Lignin Characteristic Observation And Transcriptome Analysis Of P. Lactiflora Inflorescence Stem

P.lactiflora(Paeonia lactiflora Pall.)is a traditional flower in China,and it is also a new popular high-end cut flower in the current international market.At present,the industry of.lactiflora cut flower in China is still in its infancy,and the cultivars used for cut flower production are mostly screened from traditional cultivars.Most of them have slender and insufficient strength stems,which are easy to cause the inflorescence stem to bend and not straight,which seriously affects the quality of cut flowers.Therefore,it is of great significance to study the mechanism of the formation of the P.lactiflora inflorescence stem for improving the quality of cut flowers.It has been clarified that lignin is an important chemical substance that affects the formation of the P.lactiflora inflorescence stem,but it lacks systematic and in-depth research.In this study,the P.lactiflora cultivars with different inflorescence stem strengths were used as materials,and their anatomical structures of the inflorescence stems were observed,and the dynamic developmental changes of lignin characteristics in inflorescence stem were analyzed,the lignin synthesis related genes of P.lactiflora inflorescence stem were screened based on transcriptome sequencing.The main results are as follows(1)The morphological indexes and anatomical structures of eight P.lactiflora cultivars with different inflorescence stem strengths were observed and measured.It was found that the stem diameter was significantly positively correlated with the inflorescence stem strength,and their microstructure of inflorescence stem was basically the same.The number of vascular bundles,the total vascular bundle area,the number of catheter perforations,the ratio of xylem to the cross-section,the number of cell layers thickened by the secondary cell wall and the thickness of the secondary cell wall of the P.lactiflora cultivars with high inflorescence stem strength were higher than those of the P.lactiflora cultivars with low inflorescence stem strength.Moreover,the inflorescence stem strength was significantly positively correlated with the number of vascular bundles,the total area of vascular bundles,the number of catheter perforations,the ratio of xylem to cross-section,and the thickness of secondary cells.In addition,by phloroglucin staining,it was found that the lignin in the P.lactiflora inflorescence stem was mainly deposited in the cell wall of the secondary xylem.And the P.lactiflora cultivars with high inflorescence stem strength had larger coloring area and darker color,and it was consistent with the measured lignin content.(2)The P.lactiflora cultivars tHongfeng' with high stem strength and the P.lactiflora cultivars 'Xiaxia Yingxue' with low stem strength were compared and analyzed.It was found that their morphological indexes including stem diameter,flower diameter,flower weight and inflorescence stem strength increased with the development of the plants.Besides the stem diameter and stem intensity of 'Hongfeng' were always higher than that of 'Xixia Yingxue'.In the anatomical observation and histochemical staining,it was found that the secondary cell walls of the 'Hongfeng' and 'Xixia Yingxue' inflorescence stems were continuously thickened with the development of the plants,and the number of cell layers thickened by the secondary cell wall was increasing.At the same time,the degree of lignification of the cell wall and the distribution and quantity of lignin of inflorescence stems also increased with the development of the plant,and they were significantly higher in 'Hongfeng' than 'Xixia Yingxue'.In addition,the lignin content and activity of lignin synthesis related enzymes including PAL?C4H?CAD and POD in the inflorescence stems of two P.lactiflora cultivars showed an upward trend with the development of the plants.(3)The lignin structure of 'Hongfeng' and 'Xixia Yingxue' inflorescence stems was analyzed by FTIR and 2D-HSQC.It was found that the lignin of inflorescence stem consists of three types:G-lignin,S-lignin and H-lignin.By HPLC quantitative analysis,it was found that G-lignin and S-lignin were the main lignin constituents of the two P.lactiflora cultivars inflorescence stems,and their content in the 'Hongfeng' was basically higher than that in 'Xixia Yingxue'.With the development of the plants,the S/G values of the two cultivars increased continuously,and the S/G value of 'Hongfeng' was always higher than that of 'Xixia Yingxue'.(4)The mixed samples of 'Xixia Yingxue' and 'Hongfeng' inflorescence stem in bloom stage were sequenced by the third-generation full-length transcriptome sequencing technology respectively,and a total of 191336 Polished consensus sequences were obtained.After de-redundancy,113974 transcripts were obtained,of which 105382(92.46%)Unigenes were annotated.Among them,the NR database is annotated to 103413 Unigenes,the Pfam database was annotated to 64611 Unigenes,the KOG database was annotated to 68273 Unigenes,the Swiss-prot database was annotated to 90856 Unigenes,the KEGG database was annotated to 102807 Unigenes,the GO database was annotated to 64611 Unigenes,and the NT database was annotated to 87807 Unigenes.On this basis,the second generation transcriptome sequencing was performed on the inflorescence stems of the three developmental stages of 'Xixia Yingxue,and'Hongfeng'.The average yield of each sample was 68.2 M clean reads.The average ratio of clean reads alignment to the reference gene set was 80.25%.By comparing the transcriptomes of the three developmental stages of 'Xixia Yingxue' and 'Hongfeng' inflorescence stems,34703,36336 and 34512 DEGs were obtained respectively.(5)The expression patterns of 9 randomly selected DEGs were verified by qRT-PCR.It was found that they were basically consistent with the transcriptome sequencing results in the inflorescence stems of 'Xixia Yingxue' and 'Hongfeng' in three different developmental stages.In the annotated metabolic pathway,phenylpropane biosynthesis pathway was screened which related to lignin synthesis.And 11 DEGs involved in lignin synthesis including PAL,C4H,C3'H,HCT,F5H,CCoAMOAT,COMT,4CL,CCR,CAD and POD was found in it.The expression levels of 11 DEGs involved in lignin synthesis of the developmental inflorescence stems were shown and found to have different expression patterns,and different expression patterns existed in different family members of the same gene.In this study,a total of gene family members whose expression patterns were consistent with the trend of lignin content in inflorescence stems included 3 PAL,2 C4H,5 4CL,2 HCT,1 C3'H,1 CCoAOMT,3 COMT,1 CCR,5 CAD and 3 POD.Their expression level in the two P.lactiflora cultivars developmental inflorescence stems showed an upward trend,besides the expression level in 'Hongfeng' was higher than that in'Xixia Yingxue'.