Accumulation Pattern And Regulatory Mechanisms Of Fatty And Acid In Different Safflower (carthamus Tinctorius L.) Tissues

The Linoleic acid (LA, C18:2Δ9,12) andα-linolenic acid(ALA, C18:2Δ9,12,15) are essential fatty acids that cannot be synthesized by mammals and therefore must be obtained from dietary sources and have the role of lower the plasma cholesterol levels, low-density lipoproteins and reducing blood cholesterol levels in human body. 18:2 and 18:3 were the main structural components of membrane lipids and storage lipids in plant. They contribute to inducible stress resistance through the remodeling of membrane fluidity when plants encounter the biotic and abiotic stress.18:2 and 18:3 are synthesized through both prokaryotic (chloroplast) and eukaryotic (ER) pathways by a group ofω-6 andω-3 fatty acid desaturases. The membrane-boundω-6 desaturase (codified by the microsomal FAD2 and the plastidial FAD6 genes) inserts a double bond between carbons 12 and 13 of 18:1 to generate di-unsaturated linoleic acid (18:2).ω-3 desaturase (codified by one microsomal FAD3 and two plastidial FAD7 and FAD8 genes) further catalyzes the introduction of a third bond between carbons 15 and 16 to form tri-unsaturated a-linolenic acid (18:3). Safflower oil has been traditionally characterized by a high polyunsaturation level with linoleic acid (18:2) representing more than 70% of total fatty acid. Although the lipid contents of safflower seed oil and its commercial values have been well documented, the molecular regulation of lipid biosynthesis in safflower seeds has not been explored. The process of linoleic accumulation in safflower is still much of a mystery. Our main results about safflowerω-6 andω-3 fatty acid desaturases genes and regulatory mechanisims were described as follows: 1. Eight different microsomalω-6 fatty acid desaturases and one plastidialω-6 fatty acid desaturase cDNA sequences, designated CtFAD2-1, CtFAD2-2, CtFAD2-3, CtFAD2-4, CtFAD2-5, CtFAD2-6, CtFAD2-7, CtFAD2-8 and CtFAD6 have been isolated from safflower(Carthamus tinctorius L.) using a PCR approach. All of safflower deduced amino acid sequences showed the three histidine boxes characteristic of all membrane-bound desaturases, and CtFAD2 contain a C-terminal ER retrieval motif, where as CtFAD6 possess a putative N-termianl signal peptide. Our Blast searches of the deduced aa sequences revealed that the deduced amino acid sequences of CtFAD2-1 and CtFAD2-8 showed higher similarities to other plant seed type microsomalω-6 fatty acid desaturases, where as OFAD2-2 showed higher similarities to constitutively-expressed type. The other microsomalω-6 fatty acid desaturase genes showed much lower identities to other plant FAD2. The CtFAD6 showed higher similarity with other plant plastidialω-6 fatty acid desaturases. In hydropathy analysis showed that the encoded polypeptide contains six putative membrane- spaning domains. Transmembrane analysis indicated that all the safflower fatty acid desaturases contained four to six putative membrane-spanning domains except CtFAD6, Protein second-structure indicated that safflowerω-6 fatty acid desaturase is composed by a-helix andβ-sheet2. One microsomalω-3 fatty acid desaturase gene fragment and two plastidialω-3 fatty acid desaturases cDNA and genomic sequences, designated CtFAD3, CtFAD7 and CtFAD8 have been isolated from safflower(Carthamus tinctorius L.) and submitted to GenBank. All of safflower deduced amino acid sequences showed the three histidine boxes (HDCGH, HXXXXXHRTHH and HVIHH) characteristic of all membrane-bound desaturases, and CtFAD7 and CtFAD8 possess a putative N-termianl signal peptide,6 and 27 aa respectively. Our Blast searches of the deduced aa sequences revealed that the deduced amino acid sequences of CtFAD7 and CtFAD8 showed higher similarities to other plant plastidialω-3 fatty acid desaturases (61-79%,63-78%, respectively), while OFAD3 showed higher similarity with other plant microsomalω-3 fatty acid desaturase (60-93%). In hydropathy and transmembrane analysis showed that the encoded polypeptides contain four putative hydropathy regions and transmenmbrane one to three times. Protein second-structure indicated that safflowerω-3 fatty acid desaturase were composed byα-helix andβ-sheet. Compared with the genome structures of safflower CtgFAD7 and CtgFAD8 and other plant plastidialω-3 desaturase genes, all the sequences contained 8 extons and 7 introns. The sizes of the internal 6 exons (from 2nd to 7th) were maintained in FAD7 and FAD8 from all the plant species.3. To investigate the regulatory mechanisms of the accumulation of fatty acids among the different safflower tissues, we studied the fatty acid composition and relative expression levels of theω-6 andω-3 fatty acid desaturase genes in roots, stems, petioles, leaves, flowers and developing seeds from safflower. Safflower vegetative tissues contained two main PUFAs,18:3 and 18:2, and two kinds of saturated fatty acids, palmitic acid (16:0) and stearic acid (18:0). Thus, safflower belongs to the group of so-called "18:3 plants". Difference between safflower and other 18:3 plant species was that no 18:1 presented in all the tested vegetative tissues. On contrary, large amount of 18:1 was detected in developing seeds while no 18:3 was found in this tissue. In roots, a new component, C18:3Δ9,12,15 alcohol, was detected in roots represented more than 20% when compared with fatty acids. The transcript analysis observed that all of theω-3 fatty acid desaturase genes were constitutively expressed. CtFAD3 was expressed in all the tissues except developing seeds, with highest mRNA accumulation in flower, followed by leaves, while CtFAD7 and CtFAD8 were mainly expressed in leaves. The relative percentages of 16:0 and 18:0 were decreased during seed development. The content of 18:2 decreased rapidly during the first 10 days of development, remaining steady afterwards till 15 DAF, and then it showed a fast and important increase of 18:2 in the later periods. CtFAD2-1, CtFAD2-3 and CtFAD2-8 are the main genes expressed in developing seeds and all of them show highest transcripts at the 10 day after flowering. Different from other plant, CtFAD3 did not express in developing seeds. At low temperature (5℃), both of 18:2 and 18:3 were increased in stems and petioles. In leaves, the percentages of 18:3 in leaves increased slightly (from 63.31 to 67.27%), with the compensation of 18:2, decreased from 12.73% to 8.70%. In roots, both of the percentages of 18:2 and 18:3 decreased, while interesting is the C18:3Δ9,12,15 alcohol was significantly increased from 22.41 to 32.13%. Express analysis indicated that the mechanism of temperature-dependent alterations of PUFAs composition in safflower membrane lipids is controlled at transcriptional and post-transcriptional level ofω-6 andω-3 fatty acid desaturase.4. Higher proportion of 18:2 in oil increases the chances of oxidation, which leads to unpleasant odors and tastes, thus limiting the storability of the oil. On the contrary, oils with high oleic acid (18:1) are less prone to oxidation and off-flavors and also extend the shelf life by delaying the development of rancidity. Hence recent research efforts are directed towards an improvement of oleic acid in oil crops. By RT-PCR method, the full-length cDNAs of CtFAD2-1 was isolated from safflower genotypes with normal and high ratio of oleic to linoleic acid, which were designated CtFAD2-1 and CtFAD2-1', respectively. Sequence alignment of their coding regions revealed that a deletion of cytosine (C) exists at the position+603 bp of CtFAD2' sequence of high oleic acid genotypes, which resulted in the shift of open reading frame (ORF) and truncated protein CtFAD2', with the loss of the third box involved in metal ion complex required for the reduction of oxygen. Analysis of transcript level showed that the expression of CtFAD2'in high oleic acid genotype is significant lower than CtFAD2 in normal genotypes during seed development. CtFAD2-1 and CtFAD2-1' were cloned into the expression vector, Pet30a and subsequently transformed into expression E.coli BL21 (DE3) pLysS. SDS-PAGE analysis showed that the 43 kDa target protein was visualized clearly in the cell membrane protein containing the CtFAD2-1, while the cells protein with CtFAD2-1' did not showed this band. The enzyme activity experiment of yeast (Saccharomyces cerevisiae) cell transformed with CtFAD2-1 and CtFAD2-1' proved that only CtFAD2-1 gene product showed significant microsomal oleate desaturase activity, partially convert 18:1 to 18:2. These results suggested that the change of CtFAD2' gene sequence results in the deactivation and lower transcription of delta-12 fatty acid desaturase in high oleic safflower genotypes.5. The deduced amino acid sequences ofω-6 andω-3 fatty acid desaturase genes have been compared in order to infer their phylogentic relationships and functional diverge. All the deduced proteins shared three highly conserved histidine rich motifs suggesting a common origin. The histidine rich motifs in the sequences from higher plant were more conserve than that of from prokaryotes. All of the plastidialω-6 andω-3 fatty acid desaturase possess a putative N-termianl signal peptide with different amino acids. And we identified the functional region of the peptide with hydrophobic or neutral amino acids. Most of plant microsomalω-6 andω-3 fatty acid desaturase (FAD2 and FAD3) contained a KKXX-like motif at the C-terminal, while safflower CtFAD2-3, CtFAD2-4, CtFAD2-5, CtFAD2-6 and CtFAD2-7 did not contain this motif, instead an aromatic aa enriched signal (YKNK) was found at the C-terminus of these amino sequences and such signal peptide has been reported to be both necessary and sufficient for maintaining localization of the enzymes in the ER. The phylogenetic analysis revealed four distinct clusters within the membrane desaturases. One cluster consisted of Stearic-ACP desaturase, the second group included plant plastidialω-6 fatty acid desaturase, the third cluster comprised the Eukaryotes FAD2, and the fourth contained all of the plastidial and microsomalω-3 fatty acid desaturase. This arrangement of clusters suggested thatω-3 fatty acid desaturases originated in a prokaryotic lineage from aω-6 fatty acid desaturase gene. The diverging time of plastidial and microsomalω-3 fatty acid desaturase, seed type and housekeeping type FAD2 were after the formation of dicotyledonous and monocotyledonous plants. The statistical evidence of functional divergence between plastidial and microsomalω-3 fatty acid desaturase, seed type and housekeeping type FAD2 was found in this analysis. Further more, the site for functional divergence were identified and distributed near the HisboxⅠand HisboxⅡ. These results indicated that evidence of functional divergence in theω-6 andω-3 fatty acid desaturase gene family during the long evolutionary period.