CmWRKY6-1-CmWRKY15-like transcriptional cascade negatively regulates the resistance to fusarium oxysporum infection in Chrysanthemum morifolium

Chrysanthemum Fusarium wilt is a soil-borne disease that causes serious economic losses to the chrysanthemum industry.However,the molecular mechanism underlying the response of chrysanthemum WRKY to Fusarium oxysporum infection remains largely unknown.In this study,we isolated CmWRKY6–1 from chrysanthemum‘Jinba’and identified it as a transcriptional repressor localized in the nucleus via subcellular localization and transcriptional activation assays.We found that CmWRKY6–1 negatively regulated resistance to F.oxysporum and affected reactive oxygen species(ROS)and salicylic acid(SA)pathways using transgenic experiments and transcriptomic analysis.Moreover,CmWRKY6–1 bound to the W-box element on the CmWRKY15-like promoter and inhibited its expression.Additionally,we observed that CmWRKY15-like silencing in chrysanthemum reduced its resistance to F.oxysporum via transgenic experiments.In conclusion,we revealed the mechanism underlying the CmWRKY6–1–CmWRKY15-like cascade response to F.oxysporum infection in chrysanthemum and demonstrated that CmWRKY6–1 and CmWRKY15-like regulates the immune system.

Interaction between MAPKs and MKPs in hexaploid chrysanthemum illuminates functional paralogue diversification in polyploids

Mitogen-activated protein kinases(MAPKs,also known as MPKs)regulate diverse cellular and physiological functions,and dual-specificity MAPK phosphatases(MKPs)modulate MAPK signalling through MAPK dephosphorylation and inactivation.Due to lacking of overall understanding for the regulatory networks between Chrysanthemum morifolium MKPs(CmMKPs)and C.morifolium MAPKs(CmMPKs),we systematically studied the interactions between four groups of CmMPKs and eight identified CmMKPs in chrysanthemum and found that the interaction between the specific CmMKP and the specific CmMPK differed from those in other plants.Furthermore,the expression of CmMKP1 and CmMKP1-LIKE1showed opposite trends during the development of chrysanthemum flower buds under salt treatment and Alternaria alternata inoculation,but these genes could interact with the same CmMPKs,providing insight into the subfunctionalization of paralogues.Amino acid variations(M87V,T277P and V6L)in dual-specificity protein phosphatases(DsPTP1)-LIKE1/2/3 changed the interactions of these proteins with the four CmMPK groups in chrysanthemum,providing evidence for the de/neofunctionalization of paralogues in polyploids,suggesting that we can identify the key functional sites of proteins by studying polyploid paralogues.

电位法测定茶水中氟含量的实验改进探索

通过对被测溶液中加入与不加总离子强度缓冲调节剂,及对不同茶叶的泡制水温、泡制时间等影响条件进行定量测定研究,对原有实验方案得以进一步优化改进。其目的是使学生能较深入理解控制测定条件的重要性,以便进一步激发学生对化学实验科学性、趣味性和可操作性的兴趣。

Genome-wide association study identifies favorable SNP alleles and candidate genes for waterlogging tolerance in chrysanthemums

Chrysanthemums are sensitive to waterlogging stress,and the development of screening methods for tolerant germplasms or genes and the breeding of tolerant new varieties are of great importance in chrysanthemum breeding.To understand the genetic basis of waterlogging tolerance(WT)in chrysanthemums,we performed a genome-wide association study(GWAS)using 92,811 single nucleotide polymorphisms(SNPs)in a panel of 88 chrysanthemum accessions,including 64 spray cut and 24 disbud chrysanthemums.The results showed that the average MFVW(membership function value of waterlogging)of the disbud type(0.65)was significantly higher than that of the spray type(0.55)at P<0.05,and the MFVW of the Asian accessions(0.65)was significantly higher than that of the European accessions(0.48)at P<0.01.The GWAS performed using the general linear model(GLM)and mixed linear model(MLM)identified 137 and 14 SNP loci related to WT,respectively,and 11 associations were commonly predicted.By calculating the phenotypic effect values for 11 common SNP loci,six highly favorable SNP alleles that explained 12.85—21.85%of the phenotypic variations were identified.Furthermore,the dosage-pyramiding effects of the favorable alleles and the significant linear correlations between the numbers of highly favorable alleles and phenotypic values were identified(r2=0.45;P<0.01).A major SNP locus(Marker6619-75)was converted into a derived cleaved amplified polymorphic sequence(dCAPS)marker that cosegregated with WT with an average efficiency of 78.9%.Finally,four putative candidate genes in the WT were identified via quantitative real-time PCR(qRT-PCR).The results presented in this study provide insights for further research on WT mechanisms and the application of molecular marker-assisted selection(MAS)in chrysanthemum WT breeding programs.

Overexpression of CmMYB15 provides chrysanthemum resistance to aphids by regulating the biosynthesis of lignin

MYB transcription factors are widely involved in the development of and physiological processes in plants.Here,we isolated the chrysanthemum R2R3-MYB family transcription factor CmMYB15,a homologous gene of AtMYB15.It was demonstrated that CmMYB15 expression was induced by aphids and that CmMYB15 could bind to AC elements,which usually exist in the promoter of lignin biosynthesis genes.Overexpression of CmMYB15 in chrysanthemum enhanced the resistance of aphids.Additionally,the content of lignin and the expression of several lignin biosynthesis genes increased.In summary,the results indicate that CmMYB15 regulates lignin biosynthesis genes that enhance the resistance of chrysanthemum to aphids.

A temporal gene expression map of Chrysanthemum leaves infected with Alternaria alternata reveals different stages of defense mechanisms

Chrysanthemum(Chrysanthemum morifolium)black spot disease(CBS)poses a major threat to Chrysanthemum cultivation owing to suitable climate conditions and current lack of resistant cultivars for greenhouse cultivation.In this study,we identified a number of genes that respond to Alternaria alternata infection in resistant and susceptible Chrysanthemum cultivars.Based on RNA sequencing technology and a weighted gene coexpression network analysis(WGCNA),we constructed a model to elucidate the response of Chrysanthemum leaves to A.alternata infection at different stages and compared the mapped response of the resistant cultivar‘Jinba’to that of the susceptible cultivar‘Zaoyihong’.In the early stage of infection,when lesions had not yet formed,abscisic acid(ABA),salicylic acid(SA)and EDS1-mediated resistance played important roles in the Chrysanthemum defense system.With the formation of necrotic lesions,ethylene(ET)metabolism and the Ca^(2+)signal transduction pathway strongly responded to A.alternata infection.During the late stage,when necrotic lesions continued to expand,members of the multidrug and toxic compound extrusion(MATE)gene family were highly expressed,and their products may be involved in defense against A.alternata invasion by exporting toxins produced by the pathogen,which plays important roles in the pathogenicity of A.alternata.Furthermore,the function of hub genes was verified by qPCR and transgenic assays.The identification of hub genes at different stages,the comparison of hub genes between the two cultivars and the highly expressed genes in the resistant cultivar‘Jinba’provide a theoretical basis for breeding cultivars resistant to CBS.

CmBES1 is a regulator of boundary formation in chrysanthemum ray florets

Chrysanthemum(Chrysanthemum morifolium)is an ideal model species for studying petal morphogenesis because of the diversity in the flower form across varieties;however,the molecular mechanisms underlying petal development are poorly understood.Here,we show that the brassinosteroid transcription factor BRI1-EMS-SUPPRESSOR 1(CmBES1)in chrysanthemum(C.morifolium cv.Jinba)is important for organ boundary formation because it represses organ boundary identity genes.Chrysanthemum plants overexpressing CmBES1 displayed increased fusion of the outermost ray florets due to the loss of differentiation of the two dorsal petals,which developed simultaneously with the ventral petals.RNA-seq analysis of the overexpression lines revealed potential genes and pathways involved in petal development,such as CUP-SHAPED COTYLEDON(CUC2),CYCLOIDEA 4(CYC4),genes encoding MADS-box transcription factors and homeodomain-leucine zippers(HD-Zips)and auxin pathway-related genes.This study characterizes the role of CmBES1 in ray floret development by its modulation of flower development and boundary identity genes in chrysanthemum.

The over-expression of a chrysanthemum gene encoding an RNA polymerase II CTD phosphatase-like 1 enzyme enhances tolerance to heat stress

The enzyme RNAPII CTD phosphatase-like 1 is known as a transcriptional regulator of the plant response to various abiotic stresses.Here,the isolation of CmCPL1,a chrysanthemum(Chrysanthemum morifolium)gene encoding this enzyme is described.Its predicted 955 residue gene product includes the FCPH catalytic domain,two double-stranded RNA binding motifs,and a nuclear localization signal.A sub-cellular localization assay confirmed that CmCPL1 was expressed in the nucleus.CmCPL1 transcription was shown to be significantly inducible by heat stress.The over-expression and knockdown of CmCPL1,respectively,increased and diminished the tolerance of chrysanthemum to heat stress,which maybe dependent on the regulation of CmCPL1 and on the expression of downstream heat stress-responsive genes.

Roles of endoplasmic reticulum stress and apoptosis signaling pathways in gynecologic tumor cells：A systematic review

Efficient functioning of the endoplasmic reticulum(ER) is very important for most cellular activities, such as protein folding and modification. The ER closely interacts with other organelles, including the Golgi body, endosome, membrane, and mitochondria, providing lipids and proteins for the repair of these organelles. ER stress can be induced by various abnormal materials in the cell. ER stress is a compensatory intracellular environment disorder that occurs during areaction. ER can sense the stress and respond to it through translational attenuation, upregulation of the genes for ER chaperones and related proteins, and degradation of unfolded proteins by a quality-control system, but excessive ER activation can cause cell death. The Pubmed and Web of Science databases were searched for full-text articles, and the terms "endoplasmic reticulum stress/unfolded protein response/gynecologic tumor cell apoptosis" were used as key words. Thirty-five studies of ER stress and unfolded protein response published from 2000 to 2016 were analyzed. Stress triggers apoptosis through a variety of signaling pathways. Increasing evidence has shown that the ER plays an important role in tumor cell diseases. The present review discusses the molecular mechanisms underlying unfolded protein response and its ability to promote survival and proliferation in gynecologic tumor cells.

CmMLO17 and its partner CmKIC potentially support Alternaria alternata growth in Chrysanthemum morifolium

The Mildew Resistance Locus O(MLO)gene family has been investigated in many species.However,there are few studies on chrysanthemum MLO genes.We report in this study that CmMLO17 in Chrysanthemum morifolium was upregulated after Alternaria alternata infection.Silencing of CmMLO17 by artificial microRNA resulted in reduced susceptibility of chrysanthemum to A.alternata infection.Genes in the abscisic acid(ABA)and Ca 2+signaling pathways were upregulated in the CmMLO17-silenced line R20 compared to the wild-type plants.We speculated that CmMLO17-silenced plants had a faster and stronger defense response that was mediated by the ABA and Ca 2+signaling pathways,resulting in reduced susceptibility of chrysanthemum to A.alternata infection.In addition,a candidate gene,CmKIC,that may interact with CmMLO17 was discovered by the yeast two-hybrid assay.The interaction between CmMLO17 and CmKIC was confirmed using the yeast two-hybrid assay and bimolecular fluorescence complementation(BiFC)analysis.CmMLO17 and CmKIC were both located on the plasma membrane,and CmKIC was also located on the nucleus.CmKIC overexpression increased the susceptibility of chrysanthemum to A.alternata,whereas CmKIC silencing resulted in reduced susceptibility.Therefore,CmMLO17 and CmKIC may work together in C.morifolium to support the growth of A.alternata.The results of this study will provide insight into the potential function of MLO and improve the understanding of plant defense responses to necrotrophic pathogens.

CmRCD1 represses flowering by directly interacting with CmBBX8 in summer chrysanthemum

The CmBBX8-CmFTL1 regulatory module is a key determinant in the transition from vegetative growth to reproductive development in summer-flowering chrysanthemum.However,the detailed regulatory mechanism of CmBBX8-mediated flowering remains elusive.In this study,we revealed that RADICAL-INDUCED CELL DEATH 1(CmRCD1)physically associated with CmBBX8 through bimolecular fluorescence complementation(BiFC),pulldown and Coimmunoprecipitation(CoIP)assays.Furthermore,the RCD1-SRO1-TAF4(RST)domain of CmRCD1 and the B-box of CmBBX8 mediated their interaction.In addition,Luciferase(LUC)assays and electrophoretic mobility shift assay(EMSAs)showed that CmRCD1 repressed the transcriptional activity of CmBBX8 and interfered with its binding to the CmFTL1 promoter,thereby leading to delayed flowering in the summer chrysanthemum‘Yuuka’.These results provide insight into the molecular framework of CmRCD1-CmBBX8-mediated flowering in chrysanthemum.

Chrysanthemum CmWRKY53 negatively regulates the resistance of chrysanthemum to the aphid Macrosiphoniella sanborni

Chrysanthemum is frequently attacked by aphids,which greatly hinders the growth and ornamental value of this plant species.WRKY transcription factors play an important role in the response to biotic stresses such as pathogen and insect stresses.Here,chrysanthemum CmWRKY53 was cloned,and its expression was induced by aphid infestation.To verify the role of CmWRKY53 in resistance to aphids,CmWRKY53 transgenic chrysanthemum was generated.CmWRKY53 was found to mediate the susceptibility of chrysanthemum to aphids.The expression levels of secondary metabolite biosynthesis genes,such as peroxidase-and polyphenol oxidase-encoding genes,decreased in CmWRKY53-overexpressing(CmWRKY53-Oe)plants but dramatically increased in chimeric dominant repressor(CmWRKY53-SRDX)plants,suggesting that CmWRKY53 contributes to the susceptibility of chrysanthemum to aphids,possibly due to its role in the regulation of secondary metabolites.

Generation of purple-violet chrysanthemums via anthocyanin B-ring hydroxylation and glucosylation introduced from Osteospermum hybrid F3'5'H and Clitoria ternatea A3'5'GT

Chrysanthemums possess no metabolic pathway to synthesize delphinidin because of the lack of endogenous F3'5'H gene encoding the key enzyme in its biosynthetic pathway;therefore,there are no blue or blue-purple chrysanthemums occurring naturally.Currently,the introduction of exogenous F3'5'H into chrysanthemums is an efficient method for breeding bluish chrysanthemums.In this study,we explored the effects of the introduction of mutant CmF3'H(generated via site-directed mutagenesis,T485S,CmF3'Hm)and exogenous Osteospermum hybrid F3'5'H(OhF3'5'H)genes combined with Clitoria ternatea A3'5'GT(CtA3'5'GT)on delphinidin synthesis in chrysanthemum.Among the F3'5'H transgenic lines,those overexpressing endogenous CmF3'Hm could not generate blue flower color,although red color was changed to light pink due to CtA3'5'GT function.Meanwhile,OhF3'5'H introduction promoted the accumulation of delphinidin and its derivatives in chrysanthemum,changing the flower color from red-purple to purple-violet.These results indicate the applicability of exogenous OhF3'5'H and CtA3'5'GT transformation for promoting delphinidin synthesis during the molecular breeding of violet/blue chrysanthemums.

Constitutive expression of a chrysanthemum phospholipase Dα gene in Chrysanthemum morifolium enhances drought tolerance

Drought causes water shortage and consequent retardation of plants growth and development.Therefore,improving the drought tolerance of plants is necessary for expanding cultivation and resource promotion.Increasing evidence indicates that phospholipase is involved in the response of plants to drought stress.The objective of this study was to create new drought-tolerant chrysanthemum germplasm,which lays a foundation for the study of the molecular mechanism of phospholipase mediated stress response in chrysanthemum.CmPLDαhas the closest relationship with sunflower HaPLDα,and belongs to the PLDαfamily.CmPLDαover-expressing plants showed a slight shrinking under 20%PEG6000 treatment.The survival rate increased significantly by 1.7−1.8 times that of the wild type.Relative water content(RWC)of CmPLDαover-expressing plants were nearly 10%higher than that of the wild type.Relative electrical conductivity and MDA content were significantly lower than those of the wild type.ABA content of the over-expression lines Z1,Z2 were 1.3 and 1.22 times that of wild type,but ABA content of antisense lines F1,F2 was approximately 0.83 and 0.81 of those of wild type.Most plants of antisense transgenic lines F1,F2 were wrinkled,with a wilting index of 5 and 6,and the survival rate was also lower than that of the wild type after recovery growth.RWC of antisense lines were lower than over-expression lines,relative electrical conductivity and MDA content were significantly higher than those of the wild type.In summary,CmPLDαcould enhance tolerance of chrysanthemum to drought conditions.

Variation for Resistance to Alternaria tenuissima and Potential Structural Mechanism among Different Cultivars of Chrysanthemum morifolium

Black spot disease,caused by the necrotrophic fungus Alternaria tenuissima(Fr.)Wiltsh(A.tenuissima),is considered a highly destructive disease of Chrysanthemum(Chrysanthemum morifolium Ramat.).A set of 17 accessions of commercial chrysanthemum cultivars were evaluated for resistance to A.tenuissima by seedling artificial inoculation.It was found that the reaction of the accessions to artificial inoculation ranged from resistant to highly susceptible.Five varieties of chrysanthemum(‘Zhongshan Taogui’,‘Jinba’,‘Zhongshan Jinguan’,‘Jinling Wanhuang’and‘Jinling Yangguang’)were resistant;two varieties of chrysanthemum(‘Zhongshan Xinggui’and‘Zhongshan Jinkui’)were moderately resistant;and others were susceptible to various degrees,four varieties of chrysanthemum(‘Zhongshan Zihe’,‘Zhongshan Jiuhong’,‘Zaoyihong’and‘Jinling Jiaohuang’)were highly susceptible,especially.Some leaf morphological features of two resistant and two highly susceptible cultivars were further researched.Trichome density,length,height,gland size and stomata density were found to be associated with plant passive resistance.Resistant varieties that were identified in present study will be promising germplasm for exploitation of breeding programmes aimed at developing A.tenuissima-resistant cultivars and increasing genetic diversity.

Living status and support system of children orphaned by AIDS in central China—Description and policy recommendations

This study aimed to research the living status and support system of children orphaned by AIDS in rural Henan Province. The approach of face-to-face questionnaires research was used to assess 501 children’s current situation while in-depth interview was conducted for the support system research. The age range of the children orphaned by AIDS was 2 - 15 years old and the mean age was 11.10 years. Most children among 2 - 6 years had communication skills with temper control and psychological problems. Children of 7 - 15 years old had the ability to take care of themselves and family members but could not do self-regulation. The support system for children orphaned by AIDS included social support, policies support, education support, and health care support but improvement are needed in the future. Support system can be improved through much more feasible and concrete policies and strategies to guarantee these children’s basic needs and comprehensive development.

Contrasting responses to drought stress between Chrysanthemum japonense and C. nankingense

The response of Chrysanthemum japonense and C.nankingense to drought stress induced by polyethylene glycol was characterized at the level of leaf water status,leaf surface morphology and cuticular wax(quantity and composition),the activity of antioxidant enzymes,the extent of membrane lipid peroxidation,the accumulation of proline,photosynthesis performance and abscisic acid(ABA)accumulation.The more tolerant species C.japonense maintained its water status more effectively than C.nankingense,probably because its leaves form more cuticular wax and are able to accumulate higher levels of ABA.Superoxide dismutase activity was higher in C.japonense than in C.nankingense,as was that of catalase and ascorbate peroxidase during the later part of the stress episode,but levels of peroxidase were not differentiated at the end of the stress period.Membrane damage,as measured by electrolyte leakage and malondialdehyde accumulation,was less severe in C.japonense,which was also able to generate higher levels of free proline after a 10 h exposure to stress.Thus the superior response of C.japonense also reflects a more adapted system of osmoprotection and antioxidation.As a result,photosynthesis was compromised less by drought stress in C.japonense than in C.nankingense.That provides a scientific basis for the development and application of drought tolerance resources of chrysanthemum.

Screening and functional analysis of potential S genes in Chrysanthemum morifolium

S genes are the key genes that cause plant self-incompatibility,to find out the key S genes and understand molecular mechanism of self-incompatibility in chrysanthemum,the stigmas and anthers at different developmental stages of'Q10-22-2'—a self-incompatible chrysanthemum cultivar,were used for RNA sequencing.After bioinformatics analysis,13 candidate pistil S genes and five candidate pollen S genes were excavated.A potential pistil S gene was cloned and named as CmSRK1.Meanwhile,a potential pollen S gene was cloned and named as CmPCP1.qRT-PCR revealed that CmSRK1 was specifically expressed in mature stigmas,and CmPCP1 was specifically expressed in anthers 3 d before maturation.Subcellular localization showed that both CmSRK1 and CmPCP1 were located in the nucleus and the cell membrane.Transcriptional activation activity analysis indicated that both of the two proteins had no transcriptional activation activity.Yeast two hybrid assay showed that there was no interaction between CmSRK1 and CmPCP1.CmSRK1 was constructed on the expression vector containing stigma-specific promoter,and CmPCP1 was constructed on the expression vector containing pollen-specific promoter,they were then transformed into Arabidopsis thaliana.Artificial hybridization was performed with transgenic lines containing CmSRK1 as the female parents,and transgenic lines containing CmPCP1 as the male parents.The hybridization results showed that seed sets of two transgenic lines were 19.62%and 11.64%,respectively,while cross-pollinated seed sets of Col-0 was 84.43%.Therefore,it was speculated that CmSRK1 and CmPCP1 might be pistil and pollen S genes of chrysanthemum,respectively,and SI of chrysanthemum belonged to SSI.Citation:Wang F,Xu S,Wu Z,Zhong X,Fang W,et al.2021.Screening and functional analysis of potential S genes in Chrysanthemum morifolium.

Cytological and Molecular Characteristics of Pollen Abortion in Lily with Dysplastic Tapetum

Lily was grown worldwide as a fresh cutting flower because of its colorful petals, but its anther contained a large number of pollen grains that cause serious pollen contamination, however, pollen abortion can effectively reduce the level of pollen pollution. Our analysis aims to use cytological observation to detect the critical stage when pollen abortion occurs and to provide comprehensive gene expression information at the transcriptional level. The result showed that pollen abortion in ‘Little Kiss’ began at the mononuclear stage and the callose that covers the microspores failed to degenerate when young pollens were released from the tetrads. In addition, compared with the normally developed one,the tapetum of ‘Little Kiss’ degraded in advance while the degradation of callose was delayed. Furthermore, 103 differentially expressed genes(DEGs) related to the advance degeneration of tapetum cells and callose were found in the expression levels, including 22 transcription factors(TFs). In particular, two β-glucanase genes(endo-1,3(4)-β-glucanase, exo-β-glucanase) responsible for callose degeneration were significantly down-regulated. These results suggested that pollen abortion may occur at mononuclear stage and that early degeneration of tapetum cells resulted in a significant down-regulation of β-glucanase genes. As a result, the callose to cover microspores impedes the formation of pollen walls, which may possibly lead to pollen abortion.

A multifunctional nanotheranostic agent potentiates erlotinib to EGFR wild-type non-small cell lung cancer

Epidermal growth factor receptor(EGFR)tyrosine kinase inhibitors(TKI),such as Erlotinib,have demonstrated remarkable efficacy in the treatment of non-small cell lung cancer(NSCLC)patients with mutated EGFR.However,the efficacy of EGFR-TKIs in wild-type(wt)EGFR tumours has been shown to be marginal.Methods that can sensitize Erlotinib to EGFR wild-type NSCLC remain rare.Herein,we developed a multifunctional superparamagnetic nanotheranostic agent as a novel strategy to potentiate Erlotinib to EGFR-wt NSCLCs.Our results demonstrate that the nanoparticles can co-escort Erlotinib and a vascular epithermal growth factor(VEGF)inhibitor,Bevacizumab(Bev),to EGFR-wt tumours.The nanotheranostic agent exhibits remarkable effects as an inhibitor of EGFR-wt tumour growth.Moreover,Bev normalizes the tumour embedded vessels,further promoting the therapeutic efficacy of Erlotinib.In addition,the tumour engagement of the nanoparticles and the vascular normalization could be tracked by magnetic resonance imaging(MRI).Collectively,our study,for the first time,demonstrated that elaborated nanoparticles could be employed as a robust tool to potentiate Erlotinib to EGFR-wt NSCLC,paving the way for imaging-guided nanotheranostics for refractory NSCLCs expressing EGFR wild-type genes.