Structural characterization of Linum usitatissimum hydroxynitrile lyase: A new cyanohydrin decomposition mechanism involving a cyano-zinc complex.

Hydroxynitrile lyase from Linum usitatissimum (LuHNL) is an enzyme involved in the catabolism of cyanogenic glycosides to release hydrogen cyanide upon tissue damage. This enzyme strictly conserves the substrate- and NAD(H)-binding domains of Zn2+-containing alcohol dehydrogenase (ADH); however, there is no evidence suggesting that LuHNL possesses ADH activity. Herein, we determined the ligand-free 3D structure of LuHNL and its complex with acetone cyanohydrin and (R)-2-butanone cyanohydrin using X-ray crystallography. These structures reveal that an A-form NAD+ is tightly but not covalently bound to each subunit of LuHNL. The restricted movement of the NAD+ molecule is due to the "sandwich structure" on the adenine moiety of NAD+. Moreover, the structures and mutagenesis analysis reveal a novel reaction mechanism for cyanohydrin decomposition involving the cyano-zinc complex and hydrogen-bonded interaction of the hydroxyl group of cyanohydrin with Glu323/Thr65 and H2O/Lys162 of LuHNL. The deprotonated Lys162 and protonated Glu323 residues are presumably stabilized by a partially desolvated microenvironment. In summary, the substrate binding geometry of LuHNL provides insights into the differences in activities of LuHNL and ADH, and identifying this novel reaction mechanism is an important contribution to the study of hydroxynitrile lyases.

Verification of the versatility of the in vitro enzymatic reaction giving (+)-cis-12-Oxo-phytodienoic acid.

Jasmonic acid (JA) is a plant hormone involved in the defense response against insects and fungi. JA is synthesized from α-linolenic acid (LA) by the octadecanoid pathway in plants. 12-oxo-Phytodienoic acid (OPDA) is one of the biosynthetic intermediates in this pathway. The reported stereo selective total synthesis of cis-(+)-OPDA is not very efficient due to the many steps involved in the reaction as well as the use of water sensitive reactions. Therefore, we developed an enzymatic method for the synthesis of OPDA using acetone powder of flax seed and allene oxide cyclase (PpAOC2) from Physcomitrella patens. From this method, natural cis-(+)-OPDA can be synthesized in the high yield of approximately 40%. In this study, we investigated the substrate specificity of the enzymatic synthesis of other OPDA analogs with successions to afford OPDA amino acid conjugates, dinor-OPDA (dn-OPDA), and OPDA monoglyceride, and it was suggested that the biosynthetic pathway of arabidopsides could occur via MGDG.

A transferase interactome that may facilitate channeling of polyunsaturated fatty acid moieties from phosphatidylcholine to triacylglycerol.

Polyunsaturated fatty acids (PUFAs) such as α-linolenic acid (ALA, 18:3Δ9cis,12cis,15cis ) have high nutritional and industrial values. In oilseed crops, PUFAs are synthesized on phosphatidylcholine (PC) and accumulated in triacylglycerol (TAG). Therefore, exploring the mechanisms that route PC-derived PUFA to TAG is essential for understanding and improving PUFA production. The seed oil of flax (Linum usitatissimum) is enriched in ALA, and this plant has many lipid biosynthetic enzymes that prefer ALA-containing substrates. In this study, using membrane yeast two-hybrid and bimolecular fluorescence complementation assays, we probed recombinant flax transferase enzymes, previously shown to contribute to PUFA enrichment of TAG, for physical interactions with each other under in vivo conditions. We found that diacylglycerol acyltransferases, which catalyze the final reaction in acyl-CoA-dependent TAG biosynthesis, interact with the acyl-editing enzymes phosphatidylcholine: diacylglycerol cholinephosphotransferase, and lysophosphatidylcholine acyltransferase. Physical interactions among the acyl-editing enzymes were also identified. These findings reveal the presence of an assembly of interacting transferases that may facilitate the channeling of PUFA from PC to TAG in flax and possibly also in other oleaginous plants that produce seeds enriched in PC-modified fatty acids.

Genetic diversity of SAD and FAD genes responsible for the fatty acid composition in flax cultivars and lines.

BACKGROUND: Flax (Linum usitatissimum L.) is grown for fiber and seed in many countries. Flax cultivars differ in the oil composition and, depending on the ratio of fatty acids, are used in pharmaceutical, food, or paint industries. It is known that genes of SAD (stearoyl-ACP desaturase) and FAD (fatty acid desaturase) families play a key role in the synthesis of fatty acids, and some alleles of these genes are associated with a certain composition of flax oil. However, data on genetic polymorphism of these genes are still insufficient. RESULTS: On the basis of the collection of the Institute for Flax (Torzhok, Russia), we formed a representative set of 84 cultivars and lines reflecting the diversity of fatty acid composition of flax oil. An approach for the determination of full-length sequences of SAD1, SAD2, FAD2A, FAD2B, FAD3A, and FAD3B genes using the Illumina platform was developed and deep sequencing of the 6 genes in 84 flax samples was performed on MiSeq. The obtained high coverage (about 400x on average) enabled accurate assessment of polymorphisms in SAD1, SAD2, FAD2A, FAD2B, FAD3A, and FAD3B genes and evaluation of cultivar/line heterogeneity. The highest level of genetic diversity was observed for FAD3A and FAD3B genes - 91 and 62 polymorphisms respectively. Correlation analysis revealed associations between particular variants in SAD and FAD genes and predominantly those fatty acids whose conversion they catalyze: SAD - stearic and oleic acids, FAD2 - oleic and linoleic acids, FAD3 - linoleic and linolenic acids. All except one low-linolenic flax cultivars/lines contained both the substitution of tryptophan to stop codon in the FAD3A gene and histidine to tyrosine substitution in the FAD3B gene, while samples with only one of these polymorphisms had medium content of linolenic acid and cultivars/lines without them were high-linolenic. CONCLUSIONS: Genetic polymorphism of SAD and FAD genes was evaluated in the collection of flax cultivars and lines with diverse oil composition, and associations between particular polymorphisms and the ratio of fatty acids were revealed. The achieved results are the basis for the development of marker-assisted selection and DNA-based certification of flax cultivars.

Flaxseed Cysteine Protease Exhibits Strong Anticoagulant, Antiplatelet, and Clot-Dissolving Properties.

In this study, we purified and characterized flaxseed cysteine protease (FSCP) with strong anticoagulant, antiplatelet, and clot-dissolving properties. The enzyme was purified to homogeneity by a combination of gel permeation and ion-exchange column chromatography techniques. The purity of the enzyme was evaluated by SDS-PAGE, RP-HPLC, and MALDI-TOF. FSCP was observed as a single band of approximately 160 kDa in SDS-PAGE under reducing and non-reducing conditions. The exact molecular mass of FSCP was found to be 168 kDa by MALDI-TOF spectrometry. The CD spectra of FSCP revealed the presence of 25.6% helices, 25.8% turns, and 48% random coils with no beta-sheet structures. FSCP hydrolyzed both casein and gelatin with a specific activity of 3.5 and 4.2 unit/mg min respectively. The proteolytic activity of FSCP was completely abolished by iodoacetic acid (IAA), suggesting FSCP is a cysteine protease. The pH optimum for the proteolytic activity of FSCP was pH 6.0; the temperature optimum was 30°C. FSCP exhibited strong anticoagulant effect in both platelet-rich plasma (PRP) and platelet-poor plasma (PPP) by extending the clotting time from 222 to 1100 s and from 256 to 1210 s, respectively. FSCP degraded human fibrinogen and fibrin clots. The products of fibrinogen degradation by thrombin and FSCP were different. Furthermore, FSCP inhibited aggregation of washed platelets triggered by ADP, epinephrine, thrombin, collagen, arachidonic acid, and platelet activating factor (PAF). FSCP was found to be nontoxic as it did not damage the membrane of red blood cells (RBCs) and did not induce hemorrhage and edema in experimental mice.

Effect of mcl-PHA synthesis in flax on plant mechanical properties and cell wall composition.

The high demand for new biomaterials makes synthesis of polyhydroxyalkanoates (PHA) in plants an interesting and desirable achievement. Production of polymers in plants is an example of application of biotechnology for improving the properties of plants, e.g. industrial properties, but it can also provide knowledge about plant physiology and metabolism. The subject of the present study was an industrially important plant: flax, Linum usitatissimum L., of a fibre cultivar (cv Nike). In the study the gene encoding PHA synthase from Pseudomonas aeruginosa, fused to a peroxisomal targeting signal, was expressed in flax plants with the aim of modifying the mechanical properties of plants. Medium-chain-length (mcl) hydroxy acids in flax plants from tissue cultures were detected by GC-FID and FTIR method. The introduced changes did not affect fatty acid content and composition in generated flax plants. Since mcl-PHA are known as elastomers, the mechanical properties of created plants were examined. Modified plants showed increases in the values of all measured parameters (except strain at break evaluated for one modified line). The largest increase was noted for tensile stiffness, which was 2- to 3-fold higher than in wild-type plants. The values estimated for another parameter, Young's modulus, was almost at the same level in generated flax plants, and they were about 2.7-fold higher when compared to unmodified plants. The created plants also exhibited up to about 2.4-fold higher tensile strength. The observed changes were accompanied by alterations in the expression of selected genes, related to cell wall metabolism in line with the highest expression of phaC1 gene. Biochemical data were confirmed by spectroscopic methods, which also revealed that crystallinity index values of cellulose in modified flax plants were increased in comparison to wild-type flax plants and correlated with biomechanical properties of plants.

Flax rhamnogalacturonan lyases: phylogeny, differential expression and modeling of protein structure.

Rhamnogalacturonan lyases (RGLs; EC 4.2.2.23) degrade the rhamnogalacturonan I (RG-I) backbone of pectins present in the plant cell wall. These enzymes belong to polysaccharide lyase family 4, members of which are mainly from plants and plant pathogens. RGLs are investigated, as a rule, as pathogen 'weapons' for plant cell wall degradation and subsequent infection. Despite the presence of genes annotated as RGLs in plant genomes and the presence of substrates for enzyme activity in plant cells, evidence supporting the involvement of this enzyme in certain processes is limited. The differential expression of some RGL genes in flax (Linum usitatissimum L.) tissues, revealed in our previous work, prompted us to carry out a total revision (phylogenetic analysis, analysis of expression and protein structure modeling) of all the sequences of flax predicted as coding for RGLs. Comparison of the expressions of LusRGL in various tissues of flax stem revealed that LusRGLs belong to distinct phylogenetic clades, which correspond to two co-expression groups. One of these groups comprised LusRGL6-A and LusRGL6-B genes and was specifically upregulated in flax fibers during deposition of the tertiary cell wall, which has complex RG-I as a key noncellulosic component. The results of homology modeling and docking demonstrated that the topology of the LusRGL6-A catalytic site allowed binding to the RG-I ligand. These findings lead us to suggest the presence of RGL activity in planta and the involvement of special isoforms of RGLs in the modification of RG-I of the tertiary cell wall in plant fibers.

UGT74S1 is the key player in controlling secoisolariciresinol diglucoside (SDG) formation in flax.

BACKGROUND: Flax lignan, commonly known as secoisolariciresinol (SECO) diglucoside (SDG), has recently been reported with health-promoting activities, including its positive impact in metabolic diseases. However, not much was reported on the biosynthesis of SDG and its monoglucoside (SMG) until lately. Flax UGT74S1 was recently reported to sequentially glucosylate SECO into SMG and SDG in vitro. However, whether this gene is the only UGT achieving SECO glucosylation in flax was not known. RESULTS: Flax genome-wide mining for UGTs was performed. Phylogenetic and gene duplication analyses, heterologous gene expression and enzyme assays were conducted to identify family members closely related to UGT74S1 and to establish their roles in SECO glucosylation. A total of 299 different UGTs were identified, of which 241 (81%) were duplicated. Flax UGTs diverged 2.4-153.6 MYA and 71% were found to be under purifying selection pressure. UGT74S1, a single copy gene located on chromosome 7, displayed no evidence of duplication and was deemed to be under positive selection pressure. The phylogenetic analysis identified four main clusters where cluster 4, which included UGT74S1, was the most diverse. The duplicated UGT74S4 and UGT74S3, located on chromosomes 8 and 14, respectively, were the most closely related to UGT74S1 and were differentially expressed in different tissues. Heterologous expression levels of UGT74S1, UGT74S4 and UGT74S3 proteins were similar but UGT74S4 and UGT74S3 glucosylation activity towards SECO was seven fold less than UGT74S1. In addition, they both failed to produce SDG, suggesting neofunctionalization following their divergence from UGT74S1. CONCLUSIONS: We showed that UGT74S1 is closely related to two duplicated genes, UGT74S4 and UGT74S3 which, unlike UGT74S1, failed to glucosylate SMG into SDG. The study suggests that UGT74S1 may be the key player in controlling SECO glucosylation into SDG in flax although its closely related genes may also contribute to a minor extent in supplying the SMG precursor to UGT74S1.

Spatial regulation of monolignol biosynthesis and laccase genes control developmental and stress-related lignin in flax.

BACKGROUND: Bast fibres are characterized by very thick secondary cell walls containing high amounts of cellulose and low lignin contents in contrast to the heavily lignified cell walls typically found in the xylem tissues. To improve the quality of the fiber-based products in the future, a thorough understanding of the main cell wall polymer biosynthetic pathways is required. In this study we have carried out a characterization of the genes involved in lignin biosynthesis in flax along with some of their regulation mechanisms. RESULTS: We have first identified the members of the phenylpropanoid gene families through a combination of in silico approaches. The more specific lignin genes were further characterized by high throughput transcriptomic approaches in different organs and physiological conditions and their cell/tissue expression was localized in the stems, roots and leaves. Laccases play an important role in the polymerization of monolignols. This multigenic family was determined and a miRNA was identified to play a role in the posttranscriptional regulation by cleaving the transcripts of some specific genes shown to be expressed in lignified tissues. In situ hybridization also showed that the miRNA precursor was expressed in the young xylem cells located near the vascular cambium. The results obtained in this work also allowed us to determine that most of the genes involved in lignin biosynthesis are included in a unique co-expression cluster and that MYB transcription factors are potentially good candidates for regulating these genes. CONCLUSIONS: Target engineering of cell walls to improve plant product quality requires good knowledge of the genes responsible for the production of the main polymers. For bast fiber plants such as flax, it is important to target the correct genes from the beginning since the difficulty to produce transgenic material does not make possible to test a large number of genes. Our work determined which of these genes could be potentially modified and showed that it was possible to target different regulatory pathways to modify lignification.

Functional characterization of the pinoresinol-lariciresinol reductase-2 gene reveals its roles in yatein biosynthesis and flax defense response.

MAIN CONCLUSION: This study provides new insights into the biosynthesis regulation and in planta function of the lignan yatein in flax leaves. Pinoresinol-lariciresinol reductases (PLR) catalyze the conversion of pinoresinol into secoisolariciresinol (SECO) in lignan biosynthesis. Several lignans are accumulated in high concentrations, such as SECO accumulated as secoisolariciresinol diglucoside (SDG) in seeds and yatein in aerial parts, in the flax plant (Linum usitatissimum L.) from which two PLR enzymes of opposite enantioselectivity have been isolated. While LuPLR1 catalyzes the biosynthesis of (+)-SECO leading to (+)-SDG in seeds, the role(s) of the second PLR (LuPLR2) is not completely elucidated. This study provides new insights into the in planta regulation and function of the lignan yatein in flax leaves: its biosynthesis relies on a different PLR with opposite stereospecificity but also on a distinct expression regulation. RNAi technology provided evidence for the in vivo involvement of the LuPLR2 gene in the biosynthesis of (-)-yatein accumulated in flax leaves. LuPLR2 expression in different tissues and in response to stress was studied by RT-qPCR and promoter-reporter transgenesis showing that the spatio-temporal expression of the LuPLR2 gene in leaves perfectly matches the (-)-yatein accumulation and that LuPLR2 expression and yatein production are increased by methyl jasmonate and wounding. A promoter deletion approach yielded putative regulatory elements. This expression pattern in relation to a possible role for this lignan in flax defense is discussed.

Ectopic lignification in the flax lignified bast fiber1 mutant stem is associated with tissue-specific modifications in gene expression and cell wall composition.

Histochemical screening of a flax ethyl methanesulfonate population led to the identification of 93 independent M2 mutant families showing ectopic lignification in the secondary cell wall of stem bast fibers. We named this core collection the Linum usitatissimum (flax) lbf mutants for lignified bast fibers and believe that this population represents a novel biological resource for investigating how bast fiber plants regulate lignin biosynthesis. As a proof of concept, we characterized the lbf1 mutant and showed that the lignin content increased by 350% in outer stem tissues containing bast fibers but was unchanged in inner stem tissues containing xylem. Chemical and NMR analyses indicated that bast fiber ectopic lignin was highly condensed and rich in G-units. Liquid chromatography-mass spectrometry profiling showed large modifications in the oligolignol pool of lbf1 inner- and outer-stem tissues that could be related to ectopic lignification. Immunological and chemical analyses revealed that lbf1 mutants also showed changes to other cell wall polymers. Whole-genome transcriptomics suggested that ectopic lignification of flax bast fibers could be caused by increased transcript accumulation of (1) the cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, and caffeic acid O-methyltransferase monolignol biosynthesis genes, (2) several lignin-associated peroxidase genes, and (3) genes coding for respiratory burst oxidase homolog NADPH-oxidases necessary to increase H2O2 supply.

IMPACT OF FABRICS FROM TRANSGENIC FLAX PLANT ON HUMAN DERMAL FIBROBLASTS IN VITRO PROLIFERATION.

Previously it was documented that transgenic flax plants, which contained an increased level of polyphenolic compounds, significantly improved healing of skin wounds lesions. In order to recognize mechanisms of beneficial action of transgenic flax fabrics on wound healing, in the present study the impact of flax fabric pieces/cuts from three types of transgenic flax on normal human dermal fibroblasts primary culture (NHDF) was investigated. NHDF cell cultures were exposed for 48 h to specific area of flax fabric cuts, made from M50, B 14 and M50+B14 (intertwined fibers of M and B), or parallely, extracts from fibers of the tested flax materials to cell culture medium. Cultures were inspected for cell viability, proliferation, cell cycle changes and for their resistance to oxidative stress (consecutive addition of H2,O2, to harvested cell cultures). None of the tested flax fabrics were cytotoxic to fibroblast cultures and also did not increase significantly a frequency of apoptotic cells in cultures. In the comet assay, the tested flax fabrics revealed significant protective effect on DNA damage ciused by addition of H202 to the cultures at the end of incubation time. Fabrics from transgenic flax significantly enhanced fibroblasts proliferation in vitro estimated with the SRB test. Flow cytometric analysis revealed higher frequency of cells in the S phase, in the presence of transgenic flax fabrics. Fabrics from B14 and M50+B14 flax are the most potent activators of NHDF cells in applied in vityo tests, hence they could be recommended for elaboration of new type bandage, able to improve skin wound healing.

In Vivo and in Vitro Evidence for Biochemical Coupling of Reactions Catalyzed by Lysophosphatidylcholine Acyltransferase and Diacylglycerol Acyltransferase.

Seed oils of flax (Linum usitatissimum L.) and many other plant species contain substantial amounts of polyunsaturated fatty acids (PUFAs). Phosphatidylcholine (PC) is the major site for PUFA synthesis. The exact mechanisms of how these PUFAs are channeled from PC into triacylglycerol (TAG) needs to be further explored. By using in vivo and in vitro approaches, we demonstrated that the PC deacylation reaction catalyzed by the reverse action of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) can transfer PUFAs on PC directly into the acyl-CoA pool, making these PUFAs available for the diacylglycerol acyltransferase (DGAT)-catalyzed reaction for TAG production. Two types of yeast mutants were generated for in vivo and in vitro experiments, respectively. Both mutants provide a null background with no endogenous TAG forming capacity and an extremely low LPCAT activity. In vivo experiments showed that co-expressing flax DGAT1-1 and LPCAT1 in the yeast quintuple mutant significantly increased 18-carbon PUFAs in TAG with a concomitant decrease of 18-carbon PUFAs in phospholipid. We further showed that after incubation of sn-2-[(14)C]acyl-PC, formation of [(14)C]TAG was only possible with yeast microsomes containing both LPCAT1 and DGAT1-1. Moreover, the specific activity of overall LPCAT1 and DGAT1-1 coupling process exhibited a preference for transferring (14)C-labeled linoleoyl or linolenoyl than oleoyl moieties from the sn-2 position of PC to TAG. Together, our data support the hypothesis of biochemical coupling of the LPCAT1-catalyzed reverse reaction with the DGAT1-1-catalyzed reaction for incorporating PUFAs into TAG. This process represents a potential route for enriching TAG in PUFA content during seed development in flax.

Sequence characterization and in silico structure prediction of fatty acid desaturases in linseed varieties with differential fatty acid composition.

BACKGROUND: Linseed is the richest agricultural source of α-linolenic acid (ALA), an ω-3 fatty acid (FA) that offers several nutritional benefits. In the present study, sequence characterization of six desaturase genes (SAD1, SAD2, FAD2, FAD2-2, FAD3A and FAD3B) and 3D structure prediction of their proteins from ten Indian linseed varieties differing in ALA content were performed to determine whether the nucleotide and amino acid (AA) sequence variants have any functional implications in differential accumulation of ALA or other FAs in linseed. RESULTS: The SAD and FAD2 genes exhibited few sequence variations among the ten varieties, forming only one or two protein isoforms. In contrast, the FAD3A and FAD3B genes showed more sequence variations and three or four protein isoforms. Interestingly, the two high-ALA varieties NL260 and Padmini had the same FAD3B nucleotide and protein isoforms, which differed from all other varieties. Surprisingly, no AA changes altered the 3D structures of the desaturase proteins. CONCLUSION: Several nucleotide and AA sequence variations in desaturase genes were observed; however, they did not alter the 3D structure of any desaturase protein and were not correlated with FA levels among the ten linseed varieties, which had different ALA contents. This suggests a complex regulatory process of biosynthesis of FAs in linseed. © 2016 Society of Chemical Industry.

Functional analyses of cellulose synthase genes in flax (Linum usitatissimum) by virus-induced gene silencing.

Flax (Linum usitatissimum) bast fibres are located in the stem cortex where they play an important role in mechanical support. They contain high amounts of cellulose and so are used for linen textiles and in the composite industry. In this study, we screened the annotated flax genome and identified 14 distinct cellulose synthase (CESA) genes using orthologous sequences previously identified. Transcriptomics of 'primary cell wall' and 'secondary cell wall' flax CESA genes showed that some were preferentially expressed in different organs and stem tissues providing clues as to their biological role(s) in planta. The development for the first time in flax of a virus-induced gene silencing (VIGS) approach was used to functionally evaluate the biological role of different CESA genes in stem tissues. Quantification of transcript accumulation showed that in many cases, silencing not only affected targeted CESA clades, but also had an impact on other CESA genes. Whatever the targeted clade, inactivation by VIGS affected plant growth. In contrast, only clade 1- and clade 6-targeted plants showed modifications in outer-stem tissue organization and secondary cell wall formation. In these plants, bast fibre number and structure were severely impacted, suggesting that the targeted genes may play an important role in the establishment of the fibre cell wall. Our results provide new fundamental information about cellulose biosynthesis in flax that should facilitate future plant improvement/engineering.

Impact of CAD-deficiency in flax on biogas production.

Global warming and the reduction in our fossil fuel reservoir have forced humanity to look for new means of energy production. Agricultural waste remains a large source for biofuel and bioenergy production. Flax shives are a waste product obtained during the processing of flax fibers. We investigated the possibility of using low-lignin flax shives for biogas production, specifically by assessing the impact of CAD deficiency on the biochemical and structural properties of shives. The study used genetically modified flax plants with a silenced CAD gene, which encodes the key enzyme for lignin synthesis. Reducing the lignin content modified cellulose crystallinity, improved flax shive fermentation and optimized biogas production. Chemical pretreatment of the shive biomass further increased biogas production efficiency.

Development of high oleic oil crop platform in flax through RNAi-mediated multiple FAD2 gene silencing.

KEY MESSAGE: Simultaneous gene silencing of both FAD2 genes in high linoleic acid flax leads to high level of oleic acid, which is stable across multiple generations. High oleic oil is one of the preferred traits in oil crop engineering due to its stability and multiple applications as an industrial feedstock. Flax possesses two isoforms of FAD2 enzymes that desaturate monounsaturated oleic acid to polyunsaturated linoleic acid. These two enzymes are encoded by two FAD2 genes. By simultaneous gene silencing both FAD2 genes in high linoleic acid flax, Linola, high level of oleic acid up to 80% was achieved in 69 silencing lines. The high oleic trait was stable across multiple generations with oleic acid reaching up to 77% in homozygote T3 progeny. The RNAi-mediated gene-silencing approach generated high oleic linseed oil, as well as a high oleic platform that can be exploited for further fatty acid engineering.

Isolation and characterization of two geometric allene oxide isomers synthesized from 9S-hydroperoxylinoleic acid by cytochrome P450 CYP74C3: stereochemical assignment of natural fatty acid allene oxides.

Specialized cytochromes P450 or catalase-related hemoproteins transform fatty acid hydroperoxides to allene oxides, highly reactive epoxides leading to cyclopentenones and other products. The stereochemistry of the natural allene oxides is incompletely defined, as are the structural features required for their cyclization. We investigated the transformation of 9S-hydroperoxylinoleic acid with the allene oxide synthase CYP74C3, a reported reaction that unexpectedly produces an allene oxide-derived cyclopentenone. Using biphasic reaction conditions at 0 °C, we isolated the initial products and separated two allene oxide isomers by HPLC at -15 °C. One matched previously described allene oxides in its UV spectrum (λmax 236 nm) and NMR spectrum (defining a 9,10-epoxy-octadec-10,12Z-dienoate). The second was a novel stereoisomer (UV λmax 239 nm) with distinctive NMR chemical shifts. Comparison of NOE interactions of the epoxy proton at C9 in the two allene oxides (and the equivalent NOE experiment in 12,13-epoxy allene oxides) allowed assignment at the isomeric C10 epoxy-ene carbon as Z in the new isomer and the E configuration in all previously characterized allene oxides. The novel 10Z isomer spontaneously formed a cis-cyclopentenone at room temperature in hexane. These results explain the origin of the cyclopentenone, provide insights into the mechanisms of allene oxide cyclization, and define the double bond geometry in naturally occurring allene oxides.

Identification of a pair of phospholipid:diacylglycerol acyltransferases from developing flax (Linum usitatissimum L.) seed catalyzing the selective production of trilinolenin.

The oil from flax (Linum usitatissimum L.) has high amounts of α-linolenic acid (ALA; 18:3(cis)(Δ9,12,15)) and is one of the richest sources of omega-3 polyunsaturated fatty acids (ω-3-PUFAs). To produce ∼57% ALA in triacylglycerol (TAG), it is likely that flax contains enzymes that can efficiently transfer ALA to TAG. To test this hypothesis, we conducted a systematic characterization of TAG-synthesizing enzymes from flax. We identified several genes encoding acyl-CoA:diacylglycerol acyltransferases (DGATs) and phospholipid:diacylglycerol acyltransferases (PDATs) from the flax genome database. Due to recent genome duplication, duplicated gene pairs have been identified for all genes except DGAT2-2. Analysis of gene expression indicated that two DGAT1, two DGAT2, and four PDAT genes were preferentially expressed in flax embryos. Yeast functional analysis showed that DGAT1, DGAT2, and two PDAT enzymes restored TAG synthesis when produced recombinantly in yeast H1246 strain. The activity of particular PDAT enzymes (LuPDAT1 and LuPDAT2) was stimulated by the presence of ALA. Further seed-specific expression of flax genes in Arabidopsis thaliana indicated that DGAT1, PDAT1, and PDAT2 had significant effects on seed oil phenotype. Overall, this study indicated the existence of unique PDAT enzymes from flax that are able to preferentially catalyze the synthesis of TAG containing ALA acyl moieties. The identified LuPDATs may have practical applications for increasing the accumulation of ALA and other polyunsaturated fatty acids in oilseeds for food and industrial applications.

Manipulating cinnamyl alcohol dehydrogenase (CAD) expression in flax affects fibre composition and properties.

BACKGROUND: In recent decades cultivation of flax and its application have dramatically decreased. One of the reasons for this is unpredictable quality and properties of flax fibre, because they depend on environmental factors, retting duration and growing conditions. These factors have contribution to the fibre composition, which consists of cellulose, hemicelluloses, lignin and pectin. By far, it is largely established that in flax, lignin reduces an accessibility of enzymes either to pectin, hemicelluloses or cellulose (during retting or in biofuel synthesis and paper production).Therefore, in this study we evaluated composition and properties of flax fibre from plants with silenced CAD (cinnamyl alcohol dehydrogenase) gene, which is key in the lignin biosynthesis. There is evidence that CAD is a useful tool to improve lignin digestibility and/or to lower the lignin levels in plants. RESULTS: Two studied lines responded differentially to the introduced modification due to the efficiency of the CAD silencing. Phylogenetic analysis revealed that flax CAD belongs to the "bona-fide" CAD family. CAD down-regulation had an effect in the reduced lignin amount in the flax fibre cell wall and as FT-IR results suggests, disturbed lignin composition and structure. Moreover introduced modification activated a compensatory mechanism which was manifested in the accumulation of cellulose and/or pectin. These changes had putative correlation with observed improved fiber's tensile strength. Moreover, CAD down-regulation did not disturb at all or has only slight effect on flax plants' development in vivo, however, the resistance against flax major pathogen Fusarium oxysporum decreased slightly. The modification positively affected fibre possessing; it resulted in more uniform retting. CONCLUSION: The major finding of our paper is that the modification targeted directly to block lignin synthesis caused not only reduced lignin level in fibre, but also affected amount and organization of cellulose and pectin. However, to conclude that all observed changes are trustworthy and correlated exclusively to CAD repression, further analysis of the modified plants genome is necessary. Secondly, this is one of the first studies on the crop from the low-lignin plants from the field trail which demonstrates that such plants could be successfully cultivated in a field.