| Herbaceous peony(Paeonia lactiflora Pall.)is a perennial herbs that belongs to the family Paeoniaceae,which is called "flower king" and "flower vizier" together with peony.Because of its large flower,bright color,elegant flower shape and fragrant fragrance,P.lactiflora is often used as a wedding flower and it has become a popular high-grade cut flower in the international market.The straightenness degree of inflorescence stem is an important appearance index to evaluate the quality of P.lactiflora cut flowers,while a large number of traditional P.lactiflora varieties with excellent flower color and pattern have poor stem quality and straightenness,prone to bending and falling.How to improve inflorescence stem strength and stem straightenness degree is an important problem should be solved urgently in China cut flower production of P.lactiflora.As a natural plant growth regulator,melatonin has many biological functions,while there have been no reports on its regulation of plants stem strength.In order to clarify that melatonin can regulate the inflorescence stem strength of P.lactiflora,in this study,we firstly measured the melatonin contents of inflorescence stems in high and low two groups of P.lactiflora varieties with significant difference in stem strength,and analyzed the relationship between inflorescence stem strength and melatonin content;secondly,exogenous melatonin was applied to P.lactiflora foliage to investigate the regulation effect of exogenous melatonin on inflorescence stem strength;moreover,based on transcriptome sequencing,we screened the related genes responsive to the regulation of exogenous melatonin,cloned and validated the candidate gene PlCOMT1 to clarify the important role of PlCOMT1 in the regulation of stem strength.The main results were as follows:(1)Compared with P.lactiflora varieties with low inflorescence stem strength,the Paeonia lactiflora with high inflorescence stem strength had higher stem diameter,stem weight,net photosynthetic rate(Pn),stomatal conductance(Gs),intercellular CO2 concentration(Ci),transpiration rate(Tr),stem lignin contents and melatonin contents.In addition,there was a positive correlation between inflorescence stem strength and melatonin content,both of which were significantly positively correlated with lignin content,suggesting that melatonin may indirectly affect P.lactiflora inflorescence stem strength by regulating lignin synthesis.(2)Exogenous melatonin significantly enhanced the inflorescence stem strength of P.lactiflora.Compared with the control,exogenous melatonin increased the stem diameter,stem weight,flower diameter and flower weight of P.lactiflora,especially the most significant increase in the stem diameter;besides,the thickness of xylem secondary cell wall,the number of thickened xylem secondary cell wall,the content of endogenous melatonin,the content and distribution range of lignin in P.lactiflora inflorescence stem were significantly increased.According to the FTIR and 2D-HSQC analyse on lignin structure of P.lactiflora inflorescence stem,it was found that exogenous melatonin could promote the synthesis of G-lignin and S-lignin of inflorescence stem,especially S-lignin synthesis,which increased the S/G ratio.Moreover,exogenous melatonin also significantly increased the lignin biosynthesis enzymes’ activities of P.lactiflora inflorescence stem including phenylalanine ammonia-lyase(PAL),cinnamate-4-hydroxylase(C4H),cinnamyl alcohol dehydrogenase(CAD)and peroxidase(POD).(3)A total of 51.2 Gb of sequence data were obtained by transcriptome sequencing performed on control and melatonin-treated inflorescence stems of P.lactiflora at flower-bud stage(S1)and flower-bud stage(S4).Then,a total of 90,857 genes were obtained by comparison with reference gene sets,with an average comparison rate of 71.02%.By comparison,28,309 differentially expressed genes(DEGs)were screened by CK-S1 vs MT-S1,and 25,507 DEGs were screened by CK-S4 vs MT-S4,then 9 DEGs were selected randomly to verify the accuracy and reliability of RNA-seq data by real-time quantitative PCR(qRT-PCR).Pathway analysis of DEGs showed that there were 22037,19,958 DEGs annotated into 135 metabolic pathways in S1 and S4 groups respectively and 9 metabolic pathways were co-enriched,in which satisfying QValue<0.05 were glycerolipid metabolism,RNA transport,sulfur relay system,ascorbate and aldarate metabolism,diterpenoid biosynthesis,phenylpropanoid biosynthesis and RNA polymerase.,in which phenylpropanoid biosynthesis is the main way of lignin biosynthesis.In lignin biosynthesis,exogenous melatonin induced a large number of expression of five kinds of DEGs,including two phenylalanine aminolysis genes(PAL),two cinnamoyl-CoA reductase genes(CCR),three cinnamyl alcohol dehydrogenase genes(CAD),two peroxidase genes(POD)and four caffeic acid O-methyltransferase genes(COMT);inhibited the expression of hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase(HCT)was inhibited.In melatonin biosynthesis,other than COMT,exogenous melatonin also induces a large amount of expression of tryptophan decarboxylase gene(TDC).In addition,MYB,NAC,AP2 and C3H transcription factor families were the most expressed induced by exogenous melatonin.(4)The RACE cloning technology was adopted to clone candidate gene COMT,and the PlCOMT1 gene DNA sequence was 1608 bp,cDNA sequence was 1346 bp,has three introns,the open reading frame was 1077 bp,359 amino acids encoded,having high homology with other plants COMT nucleotide and amino acids sequence.Based on the construction of an overexpression vector pBWA(V)HS-PlCOMT1-GLosgfp containing PlCOMT1 gene sequence,it was found to be mainly localized in the cytoplasm through tobacco subcellular localization observation.The over-expression vector was transferred into tobacco by agrobacterium-mediated leaf-plate method,and positive transgenic plants were obtained by PCR and qRT-PCR detection.(5)Compared with wild type tobacco,the stem strength of transgenic tobacco was increased by 29.6%,together with plant height,stem diameter,stem weight,stem melatonin content,tryptophan decarboxylase(TDC)activity,leaf photosynthetic parameters Pn,GS,Ci,Tr were all significantly increased.Secondly,the xylem secondary cell wall thickness and number of xylem thickened secondary cell wall of transgenic tobacco stem were significantly increased compared with wild type tobacco.In addition,the total amount of lignin,content of G-lignin and S-lignin,ratio of G/S,and expression levels of lignin biosynthesis gene PAL、COMT、CAD、CCR and POD in transgenic tobacco stem were significantly increased compared with those in wild type tobacco. |
PDF Full Text Request