Membrane-bound Desaturases Research Articles

Consensus mutagenesis and computational simulation provide insight into the desaturation catalytic mechanism for delta 6 fatty acid desaturase

Fatty acid desaturase (FADS) generates double bond at a certain position of the corresponding polyunsaturated fatty acids (PUFAs) with high selectivity, the enzyme activity and PUFAs products of which are essential to biological systems and are associated with a variety of physiological diseases. Little is known about the structure of FADSs and their amino acid residues related to catalytic activities. Identifying key residues of Micromonas pusilla delta 6 desaturase (MpFADS6) provides a point of departure for a better understanding of desaturation. In this study, conserved amino acids were anchored through gene consensus analysis, thereby generating corresponding variants by site-directed mutagenesis. To achieve stable and high-efficiency expression of MpFADS6 and its variants in Saccharomyces cerevisiae, the key points of induced expression were optimized. The contribution of conserved residues to the function of enzyme was determined by analyzing enzyme activity of the variants. Molecular modeling indicated that these residues are essential to catalytic activities, or substrate binding. Mutants MpFADS6[Q409R] and MpFADS6[M242P] abolished desaturation, while MpFADS6[F419V] and MpFADS6[A374Q] significantly reduced catalytic activities. Given that certain residues have been identified to have a significant impact on MpFADS6 activities, it is put forward that histidine-conserved region III of FADS6 is related to electronic transfer during desaturation, while histidine-conserved regions I and II are related to desaturation. These findings provide new insights and methods to determine the structure, mechanism and directed transformation of membrane-bound desaturases.

In Silico Analysis of Fatty Acid Desaturases Structures in Camelina sativa, and Functional Evaluation of Csafad7 and Csafad8 on Seed Oil Formation and Seed Morphology.

Fatty acid desaturases add a second bond into a single bond of carbon atoms in fatty acid chains, resulting in an unsaturated bond between the two carbons. They are classified into soluble and membrane-bound desaturases, according to their structure, subcellular location, and function. The orthologous genes in Camelina sativa were identified and analyzed, and a total of 62 desaturase genes were identified. It was revealed that they had the common fatty acid desaturase domain, which has evolved separately, and the proteins of the same family also originated from the same ancestry. A mix of conserved, gained, or lost intron structure was obvious. Besides, conserved histidine motifs were found in each family, and transmembrane domains were exclusively revealed in the membrane-bound desaturases. The expression profile analysis of C. sativa desaturases revealed an increase in young leaves, seeds, and flowers. C. sativa ω3-fatty acid desaturases CsaFAD7 and CsaDAF8 were cloned and the subcellular localization analysis showed their location in the chloroplast. They were transferred into Arabidopsis thaliana to obtain transgenic lines. It was revealed that the ω3-fatty acid desaturase could increase the C18:3 level at the expense of C18:2, but decreases in oil content and seed weight, and wrinkled phenotypes were observed in transgenic CsaFAD7 lines, while no significant change was observed in transgenic CsaFAD8 lines in comparison to the wild-type. These findings gave insights into the characteristics of desaturase genes, which could provide an excellent basis for further investigation for C. sativa improvement, and overexpression of ω3-fatty acid desaturases in seeds could be useful in genetic engineering strategies, which are aimed at modifying the fatty acid composition of seed oil.

Characterization of genes encoding ω-6 desaturase PoFAD2 and PoFAD6, and ω-3 desaturase PoFAD3 for ALA accumulation in developing seeds of oil crop Paeonia ostii var. lishizhenii

Paeonia ostii var. lishizhenii has emerged as a valuable oil-producing crop with splendid characteristic of high α-linolenic acid (C18:3, ALA) content in its seed oil for healthy food supplement, but the molecular mechanism for seed ALA accumulation remains enigmatic. In our previous report, a PoSAD gene encoding stearoyl-ACP desaturase had been cloned and functional charactered for the first desaturation procedure involved in ALA biosynthesis pathway in P. ostii var. lishizhenii endosperms, while other participants have not been identified to date. In this study, full-length cDNAs of PoFAD2 (1489 bp), PoFAD6 (1638 bp), and PoFAD3 (1709 bp) were isolated based on our recent transcriptome sequencing data. Bioinformatic analyses revealed that the PoFADs were closest to their counterparts from Paeoniaceae species P. ludlowii, P. rockii, and P. suffruticosa in phylogenetic tree, which shared highly conserved histidine boxes (HXXXH, HXXHH, and HXXHH), exhibiting typical characters of membrane-bound desaturases in higher plants. Additionally, the PoFAD2 and PoFAD3 were specifically expressed and highly associated with LA and ALA accumulation in developing endosperms, whereas PoFAD6 expression has no significantly difference during whole seed developing stages. The catalytic function of these PoFADs were further analyzed by heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana. The results showed that PoFAD2 and PoFAD6 could catalyze linoleic acid (C18:2) synthesis, while PoFAD3 had ability to produce ALA. This study functional identified three PoFAD genes, which indicates their critical roles in ALA biosynthesis pathway in P. ostii var. lishizhenii, and is of great theoretical and practical meaning on breeding and cultivating new tree peony varieties to promote human health and nutrition supplement.

Membrane-bound Δ12 fatty acid desaturase (FAD12); From Brassica napus to E. coli expression system

Fatty acid desaturase catalyzes the desaturation reactions by insertion of double bonds into the fatty acyl chain, producing unsaturated fatty acids. Though soluble fatty acid desaturases have been studied widely in advanced organisms, there are very limited studies of membrane fatty acid desaturases due to the difficulty of generating recombinant desaturase. Brassica napus is a rapeseed, which possesses a range of different membrane-bound desaturases capable of producing fatty acids including Δ3, Δ4, Δ8, Δ9, Δ12, and Δ15 fatty acids. The 1155 bp open reading frame of Δ12 fatty acid desaturase (FAD12) from Brassica napus codes for 383 amino acid residues with a molecular weight of 44 kDa. It was expressed in Escherichia coli at 37 °C in soluble and insoluble forms when induced with 0.5 mM IPTG. Soluble FAD12 has been purified using Ni2+-Sepharose affinity chromatography with a total protein yield of 0.728 mg/mL. Gas chromatography–mass spectrometry (GC–MS) analysis revealed that desaturase activity of FAD12 could produce linoleic acid from oleic acid at a retention time of 17.6 with a conversion rate of 47%. Characterization of purified FAD12 revealed the optimal temperature of FAD12 was 50 °C with 2 mM preferred substrate concentration of oleic acid. Analysis of circular dichroism (CD) showed FAD12 was made up of 47.3% and 0.9% of alpha-helix and β-sheet secondary structures. The predicted Tm value was 50.2 °C.

Pseudomonas putida Δ9-fatty acid desaturase: Gene cloning, expression, and function in the cationic surfactants stress.

Pseudomonas putida counteract the fluidizing effect of cationic surfactants decreasing the content of membrane unsaturated fatty acid (UFA). A Δ9-fatty acid desaturase gene (desA) from P. putida was isolated, cloned, and successfully expressed in Escherichia coli, a Δ9 desaturase deficient organism. desA consists of 1185 bp and codes for 394 amino acids. The deduced amino acid sequence reveals three histidine clusters and a hydropathy profile, typical of membrane-bound desaturases. Validating desA expression in E. coli cells, the amount of palmitoleic acid increased from 2.05 to 7.36%, with the concomitant increase in membrane fluidity (fluorescence polarization value decrease from 0.13 ± 0.03 to 0.09 ± 0.02). Also, when DesA activity was assayed in vivo, the percentage of UFA obtained from exogenous palmitic acid [1-14 C] increased 10-fold. In contrast, when cells expressing desA were exposed 15 min at sublethal concentration of cationic surfactants, the amount of UFA was 82% lower than that detected in cells non-exposed. Thus, the decrease in UFA content to counteract the fluidizing effect of cationic surfactants can be correlated with reduction of DesA activity.

Identification and characterization of a delta-12 fatty acid desaturase gene from marine microalgae Isochrysis galbana

The cDNA of the delta-12 fatty acid desaturase gene, IgFAD2, was cloned from the marine microalgae Isochrysis galbana, a species capable of producing docosahexaenoic acid. Sequence analysis indicated that the open reading frame measured a length of 1 158 bp and encoded 386 amino acids with a predicted molecular weight of 42.8 kDa and an isoelectric point of 9.2. Computational analysis of the protein sequence of IgFAD2 showed typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membrane-spanning regions that were universally present among plant desaturases. Quantitative real-time PCR results showed that the abundance of IgFAD2 transcript was significantly upregulated under different environmental stresses including low temperature (15°C), high salinity (salinity of 62 and 93), and nitrogen starvation (220 μmol/L). Heterologous expression indicated that yeast cells transformed with a plasmid construct containing IgFAD2 could convert endogenous oleic acid (18:1Δ9, OA) into linoleic acid (18:2Δ9, 12, LA). These findings confirm that I. galbana IgFAD2 plays important roles in the biosynthetic pathways of unsaturated fatty acids.

Molecular mechanism of substrate preference for ω-3 fatty acid desaturase from Mortierella alpina by mutational analysis and molecular docking

The ω-3 fatty acid desaturase (ω3Des) is a key enzyme in the biosynthesis of polyunsaturated fatty acids (PUFAs). However, the enzyme exhibits a significant preference towards different fatty acid substrates. To examine the molecular mechanism of its substrate specificity, a series of site-directed mutants were constructed based on the membrane topology model and functionally characterised by heterologous expression in Saccharomyces cerevisiae. Our results revealed that the W106F and V137T mutations markedly decreased the enzyme activity which indicated that these two residues were associated with substrate recognition. In contrast, the A44S, M156I and W291M mutations showed significant increments (30 to 40%) of the conversion rate for AA substrate desaturation, which suggests that these residues play a pivotal role in desaturation of longer chain-length substrates. Through homology modelling of 3-dimensional structures and molecular docking of FADS15, we propose that the critical residues that bind to the CoA groups may affect substrate localisation and govern substrate preference and chain-length specificity. Our work increases the understanding of the structure-function relationships of the microbial membrane-bound desaturases. The growing knowledge of the molecular mechanism will also aid in the efficient production of value-added fatty acids.

Insight into Arthrospira platensis Δ9 desaturase: a key enzyme in poly-unsaturated fatty acid synthesis.

Membrane-bound Δ9 desaturase perform oxygenated desaturation reactions to insert the first double bonds within fatty acyl chains between C9 and C10 positions of most saturated substrates. Arthrospira platensis, a blue green microalga, is an important source of polyunsaturated fatty acids (PUFA) such as oleic, linoleic and linolenic acids lending benefits and functions in dietetics and therapeutic uses. In this paper, we report homology modeling and docking studies of a Δ9 desaturase from Arthrospira platensis strain. The protein model showed high topology resemblance compared to membrane-bound desaturases with a cytoplasmic core displaying the catalytic site and a transmembrane domain created by four α-helices. The cytoplasmic cap contained the three conserved-histidine boxes typical for all membrane bound desaturases. The protein model was used to perform protein-protein docking and the dimer structure was generated. The two monomers are tightly related with hydrophobic interactions between the transmembrane domain helices. The study highlighted also the potent role of a particular 53 residues sequence located at the N terminal end of the enzyme.

Homology modeling and docking studies of a Δ9-fatty acid desaturase from a Cold-tolerant Pseudomonas sp. AMS8.

Membrane-bound fatty acid desaturases perform oxygenated desaturation reactions to insert double bonds within fatty acyl chains in regioselective and stereoselective manners. The Δ9-fatty acid desaturase strictly creates the first double bond between C9 and 10 positions of most saturated substrates. As the three-dimensional structures of the bacterial membrane fatty acid desaturases are not available, relevant information about the enzymes are derived from their amino acid sequences, site-directed mutagenesis and domain swapping in similar membrane-bound desaturases. The cold-tolerant Pseudomonas sp. AMS8 was found to produce high amount of monounsaturated fatty acids at low temperature. Subsequently, an active Δ9-fatty acid desaturase was isolated and functionally expressed in Escherichia coli. In this paper we report homology modeling and docking studies of a Δ9-fatty acid desaturase from a Cold-tolerant Pseudomonas sp. AMS8 for the first time to the best of our knowledge. Three dimensional structure of the enzyme was built using MODELLER version 9.18 using a suitable template. The protein model contained the three conserved-histidine residues typical for all membrane-bound desaturase catalytic activity. The structure was subjected to energy minimization and checked for correctness using Ramachandran plots and ERRAT, which showed a good quality model of 91.6 and 65.0%, respectively. The protein model was used to preform MD simulation and docking of palmitic acid using CHARMM36 force field in GROMACS Version 5 and Autodock tool Version 4.2, respectively. The docking simulation with the lowest binding energy, −6.8 kcal/mol had a number of residues in close contact with the docked palmitic acid namely, Ile26, Tyr95, Val179, Gly180, Pro64, Glu203, His34, His206, His71, Arg182, Thr85, Lys98 and His177. Interestingly, among the binding residues are His34, His71 and His206 from the first, second, and third conserved histidine motif, respectively, which constitute the active site of the enzyme. The results obtained are in compliance with the in vivo activity of the Δ9-fatty acid desaturase on the membrane phospholipids.

Identification of two genes encoding microsomal oleate desaturases (FAD2) from the biodiesel plant Pongamia pinnata L.

The current study dissect microsomal oleate desaturase genes having differential expression pattern with respect to temperature from the seeds of Pongamia pinnata, and are grouped with other legumes and biofuel plants. Biofuel often is available as plant oil or products derived thereafter, such as biodiesel. In view of the anticipated fossil fuel shortage, the biochemical and genetic basis of vegetable oil biosynthesis is vital. We are focusing on the versatile oil-yielding legume tree Pongamia pinnata. Microsomal oleate desaturase (FAD2) is the key enzyme responsible for the production of linoleic acid in non-photosynthetic tissues. This work reports on the isolation of two full length cDNA clones, tissue expression and copy number detection of Pongamia FAD2 genes, and also on the transcriptional regulation at low and high temperatures. The deduced amino acid sequences of both PpFAD2 proteins share 83 % identity and display three typical histidine boxes that are characteristics of all membrane-bound microsomal oleate desaturases. Both sequences possess aromatic amino acid containing sequence motifs at the C-terminal end necessary for maintaining endoplasmic reticulum (ER) localization. Southern blot analysis is consistent with the presence of at least two copies of FAD2 in the pongamia genome. Quantitative real-time PCR analysis showed that PpFAD2-1 is expressed strongly in developing seeds and, showing very low levels of expression in vegetative tissues, whereas PpFAD2-2 is constitutively expressed in both vegetative tissues and the developing seeds, with higher transcript levels in roots, stems and leaves. In response to low temperature stress PpFAD2-1 and PpFAD2-2 were differentially expressed in various tissues. The PpFAD2-2 transcript dramatically decreased in roots, stems and leaves under low temperatures, whereas PpFAD2-1 showed a significant increase in these tissues.

Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases

Membrane-bound desaturases are physiologically and industrially important enzymes that are involved in the production of diverse fatty acids such as polyunsaturated fatty acids and their derivatives. Here, we identified amino acid residues that determine the substrate specificity of rat Δ6 desaturase (D6d) acting on linoleoyl-CoA by comparing its amino acid sequence with that of Δ5 desaturase (D5d), which converts dihomo-γ-linolenoyl-CoA. The N-terminal cytochrome b5-like domain was excluded as a determinant by domain swapping analysis. Substitution of eight amino acid residues (Ser209, Asn211, Arg216, Ser235, Leu236, Trp244, Gln245, and Val344) of D6d with the corresponding residues of D5d by site-directed mutagenesis switched the substrate specificity from linoleoyl-CoA to dihomo-γ-linolenoyl-CoA. In addition, replacement of Leu323 of D6d with Phe323 on the basis of the amino acid sequence of zebra fish Δ5/6 bifunctional desaturase was found to render D6d bifunctional. Homology modeling of D6d using recent crystal structure data of human stearoyl-CoA (Δ9) desaturase revealed that Arg216, Trp244, Gln245, and Leu323 are located near the substrate-binding pocket. To our knowledge, this is the first report on the structural basis of the substrate specificity of a mammalian front-end fatty acid desaturase, which will aid in efficient production of value-added fatty acids.

Screening and molecular identification of overproducing γ-linolenic acid fungi and cloning the delta 6-desaturase gene.

This research aims at isolating and identifying γ-linolenic acid (GLA)-producing fungi in the traditional Chinese salt-fermented soybean food, Douchi, from Yongchuan, People's Republic of China. In this study, Rhizopus oryzae DR3 was identified as a novel fungal species that produces large amounts of GLA. A full-length cDNA, designated as RoD6D, with high homology to fungal △6 fatty acid desaturase genes was isolated from R. oryzae by using the rapid amplification of cDNA ends method. It had an open reading frame of 1,176 bp encoding a deduced polypeptide of 391 amino acids. Bioinformatics analysis characterized the putative RoD6D protein as a typical membrane-bound desaturase, including three conserved histidine-rich motifs, a hydropathy profile, and a cytochrome b5 -like domain in the N-terminus. When the coding sequence was expressed in the Saccharomyces cerevisiae strain INVScl, the encoded product of RoD6D exhibited △6 fatty acid desaturase activity that led to the accumulation of GLA. The results show that Douchi contains a large natural diverse composition, and some strains could be selected as starters for functional fermented foods. This study has also laid a foundation for developing functional Douchi products for further research.

Characterization of membrane-bound fatty acid desaturases

Membrane-bound desaturases play key role in metabolism of polyunsaturated fatty acids. Characterization of these enzymes and their genes is the first step in basic understanding of their proper functioning in living cells as well as in tailor-made preparation of highly-specific and highly-productive strains of microorganisms interesting for applied biotechnology. It is also the crucial step in creation of transgenic agricultural crops with enhanced content of individual polyunsaturated fatty acids. Properties and applications of identified membrane-bound desaturases genes and enzymes are discussed in this review.

Reducing saturated fatty acids in Arabidopsis seeds by expression of aCaenorhabditis elegans16:0-specific desaturase

Plant oilseeds are a major source of nutritional oils. Their fatty acid composition, especially the proportion of saturated and unsaturated fatty acids, has important effects on human health. Because intake of saturated fats is correlated with the incidence of cardiovascular disease and diabetes, a goal of metabolic engineering is to develop oils low in saturated fatty acids. Palmitic acid (16:0) is the most abundant saturated fatty acid in the seeds of many oilseed crops and in Arabidopsis thaliana. We expressed FAT-5, a membrane-bound desaturase cloned from Caenorhabditis elegans, in Arabidopsis using a strong seed-specific promoter. The FAT-5 enzyme is highly specific to 16:0 as substrate, converting it to 16:1∆9; expression of fat-5 reduced the 16:0 content of the seed by two-thirds. Decreased 16:0 and elevated 16:1 levels were evident both in the storage and membrane lipids of seeds. Regiochemical analysis of phosphatidylcholine showed that 16:1 was distributed at both positions on the glycerolipid backbone, unlike 16:0, which is predominately found at the sn-1 position. Seeds from a plant line homozygous for FAT-5 expression were comparable to wild type with respect to seed set and germination, while oil content and weight were somewhat reduced. These experiments demonstrate that targeted heterologous expression of a desaturase in oilseeds can reduce the level of saturated fatty acids in the oil, significantly improving its nutritional value.

Molecular cloning and functional characterization of a Δ6‐fatty acid desaturase gene from Rhizopus oryzae

The objective was to screen for and isolate a novel enzyme with the specific activity of a Δ6-fatty acid desaturase from Rhizopus oryzae. In this study, R. oryzae was identified as a novel fungal species that produces large amounts of γ-linolenic acid. A full-length cDNA, designated here as RoD6D, with high homology to fungal Δ6-fatty acid desaturase genes was isolated from R. oryzae by using the rapid amplification of cDNA ends method. It had an open reading frame of 1176 bp encoding a deduced polypeptide of 391 amino acids. Bioinformatics analysis characterized the putative RoD6D protein as a typical membrane-bound desaturase, including three conserved histidine-rich motifs, a hydropathy profile, and a cytochrome b5 -like domain in the N terminus. When the coding sequence was expressed in the Saccharomyces cerevisiae strain INVScl, the encoded product of RoD6D exhibited Δ6-fatty acid desaturase activity that led to the accumulation of γ-linolenic acid. The corresponding genomic sequence of RoD6D was 1565 bp in length, with five introns. This is the first report on the characterization and gene cloning of a Δ6-fatty acid desaturase of R. oryzae from Douchi.

A Cytochrome b 5 -Containing Plastid-Located Fatty Acid Desaturase from Chlamydomonas reinhardtii

Monogalactosyldiacylglycerol (MGDG) in Chlamydomonas reinhardtii and other green algae contains hexadeca-4,7,10,13-tetraenoic acid (16:4) in the glycerol sn-2 position. While many genes necessary for the introduction of acyl chain double bonds have been functionally characterized, the Δ4-desaturase remained unknown. Using a phylogenetic comparison, a candidate gene encoding the MGDG-specific Δ4-desaturase from Chlamydomonas (CrΔ4FAD) was identified. CrΔ4FAD shows all characteristic features of a membrane-bound desaturase, including three histidine boxes and a transit peptide for chloroplast targeting. But it also has an N-terminal cytochrome b(5) domain, distinguishing it from other known plastid desaturases. Cytochrome b(5) is the primary electron donor for endoplasmic reticulum (ER) desaturases and is often fused to the desaturase domain in desaturases modifying the carboxyl end of the acyl group. Difference absorbance spectra of the recombinant cytochrome b(5) domain of CrΔ4FAD showed that it is functional in vitro. Green fluorescent protein fusions of CrΔ4FAD localized to the plastid envelope in Chlamydomonas. Interestingly, overproduction of CrΔ4FAD in Chlamydomonas not only increased levels of 16:4 acyl groups in cell extracts but specifically increased the total amount of MGDG. Vice versa, the amount of MGDG was lowered in lines with reduced levels of CrΔ4FAD. These data suggest a link between MGDG molecular species composition and galactolipid abundance in the alga, as well as a specific function for this fatty acid in MGDG.

Newly identified essential amino acid residues affecting Δ 8-sphingolipid desaturase activity revealed by site-directed mutagenesis

In order to identify amino acid residues crucial for the enzymatic activity of Δ 8-sphingolipid desaturases, a sequence comparison was performed among Δ 8-sphingolipid desaturases and Δ 6-fatty acid desaturases from various plants. In addition to the known conserved cyt b 5 (cytochrome b 5) HPGG motif and three conserved histidine boxes, they share additional 15 completely conserved residues. A series of site-directed mutants were generated using our previously isolated Δ 8-sphingolipid desaturase gene from Brassica rapa to evaluate the importance of these residues to the enzyme function. The mutants were functionally characterized by heterologous expression in yeast, allowing the identification of the products of the enzymes. The results revealed that residues H63, N203, D208, D210, and G368 were obligatorily required for the enzymatic activity, and substitution of the residues F59, W190, W345, L369 and Q372 markedly decreased the enzyme activity. Among them, replacement of the residues W190, L369 and Q372 also has significant influence on the ratio of the two enzyme products. Information obtained in this work provides the molecular basis for the Δ 8-sphingolipid desaturase activity and aids in our understanding of the structure–function relationships of the membrane-bound desaturases.

Isolation and Characterization of a Temperature-Regulated Microsomal Oleate Desaturase Gene (CtFAD2-1) from Safflower (Carthamus tinctorius L.)

A cDNA sequence, designated CtFAD2-1 and putatively encoding a delta-12 fatty acid desaturase, was isolated from total RNA of immature normal-type safflower (Carthamus tinctorius L.) seeds using a PCR approach. The deduced amino acid sequence showed three histidine boxes characteristic of all membrane-bound desaturase and a C-terminal endoplasmic reticulum retention signal. Phylogenetic analysis indicated that CtFAD2-1 is grouped with other plant FAD2 sequences which are specifically or highly expressed in developing seeds. Functional expression of the corresponding CtFAD2-1 cDNA in yeast confirmed that it encodes a microsomal oleate desaturase, partially converting oleic fatty acid to linoleic fatty acid. Our transcriptional analysis suggested that CtFAD2-1 is constitutively expressed, with maximum transcript accumulation, in developing seeds. The sequence of this microsomal oleate desaturase gene was also obtained from high oleic safflower genotypes, CtFAD2-1′. The comparison between the coding regions of CtFAD2-1 and CtFAD2-1′ revealed that a deletion of cytosine (C) exists at the position +603 bp of CtFAD2-1′. Analysis of transcript level showed that the expression of CtFAD2-1′ in high oleic acid genotype is significantly lower than that in normal genotypes during seed development. These results suggested that the change of CtFAD2-1′ gene sequence results in the deactivation and lower transcription of delta-12 fatty acid desaturase in high oleic safflower genotypes.

Molecular Cloning and Expression Analysis of Genes Encoding Two Microsomal Oleate Desaturases (FAD2) from Safflower (Carthamus tinctorius L.)

The endoplasmic reticulum (ER)-associated oleate desaturase (FAD2) is the key enzyme responsible for the production of linoleic acid in plants. In safflower, a seed-type FAD2 gene (CtFAD2-1) has been isolated previously. In this study, two different cDNA sequences, designated CtFAD2-2 and CtFAD2-3, encoding two microsomal oleate desaturases have been isolated from safflower (Carthamus tinctorius L.) using a polymerase chain reaction (PCR) approach. Both deduced amino acid sequences showed the three histidine boxes characteristic of all membrane-bound desaturases, and possess a C-terminal ER retention signal. Phylogentic analysis shows that CtFAD2-2 is grouped with other house-keeping type FAD2 sequences from oil crops, while CtFAD2-3 does not belong to seed type or house-keeping type FAD2. Real-time PCR analysis showed that CtFAD2-2 gene is constitutively expressed in both vegetative tissues and developing seeds, with higher transcript levels in roots, stems and petioles. Transcript of CtFAD2-3 was strongly detected in developing seeds, showing low levels in vegetative tissues. In response to low-temperature stress, CtFAD2-2 and CtFAD2-3 in different tissues showed a different behavior (up- or down-regulated). The transcript of CtFAD2-2 was dramatically decreased in roots, stems and petioles under low temperature, while CtFAD2-3 showed a significant increase in these tissues. On the contrary, in leaves, the strongly enhanced expression of CtFAD2-2 was observed under low temperature, while the transcript of CtFAD2-3 showed a little decrease.

A rice microsomal delta-12 fatty acid desaturase can enhance resistance to cold stress in yeast and Oryza sativa

A full-length cDNA clone of OsFAD2, which encodes a Δ-12 fatty acid desaturase, the key enzyme for the conversion of oleic acid (18:1) into linoleic acid (18:2), was isolated from rice (Oryza sativa ssp. japonica) leaves. The deduced amino acid sequence of OsFAD2 displayed three histidine boxes characteristic of all membrane-bound desaturases, and possessed a C-terminal signal for endoplasmic reticulum retention. Phylogenetic analysis showed that OsFAD2 is grouped within plant housekeeping FAD2 sequences. Expression analysis by real-time PCR showed that the gene is expressed in all tissues of rice tested, including root, seed, stem, and leaf. In situ hybridization showed that OsFAD2 mRNA accumulated in leaf mesophyll cells and in root epidermis cells when exposed to 15°C for 4 days in dark conditions. When OsFAD2 was expressed in Saccharomyces cerevisiae, the cells could convert oleic acid to linoleic acid, which wild-type yeast cells cannot do, suggesting that the isolated gene encoded a functional FAD2 enzyme. Heterologous expression of OsFAD2 enhanced the yeast cells’ cold tolerance capacity compared to wild-type yeast. OsFAD2 was also shown to be a highly active desaturase when expressed in Xenopus oocytes. In addition, when the OsFAD2 gene was transferred into an Arabidopsis thaliana fad2-1 mutant, it effectively restored wild-type fatty acid composition and growth characteristics. Stress tolerance and light regulatory elements were identified in the predicted promoter of the OsFAD2 gene. Exogenously supplied hormone affected the level of FAD2 mRNA accumulation, accompanied by a change of content of di-unsaturated fatty acid species in rice leaves. Furthermore, OsFAD2 enhanced tolerance to low temperature when overexpressed in rice at the vegetative stage. More importantly, the 35S::OsFAD2 plants showed significantly enhanced cold tolerance at the reproductive stage, increasing grain yield by 46% over controls in the greenhouse under cold conditions. These results indicated that OsFAD2 is involved in fatty acid desaturation and maintenance of the membrane lipids balance in cells, and could improve the tolerance of yeast and rice to low temperature stress.