Molecular Mechanism Of Fatty Acid Metabolism In Chinese Wild Tree Peony(Paeonia Section Moutan DC.)

The tree peony（Paeonia section Moutan DC.）belongs to section Moutan of the genus Paeonia in the family Paeoniaceae,it is a important ornamental flower and medicinal plant.Recently,it has been found that the tree peony is a new woody oil crop that has a high yield,high oil production rate and good oil quality.Theα-linolenic acid（ALA）content of tree peony seed oil is more than 40%,which can markedly improve human health.Tree peony could be sustainable exploited as an alternative source of edible oil.Simultaneously,it is a good model to dissect metabolic pathways involved in ALA biosynthesis.Now,the molecular mechanisms research of tree peony lipid metabolism has just started,cloning and identify the function of related genes of fatty acid metabolism still have scarcely reported.In this study,systematic quantitative assessment of fatty acids in 9 wild tree peony species was carried out.Simultaneously,this study provided a description of the transcriptomic changes occurring during seed development in P.rockii,P.potaninii and P.lutea.We will clone the relative genes of fatty acid metabolism by bioinformatics and RACE.The over-expression vectors will be constructed and transformed into Arabidopsis.By analysis of fatty acid content from the seeds of transgenic plants,the function of candidate genes will be determined.The aim of our research is to provide some new genes for cultivating high yield and quality tree peony by using molecular mark and it establishes the foundation for the molecular mechanism research of ALA efficient synthesis in tree peony seeds.The results are shown as follows:1.The systematic quantitative assessment of fatty acids in 9 wild tree peony species was carried out.The results showed that the 9 wild relatives were divided into three FA content levels,and the total FA content of P.rockii,P.decomposita and P.quii was generally higher than tree peony cultivars.The fatty acid compositions were analyzed by GC-MS.There were five dominant components in 9 wild tree peony species,namely,ALA,linoleic acid（LA）,oleic acid（OA）,palmitic acid（PA）,and stearic acid（SA）.The fatty acid contents varied dramatically among species,ranging from 150.09 to 271.82 mg.g^-1.Our results showed that20-60 days after pollination（DAP）is a key period of fatty acid accumulation in wild relatives of tree peony,and the rate of fatty acid synthesis at 20-60 DAP will lead to differences in the total FA content of mature seeds.The qRT-PCR results showedβ-PDHC,SAD,FATA,FAD2,FAD3 and LPAAT were higher expressed during seed development in P.rockii than P.potaninii and P.lutea,and the expression patterns are consistent with the change of total fatty acids,indicating that these genes may play important roles in seed FA synthesis.Moreover,P.rockii have more ALA than other species,mainly due to FAD3 highly expressed.2.Eighteen cDNA libraries were constructed from P.rockii,P.potaninii and P.lutea seeds collected at 20,60 and 80 days after pollination,and sequenced using Illumina sequencing platform.A total of 62,094 contigs were obtained.A comparison of the transcriptomes of different species identified 31,466 differentially expressed unigenes.Bioinformatics analysis shown that 186 unigenes implicated in fatty acid biosynthesis and triacylglycerol assemblage were identified.Moreover,the expression of several unigenes encoding fatty acid desaturase was significantly up in the period of rapid fatty acid synthesis,and the expression levels of the above unigenes were different in three species.According to analyze gene expression and the change of fatty acid content during seed development in different species,fatty acids and triacylglycerols biosynthesis in tree peony was firstly clarified.Further analyses revealed that high ALA level was closely related to the efficient PC-derivative pathway in P.rockii,and the efficient Kennedy pathway and de novo fatty-acid synthesis also played a key role in process of fatty acid synthesis and triacylglycerol assemblage.3.Two genes encoding lysophosphatidic acid acyltransferase（LPAAT）were identified and cloned,named PrLPAAT1（GenBank accession no.for KX256278）and PrLPAAT4（GenBank accession no.for KX256279）.Our data indicated that PrLPAAT1 has 1047nucleotides and encodes a protein with 348 amino acid residues,PrLPAAT4 has 1116nucleotides and encodes a protein of 371 amino acids.PrLPAAT1 and PrLPAAT4 were located in the cytomembrane by analyzing for subcellular localization,and they belong to AGPAT family,and may have acyltransferase activity by analyzing for conserved motifs.Phylogenetic analysis suggested that PrLPAAT1 is highly homologous to Aquilegia coerulea AcLPAAT1,PrLPAAT4 is highly homologous to Citrus clementina CcLPAAT4、Populus trichocarpa PtLPAAT4、Manihot esculenta MeLPAAT4 and Ricinus communis RcLPAAT4.QRT-PCR results showed that PrLPAAT1 and PrLPAAT4 were constitutively expressed in the tree peony,with higher expression in the flower and seed,and lower expression level in the root,stem and leaf.The expression of the PrLPAAT1 gene was increased at 20-70DAP,which coincided with the highest rate of FA accumulation in tree peony seeds,and the expression of the PrLPAAT4 gene has higher level at 20-70DAP,these results indicated that PrLPAAT1 and PrLPAAT4 are probably an important component in the FA accumulation process（or Kennedy pathway）,especially during the early stages of seed development.4.Seed-specific overexpression of PrLPAAT4 in Arabidopsis results in a greater-than-average seed weight and seed yield in the transgenic plants compared with the wild-type plants（Col-0）,the 100-seed weight and seed yield per plant of L4OX-22 transgenic T3 lines was 19.9%and 29.9%greater than that of wildtype.Compared with the corresponding wildtype,the total FA content increased by 3.4%to 10.2%in seeds of the T3homozygous 2S2:PrLPAAT4 lines,and the total FA content of the seeds increased by 11.8%in the T3 homozygous 2S2:PrLPAAT1 lines.Moreover,overexpression of PrLPAAT1 and PrLPAAT4 in Arabidopsis results in a change of fatty acids ratio.Compared with the Col-0,the content of C18:1 and C18:2 were significant increase,the ratio of unsaturated to saturated FAs was decrease in PrLPAAT4 overexpressor lines;and the content of C16:0 and C18:2 were significant increase,the ratio of 18-to 20-carbon FA was decrease in PrLPAAT1overexpressor lines.We measured the genes expression levels of FA biosynthesis,TAG biosynthesis,sucrose metabolism and glycolytic pathway in developing transgenic Arabidopsis siliques.Expression content of AtACP1,AtBCCP2 and AtFPA1 in PrLPAAT4overexpressor lines was higher than in Col-0,and expression content of AtACP1,AtDGAT1and AtSUS3 in PrLPAAT1 overexpressor lines was higher than in Col-0.5.Five genes encoding fatty acid desaturation（FAD）were cloned,named PrSAD,PrFAD2,PrFAD3,PrFAD6 and PrFAD7.Our data indicated that PrSAD has 678 nucleotides and encodes a protein of 225 amino acids,PrFAD2 has 1155 nucleotides and encodes a protein of 384 amino acids,PrFAD3 has 1308 nucleotides and encodes a protein of 435 amino acids,PrFAD6 has 1230 nucleotides and encodes a protein of 409 amino acids,PrFAD7 has1353 nucleotides and encodes a protein of 450 amino acids.The amino acids of five genes contain a conserved FA-acyltransferase domain.Phylogenetic analysis suggested that PrSAD,PrFAD2,PrFAD3,PrFAD6 and PrFAD7 were clustered together with Paeonia lactiflora corresponding genes,our results showed that tree peony and Paeonia lactiflora in relationship were near,that fits nicely with the classification and relationship of the genus Paeonia.QRT-PCR results showed that five FAD genes were higher expressed in the seed and pistil.PrSAD,PrFAD2 and PrFAD3 were key genes in fatty acid synthesis of P.rockii,and PrFAD6and PrFAD7 might not play a significant role in the process of the above.Our results proved again that PC-derived pathways are major metabolic pathways of the polyunsaturated FAs（PUFA）in tree peony.