Cyclopropene Fatty Acids Biosynthesis In Plant

Carbocyclic fatty acids (CFAs) containing three-carbon ring occur infrequently occur in plant and are classified into saturated cyclopropane fatty acids (CPA-FAs) and unsaturated cyclopropene fatty acids (CPE-FAs). With special physicochemical-property, carbocyclic ring makes CFAs as a green industrial stock, which carries several high industrial and medical values. So far, a limited quantity of CFAs is mainly produced from some tropical CFA-containing trees. Obviously, it is a good way that genetic strategy is used to realize the scale production of CFAs in oil crop. CPA-FA (dihydrosterculic acid, DHS) biosynthesis machanism has been clear. CPE-FAs are mainly including sterculic acid (STC) and malvalic acid (MLV). However, it remains unclear that how DHS is converted into STC and then MLV is generated from STC, which is major limitation to genetic engineering for CFAs production. Therefore, the study on CPE-FAs biosynthesis pathway and search for genes encoding key enzymes couLd provide an important theorial foundation for CFAs production in oil crop in the future.In this study, cotton young root and embryo axis were chosed as experimental material to study CPE-FAs biosynthesis pathway and search for genes encoding key enzymes involved in CPE-FAs biosynthesis, respectively. The results will be described below.In cotton root and develpoing embryo axis was detected2-OH STC (2-hydroxyl sterculic acid), which accounted for0.18%and0.11%of total fatty acids composition, respectively. Further TLC (Thin Layer Chromatography) analysis revealed that2-OH STC mainly distributed on phosphatidylcholine (PC) and diacylglycerol (DAG), which couLd interconvert into each other rapidly by the two known pathway. While analysis result confirmed that2-OH STC couLd not be formed and accumuLated on the amphiphilic sphingolipid, which was occupied with abundant hydroxylated fatty acids.With L-[methyl-14C]-Methionine, In vivo4C isotope labeling assay in cotton young root, demonstrated that DHS was formed on sn-1position of PC and sunsequently being reacylated to sn-2of PC, DHS endured desaturation reaction to form STC; finally, the a-oxidation of converting STC into MLV was not performed on PC.Accordingly, the Malvalic acid biosynthesis pathway was proposed as below:DHS was synthesized by transferring mehtyl group from SAM to double bond on oleic acid which was located on sn-1position of PC; subsequently, DHS was reacylated to sn-2from sn-1position of PC, before DHS was converted into STC by desaturation reaction; thirdly, STC was hydroxylated to form2-OH STC; fourthly,2-OH STC was transferred on CoA to become2-OH STC-CoA; finally,2-OH STC-CoA was converted into MLV by a-oxidation reaction.Transcriptomic analysis for developing cotton embryo revealed that between embryo axis and cotyledon were differentially expressed a set of genes, classified into transcription transcription factors, seed proteins and enzymes involved carbohydrate, lipid and gossypol metabolism.In embryo axis were highly and specifically expressed the three genes (ghCPS, ghFAD2-2and ghFAH2), of which The former gene was characterized to be in the charge of DHS biosynthesis and one of the highest expression genes in embryo axis, indicating key enzymes participating in CPE-FAs biosynthesis might be also specifically and highly expressed in embryo axis. Therefore, the latter two genes as candidate genes were co-expressed with ghCPS in the transient expression system of Nicotiana benthamiana leaf and with E.coli CPS in yeast expression system. The result showed that ghFAD2-2possesses typical FAD2activity conveting c16:1into C16:2and C18:1into C18:2, while no any CPE-FAs was detected by GC-MS. CFAs composition analysis for mature cotton embryo axis from two fod.2-1mutant lines demonstrated that the ratio of CPE-FAs (MLV and STC) to total CFAs (MLV, STC and DHS) is decreased to65%, compared to81%in wild type coker315. Considering77%similarity between ghFAD2-2and ghFAD2-1, partly cross-silenced ghFAD2-2resulted in CPE-FAs production decrease, which couLd be treated as one support point that ghFAD2-2could desature DHS into STC.