The Sunflower High-oleic Mutant Ol Carries Variable Tandem Repeats of FAD2-1, a Seed-specific Oleoyl-phosphatidyl Choline Desaturase

Ol, a chemically induced, incompletely dominant mutation, greatly increases oleic acid and is correlated with greatly reduced expression of a seed-specific oleoyl-phosphatidyl choline desaturase (FAD2-1) in developing seeds of sunflower (Helianthus annuus L.). FAD2-1 is duplicated in high-oleic (mutant) strains and cosegregates with Ol. Codominant RFLP markers have been developed for FAD2-1 and are diagnositic for the Ol mutation; however, the structure of the mutant FAD2-1 locus is unknown and polymorphic sequence-tagged-site (STS) DNA markers have not been developed for FAD2-1. The mutant was discovered to carry tandem repeats of FAD2-1 separated by a 2.67 kb intergenic region. The upstream repeat (FAD2-1U) carries a 1.69 kb intron in the 5′UTR, whereas the downstream repeat (FAD2-1D) is missing the first 1.54 kb of the 5′UTR and intron. Other than the deletion in FAD2-1D, no DNA polymorphisms were identified between wildtype and mutant FAD2-1 alleles among elite oilseed inbred lines. We developed dominant INDEL markers diagnostic for presence or absence of the Ol mutation (tandem FAD2-1 repeats) by targeting DNA sequences upstream of FAD2-1D, identified 49 SNPs and five INDELs (two haplotypes) in DNA sequences downstream of FAD2-1 in the wildtype and FAD2-1U in the mutant, identified polymorphic [AT]_n and [GT]_n repeats in the 3′UTR of FAD2-1, and developed codominant SSR and INDEL markers for FAD2-1. Novel FAD2-1 alleles found in exotic low-oleic genotypes could be introgressed into elite low-oleic genotypes to facilitate marker-assisted selection of Ol in mid- and high-oleic sunflower breeding programs.     

Cloning of a Novel Omega-6 Desaturase from Flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) and Its Functional Analysis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The Δ12 desaturase represents a diverse gene family in plants and is responsible for conversion of oleic acid (18:1) to linoleic acid (18:2). Several members of this family are known from plants like Arabidopsis and Soybean. Using primers from conserved C- and N-terminal regions, we have cloned a novel Δ12 desaturase gene amplified from flax genomic DNA, denoted as LuFAD2-2. This intron-less gene is 1,149-base pair long encoding 382 amino acids—putative membrane-bound Δ12 desaturase protein. Sequence comparisons show that the novel sequence has 85% similarity with previously reported flax Δ12 desaturase at amino acid level and shows typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membrane-spanning regions that are universally present among plant desaturases. The signature amino acid sequence ‘YNNKL’ was also found to be present at the N terminus of the protein, which is necessary and sufficient for ER localization of enzyme. Neighbor-Joining tree generated from the sequence alignment grouped LuFAD2-2 among the other FAD2 sequences from Ricinus, Hevea, Jatropha, and Vernicia. When LuFAD2-2 and LuFAD2 were expressed in Saccharomyces cerevisiae, they could convert the oleic acid to linoleic acid, with an average conversion rate of 5.25 and 8.85%, respectively. However, exogenously supplied linoleic acid was feebly converted to linolenic acid suggesting that LuFAD2-2 encodes a functional FAD2 enzyme and has substrate specificity similar to LuFAD2.     

Microarray analysis of differentially expressed genes between <Emphasis Type="Italic">Brassica napus</Emphasis> strains with high- and low-oleic acid contents

An increase in oleic acid (C18:1) content is a desirable trait. Despite the critical roles of the two desaturases, FAD2 and FAD3, in the control of fatty acid desaturation, a dispute remains over whether inactivation of their genes alone is sufficient enough to generate the high-oleic trait. To address this question, we employed microarray technology to investigate the difference in gene expression profile between two different Brassica napus strains with high-C18:1 (71.71%) and low-C18:1 (55.6%) contents, respectively. Our study revealed 562 differentially expressed genes, of which 194 genes were up-regulated and 368 down-regulated. Based on the Gene Ontology classification, these genes were classified into 23 functional categories. Three of the up-regulated genes represent B. napus homologs of Arabidopsis genes encoding a cytosolic isoform of pyruvate kinase (AT3G55810), Δ9 acyl-lipid desaturase (AT1G06080, ADS1) and fatty acyl-ACP thioesterase B (AT1G08510), respectively. Conversely, the homologs of two Arabidopsis sequences encoding Δ9 acyl-lipid desaturase (AT2G31360, ADS2) and FAD3 desaturase (AT2G29980) were down-regulated in the high-oleic acid strain. Furthermore, 60 differentially expressed genes were classified as associated with relevant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Collectively, our results suggest that expressing the high-oleic acid trait may require a coordinated regulation of diverse regulatory and metabolic gene networks in addition to inactivation of the FAD2 and FAD3 genes in the oilseed. A set of the differentially expressed genes identified in this study will facilitate our efforts to tap the germplasms with the potential to express the high-oleic acid trait.     

玉米FAD2基因的克隆及序列分析

高等植物中的A12脂肪酸脱饱和酶是将油酸转化为亚油酸的酶。根据已发表的其他高等植物的FAD2基因的保守序列设计同源引物,通过RT—PCR从玉米幼胚中扩增得到一个特异的cDNA基因片段。通过生物信息学分析,从玉米幼胚cDNA和基因组中均扩增得到1164 bp FAD2基因（GenBank登陆号：DQ496227）,它编码387个氨基酸,含有完整的ORF框,在ORF框内无内含子。序列联配与树状分析结果表明,FAD2推导的氨基酸序列与其他物种的A12脱饱和酶基因具有同源性。它含有3个组氨酸保守域和2段很长的疏水区,是一个跨膜4次的膜结合蛋白。半定量RT—PCR分析显示FAD2基因在玉米幼胚中表达量最高,在叶、茎、根中亦有低水平表达。     

Molecular cloning and functional analysis of two FAD2 genes from American grape (Vitis labrusca L.)

The synthesis of polyunsaturated fatty acids (PUFAs), the most abundant fatty acids in plants, begins with a reaction catalyzed by fatty acid desaturase 2 (FAD2; EC 1.3.1.35), also called microsomal oleate Δ12-desaturase. Since the FAD2 gene was first identified in Arabidopsis thaliana, FAD2 research has gained wide interest as the essential enzyme for synthesizing PUFA. Grapes are one of the most frequently cultivated fruits in the world, with most commercial growers cultivating Vitis vinifera and V. labrusca. Grapeseed oil contains a high proportion, 60–70% of linoleic acid (18:2). We cloned two putative FAD2 genes from V. labrusca cv. Campbell Early based on V. vinifera genome sequences. Deduced amino acid sequences of two putative genes showed that VlFAD2s show high similarity to Arabidopsis FAD2 and commonly contain six transmembrane domain, three histidine boxes and endoplasmic reticulum (ER) retrieval motif representing the characteristics of fatty acid desaturase. Phylogenetic analyses of various plant FAD2s showed that VlFAD2-1 and VlFAD2-2 are separately grouped with constitutive and seed-type FAD2s, respectively. Southern blot showed that one or two bands are found in each lane. Because Campbell Early is a hybrid cultivar, FAD2-1 and FAD2-2 genes may exist as one copy in V. labrusca. Expression analysis in different tissues indicated that VlFAD2-1 is a constitutive gene but VlFAD2-2 is a seed-type gene. Complementation experiments of fad2-1 mutant Arabidopsis with VlFAD2-1 or VlFAD2-2 demonstrated that VlFAD2-1 and VlFAD2-2 can restore low PUFA proportion of fad2 to normal PUFA proportion.     

The utilisation of fatty-acid substrates in triacylglycerol biosynthesis by tissue-slices of developing safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) cotyledons

Developing cotyledons of safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) readily utilised exogenously supplied ¹⁴C-labelled fatty-acid substrates for the synthesis of triacylglycerols. The other major radioactive lipids were phosphatidylcholine and diacylglycerol. In safflower cotyledons, [¹⁴C]oleate was rapidly transferred to position 2 of sn-phosphatidylcholine and concomitant with this was the appearance of radioactive linoleate. The linoleate was further utilised in the synthesis of diacyl- and triacyl-glycerol via the reactions of the so-called Kennedy pathway. Supplying [¹⁴C]linoleate, however, resulted in a more rapid labelling of the diacylglycerols than from [¹⁴C]oleate. In contrast, sunflower cotyledons readily utilised both labelled acyl substrates for rapid diacylglycerol formation as well as incorporation into position 2 of sn-phosphatidylcholine. In both species, however, [¹⁴C]palmitate largely entered sn-phosphatidylcholine at position 1 during triacylglycerol synthesis. The results support our previous in-vitro observations with isolated microsomal membrane preparations that (i) the entry of oleate into position 2 of sn-phosphatidylcholine, via acyl exchange, for desaturation to linoleate is of major importance in regulating the level of polyunsaturated fatty acids available for triacylglycerol formation and (ii) Palmitate is largely excluded from position 2 of sn-phosphatidylcholine and enters this phospholipid at position 1 probably via the equilibration with diacylglycerol. Specie differences appear to exist between safflower and sunflower in relation to the relative importance of acyl exchange and the interconversion of diacylglycerol with phosphatidylcholine as mechanisms for the entry of oleate into the phospholipid for desaturation.Abbreviations FW fresh weight - TLC thin-layer chromatography     

沙冬青AmFAD2-1基因的克隆及其功能分析

细胞膜脂的不饱和度主要由脂肪酸去饱和酶(fatty acid desaturase,FAD)决定,二者与植物抵抗低温和高盐等逆境胁迫密切相关。沙冬青(Ammopiptanthus mongolicus)是中亚荒漠区唯一的常绿旱生阔叶植物,具有很强的耐寒、耐旱和耐盐碱等特性。该研究利用RT-PCR方法克隆到沙冬青油酸去饱和酶基因AmFAD2-1的cDNA和gDNA,其中后者不含内含子。在AmFAD2-1蛋白(含382个氨基酸残基)序列中含有FAD家族必须的保守组氨酸簇和内质网定位信号及跨膜结构域。进化分析表明,AmFAD2-1与大豆(Glycine max)和柠条锦鸡儿(Caragana korshinskii)等豆科植物的FAD2距离较近,而与玉米(Zea mays)和拟南芥(Arabidopsis thaliana)等其他植物的FAD2距离较远。qRT-PCR分析表明,在室内培养的沙冬青幼苗中,AmFAD2-1的表达量在低温处理2～24h和干旱处理6～13d期间显著上调,在盐胁迫处理期间则呈现升高、降低、再升高的变化趋势;在野外生长的沙冬青成株嫩叶中,其表达量在秋季和冬季总体上明显高于春季和夏季。成功构建了植物表达载体p3300-35S-AmFAD2-1并通过根癌农杆菌介导法转化野生型拟南芥,获得16株转AmFAD2-1基因植株。耐逆性鉴定表明,转基因株系的耐冻性较野生型拟南芥显著提高,其耐旱性和耐盐性也比后者有较明显的改善。     

Low temperature and light regulate delta 12 fatty acid desaturases (FAD2) at a transcriptional level in cotton (Gossypium hirsutum)

Lipid modifying enzymes play a key role in the development of cold stress tolerance in cold-resistant plants such as cereals. However, little is known about the role of the endogenous enzymes in cold-sensitive species such as cotton. Delta 12 fatty acid desaturases (FAD2), known to participate in adaptation to low temperatures through acyl chain modifications were used in gene expression studies in order to identify parameters of plant response to low temperatures. The induction of microsomal delta 12 fatty acid desaturases at an mRNA level under cold stress in plants is shown here for first time. Quantitative PCR showed that though both delta 12 omega 6 fatty acid desaturase genes FAD2-3 and FAD2-4 identified in cotton are induced under cold stress, FAD2-4 induction is significantly higher than FAD2-3. The induction of both isoforms was light regulated, in contrast a third isoform FAD2-2 was not affected by cold or light. Stress tolerance and light regulatory elements were identified in the predicted promoters of both FAD2-3 and FAD2-4 genes. Di-unsaturated fatty acid species rapidly increased in the microsomal fraction isolated from cotton leaves, following cold stress. Expression analysis patterns were correlated with the observed increase in both total and microsomal fatty acid unsaturation levels suggesting the direct role of the FAD2 genes in membrane adaptation to cold stress.     

Fluidization of Membrane Lipids Enhances the Tolerance of Saccharomyces cerevisiae to Freezing and Salt Stress

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Δ⁹ position. We expressed two sunflower (Helianthus annuus) oleate Δ¹² desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Δ^9,12, the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp⁺ or Trp⁻ strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.     

玉米△12脂肪酸脱氢酶基因（FAD2）在酿酒酵母中的表达

玉米△12脂肪酸脱氢酶是催化油酸形成亚油酸的关键酶。将其编码基因FAD2（GenBank登陆号：DQ496227）克隆到酿酒酵母表达载体pYES2．0中,构建成重组质粒pYE／FAD2,转化到酿酒酵母进行诱导表达,同时以pYES2．0转化子为对照。气相色谱（Gc）分析表明,重组转化子亚油酸的含量占酵母总脂肪酸的1．54％,而对照未检测到亚油酸。表明FAD2基因具有编码△12脂肪酸脱氢酶的功能。为探索转译起始密码子周边序列的改变对FAD2基因表达产生的影响,将该基因的起始密码子上游序列进行修改,构建重组表达载体pYE／FAD2—1,转化酿酒酵母进行表达。GC分析表明,pYE／FAD2—1转化子的亚油酸含量占总脂肪酸含量的8．81％,是对照pYE／FAD2转化子的近5倍。     

Fluidization of membrane lipids enhances the tolerance of Saccharomyces cerevisiae to freezing and salt stress

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Delta(9) position. We expressed two sunflower (Helianthus annuus) oleate Delta(12) desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Delta(9,12), the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15 degrees C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp(+) or Trp(-) strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30 degrees C or 15 degrees C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.     

Identification,characterization and field testing of Brassica napus mutants producing high‐oleic oils

Producing healthy, high‐oleic oils and eliminating trans‐fatty acids from foods are two goals that can be addressed by reducing activity of the oleate desaturase, FAD2, in oilseeds. However, it is essential to understand the consequences of reducing FAD2 activity on the metabolism, cell biology and physiology of oilseed crop plants. Here, we translate knowledge from studies of fad2 mutants in Arabidopsis (Arabidopsis thaliana) to investigate the limits of non‐GMO approaches to maximize oleic acid in the seed oil of canola (Brassica napus), a species that expresses three active FAD2 isozymes. A series of hypomorphic and null mutations in the FAD2.A5 isoform were characterized in yeast (Saccharomyes cerevisiae). Then, four of these were combined with null mutations in the other two isozymes, FAD2.C5 and FAD2.C1. The resulting mutant lines contained 71–87% oleic acid in their seed oil, compared with 62% in wild‐type controls. All the mutant lines grew well in a greenhouse, but in field experiments we observed a clear demarcation in plant performance. Mutant lines containing less than 80% oleate in the seed oil were indistinguishable from wild‐type controls in growth parameters and seed oil content. By contrast, lines with more than 80% oleate in the seed oil had significantly lower seedling establishment and vigor, delayed flowering and reduced plant height at maturity. These lines also had 7–11% reductions in seed oil content. Our results extend understanding of the B. napusFAD2 isozymes and define the practical limit to increasing oil oleate content in this crop species.