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Waterer D.,University of Saskatchewan | Benning N.T.,University of Saskatchewan | Wu G.,Bioriginal Food and Science Corporation | Luo X.,Agrisoma Biosciences Inc. | And 4 more authors.
Molecular Breeding | Year: 2010

Abiotic stresses such as drought and extremes of temperature commonly reduce both yield and quality of potato. This study investigated the potential to use gene transfer technology to enhance the tolerance of potato to commonly encountered abiotic stresses. Agrobacterium mediated transformation was used to create lines of potato (cv. Desiree) that overexpressed either a wheat mitochondrial Mn superoxide dismutase (SOD3:1), dehydrin 4 (DHN 4) isolated from barley, a cold-inducible transcriptional factor DREB/CBF1 isolated from canola or ROB5, a stress inducible gene isolated from bromegrass that encodes for a heat stable LEA group 3-like protein. The transgenes were under the control of either a constitutive 35S promoter or a stress-induced Arabidopsis COR78 promoter. Yield potential of the transformed lines was evaluated under drought stress conditions in a greenhouse trial and under non-irrigated conditions in field trials conducted over 4 years in Saskatoon, Saskatchewan. In the years when the field trials experienced significant drought stress (2001, 2003 and 2006) many of the transformed lines produced higher yields than the control. However, under relatively cooler, wetter conditions (2005 cropping season) yields of most transformed lines were equivalent or inferior to the non-transformed parental line. Under non-stressed conditions, transformations utilizing the stress-induced COR78 promoter were higher yielding than transformations based on the constitutive 35S promoter. Combining the ROB5, DHN or SOD3.1 transgenes with the COR78 promoter all showed significant potential to enhance yields under moisture stress. All of the transgenes appeared to enhance the heat stress tolerance (44°C) of whole plants or excised leaves, with lines transformed with SOD3.1 showing the greatest effect. In low temperature stress trials conducted under controlled environment conditions and in the field, lines over-expressing SOD3:1 showed an enhanced capacity to grow at sub-optimal temperatures (10°C), while lines transformed with SOD3.1 or ROB5 had greater tolerance of freezing temperatures than the parental line. These results are encouraging as even a small degree of enhancement of stress tolerance has the potential to produce significant economic benefits in high value/stress sensitive crops such as potato. © Springer Science+Business Media B.V. 2009.


Cheng B.,Bioriginal Food and Science Corporation | Cheng B.,Agriculture and Agri Food Canada | Wu G.,Bioriginal Food and Science Corporation | Vrinten P.,Bioriginal Food and Science Corporation | And 3 more authors.
Transgenic Research | Year: 2010

Eicosapentaenoic acid (EPA, 20:5n-3) plays an important role in many aspects of human health. In our efforts towards producing high levels of EPA in plants, we investigated the effects of different host species, genes and promoters on EPA biosynthesis. Zero-erucic acid Brassica carinata appeared to be an outstanding host species for EPA production, with EPA levels in transgenic seed of this line reaching up to 25%. Two novel genes, an 18-carbon ω3 desaturase (CpDesX) from Claviceps purpurea and a 20-carbon ω3 desaturase (Pir-ω3) from Pythium irregulare, proved to be very effective in increasing EPA levels in high-erucic acid B. carinata. The conlinin1 promoter from flax functioned reasonably well in B. carinata, and can serve as an alternative to the napin promoter from B. napus. In summary, the judicious selection of host species and promoters, together with the inclusion of genes that enhance the basic very long chain polyunsaturated fatty acid biosynthetic pathway, can greatly influence the production of EPA in plants. © Springer Science+Business Media B.V. 2009.


Vrinten P.,Bioriginal Food and Science Corporation | Mavraganis I.,Bioriginal Food and Science Corporation | Mavraganis I.,National Research Council Canada | Qiu X.,University of Saskatchewan | Senger T.,BASF
Lipids | Year: 2013

Sphaeroforma arctica is a unique, recently discovered marine protist belonging to a group falling close to the yeast/animal border. S. arctica is found in cold environments, and accordingly has a fatty acid composition containing a high proportion of very long chain polyunsaturated fatty acids, including the ω3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA). Two elongases and five desaturases, representing the complete set of enzymes necessary for the synthesis of DHA from oleic acid, were isolated from this species and characterized in yeast. One elongase showed high conversion rates on a wide range of 18 and 20 carbon substrates, and was capable of sequential elongation reactions. The second elongase had a strong preference for the 20-carbon fatty acids EPA and arachidonic acid, with over 80 % of EPA converted to docosapentaenoic acid (DPA) in the heterologous yeast host. The isolation of a Δ8-desaturase, along with the detection of eicosadienoic acid in S. arctica cultures indicated that this species uses the alternate Δ8-pathway for the synthesis of long-chain polyunsaturated fatty acids. S. arctica also carried a Δ4-desaturase that proved to be very active in the production of DHA from DPA. Finally, a long chain acyl-CoA synthetase from S. arctica improved DHA uptake in the heterologous yeast host and led to an improvement in desaturation and elongation efficiencies. © 2012 AOCS.


Vrinten P.L.,Bioriginal Food and Science Corporation | Hoffman T.,Bioriginal Food and Science Corporation | Bauer J.,BASF | Qiu X.,Bioriginal Food and Science Corporation | Qiu X.,University of Saskatchewan
Biochemistry | Year: 2010

We describe a condensing enzyme from Pythium irregulare (PirELO) that shows highest activity on the 18-carbon, δ-6 desaturated fatty acids, stearidonic acid and γ-linolenic acid. However, this enzyme is also capable of elongating a number of other fatty acids including the 20-carbon, δ-5 desaturated fatty acid eicosapentaenoic acid. Surprisingly, a Phytophthora infestans condensing enzyme (PinELO) with very high homology to PirELO did not show activity with 20-carbon fatty acids. A series of chimeric proteins for these two enzymes were constructed to investigate the influence of different regions on substrate and product length. The substitution of a region from near the center of PirELO into PinELO resulted in an enzyme having EPA-elongating activity similar to that of PirELO. Only eight amino acids differed between the two proteins in this region; however, substitution of the same region from PinELO into PirELO produced a protein which was almost inactive. The addition of a small region from near the N-terminus of PinELO was sufficient to restore activity with GLA, indicating that amino acids from these two regions interact to determine protein structure or function. Predicted topology models for PirELO and PinELO placed the two regions described here near the luminal?proximal ends of the first and fourth/fifth transmembrane helixes, at the opposite end of the condensing enzyme from four conserved regions thought to form a catalytic ring. Thus, protein characteristics determined by specific luminal?proximal regions of fatty acid condensing enzymes have a major influence on substrate specificity and final product length. © 2010 American Chemical Society.


Lim Z.L.,Bioriginal Food and Science Corporation | Senger T.,BASF | Vrinten P.,Bioriginal Food and Science Corporation
Lipids | Year: 2014

Although ω3- and ω6- desaturases have been well studied in terms of substrate preference and regiospecificity, relatively little is known about the membrane-bound, "front-end" long chain fatty acid desaturases, such as δ4, Δ5 or Δ6 desaturases. The first vertebrate δ4 desaturase was recently identified in the marine teleost fish Siganus canaliculatus (S. canaliculatus), which also possesses a bifunctional Δ5/6 desaturase. These two long chain polyunsaturated fatty acid desaturases are very different in terms of regiospecificity and substrate chain-length, but share an unusually high degree of amino acid identity (83 %). We took advantage of this similarity by constructing a series of chimeric enzymes, replacing regions of one enzyme with the corresponding sequence of the other. Heterologous expression of the chimeric series of enzymes in yeast indicated that the substitution of a four amino acid region was sufficient to convert a δ4 desaturase to an enzyme with δ6 desaturase activity, and convert a δ5/6 desaturase to an enzyme with a low level of δ4 desaturase activity. In addition, enzymes having both δ4 and δ6 desaturase activities were produced by single or double amino acid substitutions within this four-amino acid region. © 2014 AOCS.


Saito M.,Japan National Agriculture and Food Research Organization | Saito M.,Nippon Flour Mills Co. | Vrinten P.,Bioriginal Food and Science Corporation | Nakamura T.,Japan National Agriculture and Food Research Organization
Japan Agricultural Research Quarterly | Year: 2010

Approximately 70% of wheat endosperm consists of starch, and variations in the quality and quantity of starch affect the processing characteristics of wheat flour. Amylose content in particular has a major effect on Asian noodle quality, and selection of wheat lines with slightly lower amylose levels is an important goal in Japanese wheat breeding programs. Accurately measuring amylose content by direct methods such as colorimetric assays was found to be problematic, suggesting there was a need for a more efficient and accurate method of screening for reduced amylose content. Therefore, we characterized mutations in the wheat waxy genes, which control amylose synthesis, and developed DNA markers for the identification of null waxy alleles. In this review, we describe the development of these markers and outline their utility for wheat breeding programs.


Ray H.,Bioriginal Food and Science Corporation | Bett K.,University of Saskatchewan | Tar'an B.,University of Saskatchewan | Vandenberg A.,University of Saskatchewan | And 2 more authors.
Crop Science | Year: 2014

The mineral content of pulses grown in Saskatchewan, Canada, was examined for magnesium, potassium, iron, zinc, manganese, copper, selenium, and in some cases nickel and calcium. Eight to 18 cultivars of each of field pea (Pisum sativum), common bean (Phaseolus vulgaris), chickpea (Cicer arietinum), and lentil (Lens culinaris) were grown at several locations in southern Saskatchewan in 2005 and 2006 in randomized complete block designs with three replicates. Mineral content was examined by atomic absorption spectrometry. The pulses were found to contain significant proportions of the recommended daily allowance (RDA) for all the tested minerals except calcium. In many cases a 100 g (dry weight) portion of the crop provided over 50% of the RDA. For selenium, pulses grown in some locations provided 100% of the RDA. The effect of location was highly significant in most instances, while that of year and cultivar were generally less so. Pairwise differences among cultivars were examined by Tukey's test. Where possible, crops grown side by side were compared. © Crop Science Society of America.


Mavraganis I.,University of Saskatchewan | Meesapyodsuk D.,University of Saskatchewan | Vrinten P.,Bioriginal Food and Science Corporation | Smith M.,National Research Council Canada | Qiu X.,University of Saskatchewan
Applied and Environmental Microbiology | Year: 2010

Claviceps purpurea, the fungal pathogen that causes the cereal disease ergot, produces glycerides that contain high levels of ricinoleic acid [(R)-12-hydroxyoctadec-cis-9-enoic acid] in its sclerotia. Recently, a fatty acid hydroxylase (C. purpurea FAH [CpFAH]) involved in the biosynthesis of ricinoleic acid was identified from this fungus (D. Meesapyodsuk and X. Qiu, Plant Physiol. 147:1325-1333, 2008). Here, we describe the cloning and biochemical characterization of a C. purpurea type II diacylglycerol acyltransferase (CpDGAT2) involved in the assembly of ricinoleic acid into triglycerides. The CpDGAT2 gene was cloned by degenerate RT-PCR (reverse transcription-PCR). The expression of this gene restored the in vivo synthesis of triacylglycerol (TAG) in the quadruple mutant strain Saccharomyces cerevisiae H1246, in which all four TAG biosynthesis genes (DGA1, LR01, ARE1, and ARE2) are disrupted. In vitro enzymatic assays using microsomal preparations from the transformed yeast strain indicated that CpDGAT2 prefers ricinoleic acid as an acyl donor over linoleic acid, oleic acid, or linolenic acid, and it prefers 1,2-dioleoyl-sn-glycerol over 1,2-dipalmitoyl-sn-glycerol as an acyl acceptor. The coexpression of CpFAH with CpDGAT2 in yeast resulted in an increased accumulation of ricinoleic acid compared to the coexpression of CpFAH with the native yeast DGAT2 (S. cerevisiae DGA1 [ScDGA1]) or the expression of CpFAH alone. Northern blot analysis indicated that CpFAH is expressed solely in Sclerotium cells, with no transcripts of this gene being detected in mycelium or conidial cells. CpDGAT2 was more widely expressed among the cell types examined, although expression was low in conidiospores. The high expression of CpDGAT2 and CpFAH in Sclerotium cells, where high levels of ricinoleate glycerides accumulate, provided further evidence supporting the roles of CpDGAT2 and CpFAH as key enzymes for the synthesis and assembly of ricinoleic acid in C. purpurea. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Shimbata T.,Nippon Flour Mills Co. | Inokuma T.,Nippon Flour Mills Co. | Sunohara A.,Nippon Flour Mills Co. | Vrinten P.,Bioriginal Food and Science Corporation | And 3 more authors.
Journal of Agricultural and Food Chemistry | Year: 2011

Sweet wheat (SW), which lacks functional granule-bound starch synthase I (GBSSI) and starch synthase IIa (SSIIa), accumulates high levels of free sugars in immature seeds. Here, we examined the effects of the lack of these two enzymes on mature kernel composition.Whole grain flour ofSW had higher levels of sugars, particularly maltose, slightly higher ash and protein content, approximately two to three times higher lipid levels, and about twice as much total dietary fiber as parental or wild-type lines. Considerably higher levels of low-molecular-weight soluble dietary fiber (LMW-SDF), largely consisting of fructan, were also detected in SW. Although there were no differences in total amino acid levels, the free amino acid content of SW was approximately 4-fold higher than that of wild type, and the levels of certain free amino acids such as proline were particularly high. Thus, we were able to clearly demonstrate that the lack of GBSSI and SSIIa caused dramatic changes in mature seed composition in SW. These compositional changes suggest that SW flour may provide health benefits when used as a food ingredient. © 2011 American Chemical Society.


PubMed | Bioriginal Food and Science Corporation
Type: Journal Article | Journal: Lipids | Year: 2013

Sphaeroforma arctica is a unique, recently discovered marine protist belonging to a group falling close to the yeast/animal border. S. arctica is found in cold environments, and accordingly has a fatty acid composition containing a high proportion of very long chain polyunsaturated fatty acids, including the 3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA). Two elongases and five desaturases, representing the complete set of enzymes necessary for the synthesis of DHA from oleic acid, were isolated from this species and characterized in yeast. One elongase showed high conversion rates on a wide range of 18 and 20 carbon substrates, and was capable of sequential elongation reactions. The second elongase had a strong preference for the 20-carbon fatty acids EPA and arachidonic acid, with over 80% of EPA converted to docosapentaenoic acid (DPA) in the heterologous yeast host. The isolation of a 8-desaturase, along with the detection of eicosadienoic acid in S. arctica cultures indicated that this species uses the alternate 8-pathway for the synthesis of long-chain polyunsaturated fatty acids. S. arctica also carried a 4-desaturase that proved to be very active in the production of DHA from DPA. Finally, a long chain acyl-CoA synthetase from S. arctica improved DHA uptake in the heterologous yeast host and led to an improvement in desaturation and elongation efficiencies.

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