A constitutive serine protease inhibitor suppresses herbivore performance in tea(Camellia sinensis)

Protease inhibitors promote herbivore resistance in diverse plant species.Although many inducible protease inhibitors have been identified,there are limited reports available on the biological relevance and molecular basis of constitutive protease inhibitors in herbivore resistance.Here,we identified a serine protease inhibitor,CsSERPIN1,from the tea plant(Camellia sinensis).Expression of CsSERPIN1 was not strongly affected by the assessed biotic and abiotic stresses.In vitro and in vivo experiments showed that CsSERPIN1 strongly inhibited the activities of digestive protease activities of trypsin and chymotrypsin.Transient or heterologous expression of CsSERPIN1 significantly reduced herbivory by two destructive herbivores,the tea geometrid and fall armyworm,in tea and Arabidopsis plants,respectively.The expression of CsSERPIN1 in Arabidopsis did not negatively influence the growth of the plants under the measured parameters.Our findings suggest that CsSERPIN1 can inactivate gut digestive proteases and suppress the growth and development of herbivores,making it a promising candidate for pest prevention in agriculture.

An NBS-LRR-encoding gene CsRPM1 confers resistance to the fungus Colletotrichum camelliae in tea plant

Nucleotide binding site,leucine-rich repeat(NBS-LRR)proteins are the main types of disease resistance proteins in plants,which can perceive plant pathogens.Anthracnose,caused by the fungus Colletotrichum camelliae,is one of the most severe diseases in tea plant.Here,we identified an NBS-LRR-encoding gene,CsRPM1,probably conferring resistance of tea plant to C.camelliae.Phylogenetic analysis showed that CsRPM1 was clustered with RPM1 in Arabidopsis and grouped into CC-NBS-LRR(CNL).It contained a signal peptide,a NB-ARC domain,and multiple LRR motifs.RNA-seq and qRT-PCR analysis showed that the transcript level of CsRPM1 was significantly up-regulated after inoculation with C.camelliae.Transiently silencing of CsRPM1 in tea leaves comprised the resistance to C.camelliae,indicating that CsRPM1 was required for plant defense against the pathogen.The subcellular localization showed that CsRPM1 protein was localized in the nucleus,cytoplasm,and cell membrane.Furthermore,the promoter region of CsRPM1 gene contained MeJA-responsive elements,and the expression of CsRPM1 was induced by exogenous methyl jasmonate,suggesting that CsRPM1 gene may be closely related to JA signaling pathway.A total of 17 transcription factors might be responsible for the expression of CsRPM1.Our data indicates that CsRPM1 is required for disease resistance to C.camelliae in tea plant.The characterization of this disease resistance gene sheds light on NLR protein function in tea plant and may facilitate breeding to control the severe anthracnose.

iTRAQ-based quantitative proteomic analysis of tea plant(Camellia sinensis(L.)O.Kuntze)during cold acclimation and de-acclimation procedures

To gain a better understanding on the mechanism of cold acclimation in tea plant[Camellia sinensis(L.)O.Kuntze]at the proteome level,an iTRAQ based quantitative proteome analysis was carried out to identify differentially accumulated proteins in the mature leaves which were collected at non-acclimated(NA),fully acclimated(CA)and de-acclimated(DA)stages.407 and 477 proteins identified from CA and DA showed significant abundance changes(at 95%confidence)compared with NA,respectively.Moreover,251 protein species changed their abundance in DA compared with CA.Those differential abundance protein species were mainly involved in metabolism,cell wall,photosynthesis,energy,protein synthesis,antioxidation,carbohydrate metabolic process and binding,and mapped to the pathways of biosynthesis of secondary metabolites,microbial metabolism in diverse environment,ribosome,metabolic pathway,sugar metabolism,protein processing,photosynthesis,and plant-pathogen interaction pathway.However,no significant correlation was detected between the identified proteins and cognate gene transcript levels by correlation analysis and qRT-PCR analysis.This study presents a comprehensive proteome in mature leaves at different cold acclimation status and provides new insights into cold acclimation mechanisms in tea plants.

Transcriptional analysis and histochemistry reveal that hypersensitive cell death and H2O2 have crucial roles in the resistance of tea plant (Camellia sinensis (L.) O. Kuntze) to anthracnose

Anthracnose causes severe losses of tea production in China.Although genes and biological processes involved in anthracnose resistance have been reported in other plants,the molecular response to anthracnose in tea plant is unknown.We used the susceptible tea cultivar Longjing 43 and the resistant cultivar Zhongcha 108 as materials and compared transcriptome changes in the leaves of both cultivars following Colletotrichum fructicola inoculation.In all,9015 and 8624 genes were differentially expressed between the resistant and susceptible cultivars and their controls(0 h),respectively.In both cultivars,the differentially expressed genes(DEGs)were enriched in 215 pathways,including responses to sugar metabolism,phytohormones,reactive oxygen species(ROS),biotic stimuli and signalling,transmembrane transporter activity,protease activity and signalling receptor activity,but DEG expression levels were higher in Zhongcha 108 than in Longjing 43.Moreover,functional enrichment analysis of the DEGs showed that hydrogen peroxide(H2O2)metabolism,cell death,secondary metabolism,and carbohydrate metabolism are involved in the defence of Zhongcha 108,and 88 key genes were identified.Protein–protein interaction(PPI)network demonstrated that putative mitogen-activated protein kinase(MAPK)cascades are activated by resistance(R)genes and mediate downstream defence responses.Histochemical analysis subsequently validated the strong hypersensitive response(HR)and H2O2 accumulation that occurred around the hyphal infection sites in Zhongcha 108.Overall,our results indicate that the HR and H2O2 are critical mechanisms in tea plant defence against anthracnose and may be activated by R genes via MAPK cascades.

Endogenous nitric oxide and hydrogen peroxide detection in indole-3-butyric acid-induced adventitious root formation in Camellia sinensis

Nitric oxide（NO）and hydrogen peroxide（H2O2）are essential signaling molecules with key roles in auxin induced adventitious root formation in many plants.However,whether they are the sole determinants for adventitious root formation is worth further study.In this study,endogenous NO and H2O2 were monitored in tea cutting with or without indole-3-butyric acid（IBA）treatment by using the fluorescent probes diaminofluorescein diacetate（DAF-2DA）and 2＇,7＇-dichlorodihydrofluorescein diacetate（DCF-DA）,respectively.The overproduction of NO and H2O2 was detected in the rooting parts of tea cuttings treated with or without IBA.But little NO and H2O2 was detected before the initiation phase of tea cuttings even with IBA treatment indicating that they might be not directly induced by IBA.Further carbon and nitrogen analysis found that the overproduction of NO and H2O2 were coincident with the consumption of soluble sugars and the assimilation of nitrogen.These results suggest that rooting phases should be taken into consideration with the hypothesis that auxin induces adventitious root formation via NO-and H2O2-dependent pathways and sink establishment might be a prerequisite for NO and H2O2 mediated adventitious root formation.

An integrative analysis of metabolomics,DNA methylation and RNASeq data reveals key genes involved in albino tea'Haishun 2'

Albino tea,a type of tea closely associated with the chlorophyll-deficient phenotype is of great interest due to its multiple benefits to human health.To explore the potential mechanisms involved in albino tea,we performed metabolomics,DNA methylation and RNA-Seq analysis of green and albino leaves in a special tea mutant'Haishun 2'.The albino leaves accumulated significantly less catechins compared with the green leaves,which is closely associated with their difference in chlorophyll formation.A total of 385 candidate genes were identified by the integrative analysis.Two NAC transcription factors were less methylated and highly up regulated in the albino leaves of'Haishun 2',which was the first identification of the key roles of NAC transcription factors in albino tea and worth further study.Many genes involved in catechin biosynthesis were down regulated in albino leaves,such as 4-coumarate-CoA ligase,flavonol synthase and leucoanthocyanidin reductase genes,suggesting chlorophyll formation is important for catechin biosynthesis.Furthermore,CsMYB5 and CsGSTF11 were found to be differentially methylated and expressed in albino and green leaves,indicating their potential roles in catechin accumulation.This was the first report of the integrative analysis of transcriptome and DNA methylation data in tea plants and the results suggest that the integrative analysis is useful for exploring key genes associated with epigenetic changes in tea plants.

Genome-wide identification and expression profiling reveal the diverse role of Methyl-CpG-binding domain proteins in tea plant Camellia sinensis

Methyl-CpG-binding domain(MBD)proteins are important DNA methylation readers that recognise methylated CpG sites and recruit histone deacetylase(HDAC)complexes and chromatin remodelling factors,leading to chromatin compaction,gene transcription,and genome integrity.Currently,MBD genes have only been identified in a few plant species and their structure and function in tea plants(Camellia sinensis)are unknown.In this study,16 C.sinensis MBD genes(CsMBD)were identified on a genome-wide level and classified into eight classes.The CsMBD genes were mapped on nine chromosomes in tea plants,and nine pairs of CsMBD genes existed.Based on conserved domain analysis,all of the identified CsMBD proteins contained at least one MBD domain.Expression analyses showed that CsMBD genes were expressed in tissue-and organ-specific patterns.We investigated the expression patterns of CsMBD genes in response to abiotic and biotic stresses and during different plant growth and development stages.Multiple pthytohormone and stress-related cis-acting was evaluated in their promoter region,such as GGTCA,TGACG,ABRE and LTR.Specific CsMBD genes were associated with environmental stresses and developmental stages,with little overlap.Overall,our findings reveal the diverse roles of CsMBD genes under different stress and developmental conditions,highlighting candidate genes for further functional studies on tea plants.

Contribution of Asian dust to soils in Southeast China estimated with Nd and Pb isotopic compositions

Asian dust has been identified in subtropical soils of China.Neodymium(Nd)and lead(Pb)elemental and isotopic geochemistry of soils in Southeast China were used to assess the significance of local versus extraneous sources.The εNd(0)values were close to the parent rocks(+2.9)in the young soils(NSJ);while their values were always negative(〜—3.7)in the old soils(OMJ),implying dust deposition.The young soils contained less Asian dust contribution(<8%)while the old soils contained more than 50%Asian dust contribution.This implied that dust contributions were gradually increasing with the soil development stage and Asian dust input had become the principal Nd source for the old soils in this area.On the other hand,Pb excesses and low 207Pb/206Pb values(〜0.8503)in near-surface soils indicated a significant anthropogenic Pb addition onto surface soils.The close relationship between the Pb content and isotopic ratios in the soil profile indicated that the excessive lead in the surface soil was exogenous.These results suggested that Asian dust made up a significant fraction in the old soils,but that local sources(i.e.,basalt and anthropogenic)were not trivial in Southeast China.

CsWRKY13,a novel WRKY transcription factor of Camellia sinensis,involved in lignin biosynthesis and accumulation

Lignin is an aromatic polymer that provides the necessary mechanical strength for the transport of water and nutrients in higher plants.Lignin biosynthesis and accumulation affect growth and development of tea plants.The degree of lignification related to the tenderness of fresh tea leaves determines the quality of tea.WRKY transcription factors play central roles in plant development and physiological processes.However,the roles of WRKY transcription factors in lignin biosynthesis of tea plants remain unclear.In this study,a WRKY gene,CsWRKY13,was cloned from tea plant'Longjing 43'.The open reading frame(ORF)of CsWRKY13 gene was 708 bp,encoding 235 amino acids.Sequence analysis showed that CsWRKY13 contained a conserved WRKYGQK amino acid sequence and a zinc-finger-like motif CX4CX23HXH.Subcellular localization showed that CsWRKY13 was localized in the nucleus.The yeast trans-activation assay showed that CsWRKY13 had no transcriptional activity.Expression analysis showed that the CsWRKY13 gene was highly expressed in the stem.Overexpression of CsWRKY13 in Arabidopsis thaliana reduced lignin content and the expression levels of genes related to lignin biosynthesis in transgenic plants.Most flavonoids pathway related genes were significantly up-regulated.This study shows that CsWRKY13 might function as a negative regulator in regulation of lignin synthesis.

Induction of priming by cold stress via inducible volatile cues in neighboring tea plants^FA

Plants have evolved sophisticated defense mechanisms to overcome their sessile nature.However,if and how volatiles from cold‐stressed plants can trigger interplant communication is still unknown.Here,we provide the first evidence for interplant communication via inducible volatiles in cold stress.The volatiles,including nerolidol,geraniol,linalool,and methyl salicylate,emitted from cold‐stressed tea plants play key role(s)in priming cold tolerance of their neighbors via a C‐repeatbinding factors‐dependent pathway.The knowledge will help us to understand how plants respond to volatile cues in cold stress and agricultural ecosystems.

Cloning and Expression of Two MYB Transcription Factors in Tea Plant(Camellia sinensis)

MYB transcription factors represent a family of genes that include the conserved MYB DNA-binding domain,and they are widely involved in the regulation of plant development and secondary metabolism.In this study,Part of sequences of two MYB transcription factors was determined through the cDNA microarray hybridization and selection of cDNA library derived from tender shoots.The full-length cDNAs of the genes were obtained with RT-PCR and RACE,and they were 1 132 bp and 1 020 bp,named as CsMYB1 and CsMYB2 (GenBank accession No.HQ660373 and HQ660374), and contained ORFs of 879 bp and 675 bp encoding 292 and 224 amino acids,respectively.Sequences analysis showed that the deduced protein molecular weight of the two genes were 32.9 ku and 25.4 ku, and the proteins contained two conserved MYB domains near the N-terminus and a conserved C1 motif near the R3 domains.The deduced amino acid sequence of CsMYB1 and CsMYB2 from tea plant showed high identity with that of other plants,for instance CsMYB1 shared 57%homology with MYB1 of Gossypium hirsutum and CsMYB2 shared 75% homology with MYBC2 of Vitis vinifera.The result of real time-PCR analysis showed the two genes were expressed constitutively in all tissues with different expression levels,e.g.the relative expression level of CsMYB2 in leaf was hundred times higher than that in root.Additionally,shading enhanced CsMYB1 expression,while the treatment did not alter the expression level of CsMYB2.