yam Dioscorea alata L.is widely cultivated globally.Purple-fleshed varieties of this important crop have enhanced market value due to their high anthocyanin contents,but how anthocyanin biosynthesis in D.alata tub...The yam Dioscorea alata L.is widely cultivated globally.Purple-fleshed varieties of this important crop have enhanced market value due to their high anthocyanin contents,but how anthocyanin biosynthesis in D.alata tubers is regulated remains poorly understood.In this study,we identified and functionally validated key transcription factors that regulate anthocyanin biosynthesis based on a comparative transcriptome and metabolome analysis of three D.alata cultivars with different colored tubers(dark purple,light purple,and white).The anthocyanin glycoside cyanidin-3-O-(2′′-O-glucosyl)glucoside was abundant during early tuber development,and we determined that its accumulation is regulated in opposite manners by two R2R3-MYB transcription factors:DaMYB75 and DaMYB56.Yeast two-hybrid and bimolecular fluorescence complementation assays in Nicotiana benthamiana and co-expression assays in D.alata demonstrated that DaMYB75 promotes anthocyanin biosynthesis by specifically activating the promoter of the late anthocyanin biosynthesis gene DaANS and enhancing its expression through an interaction with DabHLH72.By contrast,DaMYB56 is a negative regulator of anthocyanin biosynthesis that binds to the DaANS promoter together with DabHLH72.Furthermore,the methylation levels of the DaMYB75 promoter were significantly lower in purple tubers than in white tubers.These findings shed light on the regulation of anthocyanin biosynthesis by MYBs and provide the basis for genetically improving anthocyanin content in D.alata.展开更多
Iris domestica,a perennial herb of the Iridaceae family,is widely recognized for its rich isoflavone content and broad therapeutic properties.To elucidate the biosynthetic pathway of these medicinally significant comp...Iris domestica,a perennial herb of the Iridaceae family,is widely recognized for its rich isoflavone content and broad therapeutic properties.To elucidate the biosynthetic pathway of these medicinally significant compounds,we constructed a haplotype-resolved genome assembly of this species.Transcriptomic and metabolomic analyses revealed tissue-specific accumulation of isoflavone,particularly in rhizomes and roots.Functional characterization identified two candidate isoflavone synthase genes,among which IdIFS was confirmed to promote the biosynthesis of key compounds tectorigenin and irisflorentin.The high-quality genome assembly presented here provides a foundational resource for further research into the evolution,secondary metabolite,and environmental adaptation of I.domestica.展开更多
Saikosaponins are the major pharmacologically active components in Bupleurum genus and exhibit significant application potential in multiple fields such as immune regulation and anti-tumor activity.To elucidate the bi...Saikosaponins are the major pharmacologically active components in Bupleurum genus and exhibit significant application potential in multiple fields such as immune regulation and anti-tumor activity.To elucidate the biosynthetic pathway of saikosaponins,we identified two cytochrome P450 monooxygenases,CYP716A41 and CYP716Y4,in Bupleurum chinense.These enzymes catalyze the C-28 oxidation and C-16 hydroxylation of oleanane-type triterpene skeletons,respectively.The catalytic efficiency of CYP716A41 from a southern B.chinense variety was significantly higher than that from a northern variety.Molecular docking and mutagenesis experiments revealed that amino acid residues at sites 9 and 35 may contribute to this difference in catalytic efficiency.Additionally,under cold stress,the expression levels of both CYP450 genes and the saikosaponin contents in the leaves of southern varieties were significantly higher compared to those in northern varieties.The variation in the catalytic efficiency of CYP716A41 and the differential expression of the two CYP450 genes under cold stress during winter are associated with the differences in saikosaponin biosynthesis in the leaves of southern and northern B.chinense varieties.This is consistent with the distinct medicinal usage practices observed between southern and northern China.展开更多
Lignin is a significant secondary metabolite produced through the phenylpropanoid pathway.As a vital component of the plant cell wall,lignin affects various fruit characteristics,including size,seed quantity,and firmn...Lignin is a significant secondary metabolite produced through the phenylpropanoid pathway.As a vital component of the plant cell wall,lignin affects various fruit characteristics,including size,seed quantity,and firmness.In this study,we conducted comprehensive identification and phylogenetic analysis of 265 Caffeic acid O-methyltransferase(COMT)genes across ten different plant species,including Vaccinium corymbosum and four other Vaccinium species.The results reveal that VcCOMT38 is a promising structural gene for the biosynthesis of lignin in blueberry.An in vitro enzymatic assay of VcCOMT38 demonstrated that it is a special enzyme in the lignin biosynthesis pathway and prefers to use caffeic acid as a substrate over 5-hydroxyferulic acid.Transient overexpression and silencing of VcCOMT38 in Vaccinium corymbosum‘Northland’fruits demonstrated that VcCOMT38 participates in lignin biosynthesis and contributes to both an increased number of immature seeds and enhanced fruit firmness.The heterologous overexpression of VcCOMT38 in Nicotiana benthamiana revealed that this gene could increase the lignin content and the syringyl/guaiacyl(S/G)ratio,which determines the maximum monomer yield during lignin depolymerization.These results highlight VcCOMT38 as a crucial gene in lignin biosynthesis and its potential for improving lignin production in industry through genetically modified woody plants.展开更多
Flavonoids are crucial secondary metabolites widely distributed in plants,playing vital roles in diverse biological processes.Although the flavonoid biosynthesis pathway has been extensively characterized,the transcri...Flavonoids are crucial secondary metabolites widely distributed in plants,playing vital roles in diverse biological processes.Although the flavonoid biosynthesis pathway has been extensively characterized,the transcriptional regulatory mechanisms remain poorly understood.In this study,we identify the miR166–ATHB14-LIKE module comprising the miR166 and its target gene ATHB14-LIKE as a key regulator of flavonoid biosynthesis in soybean(Glycine max).Knockdown of miR166 or overexpression of ATHB14-LIKE upregulated multiple flavonoid biosynthesis genes,leading to increased flavonoid accumulation.Conversely,miR166 overexpression suppressed these genes and reduced flavonoid levels.We further show that ATHB14-LIKE directly activates specific flavonoid biosynthesis genes by binding to their promoters.Additionally,ATHB14-LIKE forms homodimers and heterodimers with homologous proteins to regulate downstream flavonoid biosynthesis genes.Together,our findings demonstrate that the miR166–ATHB14-LIKE module controls soybean flavonoid content by coordinating the expression of key biosynthetic genes.展开更多
Locust plagues result in significant agricultural and ecological damage,and the current dependence on broadspectrum chemical pesticides raises serious concerns regarding environmental sustainability and public health....Locust plagues result in significant agricultural and ecological damage,and the current dependence on broadspectrum chemical pesticides raises serious concerns regarding environmental sustainability and public health.In this study,we elucidated the biosynthetic pathway of the locust aggregation pheromone 4-vinylanisole(4VA).Through analysis of the crystal structure of the 4VPMT2-4VA-SAM complex,it was determined that 4-nitrophenol acts as a substrate analogue,effectively inhibiting 4VPMT enzyme activity and thereby preventing the formation of 4VA.This study revealed key enzymatic targets and lead inhibitors for intervention,establishing a molecular foundation for pheromone-based,environmentally sustainable locust control strategies and offering a viable alternative to reduce reliance on conventional chemical pesticides.展开更多
Background:Excessive use of inorganic trace minerals(ITMs)in swine production leads to high fecal mineral excretion and environmental risks,while most studies on organic trace minerals(OTMs)focus on single elements,wi...Background:Excessive use of inorganic trace minerals(ITMs)in swine production leads to high fecal mineral excretion and environmental risks,while most studies on organic trace minerals(OTMs)focus on single elements,with limited data on the synergistic effects and molecular mechanisms of combined OTMs(Fe,Cu,Mn,Zn)in growing-finishing pigs.Methods:This study aimed to investigate the effects of graded levels of micromineral proteinates(combined OTMs)on growth performance,mineral metabolism,and mRNA expression of mineral regulatory proteins.A total of 360 crossbred Duroc×Landrace×Large White pigs(initial body weight 47.1±4.8 kg)were randomly assigned to 6 dietary treatments:basal diet without microminerals(CON),basal diet with ITMs at commercially recommended levels(IT),and basal diets with 15%(OT 15%),25%(OT 25%),35%(OT 35%)commercially recommended levels(CRL)of combined micromineral proteinates.After a 70-day feeding trial,samples were analyzed using ICP-OES,ELISA,and RT-qPCR.Results:Results showed that reduced levels(15-35%CRL)of micromineral proteinates did not significantly affect average daily gain,average daily feed intake,or feed conversion ratio(gain-to-feed ratio)compared to IT(P>0.05),but significantly increased plasma Cu(1.73-1.83μg/mL)and Zn(1.72-1.97μg/mL)concentrations(P<0.05)and elevated activities of Cu/Zn-superoxide dismutase(32.9-35.9 U/L)and manganese superoxide dismutase(20.5-24.1 U/L)compared to CON(P<0.05),with no significant differences from IT(P>0.05).Fecal excretion of Fe,Cu,Mn,and Zn was significantly reduced by 35-50%in OT 15%-OT 35%groups compared to IT(P<0.05).OT 25%group exhibited the highest apparent absorptivity of Fe(38.5%),Cu(27.8%),and Zn(42.4%)(P<0.05),which was associated with significantly regulated mRNA expression of mineral regulatory proteins:upregulated DMT1,FPN1,ZIP4,and MT1A in the duodenum,and modulated HAMP,ATP7B,ZIP14,and ZnT1 in the liver(P<0.05).Conclusion:In conclusion,dietary supplementation with 25%CRL or less of combined micromineral proteinates can fully meet the nutritional needs of growing-finishing pigs,improve mineral absorptivity,and reduce fecal mineral excretion by regulating intestinal and hepatic mineral transport and homeostatic proteins,providing a sustainable alternative to high-dose ITMs.展开更多
Flavonoids,abundant in the fruits,are pivotal to their growth,development,and storage.In addition,they have significant beneficial effects on human health.Consequently,research is increasingly concentrating on the reg...Flavonoids,abundant in the fruits,are pivotal to their growth,development,and storage.In addition,they have significant beneficial effects on human health.Consequently,research is increasingly concentrating on the regulatory mechanisms governing flavonoid biosynthesis in fruits.Phytohormones are involved in the regulation of flavonoid biosynthesis.The abscisic acid,ethylene,jasmonic acid,cytokinins,and brassinosteroids promote flavonoid biosynthesis,while auxin negatively regulates flavonoid biosynthesis.Subsequently,transcription factors from the MYB,bHLH,WRKY,NAC,and bZIP families are pivotal in regulating flavonoid biosynthesis.In addition,non-coding RNAs(microRNA and lncRNA)also participate in the regulation of flavonoids biosynthesis.MicroRNAs are generally believed to negatively regulate flavonoid metabolism in fruits,while lncRNAs have the opposite effect.Furthermore,the interactions between plant hormones,transcription factors,and non-coding RNAs in fruit flavonoid biosynthesis were analyzed.Ultimately,a foundational regulatory network for fruit flavonoid biosynthesis was hereby established.展开更多
Proanthocyanidins(PAs)are a group of oligomeric flavonoids also known as condensed tannins.PAs in human diet have antioxidant and health-promoting effects.BSISTER(BS)promotes the accumulation of PAs,but its direct tar...Proanthocyanidins(PAs)are a group of oligomeric flavonoids also known as condensed tannins.PAs in human diet have antioxidant and health-promoting effects.BSISTER(BS)promotes the accumulation of PAs,but its direct target gene and regulation mechanism in PAs biosynthesis are not clear.Here,we reported VviBS1 and VviBS2 genes,which bind directly to the promoter of PA synthesis key genes VviANR(ANTHOCYANIDIN REDUCTASE)and VviLAR(LEUCOANTHOCYANIDIN REDUCTASE)and up-regulate their expression to promote the PAs biosynthesis in grapevine.VviBS1 and VviBS2 partially rescued the transparent testa phenotype of an Arabidopsis tt16 mutant.Overexpression of BS1 and BS2 in grapevine callus increased the content of PAs,as well as the expression of specific genes related to PA synthesis.VviBS1 and VviBS2 proteins bound directly to the promoter regions of the key PA synthesis genes VviANR1,VviANR2,and VviLAR1.Overexpression of VviBS1 and VviBS2 promotes VviANR1,VviANR2 and VviLAR1 expression and PAs biosynthesis.Some BS1-and BS2-interacting MADS-box proteins have an effect on PAs biosynthesis.This study provides insight into the regulatory mechanisms of PAs biosynthesis in grapevine,which could be effectively employed for metabolic engineering to increase PA content.展开更多
Lodging is a major constraint limiting oil flax production efficiency in northern China.Crop lodging susceptibility is closely related to stem lignin content,and the regulatory mechanisms by which nitrogen and potassi...Lodging is a major constraint limiting oil flax production efficiency in northern China.Crop lodging susceptibility is closely related to stem lignin content,and the regulatory mechanisms by which nitrogen and potassium fertilization interactively influence lignin biosynthesis in oil flax stems require further investigation.Therefore,this study aimed to enhance lodging resistance and increase grain yield in oil flax.We examined the interactive effects of different nitrogen (75,150,and 225 kg N ha^(–1)) and potassium (60 and 90 kg K_(2)O ha^(–1)) fertilizer rates on lignin metabolism,lodging resistance,and grain yield during the 2022 and 2023 growing seasons.Results indicated that nitrogen and potassium fertilizer levels and their interactions promoted lignin accumulation,improved lodging resistance,and increased grain yield.Compared to the control (CK),the75–150 kg N ha^(–1) combined with 60 kg K_(2)O ha^(–1) treatments significantly enhanced the activities of key lignin-synthesizing enzymes (tyrosine ammonia-lyase (TAL),phenylalanine ammonia-lyase (PAL),cinnamyl alcohol dehydrogenase (CAD),and peroxidase (POD)) and upregulated the expression of 4CL1 and F5H3 genes,leading to a 29.63–43.30%increase in lignin content,improved stem bending strength and lodging resistance index,and a 23.27–32.34%increase in grain yield.Correlation analysis revealed that nitrogen and potassium fertilizers positively regulated enzyme activities and gene expression related to lignin biosynthesis,thereby facilitating lignin accumulation and enhancing stem mechanical strength and lodging resistance.Positive correlations were observed among lignin-related enzyme activities,gene expression,lodging resistance traits,and grain yield.In summary,the application of 75–150 kg N ha^(–1) in conjunction with 60 kg K_(2)O ha^(–1)promoted lignin biosynthesis and accumulation,enhanced lodging resistance,and increased grain yield in oil flax grown in the dryland farming region of central Gansu,China.Furthermore,this treatment provides a technical basis for cultivating stress-tolerant and high-yield oil flax in arid regions.展开更多
Flower color is an essential trait in ornamental plant breeding. Lycoris longituba is a popular ornamental plant native to central eastern China. The decrease in anthocyanin accumulation causes L. longituba petal colo...Flower color is an essential trait in ornamental plant breeding. Lycoris longituba is a popular ornamental plant native to central eastern China. The decrease in anthocyanin accumulation causes L. longituba petal color fading during flower development, which considerably affects the ornamental value of L. longituba. However, mechanisms underlying anthocyanin biosynthesis inhibition during L. longituba petal development remain unclear. In this study, three LlDFR genes were confirmed to be involved in anthocyanin biosynthesis and LlDFRc exerted the strongest promoting effect on anthocyanin accumulation. According to the correlation analysis results, LlbHLH12 exhibited the strongest negative correlation with LlDFRc. Quantitative real-time PCR analysis showed that LlbHLH12 was highly expressed during the medium bud and full bloom stages of flower development. LlbHLH12 was identified as a member of subgroup XII of bHLH transcription factor family. Subcellular localization and transcriptional activation ability assay revealed that LlbHLH12 was located in the nucleus without transcriptional activation activity. Overexpression of LlbHLH12 in Nicotiana tabacum and L. longituba inhibited anthocyanin accumulation by suppressing the expression of anthocyanin biosynthetic pathway genes. Furthermore, yeast one-hybrid, dual-luciferase, and β-glucuronidase activity assays showed that LlbHLH12 directly bound to the promoters of LlPAL and LlDFRc and suppressed their expression to inhibit anthocyanin biosynthesis. Overall, our study identified a novel bHLH repressor negatively regulating anthocyanin biosynthesis and provided new insights into the molecular mechanisms underlying color fading in L. longituba petals.展开更多
Spinal cord injury is a severe neurological condition with limited neuronal regeneration and functional recovery.Currently,no effective treatments exist to improve spinal cord injury prognosis.Neuronal guidance protei...Spinal cord injury is a severe neurological condition with limited neuronal regeneration and functional recovery.Currently,no effective treatments exist to improve spinal cord injury prognosis.Neuronal guidance proteins are a diverse group of molecules that play crucial roles in axon and dendrite growth during nervous system development.Increasing evidence highlights their regulatory functions in spinal cord injury.This review provides a brief overview of the modulation patterns of key neuronal guidance proteins in neuronal axon growth during nervous system formation and subsequently focuses on their roles in neuronal regeneration and functional recovery following spinal cord injury.Neuronal guidance proteins include,but are not limited to,semaphorins and their receptors,plexins;netrins and their receptors,deleted in colorectal cancer and UNC5;Eph receptors and their ligands,ephrins;Slit and its receptor,Robo;repulsive guidance molecules and their receptor,neogenin;Wnt proteins and their receptor,Frizzled;and protocadherins.Localized Netrin-1 at the injury site inhibits motor axon regeneration after adult spinal cord injury while promoting oligodendrocyte growth.Slit2 enhances synapse formation in the injured spinal cord of rats.EphA7 regulates acute apoptosis in the early pathophysiological stages of spinal cord injury,while ephrinA1 plays a role in the nervous system’s injury response,with its reduced expression leading to impaired motor function in rats.EphA3 is upregulated following spinal cord injury,promoting an inhibitory environment for axonal regeneration.After spinal cord injury,bidirectional activation of ephrinB2 and EphB2 in astrocytes and fibroblasts results in the formation of a dense astrocyte-meningeal fibroblast scar.EphB1/ephrinB1 signaling mediates pain processing in spinal cord injury by regulating calpain-1 and caspase-3 in neurons.EphB3 expression increases in white matter after spinal cord injury,further inhibiting axon regeneration.Sema3A,expressed by neurons and fibroblasts in the scar surrounding the injury,inhibits motor neuron and sensory nerve growth after spinal cord injury.Sema4D suppresses neuronal axon myelination and axon regeneration,while its inhibition significantly enhances axon regeneration and motor recovery.Sema7A is involved in glial scar formation and may influence serotonin channel remodeling,thereby affecting motor coordination.Given these findings,the local or systemic application of neuronal guidance proteins represents a promising avenue for spinal cord injury treatment.展开更多
The study of target proteins is crucial for understanding molecular interactions and developing analytical platforms,therapeutic agents and functional tools.Herein,we present a novel nanoplatform activated by near-inf...The study of target proteins is crucial for understanding molecular interactions and developing analytical platforms,therapeutic agents and functional tools.Herein,we present a novel nanoplatform activated by near-infrared(NIR) light for triple-modal proteins study,which enabling target protein labeling,enrichment and visualization.Azido-naphthalimide-coated upconversion nanoparticles(UCNPs) serve as NIR light-responsive nanoplatforms,showing promising applications in studying interactions between various bioactive molecules and proteins in living systems.Under NIR light irradiation,azido-naphthalimides are activated by ultraviolet(UV) and blue light emitted from UCNPs and the resulting amino-naphthalimides intermediate not only crosslink nearby target proteins but also enable imaging performance.We demonstrate that this nanoplatform is capable of selective protein labeling and imaging in complex protein environments,achieving specific labeling and imaging of both intracellular and extracellular proteins in mammalian cells as well as bacteria.Furthermore,in vivo protein labeling has been achieved using this novel NIR light-activatable nanoplatform.This technique will open new avenues for discoveries and mechanistic interrogation in chemical biology.展开更多
HPPD(4-hydroxyphenylpyruvate dioxygenase)inhibitor are widely used in agriculture due to their high efficacy and environmental friendliness.However,many important crops,such as rice,wheat,and soybean,are naturally sen...HPPD(4-hydroxyphenylpyruvate dioxygenase)inhibitor are widely used in agriculture due to their high efficacy and environmental friendliness.However,many important crops,such as rice,wheat,and soybean,are naturally sensitive to these herbicides.In this study,we employed a directed evolution strategy to enhance the metabolic capacity of OsHSL2,OsHSL4,OsHSL6,and SbHSL1 proteins toward HPPD inhibitors,providing a new technological approach as well as theoretical foundation for molecular breeding of herbicide-resistant crops.By combining AlphaFold 3 protein models with crystal structures,we systematically redesigned key residues to resemble the active residues found in HIS1.Catalytic activity assays demonstrated that specific mutations significantly improved the metabolic activity of HSLs proteins toward various HPPD inhibitors.Notably,the OsHSL2-M4 mutant exhibited enhanced metabolic activity for BBC-OH and methyl-benquitrione,while the OsHSL4-M5 mutant completely metabolized BBC-OH and topramezone.Additionally,the SbHSL1-M4 mutant showed significant improvement in the metabolism of BBC-OH and several other herbicides,providing strong evidence to support the use of structure-guided HSL mutations to enhance crop resistance to HPPD inhibitors.展开更多
Multienzyme cascades enable the sequential synthesis of complex chemicals by combining multiple catalytic processes in one pot,offering considerable time and cost savings compared to a series of separate batch reactio...Multienzyme cascades enable the sequential synthesis of complex chemicals by combining multiple catalytic processes in one pot,offering considerable time and cost savings compared to a series of separate batch reactions.However,challenges related to coordination and regulatory interplay among multiple enzymes reduce the catalytic efficiency of such cascades.Herein,we genetically programmed a scaffold framework that selectively and orthogonally recruits enzymes as designed.The system was then used to generate multienzyme complexes of D-allulose 3-epimerase(DAE),ribitol dehydrogenase(RDH),and formate dehydrogenase(FDH)for rare sugar production.This scaffolded multienzymatic assembly achieves a 10.4-fold enhancement in the catalytic performance compared to its unassembled counterparts,obtaining allitol yield of more than 95%.Molecular dynamics simulations revealed that shorter distances between neighboring enzymes in scaffold-mounted complexes facilitated the transfer of reaction intermediates.A dual-module catalytic system incorporating(1)scaffold-bound complexes of DAE,RDH,and FDH and(2)scaffold-bound complexes of alcohol dehydrogenase and NADH oxidase expressed intracellularly in E.coli was used to synthesize D-allulose from D-fructose.This system synthesized 90.6%D-allulose from 300 g L^(−1)D-fructose,with a space-time yield of 13.6 g L^(−1)h^(−1).Our work demonstrates the programmability and versatility of scaffold-based strategies for the advancement of multienzyme cascades.展开更多
<span style="font-family:;" "=""><span style="font-family:Verdana;">Methane production by archaea depends on tetrahydromethanopterin (H</span><sub><span st...<span style="font-family:;" "=""><span style="font-family:Verdana;">Methane production by archaea depends on tetrahydromethanopterin (H</span><sub><span style="font-family:Verdana;">4</span></sub><span style="font-family:Verdana;">MPT), a pterin-containing cofactor that carries one-carbon units. Two redox reactions within the nine steps of H</span><sub><span style="font-family:Verdana;">4</span></sub><span style="font-family:Verdana;">MPT side chain biosynthesis have been hypothesized. Biochemical assays have demonstrated that the archaeal iron-sulfur flavoprotein dihydromethanopterin reductase X (DmrX or MM1854) catalyzes the final reaction of the pathway, the reduction of dihydromethanopterin to H</span><sub><span style="font-family:Verdana;">4</span></sub><span style="font-family:Verdana;">MPT</span></span><span style="font-family:Verdana;">,</span><span style="font-family:;" "=""><span style="font-family:Verdana;"> using dithiothreitol (DTT) as an artificial electron donor. The crystal structure of DmrB, a bacterial DmrX homolog that lacks iron-sulfur clusters, has led to a proposed ping-pong mechanism of electron transfer between FMNH</span><sub><span style="font-family:Verdana;">2</span></sub><span style="font-family:Verdana;"> and the FMN prosthetic group of DmrB. However, an enzymatic assay to test the hypothetical DmrB mechanism is lacking because a suitable electron donor has not previously been identified. Furthermore, a second uncharacterized archaeal flavoprotein (MM1853) has been hypothesized to function in H</span><sub><span style="font-family:Verdana;">4</span></sub><span style="font-family:Verdana;">MPT side chain biosynthesis. In this </span><span style="font-family:Verdana;">work, to facilitate the development of assays to elucidate the functions of DmrB </span><span style="font-family:Verdana;">and MM1853, we tested a variety of electron donors, including dithiothreitol, ferredoxin, and a system consisting of NADH and an NADH-dependent fla</span><span style="font-family:Verdana;">vin-reducing enzyme (Fre).</span><span style="font-family:Verdana;"> Reduction of the DmrB prosthetic group (FMN) was measured as a decrease in absorbance at 460 nm. NADPH, NADH, and </span><span style="font-family:Verdana;">DTT were unable to reduce DmrB. However, NADH/Fre was able to reduce </span><span style="font-family:Verdana;">DmrB within 70 min (initial rate of 1.3 μM/min), providing the basis for a future DmrB activity assay. Carbon monoxide (CO)/CO dehydrogenase/ferredoxin reduced DmrB more rapidly within 6 min. Both electr</span><span style="font-family:Verdana;">on transfer systems reduced a second flavin-containing archaeal protein MM1853, which is predicted to catalyze the third step of H</span><sub><span style="font-family:Verdana;">4</span></sub><span style="font-family:Verdana;">MPT biosynthesis. While NADH and NADPH were incapable of directly reducing the FMN cofactor of MM1853, DTT or NADH/Fre could eliminate the FMN peaks. These results establish the basis for new oxidoreductase assays that will facilitate testing several proposed DmrB mechanisms and defining the specific function of MM1853 in methanogen cofactor biosynthesis.</span></span>展开更多
Our previous studies showed a predominance of high molecular weight protein group in tumor nuclear matrices. Contrary to normal cells, proteins of this group are preferentially phosphorylated. Phosphoproteins of hepat...Our previous studies showed a predominance of high molecular weight protein group in tumor nuclear matrices. Contrary to normal cells, proteins of this group are preferentially phosphorylated. Phosphoproteins of hepatoma nuclear matrix are selectively subjected to rapid proteolysis. By alkali treatment and a monoclonal antibody against phosphotyrosyl residue the presence of two high molecular weight bands of phosphotyrosyl-containing proteins was detected in nuclear matrices of tumor but not of normal liver cells. High molecular weight protein group of tumor nuclear matrices revealed also a rapid turnover and preferential incorporation of labeled amino acids selectively inhibited by chloramphenicol.展开更多
Chitin is an abundant aminopolysaccharide found in insect pests and phytopathogenic microorganisms but absent in higher plants and vertebrates. It is crucial for mitigating threats posed by chitin-containing organisms...Chitin is an abundant aminopolysaccharide found in insect pests and phytopathogenic microorganisms but absent in higher plants and vertebrates. It is crucial for mitigating threats posed by chitin-containing organisms to human health, food safety, and agriculture. Therefore, targeting the chitin biosynthesisassociated bioprocess holds a promise for developing human-safe and eco-friendly antifungal agents or pesticides. Chitin biosynthesis requires chitin synthase and associated factors, which are involved in the modification, regulation, organization or turnover of chitin during its biosynthesis. A number of enzymes such as chitinases, hexosaminidases, chitin deacetylases are closely related and therefore are promising targets for designing novel agrochemicals that target at chitin biosynthesis. This review summarizes the advances in understanding chitin biology over the past decade by our research group and collaborates,specifically regarding essential proteins linked to chitin biosynthesis that can be exploited as promising pesticide targets. Examples of small bioactive molecules that against the activity of these targets are given.展开更多
Xylo-oligosaccharides(XOSs)are a category of functional oligosaccharides primarily composed of 2-7 xylose units linked byβ-1,4 glycosidic bonds.They are recognized as soluble dietary fibers with prebiotic properties....Xylo-oligosaccharides(XOSs)are a category of functional oligosaccharides primarily composed of 2-7 xylose units linked byβ-1,4 glycosidic bonds.They are recognized as soluble dietary fibers with prebiotic properties.Recently, there has been significant interest in manufacturing XOSs from xylan extracted from lignocellulosic biomass using enzyme catalysis under mild conditions. In this work, the arabinofuranosidase Abf62A gene was cloned from Aspergillus usamii genomic DNA through sequential molecular processes and expressed in Pichia pastoris X33. The xylan (100 g/L) extracted xylan in wheat straw (WS) was biologically hydrolyzed into 50.32 g/L of XOSs by xylanase Xyn11A (300 U/g substrate) and arabinofuranase Abf62A (20 U/g substrate), which indicated a notable synergistic effect compared to the 34.42 g/L XOSs produced via Xyn11A. The 50.32 g/L of XOSs products comprised xylobiose (31.71 g/L), xylotriose (15.92 g/L), xylotetraose (1.65 g/L) and xylopentaose (1.04 g/L). Notably, the combined content of xylobiose and xylotriose accounted for up to 94.7%. The XOSs purified from the enzyme hydrolysate could effectually scavenge free radicals, and the antioxidant activity was more than 90%. In summary, XOSs were biologically manufactured from wheat straw xylan through the synergistic biocatalysis via xylanase and arabinofuranosidase Abf62A in a green and sustainable way, rending one kind of prebiotic oligosaccharides with substantial positive effects on human and animal health.展开更多
基金supported partially by the Kentucky Tobacco Research and Development Center(KTRDC),University of Kentucky.
文摘The evolutionarily conserved mitogen-activated protein kinase(MAPK)cascades relay extracellular signals into cells,triggering a variety of cellular responses.We previously revealed NtMPK4 as a positive regulator of nicotine biosynthesis;however,its upstream regulation remains unclear.Here,we characterized a MAPK cascade,comprising NtMEKK1b,NtMPKK2a,and NtMPK4,that promotes nicotine biosynthesis.This signaling module transduces external cues,including jasmonate and pathogen elicitors such as flg22,into post-translational modifications that enhance transcriptional activity and pathway gene expression.NtMPKK2a physically interacts with and phosphorylates NtMPK4 in vivo,confirming its role as an upstream kinase.RNAi-mediated silencing of NtMPKK2a significantly reduced the expression of nicotine pathway genes and decreased nicotine accumulation,whereas induced-overexpression of NtMPKK2a upregulated nicotine pathway genes and increased nicotine contents in tobacco hairy roots.Overexpression of NtMPKK2a in tobacco cells enhanced the transactivation activity of a NIC2-locus Ethylene Response Factor NtERF221 on Putrescine N-methyltransferase(NtPMT)promotor,further supporting its role in promoting nicotine biosynthesis.Furthermore,we identified NtMEKK1b,a tobacco MEKK that interacts with NtMAPKK2a in yeast cells.Knock-down of NtMEKK1b in transgenic tobacco plants attenuated the expression of nicotine pathway genes and reduced nicotine contents,whereas induced-overexpression of NtMEKK1b upregulated gene expression and nicotine accumulation.Our findings uncover a previously uncharacterized MAPK cascade module,NtMEKK1b-NtMPKK2a-NtMPK4,that regulates nicotine biosynthesis,highlighting the importance of posttranslational regulation in nicotine biosynthesis.
基金supported by the National Natural Science Foundation of China(32460767)Jiangxi Provincial Key Research and Development Program(20232BBF60007)Jiangxi Provincial Natural Science Foundation(20224BAB205024).
文摘The yam Dioscorea alata L.is widely cultivated globally.Purple-fleshed varieties of this important crop have enhanced market value due to their high anthocyanin contents,but how anthocyanin biosynthesis in D.alata tubers is regulated remains poorly understood.In this study,we identified and functionally validated key transcription factors that regulate anthocyanin biosynthesis based on a comparative transcriptome and metabolome analysis of three D.alata cultivars with different colored tubers(dark purple,light purple,and white).The anthocyanin glycoside cyanidin-3-O-(2′′-O-glucosyl)glucoside was abundant during early tuber development,and we determined that its accumulation is regulated in opposite manners by two R2R3-MYB transcription factors:DaMYB75 and DaMYB56.Yeast two-hybrid and bimolecular fluorescence complementation assays in Nicotiana benthamiana and co-expression assays in D.alata demonstrated that DaMYB75 promotes anthocyanin biosynthesis by specifically activating the promoter of the late anthocyanin biosynthesis gene DaANS and enhancing its expression through an interaction with DabHLH72.By contrast,DaMYB56 is a negative regulator of anthocyanin biosynthesis that binds to the DaANS promoter together with DabHLH72.Furthermore,the methylation levels of the DaMYB75 promoter were significantly lower in purple tubers than in white tubers.These findings shed light on the regulation of anthocyanin biosynthesis by MYBs and provide the basis for genetically improving anthocyanin content in D.alata.
文摘Iris domestica,a perennial herb of the Iridaceae family,is widely recognized for its rich isoflavone content and broad therapeutic properties.To elucidate the biosynthetic pathway of these medicinally significant compounds,we constructed a haplotype-resolved genome assembly of this species.Transcriptomic and metabolomic analyses revealed tissue-specific accumulation of isoflavone,particularly in rhizomes and roots.Functional characterization identified two candidate isoflavone synthase genes,among which IdIFS was confirmed to promote the biosynthesis of key compounds tectorigenin and irisflorentin.The high-quality genome assembly presented here provides a foundational resource for further research into the evolution,secondary metabolite,and environmental adaptation of I.domestica.
基金supported by CARS(CARS-21),the CAMS Innovation Fund for Medical Sciences(2021-I2M-1-032)the Science and Technology Department of Xizang(XZ202401ZY0020)+2 种基金the Science and Technology Department of Sichuan Province(2023YFH0044,2023YFH0018)the Sichuan Province Science Foundation for Distinguished Young Scholars(2022JDJQ0006)the Doctoral Fund of Southwest University of Science and Technology(19ZX7117,21ZX7116).
文摘Saikosaponins are the major pharmacologically active components in Bupleurum genus and exhibit significant application potential in multiple fields such as immune regulation and anti-tumor activity.To elucidate the biosynthetic pathway of saikosaponins,we identified two cytochrome P450 monooxygenases,CYP716A41 and CYP716Y4,in Bupleurum chinense.These enzymes catalyze the C-28 oxidation and C-16 hydroxylation of oleanane-type triterpene skeletons,respectively.The catalytic efficiency of CYP716A41 from a southern B.chinense variety was significantly higher than that from a northern variety.Molecular docking and mutagenesis experiments revealed that amino acid residues at sites 9 and 35 may contribute to this difference in catalytic efficiency.Additionally,under cold stress,the expression levels of both CYP450 genes and the saikosaponin contents in the leaves of southern varieties were significantly higher compared to those in northern varieties.The variation in the catalytic efficiency of CYP716A41 and the differential expression of the two CYP450 genes under cold stress during winter are associated with the differences in saikosaponin biosynthesis in the leaves of southern and northern B.chinense varieties.This is consistent with the distinct medicinal usage practices observed between southern and northern China.
文摘Lignin is a significant secondary metabolite produced through the phenylpropanoid pathway.As a vital component of the plant cell wall,lignin affects various fruit characteristics,including size,seed quantity,and firmness.In this study,we conducted comprehensive identification and phylogenetic analysis of 265 Caffeic acid O-methyltransferase(COMT)genes across ten different plant species,including Vaccinium corymbosum and four other Vaccinium species.The results reveal that VcCOMT38 is a promising structural gene for the biosynthesis of lignin in blueberry.An in vitro enzymatic assay of VcCOMT38 demonstrated that it is a special enzyme in the lignin biosynthesis pathway and prefers to use caffeic acid as a substrate over 5-hydroxyferulic acid.Transient overexpression and silencing of VcCOMT38 in Vaccinium corymbosum‘Northland’fruits demonstrated that VcCOMT38 participates in lignin biosynthesis and contributes to both an increased number of immature seeds and enhanced fruit firmness.The heterologous overexpression of VcCOMT38 in Nicotiana benthamiana revealed that this gene could increase the lignin content and the syringyl/guaiacyl(S/G)ratio,which determines the maximum monomer yield during lignin depolymerization.These results highlight VcCOMT38 as a crucial gene in lignin biosynthesis and its potential for improving lignin production in industry through genetically modified woody plants.
基金the Projects of Science and Technology of Shanghai(22N11900400)Key Projects of Beijing Institute of Life Science and Technology(2024400CB0050)+1 种基金National Natural Science Foundation of China(32488102)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA24030303)。
文摘Flavonoids are crucial secondary metabolites widely distributed in plants,playing vital roles in diverse biological processes.Although the flavonoid biosynthesis pathway has been extensively characterized,the transcriptional regulatory mechanisms remain poorly understood.In this study,we identify the miR166–ATHB14-LIKE module comprising the miR166 and its target gene ATHB14-LIKE as a key regulator of flavonoid biosynthesis in soybean(Glycine max).Knockdown of miR166 or overexpression of ATHB14-LIKE upregulated multiple flavonoid biosynthesis genes,leading to increased flavonoid accumulation.Conversely,miR166 overexpression suppressed these genes and reduced flavonoid levels.We further show that ATHB14-LIKE directly activates specific flavonoid biosynthesis genes by binding to their promoters.Additionally,ATHB14-LIKE forms homodimers and heterodimers with homologous proteins to regulate downstream flavonoid biosynthesis genes.Together,our findings demonstrate that the miR166–ATHB14-LIKE module controls soybean flavonoid content by coordinating the expression of key biosynthetic genes.
基金support of the National Natural Science Foundation of China(32472594).
文摘Locust plagues result in significant agricultural and ecological damage,and the current dependence on broadspectrum chemical pesticides raises serious concerns regarding environmental sustainability and public health.In this study,we elucidated the biosynthetic pathway of the locust aggregation pheromone 4-vinylanisole(4VA).Through analysis of the crystal structure of the 4VPMT2-4VA-SAM complex,it was determined that 4-nitrophenol acts as a substrate analogue,effectively inhibiting 4VPMT enzyme activity and thereby preventing the formation of 4VA.This study revealed key enzymatic targets and lead inhibitors for intervention,establishing a molecular foundation for pheromone-based,environmentally sustainable locust control strategies and offering a viable alternative to reduce reliance on conventional chemical pesticides.
基金financially supported by the Hainan Province Science and Technology Special Fund(Grant no:ZDYF2024XDNY187).
文摘Background:Excessive use of inorganic trace minerals(ITMs)in swine production leads to high fecal mineral excretion and environmental risks,while most studies on organic trace minerals(OTMs)focus on single elements,with limited data on the synergistic effects and molecular mechanisms of combined OTMs(Fe,Cu,Mn,Zn)in growing-finishing pigs.Methods:This study aimed to investigate the effects of graded levels of micromineral proteinates(combined OTMs)on growth performance,mineral metabolism,and mRNA expression of mineral regulatory proteins.A total of 360 crossbred Duroc×Landrace×Large White pigs(initial body weight 47.1±4.8 kg)were randomly assigned to 6 dietary treatments:basal diet without microminerals(CON),basal diet with ITMs at commercially recommended levels(IT),and basal diets with 15%(OT 15%),25%(OT 25%),35%(OT 35%)commercially recommended levels(CRL)of combined micromineral proteinates.After a 70-day feeding trial,samples were analyzed using ICP-OES,ELISA,and RT-qPCR.Results:Results showed that reduced levels(15-35%CRL)of micromineral proteinates did not significantly affect average daily gain,average daily feed intake,or feed conversion ratio(gain-to-feed ratio)compared to IT(P>0.05),but significantly increased plasma Cu(1.73-1.83μg/mL)and Zn(1.72-1.97μg/mL)concentrations(P<0.05)and elevated activities of Cu/Zn-superoxide dismutase(32.9-35.9 U/L)and manganese superoxide dismutase(20.5-24.1 U/L)compared to CON(P<0.05),with no significant differences from IT(P>0.05).Fecal excretion of Fe,Cu,Mn,and Zn was significantly reduced by 35-50%in OT 15%-OT 35%groups compared to IT(P<0.05).OT 25%group exhibited the highest apparent absorptivity of Fe(38.5%),Cu(27.8%),and Zn(42.4%)(P<0.05),which was associated with significantly regulated mRNA expression of mineral regulatory proteins:upregulated DMT1,FPN1,ZIP4,and MT1A in the duodenum,and modulated HAMP,ATP7B,ZIP14,and ZnT1 in the liver(P<0.05).Conclusion:In conclusion,dietary supplementation with 25%CRL or less of combined micromineral proteinates can fully meet the nutritional needs of growing-finishing pigs,improve mineral absorptivity,and reduce fecal mineral excretion by regulating intestinal and hepatic mineral transport and homeostatic proteins,providing a sustainable alternative to high-dose ITMs.
基金supported by the China Agricultural Research System(Grant No.CARS-09)the Central Government Guiding Local Science and Technology Development Project(Grant No.YDZX2023029)the Gansu Planning Projects on Science and Technology(Grant No.23CXNJ0013).
文摘Flavonoids,abundant in the fruits,are pivotal to their growth,development,and storage.In addition,they have significant beneficial effects on human health.Consequently,research is increasingly concentrating on the regulatory mechanisms governing flavonoid biosynthesis in fruits.Phytohormones are involved in the regulation of flavonoid biosynthesis.The abscisic acid,ethylene,jasmonic acid,cytokinins,and brassinosteroids promote flavonoid biosynthesis,while auxin negatively regulates flavonoid biosynthesis.Subsequently,transcription factors from the MYB,bHLH,WRKY,NAC,and bZIP families are pivotal in regulating flavonoid biosynthesis.In addition,non-coding RNAs(microRNA and lncRNA)also participate in the regulation of flavonoids biosynthesis.MicroRNAs are generally believed to negatively regulate flavonoid metabolism in fruits,while lncRNAs have the opposite effect.Furthermore,the interactions between plant hormones,transcription factors,and non-coding RNAs in fruit flavonoid biosynthesis were analyzed.Ultimately,a foundational regulatory network for fruit flavonoid biosynthesis was hereby established.
基金funded by the Key R&D Program of Shaanxi province(Grant Nos.2023-ZDLNY-23 and 2023XCZX2-18)the National Key R&D Program of China(Grant No.2019YFD1001405).
文摘Proanthocyanidins(PAs)are a group of oligomeric flavonoids also known as condensed tannins.PAs in human diet have antioxidant and health-promoting effects.BSISTER(BS)promotes the accumulation of PAs,but its direct target gene and regulation mechanism in PAs biosynthesis are not clear.Here,we reported VviBS1 and VviBS2 genes,which bind directly to the promoter of PA synthesis key genes VviANR(ANTHOCYANIDIN REDUCTASE)and VviLAR(LEUCOANTHOCYANIDIN REDUCTASE)and up-regulate their expression to promote the PAs biosynthesis in grapevine.VviBS1 and VviBS2 partially rescued the transparent testa phenotype of an Arabidopsis tt16 mutant.Overexpression of BS1 and BS2 in grapevine callus increased the content of PAs,as well as the expression of specific genes related to PA synthesis.VviBS1 and VviBS2 proteins bound directly to the promoter regions of the key PA synthesis genes VviANR1,VviANR2,and VviLAR1.Overexpression of VviBS1 and VviBS2 promotes VviANR1,VviANR2 and VviLAR1 expression and PAs biosynthesis.Some BS1-and BS2-interacting MADS-box proteins have an effect on PAs biosynthesis.This study provides insight into the regulatory mechanisms of PAs biosynthesis in grapevine,which could be effectively employed for metabolic engineering to increase PA content.
基金funded by the National Natural Science Foundation of China (31760363)the Earmarked Fund for CARS (CARS-14-1-16)+1 种基金the Gansu Education Science and Technology Innovation Industry Support Program,China (2021CYZC-38)the Gansu Provincial Key Laboratory of Arid Land Crop Science,Gansu Agricultural University,China (GSCS-2020-Z6)。
文摘Lodging is a major constraint limiting oil flax production efficiency in northern China.Crop lodging susceptibility is closely related to stem lignin content,and the regulatory mechanisms by which nitrogen and potassium fertilization interactively influence lignin biosynthesis in oil flax stems require further investigation.Therefore,this study aimed to enhance lodging resistance and increase grain yield in oil flax.We examined the interactive effects of different nitrogen (75,150,and 225 kg N ha^(–1)) and potassium (60 and 90 kg K_(2)O ha^(–1)) fertilizer rates on lignin metabolism,lodging resistance,and grain yield during the 2022 and 2023 growing seasons.Results indicated that nitrogen and potassium fertilizer levels and their interactions promoted lignin accumulation,improved lodging resistance,and increased grain yield.Compared to the control (CK),the75–150 kg N ha^(–1) combined with 60 kg K_(2)O ha^(–1) treatments significantly enhanced the activities of key lignin-synthesizing enzymes (tyrosine ammonia-lyase (TAL),phenylalanine ammonia-lyase (PAL),cinnamyl alcohol dehydrogenase (CAD),and peroxidase (POD)) and upregulated the expression of 4CL1 and F5H3 genes,leading to a 29.63–43.30%increase in lignin content,improved stem bending strength and lodging resistance index,and a 23.27–32.34%increase in grain yield.Correlation analysis revealed that nitrogen and potassium fertilizers positively regulated enzyme activities and gene expression related to lignin biosynthesis,thereby facilitating lignin accumulation and enhancing stem mechanical strength and lodging resistance.Positive correlations were observed among lignin-related enzyme activities,gene expression,lodging resistance traits,and grain yield.In summary,the application of 75–150 kg N ha^(–1) in conjunction with 60 kg K_(2)O ha^(–1)promoted lignin biosynthesis and accumulation,enhanced lodging resistance,and increased grain yield in oil flax grown in the dryland farming region of central Gansu,China.Furthermore,this treatment provides a technical basis for cultivating stress-tolerant and high-yield oil flax in arid regions.
基金supported by the National Natural Science Foundation of China(Grant Nos.31870695,32071828)the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD).
文摘Flower color is an essential trait in ornamental plant breeding. Lycoris longituba is a popular ornamental plant native to central eastern China. The decrease in anthocyanin accumulation causes L. longituba petal color fading during flower development, which considerably affects the ornamental value of L. longituba. However, mechanisms underlying anthocyanin biosynthesis inhibition during L. longituba petal development remain unclear. In this study, three LlDFR genes were confirmed to be involved in anthocyanin biosynthesis and LlDFRc exerted the strongest promoting effect on anthocyanin accumulation. According to the correlation analysis results, LlbHLH12 exhibited the strongest negative correlation with LlDFRc. Quantitative real-time PCR analysis showed that LlbHLH12 was highly expressed during the medium bud and full bloom stages of flower development. LlbHLH12 was identified as a member of subgroup XII of bHLH transcription factor family. Subcellular localization and transcriptional activation ability assay revealed that LlbHLH12 was located in the nucleus without transcriptional activation activity. Overexpression of LlbHLH12 in Nicotiana tabacum and L. longituba inhibited anthocyanin accumulation by suppressing the expression of anthocyanin biosynthetic pathway genes. Furthermore, yeast one-hybrid, dual-luciferase, and β-glucuronidase activity assays showed that LlbHLH12 directly bound to the promoters of LlPAL and LlDFRc and suppressed their expression to inhibit anthocyanin biosynthesis. Overall, our study identified a novel bHLH repressor negatively regulating anthocyanin biosynthesis and provided new insights into the molecular mechanisms underlying color fading in L. longituba petals.
基金supported by Shenzhen University General Hospital Scientific Research Project,No.SUGH2019QD002Shenzhen Science and Technology Development Foundation,No.20220810173216001(both to ZS).
文摘Spinal cord injury is a severe neurological condition with limited neuronal regeneration and functional recovery.Currently,no effective treatments exist to improve spinal cord injury prognosis.Neuronal guidance proteins are a diverse group of molecules that play crucial roles in axon and dendrite growth during nervous system development.Increasing evidence highlights their regulatory functions in spinal cord injury.This review provides a brief overview of the modulation patterns of key neuronal guidance proteins in neuronal axon growth during nervous system formation and subsequently focuses on their roles in neuronal regeneration and functional recovery following spinal cord injury.Neuronal guidance proteins include,but are not limited to,semaphorins and their receptors,plexins;netrins and their receptors,deleted in colorectal cancer and UNC5;Eph receptors and their ligands,ephrins;Slit and its receptor,Robo;repulsive guidance molecules and their receptor,neogenin;Wnt proteins and their receptor,Frizzled;and protocadherins.Localized Netrin-1 at the injury site inhibits motor axon regeneration after adult spinal cord injury while promoting oligodendrocyte growth.Slit2 enhances synapse formation in the injured spinal cord of rats.EphA7 regulates acute apoptosis in the early pathophysiological stages of spinal cord injury,while ephrinA1 plays a role in the nervous system’s injury response,with its reduced expression leading to impaired motor function in rats.EphA3 is upregulated following spinal cord injury,promoting an inhibitory environment for axonal regeneration.After spinal cord injury,bidirectional activation of ephrinB2 and EphB2 in astrocytes and fibroblasts results in the formation of a dense astrocyte-meningeal fibroblast scar.EphB1/ephrinB1 signaling mediates pain processing in spinal cord injury by regulating calpain-1 and caspase-3 in neurons.EphB3 expression increases in white matter after spinal cord injury,further inhibiting axon regeneration.Sema3A,expressed by neurons and fibroblasts in the scar surrounding the injury,inhibits motor neuron and sensory nerve growth after spinal cord injury.Sema4D suppresses neuronal axon myelination and axon regeneration,while its inhibition significantly enhances axon regeneration and motor recovery.Sema7A is involved in glial scar formation and may influence serotonin channel remodeling,thereby affecting motor coordination.Given these findings,the local or systemic application of neuronal guidance proteins represents a promising avenue for spinal cord injury treatment.
基金supported by the National Natural Science Foundation of China (No.22007008)the LiaoNing Revitalization Talents Program (No.XLYC1907021)the Fundamental Research Funds for the Central Universities (Nos.DUT23YG120,DUT19RC(3)009)。
文摘The study of target proteins is crucial for understanding molecular interactions and developing analytical platforms,therapeutic agents and functional tools.Herein,we present a novel nanoplatform activated by near-infrared(NIR) light for triple-modal proteins study,which enabling target protein labeling,enrichment and visualization.Azido-naphthalimide-coated upconversion nanoparticles(UCNPs) serve as NIR light-responsive nanoplatforms,showing promising applications in studying interactions between various bioactive molecules and proteins in living systems.Under NIR light irradiation,azido-naphthalimides are activated by ultraviolet(UV) and blue light emitted from UCNPs and the resulting amino-naphthalimides intermediate not only crosslink nearby target proteins but also enable imaging performance.We demonstrate that this nanoplatform is capable of selective protein labeling and imaging in complex protein environments,achieving specific labeling and imaging of both intracellular and extracellular proteins in mammalian cells as well as bacteria.Furthermore,in vivo protein labeling has been achieved using this novel NIR light-activatable nanoplatform.This technique will open new avenues for discoveries and mechanistic interrogation in chemical biology.
基金supported by the National Key Research and Development Program of China(No.2024YFE0214300)Hubei Provincial Science and Technology Plan Project(2022BEC051)selfdetermined research funds of CCNU from the colleges'basic research and operation of MOE(No.CCNU24JCPT023).
文摘HPPD(4-hydroxyphenylpyruvate dioxygenase)inhibitor are widely used in agriculture due to their high efficacy and environmental friendliness.However,many important crops,such as rice,wheat,and soybean,are naturally sensitive to these herbicides.In this study,we employed a directed evolution strategy to enhance the metabolic capacity of OsHSL2,OsHSL4,OsHSL6,and SbHSL1 proteins toward HPPD inhibitors,providing a new technological approach as well as theoretical foundation for molecular breeding of herbicide-resistant crops.By combining AlphaFold 3 protein models with crystal structures,we systematically redesigned key residues to resemble the active residues found in HIS1.Catalytic activity assays demonstrated that specific mutations significantly improved the metabolic activity of HSLs proteins toward various HPPD inhibitors.Notably,the OsHSL2-M4 mutant exhibited enhanced metabolic activity for BBC-OH and methyl-benquitrione,while the OsHSL4-M5 mutant completely metabolized BBC-OH and topramezone.Additionally,the SbHSL1-M4 mutant showed significant improvement in the metabolism of BBC-OH and several other herbicides,providing strong evidence to support the use of structure-guided HSL mutations to enhance crop resistance to HPPD inhibitors.
文摘Multienzyme cascades enable the sequential synthesis of complex chemicals by combining multiple catalytic processes in one pot,offering considerable time and cost savings compared to a series of separate batch reactions.However,challenges related to coordination and regulatory interplay among multiple enzymes reduce the catalytic efficiency of such cascades.Herein,we genetically programmed a scaffold framework that selectively and orthogonally recruits enzymes as designed.The system was then used to generate multienzyme complexes of D-allulose 3-epimerase(DAE),ribitol dehydrogenase(RDH),and formate dehydrogenase(FDH)for rare sugar production.This scaffolded multienzymatic assembly achieves a 10.4-fold enhancement in the catalytic performance compared to its unassembled counterparts,obtaining allitol yield of more than 95%.Molecular dynamics simulations revealed that shorter distances between neighboring enzymes in scaffold-mounted complexes facilitated the transfer of reaction intermediates.A dual-module catalytic system incorporating(1)scaffold-bound complexes of DAE,RDH,and FDH and(2)scaffold-bound complexes of alcohol dehydrogenase and NADH oxidase expressed intracellularly in E.coli was used to synthesize D-allulose from D-fructose.This system synthesized 90.6%D-allulose from 300 g L^(−1)D-fructose,with a space-time yield of 13.6 g L^(−1)h^(−1).Our work demonstrates the programmability and versatility of scaffold-based strategies for the advancement of multienzyme cascades.
文摘<span style="font-family:;" "=""><span style="font-family:Verdana;">Methane production by archaea depends on tetrahydromethanopterin (H</span><sub><span style="font-family:Verdana;">4</span></sub><span style="font-family:Verdana;">MPT), a pterin-containing cofactor that carries one-carbon units. Two redox reactions within the nine steps of H</span><sub><span style="font-family:Verdana;">4</span></sub><span style="font-family:Verdana;">MPT side chain biosynthesis have been hypothesized. Biochemical assays have demonstrated that the archaeal iron-sulfur flavoprotein dihydromethanopterin reductase X (DmrX or MM1854) catalyzes the final reaction of the pathway, the reduction of dihydromethanopterin to H</span><sub><span style="font-family:Verdana;">4</span></sub><span style="font-family:Verdana;">MPT</span></span><span style="font-family:Verdana;">,</span><span style="font-family:;" "=""><span style="font-family:Verdana;"> using dithiothreitol (DTT) as an artificial electron donor. The crystal structure of DmrB, a bacterial DmrX homolog that lacks iron-sulfur clusters, has led to a proposed ping-pong mechanism of electron transfer between FMNH</span><sub><span style="font-family:Verdana;">2</span></sub><span style="font-family:Verdana;"> and the FMN prosthetic group of DmrB. However, an enzymatic assay to test the hypothetical DmrB mechanism is lacking because a suitable electron donor has not previously been identified. Furthermore, a second uncharacterized archaeal flavoprotein (MM1853) has been hypothesized to function in H</span><sub><span style="font-family:Verdana;">4</span></sub><span style="font-family:Verdana;">MPT side chain biosynthesis. In this </span><span style="font-family:Verdana;">work, to facilitate the development of assays to elucidate the functions of DmrB </span><span style="font-family:Verdana;">and MM1853, we tested a variety of electron donors, including dithiothreitol, ferredoxin, and a system consisting of NADH and an NADH-dependent fla</span><span style="font-family:Verdana;">vin-reducing enzyme (Fre).</span><span style="font-family:Verdana;"> Reduction of the DmrB prosthetic group (FMN) was measured as a decrease in absorbance at 460 nm. NADPH, NADH, and </span><span style="font-family:Verdana;">DTT were unable to reduce DmrB. However, NADH/Fre was able to reduce </span><span style="font-family:Verdana;">DmrB within 70 min (initial rate of 1.3 μM/min), providing the basis for a future DmrB activity assay. Carbon monoxide (CO)/CO dehydrogenase/ferredoxin reduced DmrB more rapidly within 6 min. Both electr</span><span style="font-family:Verdana;">on transfer systems reduced a second flavin-containing archaeal protein MM1853, which is predicted to catalyze the third step of H</span><sub><span style="font-family:Verdana;">4</span></sub><span style="font-family:Verdana;">MPT biosynthesis. While NADH and NADPH were incapable of directly reducing the FMN cofactor of MM1853, DTT or NADH/Fre could eliminate the FMN peaks. These results establish the basis for new oxidoreductase assays that will facilitate testing several proposed DmrB mechanisms and defining the specific function of MM1853 in methanogen cofactor biosynthesis.</span></span>
文摘Our previous studies showed a predominance of high molecular weight protein group in tumor nuclear matrices. Contrary to normal cells, proteins of this group are preferentially phosphorylated. Phosphoproteins of hepatoma nuclear matrix are selectively subjected to rapid proteolysis. By alkali treatment and a monoclonal antibody against phosphotyrosyl residue the presence of two high molecular weight bands of phosphotyrosyl-containing proteins was detected in nuclear matrices of tumor but not of normal liver cells. High molecular weight protein group of tumor nuclear matrices revealed also a rapid turnover and preferential incorporation of labeled amino acids selectively inhibited by chloramphenicol.
基金supported by the National Key Research and Development Program of China (No. 2022YFD1700200)the National Natural Science Foundation of China (Nos. 32161133010, 3230170969)+1 种基金the Innovation Program of Chinese Academy of Agricultural Sciences, the Shenzhen Science and Technology Program (No. KQTD20180411143628272)the Special Funds for Science Technology Innovation and Industrial Development of Shenzhen Dapeng New District (No. PT202101–02)。
文摘Chitin is an abundant aminopolysaccharide found in insect pests and phytopathogenic microorganisms but absent in higher plants and vertebrates. It is crucial for mitigating threats posed by chitin-containing organisms to human health, food safety, and agriculture. Therefore, targeting the chitin biosynthesisassociated bioprocess holds a promise for developing human-safe and eco-friendly antifungal agents or pesticides. Chitin biosynthesis requires chitin synthase and associated factors, which are involved in the modification, regulation, organization or turnover of chitin during its biosynthesis. A number of enzymes such as chitinases, hexosaminidases, chitin deacetylases are closely related and therefore are promising targets for designing novel agrochemicals that target at chitin biosynthesis. This review summarizes the advances in understanding chitin biology over the past decade by our research group and collaborates,specifically regarding essential proteins linked to chitin biosynthesis that can be exploited as promising pesticide targets. Examples of small bioactive molecules that against the activity of these targets are given.
文摘Xylo-oligosaccharides(XOSs)are a category of functional oligosaccharides primarily composed of 2-7 xylose units linked byβ-1,4 glycosidic bonds.They are recognized as soluble dietary fibers with prebiotic properties.Recently, there has been significant interest in manufacturing XOSs from xylan extracted from lignocellulosic biomass using enzyme catalysis under mild conditions. In this work, the arabinofuranosidase Abf62A gene was cloned from Aspergillus usamii genomic DNA through sequential molecular processes and expressed in Pichia pastoris X33. The xylan (100 g/L) extracted xylan in wheat straw (WS) was biologically hydrolyzed into 50.32 g/L of XOSs by xylanase Xyn11A (300 U/g substrate) and arabinofuranase Abf62A (20 U/g substrate), which indicated a notable synergistic effect compared to the 34.42 g/L XOSs produced via Xyn11A. The 50.32 g/L of XOSs products comprised xylobiose (31.71 g/L), xylotriose (15.92 g/L), xylotetraose (1.65 g/L) and xylopentaose (1.04 g/L). Notably, the combined content of xylobiose and xylotriose accounted for up to 94.7%. The XOSs purified from the enzyme hydrolysate could effectually scavenge free radicals, and the antioxidant activity was more than 90%. In summary, XOSs were biologically manufactured from wheat straw xylan through the synergistic biocatalysis via xylanase and arabinofuranosidase Abf62A in a green and sustainable way, rending one kind of prebiotic oligosaccharides with substantial positive effects on human and animal health.
