BACKGROUND
seed oils provide an important source of dietary fats in both human and livestock nutrition  <cit>  and are becoming increasingly attractive in nutraceutical and bio-based industrial applications, as well as biofuel production  <cit> .
the functional qualities of the various seed oils, and thus their suitability for a particular application, are primarily determined by their fatty acid  content and composition.
seed oil from flax  is enriched in α-linolenic acid   <cit> , with conventional varieties containing 45% to 65% of this essential dietary fa.
ala is a precursor for the synthesis of very long chain omega- <dig> polyunsaturated fatty acids , which are responsible for the myriad of health benefits that have been attributed to flax oil, including positive effects with respect to cardiovascular health and inflammatory diseases, as well as anticancer properties  <cit> .
as a result of this, flax seeds have been widely used in animal feed to increase the ala content of eggs and meat  <cit> , thus altering their fa profiles and rendering them more nutritionally attractive  <cit> .
furthermore, the remarkably high ala content of flax seed oil also provides it with a superior “drying” quality, making it very suitable for widespread use in industrial and domestic products  <cit> .
fa biosynthesis within developing seeds of oleaginous crops occurs in the plastids, with the resulting fas being subsequently released into the cytosol predominantly as oleic acid , along with minor amounts of palmitic  and stearic  acids in the form of acyl-coenzyme a   <cit> .
oa-coa can be further elongated on the endoplasmic reticulum  or used in the acylation of sn-glycerol-3-phosphate.
in a large number of oilseeds, the majority of oa enters the membrane lipid phosphatidylcholine , where a second and third double bond can be added at the sn- <dig> position via the catalytic action of er-localized fatty acid desaturases , fad <dig> and fad <dig>  to produce the pufas linoleic acid  and ala. these pufas, present on pc, can then be incorporated into triacylglycerol , which is the most common form of storage lipid in the seeds of many plant species and serves as a vital energy source for a number of biological functions.
the pufas produced in pc are known to end up in tag via various possible metabolic routes  <cit> .
firstly, pufas can be cleaved from pc through the catalytic action of phospholipase a, with the resulting free fas being esterified to coenzyme a  through the catalytic action of long chain acyl-coa synthetase.
pufas may also enter the acyl-coa pool through acyl-exchange with pc catalyzed by lysophosphatidylcholine acyltransferase .
in both cases, the resulting pufa-coas are then available as a source of fatty acyl chains for incorporation into tag via the acyl-coa-dependent sn-glycerol-3-phosphate pathway whereby diacylglycerol acyltransferase  catalyzes the acylation of diacylglycerol  to form tag.
alternatively, pufas can be directly transferred from the sn- <dig> position of pc onto dag to generate tag through the catalytic action of phospholipid:diacylglycerol acyltransferase  in an acyl-coa-independent manner.
in the case of phospholipase a and pdat action, the resulting lysophosphatidylcholine can be reacylated to pc via the forward reaction catalyzed by lpcat.
a more recently discovered metabolic route for channelling pufa into tag involves the conversion of pufa-enriched pc to pufa-enriched dag through the removal of the phosphocholine headgroup, which has been suggested to be the predominant pathway of dag production  <cit> .
this pc-derived dag appears to be mainly produced through the catalytic action of phosphatidylcholine diacylglycerol cholinephosphotransferase   <cit> , although smaller proportions may also be generated via the reverse action of cdp-choline: diacylglycerol cholinephosphotransferase   <cit>  or by the action of phospholipase c and/or d through a lipase-mediated pathway  <cit> .
furthermore, in a similar manner to its involvement in desaturation, pc also acts as the substrate for the production of epoxy-, conjugated-, hydroxy-, acetylenic-, and other unusual fas  <cit> , with acyl moieties on dag entering pc through the action of pdct, which are then modified and returned to dag for further acylation catalyzed by dgat in the sn-glycerol-3-phosphate pathway.
thus, while it is possible that the metabolic channelling of pufas and unusual fas from pc to tag involves more than one of the aforementioned mechanisms and may vary based on the plant species, it seems that pdct in particular is likely to play a key role in the determination of seed oil fa composition.
in this paper, we report the isolation of two embryo-expressed pdct genes from flax that share a very high level of sequence identity with one another.
functional characterization of these two genes in yeast and plant systems demonstrated that they both encode functional enzymes.
furthermore, heterologous expression of both flax genes increased pufa levels in metabolically engineered yeast and transgenic a. thaliana.
while mutation of a pdct from arabidopsis  has previously been shown to result in decreased pufa accumulation in that species  <cit> , this is the first instance in which the heterologous expression of a pdct has been linked with enhanced production of pufas, providing further evidence that pdct plays a crucial role in determining the fa profile of seed lipids.
since the use of biotechnology to improve the fa composition of seed oils to match the demand of a particular industry is fast becoming a popular approach  <cit> , the flax pdct genes hold great promise for the future modification of pufa levels in a wide range of oil crops.
RESULTS
isolation of pdct homologs in flax
the a. thaliana rod <dig>  sequence  <cit>  was used to query the flax genomic sequence database   <cit>  using the basic local alignment search tool   <cit> .
by analyzing the alignments of the positive hits, two pdct homologs were identified .
the full length cdna sequences of both flax genes were cloned and subsequent sequence analysis indicated that they displayed approximately 97% identity at the nucleotide level, while the deduced amino acid sequences displayed approximately 98% identity .
when compared to atrod <dig>  lupdct <dig> and lupdct <dig> exhibited  <dig> % and  <dig> % identity at the nucleotide level, and  <dig> % and  <dig> % identity at the amino acid level, respectively.
pdct homologs have also been identified in a range of other plant species, and their deduced polypeptide sequences shared between  <dig> % and  <dig> % identity with lupdct <dig> and lupdct <dig> .figure  <dig> comparison of pdct homologs from higher plants.
alignment of deduced amino acid sequences from lupdct <dig>  lupdct <dig>  rcrod <dig> and atrod <dig>  sequences were aligned using clustalw and shading was applied using dna boxshade.
identical amino acids are shaded in black, while conserved substitutions are shaded in gray.
putative transmembrane domains are underlined.
the five highly conserved residues in the c <dig> and c <dig> domains of the lpt family are denoted with arrows and the catalytic triads  are indicated with triangles.
protein sequences were deduced from genbank accession numbers: ricinus communis , xm_002517597; and arabidopsis thaliana , at3g <dig>   phylogenetic tree showing the relationship between deduced pdct proteins from different plant species.
the amino acid sequences were deduced from genbank accession numbers: ricinus communis , xm_002517597; arabidopsis thaliana , at3g15820; brachypodium distachyon, xp_003563650; glycine max, xp_003528315; hordeum vulgare, bak03357; medicago truncatula, xp_003604371; picea sitchensis, abk25679; populus trichocarpa, xp_002327418; sorghum bicolor, xp_002437259; zea maize, np_001145186; and oryza sativa, np_ <dig>  the brassica napus, gossypum spp., and helianthus annuus sequences were obtained from the computational biology and functional genomic website  with the following gene identities: bn, tc171107; gs, tc240631; and ha, tc <dig>  the amino acid sequences were aligned and the phylogenetic tree was constructed using genious v.  <dig>  .
topology prediction software including hmmtop  <cit>  and tmpred  <cit>  identified both lupdct <dig> and lupdct <dig> as integral-membrane proteins with five transmembrane regions .
this finding is consistent with pdct proteins identified previously in other plant species, such as a. thaliana and castor  rod <dig> proteins, which were predicted to have six transmembrane regions  <cit> .
furthermore, pdct belongs to a large family of lipid phosphatase/phosphotransferase  proteins  <cit> , which contain five highly conserved residues in the c <dig> and c <dig> domains, as well as a catalytic triad .
as expected, all of these conserved residues were also found in both deduced flax pdct polypeptides .
other pdct homologs share  <dig> % to  <dig> % identity with the deduced amino acid sequences of flax pdcts .
phylogenetic analysis of the two lupdct proteins in relation to other higher plant pdcts, including functionally tested enzymes from a. thaliana  <cit>  and castor  <cit> , indicated that they are more closely related to protein homologs from plants such as sitka spruce  and castor than they are to that from the model plant, a. thaliana .
lupdct <dig> and lupdct <dig> are specifically expressed in the embryo
to gain insight into the potential roles of lupdct <dig> and lupdct <dig> in flax, their expression patterns were monitored in vegetative tissues, reproductive tissues, and at various stages of embryo development using taqman-based qrt-pcr assays .
the specificity of the primers used for pdct genes and control gene amplification were verified by separating qrt-pcr amplicons on a 2% agarose gel.
the gene-specific primers produced a single product of the desired length, confirming their specificity .figure  <dig> relative expression levels of lupdct1
or lupdct2
in flax vegetative tissues, flowers, and during embryo development.
transcript levels were compared across different tissues and at different stages of embryo development using the two reference genes, gapdh and ubi <dig>  data shown represent means ± se of three biological and three technical replicates.
s <dig>  immature stem; s <dig>  developing stem; s <dig>  mature stem; l, leaves; a, apexes; r, roots; f, flowers;  <dig> to 20d, days after anthesis of developing embryos; 25d and 40d, days after anthesis of mature seeds.
in general, lupdct <dig> and lupdct <dig> exhibited similar spatiotemporal patterns of expression; however, due to differences in their primer pair efficiencies , it was not possible to compare levels of expression between the two genes.
while both lupdct <dig> and lupdct <dig> were found to be expressed at only very low levels in all vegetative and floral tissues tested , both genes were highly expressed during the mid- to late-stages  of embryo development, peaking at approximately  <dig> daa with transcript levels decreasing dramatically after  <dig> daa to nearly undetectable levels for the remainder of seed development .
previously, it was shown that the proportion of ala in flax cultivar cdc bethune embryos increased from 30% to 43% between  <dig> and  <dig> daa, and then stabilized at approximately  <dig> daa, which corresponded with a rapid accumulation of oil that increased from 1% to 20% of fresh weight between  <dig> and  <dig> daa  <cit> .
these results suggest that the two flax pdct proteins may play an active role in increasing the pufa content of the seed oil.
lupdct <dig> and lupdct <dig> encode functional pdct enzymes
to test the enzymatic activity of the lupdct <dig> and lupdct <dig> proteins, the coding sequences of both genes were cloned into the yeast expression vector pyesbop  <cit>  under the control of the galactose-inducible gal <dig> promoter.
the resulting vectors were transformed into the saccharomyces cerevisiae mutant strain ynl 130c , which lacks cpt activity  <cit> .
since the pdct activity of the enzyme encoded by the atrod <dig> gene was confirmed previously using the yeast microsomal fraction  <cit>  and the deduced amino acid sequences of both lupdct <dig> and lupdct <dig> were predicted to have five transmembrane domains, microsomal preparations of lupdct1-, lupdct2- and empty vector-transformed cells were tested for their ability to catalyze the synthesis of pc from dag or dag from pc.
for assessing the rate of formation of pc, -glycerol-diolein was incubated with dioleoyl-pc and yeast microsomal fraction, and the production of radiolabeled-pc was monitored after 15 min by thin layer chromatography  .
radio-labeled pc was produced through the catalytic action of both lupdct <dig>  and lupdct <dig> , and the rate of production was found to be linear for up to 5 min .
in contrast, yeast microsomes bearing the empty vector control  and boiled microsomes containing the recombinant enzymes, p <dig>  and p <dig> , failed to produce radio-labeled pc.
the rate of appearance of radio-labeled pc catalyzed by microsomes containing recombinant lupdct <dig> or lupdct <dig> is shown in figure 3b; in both cases, the enzymatic reactions were linear for up to 5 min.
microsomes containing recombinant lupdct <dig> or lupdct <dig> were also capable of catalyzing the formation of radio-labeled dag when supplied with 1-palmitoyl-2- oleoyl-pc and diolein .
in this latter case, time courses were not conducted and radio-labeled dag production was only determined after 15 min of incubation.
small quantities of tag were also produced during the reactions where production of radio-labeled dag was monitored .figure  <dig> analysis of pdct activity of s. cerevisiae microsomes producing recombinant lupdct <dig> or lupdct <dig>   production of radio-labeled phosphatidylcholine  catalyzed by lupdct <dig> or lupdct <dig> following 15 min incubation.
radio-labeled substrate and product were separated using radio-thin layer chromatography  and visualized by phosphor-imaging.
microsomes from yeast ynl 130c cells transformed with pyes-lupdct <dig> , pyes-lupdct <dig>  or pyes  were incubated with -glycerol- diolein in the presence of dioleoyl-pc.
p <dig>  and p <dig> , respectively, indicate microsomes from pyes-lupdct <dig> and pyes-lupdct <dig> that were boiled prior to the assay.
the phosphor image is a composite of five representative samples selected from two tlc plates which were developed under identical conditions.
time course production of pc catalyzed by lupdct <dig>  lupdct <dig> or ev microsomes.
data shown represent means ± se based on three replicates.
lupdct <dig> and lupdct <dig> enhance pufa accumulation in pc, dag and tag in yeast
s. cerevisiae has been widely used as a model organism for studying eukaryotic cellular and molecular functions  <cit> , and yeast expression systems have commonly been used for functional characterization of plant integral membrane fatty acid desaturases such as fad <dig>  <cit>  and fad <dig>  <cit> .
however, s. cerevisiae does not synthesize pufas due to a lack of enzymes capable of introducing more than one double bond into its fas  <cit> , thus limiting its use in oil biosynthesis-related research involving the generation of pufas.
to circumvent this problem, we engineered s. cerevisiae to produce la and ala by sequentially introducing expression cassettes containing cdnas encoding lufad <dig>  <cit>  and lufad3b  <cit> .
analysis of the different lipid classes revealed that the pc fraction from wild-type  yeast cells transformed with lufad <dig> and lufad3b accumulated small but significant quantities of la  and ala , with minute quantities of pufas  observed in tag and dag fractions .
these results demonstrate that transgenic yeast cells carrying lufad <dig> and lufad3b are capable of producing la and ala, particularly in pc, which are fas not normally present in yeast.
we also detected small quantities of  <dig>  15-octadecadienoic acid  in the pc fraction of these cells.
this is consistent with previous reports  <cit>  in which plant fad <dig> genes have been expressed in yeast, and is very likely due to the minor catalytic activity of higher plant fad <dig> enzymes in the desaturation of monounsaturated fas.figure  <dig> engineering s. cerevisiae to produce polyunsaturated fatty acids .
gas chromatography–mass spectrometry analysis of fa methyl esters derived from the phosphatidylcholine fraction of wild-type yeast cultures transformed with the empty pyesbop vector , pyes + f2f <dig>  and pyes + f2f3 + p <dig>  constructs.
pyes + f2f3 + p <dig> produced similar results to that of pyes + f2f3 + p <dig>
fatty acid compositions of lipid classes from transgenic
s. cerevisae
construct
fa composition of pc
c16:0
c16:1
c16:2
c18:0
c18:1
c18:2
c18:3
adata represent results from three independent yeast colonies .
bnot detected.
fatty acid composition of phosphatidylcholine , diacylglycerol  and triacylglycerol  from transgenic yeast expressing lufad <dig>  or lufad3b  alone, or along with lupdct <dig>  or lupdct <dig> , as well as wild-type yeast transformed with empty pyesbop vector .
to further our understanding of the role of pdct in pufa accumulation, lupdct <dig> and lupdct <dig> were independently co-expressed with lufad <dig> and lufad3b in wt yeast under the control of galactose inducible promoters .
intriguingly, the combined level of la and ala increased approximately 6- and 4-fold in the pc fraction when lupdct <dig> and lupdct <dig> were co-expressed with the aforementioned desaturases, respectively .
similarly, expression of the lupdct genes in this context also resulted in a substantial increase in the accumulation of pufas in both the tag  and dag  fractions .
interestingly, a concomitant decrease in oa was observed in all three of the lipid fractions in both lupdct <dig> and lupdct2-transformed cells .
furthermore, an accumulation of 16:2cisΔ <dig>  in both the pc and tag fractions, but not dag, were observed only in those cells harbouring the lupdct genes .
expression of lupdct <dig> and lupdct <dig> restores the fa composition of the arabidopsis rod <dig> mutant
to provide additional evidence that lupdct <dig> and lupdct <dig> encode functional proteins, their corresponding open reading frames were expressed in the seeds of the a. thaliana rod <dig> mutant, which exhibits a marked decrease in 18: <dig> and 18: <dig> pufas, along with a concomitant increase in 18: <dig> relative to wt plants  <cit> .
since genome position-related expression variation can occur in transgenic plants due to the random nature of transgenic insertions  <cit> , we analyzed at least ten independent lines bearing each construct, respectively.
analysis of t <dig> -segregating transgenic seeds revealed that both lupdct <dig> and lupdct <dig> were capable of restoring the fa composition of the rod <dig> mutant to wt levels in the majority of lines analyzed, whereas empty vector-transformed plants exhibited a fa profile similar to that of the rod <dig> mutant .
on average, expression of either lupdct gene caused the combined proportion of la and ala  to increase to over 41% of the total fa content from approximately 27% in the rod <dig> mutant, while oa levels decreased to nearly 21% from 33% .
this corresponds well with wild-type oa, la and ala proportions, which make up  <dig> %,  <dig> % and  <dig> % of the total fa content, respectively .
this compensation of pufa levels at the expense of oa further confirms that both lupdct <dig> and lupdct <dig> encode functional pdct enzymes.figure  <dig> expression of lupdct <dig> or lupdct <dig> restores the fatty acid composition of the a. thaliana rod <dig> mutant.
fatty acid composition of the a. thaliana rod <dig> mutant bearing lupdct <dig> or lupdct <dig> expression cassettes, respectively.
proportions of c18: <dig>  c18: <dig>  and c18: <dig> in untransformed wild-type seeds , rod <dig> mutant seeds , t <dig> seeds of the rod <dig> mutant transformed with empty vector , and t <dig> seeds of the rod <dig> mutant bearing either lupdct <dig>  or lupdct <dig>  expression cassettes.
data shown represent means ± se .
heterologous expression of lupdct <dig> and lupdct <dig> increases pufa accumulation in wild-type a. thaliana
to gain insight into the role of pdct in pufa accumulation within seeds, and to determine whether the results we observed in a yeast system would translate to a plant system, lupdct <dig> and lupdct <dig> were independently expressed in wt a. thaliana.
analysis of t2-segregating seeds of independent transgenic lines bearing each construct, respectively, revealed significant changes in fa composition compared to negative control lines .
on average, the proportion of c18-pufas increased from  <dig>  ±  <dig> % in empty vector-transformed seeds to  <dig>  ±  <dig> % and  <dig>  ±  <dig> % in lupdct <dig> and lupdct <dig> transformed seeds, respectively .
furthermore, the oa levels of the lupdct-expressing lines also dropped to approximately  <dig>  ±  <dig> % from  <dig>  ±  <dig> % in empty vector-transformed seeds , which supports the premise that pdct plays a key role in pufa production by enhancing the channelling of oleoyl moieties into pc for desaturation.figure  <dig> heterologous expression of lupdct <dig> or lupdct <dig> increases polyunsaturated fatty acid  content in wild-type  a. thaliana.
fatty acid composition of wild-type a. thaliana bearing lupdct <dig> or lupdct <dig> expression cassettes.
proportions of c18: <dig> and c18-pufas  in untransformed wt seeds, t <dig> seeds of wt a. thaliana bearing empty vector , lupdct <dig>  or lupdct <dig>  expression cassettes.
data shown represent means ± se .
discussion
flax seed is a rich agricultural source of ala, and has been classified as one of the most important plant-based suppliers of pufas.
unfortunately, there is limited information concerning the metabolic pathway of oil synthesis in this species, which has significantly hindered its genetic improvement as an oilseed crop.
however, a number of genetic and biochemical studies have been initiated using this species in recent years, and they are now beginning to generate insight into this previously unknown territory.
to date, it has been assumed that flax fa desaturases were the primary enzymes required for the determination of pufa content in tag.
however, recent studies in a. thaliana have since demonstrated the importance of phosphocholine headgroup exchange between pc and dag in providing pufa for tag synthesis as this mechanism appears to control the majority of acyl flux through pc  <cit> .
pc is the enzymatic substrate for fa desaturases, and pc containing 18: <dig> at the sn- <dig> position is the initial substrate recognized by fad <dig> leading to the production of c18-pufas  <cit> .
following desaturation by fad <dig> and fad <dig>  the modified fas are moved out of pc to eventually become incorporated into tag.
thus, the efficiency of incorporation of fas into pc and successive removal of desaturated fas from pc for eventual tag synthesis could be of great importance in determining the final fa composition of tag in flax.
the two major pathways controlling the movement of fas into and out of pc are acyl editing and dag-pc interconversion via the action of pdct after de novo synthesis of dag through the kennedy pathway.
this symmetrical interconversion of pc and dag liberates pc-derived dag with modified fas to be utilized by either dgat or pdat for tag production.
the ability of pdct to catalyze the symmetrical interconversion between dag and pc through a phosphocholine headgroup exchange was first demonstrated in plants through the characterization of the a. thaliana rod <dig> mutant  <cit> .
pdct was found to act as a gatekeeper enzyme, providing a major path through which oleoyl moieties entered pc for desaturation at the sn- <dig> position.
this mutation resulted in a significant decrease in pufa levels in seed tag compared to wt   <cit> , demonstrating the importance of pdct in the efficient production of pufas in this species.
indeed, in arabidopsis seeds it has been estimated that at least 40% of pufas in tag are derived through the action of pdct alone  <cit> .
furthermore, it has recently been shown that the collective action of pdct along with acyl-coa:lysophosphatidylcholine acyltransferases  are responsible for the accumulation of nearly two-thirds of the c18-pufas in this species  <cit> .
recently, it has also been demonstrated that pdct is required for efficient hydroxy fatty acid accumulation in transgenic a. thaliana  <cit> , demonstrating the importance of pdct in channelling modified fatty acids into tag.
while pdct has been shown to play a key role in the production of tag enriched with modified fas, including pufas, in model plant species, this is the first study in which the importance of this enzyme from a crop plant specifically grown for its high levels of pufas has been analyzed.
interestingly, results from a number of recent studies have suggested that arabidopsis is not the only plant species that utilizes pc-derived dag to produce pufa-rich tag or tag enriched with other modified fas   <cit> .
for example, it has been estimated that flax utilizes more than about 70% pc-derived dag to synthesize tag  <cit> , which implies that the pdct-catalyzed reaction may be the main route of pc-derived dag synthesis in flax and may therefore be an important contributor to the high levels of pufas found in this crop species.
in an initial attempt to provide evidence that this is, indeed, the case, we have isolated cdna from two flax pdct homologs  that displayed high levels of sequence identity with one another .
flax contains a number of highly homologous gene pairs encoding enzymes involved in oil biosynthesis  <cit> , which is consistent with our results and may be the consequence of a putative whole-genome duplication that has been suggested to have occurred in this species  <cit> .
phylogenetic comparison of the two lupdct proteins with other higher plant pdcts, including functionally tested enzymes from a. thaliana  <cit>  and castor  <cit> , indicated that they are more closely related to homologous proteins from plants such as spruce and castor than they are to that from a. thaliana .
this has also been found to be the case for several other flax oil biosynthetic genes  <cit> .
the fact that the pdct homologs with the highest sequence identity to the two lupdct proteins tended to be from other high c18-pufa and unusual fa-producing plants such as sunflower , cotton  and castor   suggests that pdct likely provides an important function in the incorporation of modified fas into tag in many plant taxa.
interestingly, pdct homologs have not been found to be present in certain plant species that accumulate conjugated fas, including bitter melon  and tung tree   <cit> , which implies that pdct may not be ubiquitously distributed throughout the plant kingdom.
it would be interesting to compare flax pdct with chia  pdct, if homologs are present, once the sequence information becomes available as chia has a close c18-pufa profile to flax  <cit> .
as is the case for atrod <dig>  <cit> , lupdct <dig> and lupdct <dig> transcript levels were dramatically higher in developing flax embryos than in other parts of the plant .
this increase in transcript levels, which peaked at approximately  <dig> daa and declined to nearly undetectable levels by  <dig> daa , correlates well with the timing of rapid oil  accumulation in developing embryos of this species  <cit>  and provides strong evidence that the lupdct proteins are involved in the production of high levels of c18-pufas in flax.
we also noted very low levels of lupdct <dig> and lupdct <dig> transcripts in vegetative tissues and flowers , which is in contrast to the expression of atrod <dig> in these tissues in a. thaliana  <cit> .
these findings imply that the lupdct enzymes may provide a secondary function of pufa enrichment in non-oil-accumulating tissues in addition to tag-rich embryos in flax.
in agreement with this, the rod <dig> mutation in a. thaliana did not result in substantial fa compositional changes in leaf or root whereas pufa levels were substantially decreased in seeds  <cit> .
enzyme assays with microsomal preparations from s. cerevisiae cells expressing lupdct <dig> or lupdct <dig> demonstrated that both proteins possessed pdct activity in catalyzing both forward and reverse reactions .
when assayed for the formation of dag from pc, a small quantity of tag was also produced , which likely resulted from the catalytic action of yeast tag synthesizing enzymes present in the microsomal fraction.
interestingly, previous studies indicated that both atrod <dig>  <cit>  and rcrod <dig>  <cit>  exhibited high pdct activities at 15°c; however, both lupdct <dig> and lupdct <dig> exhibited extremely low activity at 15°c compared to 30°c .
although enzymatic activity data was not available for atrod <dig> and rcrod <dig> at 30°c and different enzyme assay conditions were utilized in the various studies, these data indicate that the flax pdct proteins may possess different enzymatic properties than those of a. thaliana or castor.
direct evidence for the role of lupdct enzymes in c18-pufa enrichment was obtained through the expression of the corresponding genes in fad2/fad3-expressing yeast, where major compositional changes were observed in the different lipid classes compared to yeast lacking the lupdct coding sequences .
metabolic engineering of yeast with lufad <dig> and lufad3b expression cassettes provided an excellent system with which to study the effects of pdct expression on pufa accumulation in pc, dag and tag fractions without having to resort to the feeding of yeast cultures with la and ala, which are not produced naturally by s. cerevisiae.
fa feeding in an attempt to introduce exogenous acyl moieties to yeast cells is often utilized in such situations, but fa uptake into yeast cells can at times lead to deleterious effects  <cit> , making the system developed here a much more desirable option.
when only the fads were expressed, pufas were observed in the pc fraction, with only minute quantities being present in dag and tag fractions.
co-expression with each lupdct, however, resulted in a marked increase in pufa levels in both dag and tag fractions , providing further support for the premise that pdct plays a critical role in channelling pufas to dag, and subsequently tag, following production on pc.
moreover, c18-pufa content in the pc fraction was also substantially increased  at the concomitant expense of 18: <dig>  taken together, these data suggest that pdct enhances the channelling of oleyol acyl moieties into pc by rapidly converting dag to pc, thus increasing substrate availability for fads.
this dual mechanism of pdct  appears to result in a synergy leading to increased pufa accumulation in this yeast system.
these results were confirmed in a plant system, wherein both wild-type and rod <dig> mutant a. thaliana were transformed with vectors designed to express lupdct <dig> and lupdct <dig>  respectively.
in the case of the transgenic rod <dig> mutant plants, either lupdct <dig> or lupdct <dig> was capable of restoring pufa levels to more wt levels .
there was some variation in the level of complementation of the fa composition in the transgenic lines , which was likely due to positional effects of transgene insertion; this is a common phenomenon in the generation of transgenic plants.
similar variations among independent transformants were observed when the castor fatty acid hydroxylase gene  was expressed in the a. thaliana fad2/fae <dig> double mutant  <cit>  or fatty acid elongase  gene from nasturtium  was overexpressed in wt a. thaliana  <cit> .
in line with results obtained with the rod <dig> mutant, expression of the two flax pdct genes in wt a. thaliana substantially increased pufa production in transgenic lines.
on average, the proportion of c18-pufa increased by  <dig> % and  <dig> % in lupdct1- or lupdct2-expressing lines, respectively, at the concomitant expense of 18: <dig> .
these data provide further support that pdct increases pufa accumulation by enhancing the channelling of monounsaturated acyl moieties through desaturation on pc and making available the resulting polyunsaturated acyl moieties for tag synthesis.
compositional analysis of flax seed lipids has previously revealed that approximately 95% of its tag is enriched with pufas at any one of the sn-positions, whereas only approximately 40% of its tag contains c18-pufas at all three sn-positions  <cit> , indicating that there is a vast potential to increase the proportion of pufa-rich tag in this species.
CONCLUSIONS
we have identified two novel pdct homologs from flax, which encode enzymes catalyzing increases in c18-pufa levels substantially in metabolically engineered yeast and transgenic a. thaliana seeds.
these flax pdcts appear to play an important dual role in the determination of pufa content by efficiently channelling monounsaturated fas into pc for desaturation and moving the resulting pufas out of pc for subsequent use in tag synthesis.
these results have distinguished pdct as an ideal candidate gene for future bioengineering of flax and other oil crops to produce improved levels of pufas for widespread use in industrial and nutraceutical applications.

 seed oils provide an important source of dietary fats in both human and livestock nutrition and are becoming increasingly attractive in nutraceutical and bio-based industrial applications, as well as biofuel production. the functional qualities of the various seed oils, and thus their suitability for a particular application, are primarily determined by their fatty acid content and composition. seed oil from flax is enriched in α-linolenic acid, with conventional varieties containing 45% to 65% of this essential dietary fa. ala is a precursor for the synthesis of very long chain omega- polyunsaturated fatty acids, which are responsible for the myriad of health benefits that have been attributed to flax oil, including positive effects with respect to cardiovascular health and inflammatory diseases, as well as anticancer properties. as a result of this, flax seeds have been widely used in animal feed to increase the ala content of eggs and meat, thus altering their fa profiles and rendering them more nutritionally attractive. furthermore, the remarkably high ala content of flax seed oil also provides it with a superior “drying” quality, making it very suitable for widespread use in industrial and domestic products. fa biosynthesis within developing seeds of oleaginous crops occurs in the plastids, with the resulting fas being subsequently released into the cytosol predominantly as oleic acid, along with minor amounts of palmitic and stearic acids in the form of acyl-coenzyme a. oa-coa can be further elongated on the endoplasmic reticulum or used in the acylation of sn-glycerol-3-phosphate. in a large number of oilseeds, the majority of oa enters the membrane lipid phosphatidylcholine, where a second and third double bond can be added at the sn- position via the catalytic action of er-localized fatty acid desaturases, fad and fad to produce the pufas linoleic acid and ala. these pufas, present on pc, can then be incorporated into triacylglycerol, which is the most common form of storage lipid in the seeds of many plant species and serves as a vital energy source for a number of biological functions. the pufas produced in pc are known to end up in tag via various possible metabolic routes. firstly, pufas can be cleaved from pc through the catalytic action of phospholipase a, with the resulting free fas being esterified to coenzyme a through the catalytic action of long chain acyl-coa synthetase. pufas may also enter the acyl-coa pool through acyl-exchange with pc catalyzed by lysophosphatidylcholine acyltransferase. in both cases, the resulting pufa-coas are then available as a source of fatty acyl chains for incorporation into tag via the acyl-coa-dependent sn-glycerol-3-phosphate pathway whereby diacylglycerol acyltransferase catalyzes the acylation of diacylglycerol to form tag. alternatively, pufas can be directly transferred from the sn- position of pc onto dag to generate tag through the catalytic action of phospholipid:diacylglycerol acyltransferase in an acyl-coa-independent manner. in the case of phospholipase a and pdat action, the resulting lysophosphatidylcholine can be reacylated to pc via the forward reaction catalyzed by lpcat. a more recently discovered metabolic route for channelling pufa into tag involves the conversion of pufa-enriched pc to pufa-enriched dag through the removal of the phosphocholine headgroup, which has been suggested to be the predominant pathway of dag production. this pc-derived dag appears to be mainly produced through the catalytic action of phosphatidylcholine diacylglycerol cholinephosphotransferase, although smaller proportions may also be generated via the reverse action of cdp-choline: diacylglycerol cholinephosphotransferase or by the action of phospholipase c and/or d through a lipase-mediated pathway. furthermore, in a similar manner to its involvement in desaturation, pc also acts as the substrate for the production of epoxy-, conjugated-, hydroxy-, acetylenic-, and other unusual fas, with acyl moieties on dag entering pc through the action of pdct, which are then modified and returned to dag for further acylation catalyzed by dgat in the sn-glycerol-3-phosphate pathway. thus, while it is possible that the metabolic channelling of pufas and unusual fas from pc to tag involves more than one of the aforementioned mechanisms and may vary based on the plant species, it seems that pdct in particular is likely to play a key role in the determination of seed oil fa composition. in this paper, we report the isolation of two embryo-expressed pdct genes from flax that share a very high level of sequence identity with one another. functional characterization of these two genes in yeast and plant systems demonstrated that they both encode functional enzymes. furthermore, heterologous expression of both flax genes increased pufa levels in metabolically engineered yeast and transgenic a. thaliana. while mutation of a pdct from arabidopsis has previously been shown to result in decreased pufa accumulation in that species, this is the first instance in which the heterologous expression of a pdct has been linked with enhanced production of pufas, providing further evidence that pdct plays a crucial role in determining the fa profile of seed lipids. since the use of biotechnology to improve the fa composition of seed oils to match the demand of a particular industry is fast becoming a popular approach, the flax pdct genes hold great promise for the future modification of pufa levels in a wide range of oil crops.  isolation of pdct homologs in flax the a. thaliana rod sequence was used to query the flax genomic sequence database using the basic local alignment search tool. by analyzing the alignments of the positive hits, two pdct homologs were identified. the full length cdna sequences of both flax genes were cloned and subsequent sequence analysis indicated that they displayed approximately 97% identity at the nucleotide level, while the deduced amino acid sequences displayed approximately 98% identity. when compared to atrod lupdct and lupdct exhibited % and % identity at the nucleotide level, and % and % identity at the amino acid level, respectively. pdct homologs have also been identified in a range of other plant species, and their deduced polypeptide sequences shared between % and % identity with lupdct and lupdct.figure comparison of pdct homologs from higher plants. alignment of deduced amino acid sequences from lupdct lupdct rcrod and atrod sequences were aligned using clustalw and shading was applied using dna boxshade. identical amino acids are shaded in black, while conserved substitutions are shaded in gray. putative transmembrane domains are underlined. the five highly conserved residues in the c and c domains of the lpt family are denoted with arrows and the catalytic triads are indicated with triangles. protein sequences were deduced from genbank accession numbers: ricinus communis, xm_002517597; and arabidopsis thaliana, at3g phylogenetic tree showing the relationship between deduced pdct proteins from different plant species. the amino acid sequences were deduced from genbank accession numbers: ricinus communis, xm_002517597; arabidopsis thaliana, at3g15820; brachypodium distachyon, xp_003563650; glycine max, xp_003528315; hordeum vulgare, bak03357; medicago truncatula, xp_003604371; picea sitchensis, abk25679; populus trichocarpa, xp_002327418; sorghum bicolor, xp_002437259; zea maize, np_001145186; and oryza sativa, np_ the brassica napus, gossypum spp., and helianthus annuus sequences were obtained from the computational biology and functional genomic website with the following gene identities: bn, tc171107; gs, tc240631; and ha, tc the amino acid sequences were aligned and the phylogenetic tree was constructed using genious v.. topology prediction software including hmmtop and tmpred identified both lupdct and lupdct as integral-membrane proteins with five transmembrane regions. this finding is consistent with pdct proteins identified previously in other plant species, such as a. thaliana and castor rod proteins, which were predicted to have six transmembrane regions. furthermore, pdct belongs to a large family of lipid phosphatase/phosphotransferase proteins, which contain five highly conserved residues in the c and c domains, as well as a catalytic triad. as expected, all of these conserved residues were also found in both deduced flax pdct polypeptides. other pdct homologs share % to % identity with the deduced amino acid sequences of flax pdcts. phylogenetic analysis of the two lupdct proteins in relation to other higher plant pdcts, including functionally tested enzymes from a. thaliana and castor, indicated that they are more closely related to protein homologs from plants such as sitka spruce and castor than they are to that from the model plant, a. thaliana. lupdct and lupdct are specifically expressed in the embryo to gain insight into the potential roles of lupdct and lupdct in flax, their expression patterns were monitored in vegetative tissues, reproductive tissues, and at various stages of embryo development using taqman-based qrt-pcr assays. the specificity of the primers used for pdct genes and control gene amplification were verified by separating qrt-pcr amplicons on a 2% agarose gel. the gene-specific primers produced a single product of the desired length, confirming their specificity.figure relative expression levels of lupdct1 or lupdct2 in flax vegetative tissues, flowers, and during embryo development. transcript levels were compared across different tissues and at different stages of embryo development using the two reference genes, gapdh and ubi data shown represent means ± se of three biological and three technical replicates. s immature stem; s developing stem; s mature stem; l, leaves; a, apexes; r, roots; f, flowers; to 20d, days after anthesis of developing embryos; 25d and 40d, days after anthesis of mature seeds. in general, lupdct and lupdct exhibited similar spatiotemporal patterns of expression; however, due to differences in their primer pair efficiencies, it was not possible to compare levels of expression between the two genes. while both lupdct and lupdct were found to be expressed at only very low levels in all vegetative and floral tissues tested, both genes were highly expressed during the mid- to late-stages of embryo development, peaking at approximately daa with transcript levels decreasing dramatically after daa to nearly undetectable levels for the remainder of seed development. previously, it was shown that the proportion of ala in flax cultivar cdc bethune embryos increased from 30% to 43% between and daa, and then stabilized at approximately daa, which corresponded with a rapid accumulation of oil that increased from 1% to 20% of fresh weight between and daa. these results suggest that the two flax pdct proteins may play an active role in increasing the pufa content of the seed oil. lupdct and lupdct encode functional pdct enzymes to test the enzymatic activity of the lupdct and lupdct proteins, the coding sequences of both genes were cloned into the yeast expression vector pyesbop under the control of the galactose-inducible gal promoter. the resulting vectors were transformed into the saccharomyces cerevisiae mutant strain ynl 130c, which lacks cpt activity. since the pdct activity of the enzyme encoded by the atrod gene was confirmed previously using the yeast microsomal fraction and the deduced amino acid sequences of both lupdct and lupdct were predicted to have five transmembrane domains, microsomal preparations of lupdct1-, lupdct2- and empty vector-transformed cells were tested for their ability to catalyze the synthesis of pc from dag or dag from pc. for assessing the rate of formation of pc, -glycerol-diolein was incubated with dioleoyl-pc and yeast microsomal fraction, and the production of radiolabeled-pc was monitored after 15 min by thin layer chromatography. radio-labeled pc was produced through the catalytic action of both lupdct and lupdct, and the rate of production was found to be linear for up to 5 min. in contrast, yeast microsomes bearing the empty vector control and boiled microsomes containing the recombinant enzymes, p and p, failed to produce radio-labeled pc. the rate of appearance of radio-labeled pc catalyzed by microsomes containing recombinant lupdct or lupdct is shown in figure 3b; in both cases, the enzymatic reactions were linear for up to 5 min. microsomes containing recombinant lupdct or lupdct were also capable of catalyzing the formation of radio-labeled dag when supplied with 1-palmitoyl-2- oleoyl-pc and diolein. in this latter case, time courses were not conducted and radio-labeled dag production was only determined after 15 min of incubation. small quantities of tag were also produced during the reactions where production of radio-labeled dag was monitored.figure analysis of pdct activity of s. cerevisiae microsomes producing recombinant lupdct or lupdct production of radio-labeled phosphatidylcholine catalyzed by lupdct or lupdct following 15 min incubation. radio-labeled substrate and product were separated using radio-thin layer chromatography and visualized by phosphor-imaging. microsomes from yeast ynl 130c cells transformed with pyes-lupdct, pyes-lupdct or pyes were incubated with -glycerol- diolein in the presence of dioleoyl-pc. p and p, respectively, indicate microsomes from pyes-lupdct and pyes-lupdct that were boiled prior to the assay. the phosphor image is a composite of five representative samples selected from two tlc plates which were developed under identical conditions. time course production of pc catalyzed by lupdct lupdct or ev microsomes. data shown represent means ± se based on three replicates. lupdct and lupdct enhance pufa accumulation in pc, dag and tag in yeast s. cerevisiae has been widely used as a model organism for studying eukaryotic cellular and molecular functions, and yeast expression systems have commonly been used for functional characterization of plant integral membrane fatty acid desaturases such as fad and fad. however, s. cerevisiae does not synthesize pufas due to a lack of enzymes capable of introducing more than one double bond into its fas, thus limiting its use in oil biosynthesis-related research involving the generation of pufas. to circumvent this problem, we engineered s. cerevisiae to produce la and ala by sequentially introducing expression cassettes containing cdnas encoding lufad and lufad3b. analysis of the different lipid classes revealed that the pc fraction from wild-type yeast cells transformed with lufad and lufad3b accumulated small but significant quantities of la and ala, with minute quantities of pufas observed in tag and dag fractions. these results demonstrate that transgenic yeast cells carrying lufad and lufad3b are capable of producing la and ala, particularly in pc, which are fas not normally present in yeast. we also detected small quantities of 15-octadecadienoic acid in the pc fraction of these cells. this is consistent with previous reports in which plant fad genes have been expressed in yeast, and is very likely due to the minor catalytic activity of higher plant fad enzymes in the desaturation of monounsaturated fas.figure engineering s. cerevisiae to produce polyunsaturated fatty acids. gas chromatography–mass spectrometry analysis of fa methyl esters derived from the phosphatidylcholine fraction of wild-type yeast cultures transformed with the empty pyesbop vector, pyes + f2f and pyes + f2f3 + p constructs. pyes + f2f3 + p produced similar results to that of pyes + f2f3 + p fatty acid compositions of lipid classes from transgenic s. cerevisae construct fa composition of pc c16:0 c16:1 c16:2 c18:0 c18:1 c18:2 c18:3 adata represent results from three independent yeast colonies. bnot detected. fatty acid composition of phosphatidylcholine, diacylglycerol and triacylglycerol from transgenic yeast expressing lufad or lufad3b alone, or along with lupdct or lupdct, as well as wild-type yeast transformed with empty pyesbop vector. to further our understanding of the role of pdct in pufa accumulation, lupdct and lupdct were independently co-expressed with lufad and lufad3b in wt yeast under the control of galactose inducible promoters. intriguingly, the combined level of la and ala increased approximately 6- and 4-fold in the pc fraction when lupdct and lupdct were co-expressed with the aforementioned desaturases, respectively. similarly, expression of the lupdct genes in this context also resulted in a substantial increase in the accumulation of pufas in both the tag and dag fractions. interestingly, a concomitant decrease in oa was observed in all three of the lipid fractions in both lupdct and lupdct2-transformed cells. furthermore, an accumulation of 16:2cisΔ in both the pc and tag fractions, but not dag, were observed only in those cells harbouring the lupdct genes. expression of lupdct and lupdct restores the fa composition of the arabidopsis rod mutant to provide additional evidence that lupdct and lupdct encode functional proteins, their corresponding open reading frames were expressed in the seeds of the a. thaliana rod mutant, which exhibits a marked decrease in 18: and 18: pufas, along with a concomitant increase in 18: relative to wt plants. since genome position-related expression variation can occur in transgenic plants due to the random nature of transgenic insertions, we analyzed at least ten independent lines bearing each construct, respectively. analysis of t -segregating transgenic seeds revealed that both lupdct and lupdct were capable of restoring the fa composition of the rod mutant to wt levels in the majority of lines analyzed, whereas empty vector-transformed plants exhibited a fa profile similar to that of the rod mutant. on average, expression of either lupdct gene caused the combined proportion of la and ala to increase to over 41% of the total fa content from approximately 27% in the rod mutant, while oa levels decreased to nearly 21% from 33%. this corresponds well with wild-type oa, la and ala proportions, which make up %, % and % of the total fa content, respectively. this compensation of pufa levels at the expense of oa further confirms that both lupdct and lupdct encode functional pdct enzymes.figure expression of lupdct or lupdct restores the fatty acid composition of the a. thaliana rod mutant. fatty acid composition of the a. thaliana rod mutant bearing lupdct or lupdct expression cassettes, respectively. proportions of c18: c18: and c18: in untransformed wild-type seeds, rod mutant seeds, t seeds of the rod mutant transformed with empty vector, and t seeds of the rod mutant bearing either lupdct or lupdct expression cassettes. data shown represent means ± se. heterologous expression of lupdct and lupdct increases pufa accumulation in wild-type a. thaliana to gain insight into the role of pdct in pufa accumulation within seeds, and to determine whether the results we observed in a yeast system would translate to a plant system, lupdct and lupdct were independently expressed in wt a. thaliana. analysis of t2-segregating seeds of independent transgenic lines bearing each construct, respectively, revealed significant changes in fa composition compared to negative control lines. on average, the proportion of c18-pufas increased from  ±  % in empty vector-transformed seeds to  ±  % and  ±  % in lupdct and lupdct transformed seeds, respectively. furthermore, the oa levels of the lupdct-expressing lines also dropped to approximately  ±  % from  ±  % in empty vector-transformed seeds, which supports the premise that pdct plays a key role in pufa production by enhancing the channelling of oleoyl moieties into pc for desaturation.figure heterologous expression of lupdct or lupdct increases polyunsaturated fatty acid content in wild-type a. thaliana. fatty acid composition of wild-type a. thaliana bearing lupdct or lupdct expression cassettes. proportions of c18: and c18-pufas in untransformed wt seeds, t seeds of wt a. thaliana bearing empty vector, lupdct or lupdct expression cassettes. data shown represent means ± se. discussion flax seed is a rich agricultural source of ala, and has been classified as one of the most important plant-based suppliers of pufas. unfortunately, there is limited information concerning the metabolic pathway of oil synthesis in this species, which has significantly hindered its genetic improvement as an oilseed crop. however, a number of genetic and biochemical studies have been initiated using this species in recent years, and they are now beginning to generate insight into this previously unknown territory. to date, it has been assumed that flax fa desaturases were the primary enzymes required for the determination of pufa content in tag. however, recent studies in a. thaliana have since demonstrated the importance of phosphocholine headgroup exchange between pc and dag in providing pufa for tag synthesis as this mechanism appears to control the majority of acyl flux through pc. pc is the enzymatic substrate for fa desaturases, and pc containing 18: at the sn- position is the initial substrate recognized by fad leading to the production of c18-pufas. following desaturation by fad and fad the modified fas are moved out of pc to eventually become incorporated into tag. thus, the efficiency of incorporation of fas into pc and successive removal of desaturated fas from pc for eventual tag synthesis could be of great importance in determining the final fa composition of tag in flax. the two major pathways controlling the movement of fas into and out of pc are acyl editing and dag-pc interconversion via the action of pdct after de novo synthesis of dag through the kennedy pathway. this symmetrical interconversion of pc and dag liberates pc-derived dag with modified fas to be utilized by either dgat or pdat for tag production. the ability of pdct to catalyze the symmetrical interconversion between dag and pc through a phosphocholine headgroup exchange was first demonstrated in plants through the characterization of the a. thaliana rod mutant. pdct was found to act as a gatekeeper enzyme, providing a major path through which oleoyl moieties entered pc for desaturation at the sn- position. this mutation resulted in a significant decrease in pufa levels in seed tag compared to wt, demonstrating the importance of pdct in the efficient production of pufas in this species. indeed, in arabidopsis seeds it has been estimated that at least 40% of pufas in tag are derived through the action of pdct alone. furthermore, it has recently been shown that the collective action of pdct along with acyl-coa:lysophosphatidylcholine acyltransferases are responsible for the accumulation of nearly two-thirds of the c18-pufas in this species. recently, it has also been demonstrated that pdct is required for efficient hydroxy fatty acid accumulation in transgenic a. thaliana, demonstrating the importance of pdct in channelling modified fatty acids into tag. while pdct has been shown to play a key role in the production of tag enriched with modified fas, including pufas, in model plant species, this is the first study in which the importance of this enzyme from a crop plant specifically grown for its high levels of pufas has been analyzed. interestingly, results from a number of recent studies have suggested that arabidopsis is not the only plant species that utilizes pc-derived dag to produce pufa-rich tag or tag enriched with other modified fas. for example, it has been estimated that flax utilizes more than about 70% pc-derived dag to synthesize tag, which implies that the pdct-catalyzed reaction may be the main route of pc-derived dag synthesis in flax and may therefore be an important contributor to the high levels of pufas found in this crop species. in an initial attempt to provide evidence that this is, indeed, the case, we have isolated cdna from two flax pdct homologs that displayed high levels of sequence identity with one another. flax contains a number of highly homologous gene pairs encoding enzymes involved in oil biosynthesis, which is consistent with our results and may be the consequence of a putative whole-genome duplication that has been suggested to have occurred in this species. phylogenetic comparison of the two lupdct proteins with other higher plant pdcts, including functionally tested enzymes from a. thaliana and castor, indicated that they are more closely related to homologous proteins from plants such as spruce and castor than they are to that from a. thaliana. this has also been found to be the case for several other flax oil biosynthetic genes. the fact that the pdct homologs with the highest sequence identity to the two lupdct proteins tended to be from other high c18-pufa and unusual fa-producing plants such as sunflower, cotton and castor suggests that pdct likely provides an important function in the incorporation of modified fas into tag in many plant taxa. interestingly, pdct homologs have not been found to be present in certain plant species that accumulate conjugated fas, including bitter melon and tung tree, which implies that pdct may not be ubiquitously distributed throughout the plant kingdom. it would be interesting to compare flax pdct with chia pdct, if homologs are present, once the sequence information becomes available as chia has a close c18-pufa profile to flax. as is the case for atrod, lupdct and lupdct transcript levels were dramatically higher in developing flax embryos than in other parts of the plant. this increase in transcript levels, which peaked at approximately daa and declined to nearly undetectable levels by daa, correlates well with the timing of rapid oil accumulation in developing embryos of this species and provides strong evidence that the lupdct proteins are involved in the production of high levels of c18-pufas in flax. we also noted very low levels of lupdct and lupdct transcripts in vegetative tissues and flowers, which is in contrast to the expression of atrod in these tissues in a. thaliana. these findings imply that the lupdct enzymes may provide a secondary function of pufa enrichment in non-oil-accumulating tissues in addition to tag-rich embryos in flax. in agreement with this, the rod mutation in a. thaliana did not result in substantial fa compositional changes in leaf or root whereas pufa levels were substantially decreased in seeds. enzyme assays with microsomal preparations from s. cerevisiae cells expressing lupdct or lupdct demonstrated that both proteins possessed pdct activity in catalyzing both forward and reverse reactions. when assayed for the formation of dag from pc, a small quantity of tag was also produced, which likely resulted from the catalytic action of yeast tag synthesizing enzymes present in the microsomal fraction. interestingly, previous studies indicated that both atrod and rcrod exhibited high pdct activities at 15°c; however, both lupdct and lupdct exhibited extremely low activity at 15°c compared to 30°c. although enzymatic activity data was not available for atrod and rcrod at 30°c and different enzyme assay conditions were utilized in the various studies, these data indicate that the flax pdct proteins may possess different enzymatic properties than those of a. thaliana or castor. direct evidence for the role of lupdct enzymes in c18-pufa enrichment was obtained through the expression of the corresponding genes in fad2/fad3-expressing yeast, where major compositional changes were observed in the different lipid classes compared to yeast lacking the lupdct coding sequences. metabolic engineering of yeast with lufad and lufad3b expression cassettes provided an excellent system with which to study the effects of pdct expression on pufa accumulation in pc, dag and tag fractions without having to resort to the feeding of yeast cultures with la and ala, which are not produced naturally by s. cerevisiae. fa feeding in an attempt to introduce exogenous acyl moieties to yeast cells is often utilized in such situations, but fa uptake into yeast cells can at times lead to deleterious effects, making the system developed here a much more desirable option. when only the fads were expressed, pufas were observed in the pc fraction, with only minute quantities being present in dag and tag fractions. co-expression with each lupdct, however, resulted in a marked increase in pufa levels in both dag and tag fractions, providing further support for the premise that pdct plays a critical role in channelling pufas to dag, and subsequently tag, following production on pc. moreover, c18-pufa content in the pc fraction was also substantially increased at the concomitant expense of 18: taken together, these data suggest that pdct enhances the channelling of oleyol acyl moieties into pc by rapidly converting dag to pc, thus increasing substrate availability for fads. this dual mechanism of pdct appears to result in a synergy leading to increased pufa accumulation in this yeast system. these results were confirmed in a plant system, wherein both wild-type and rod mutant a. thaliana were transformed with vectors designed to express lupdct and lupdct respectively. in the case of the transgenic rod mutant plants, either lupdct or lupdct was capable of restoring pufa levels to more wt levels. there was some variation in the level of complementation of the fa composition in the transgenic lines, which was likely due to positional effects of transgene insertion; this is a common phenomenon in the generation of transgenic plants. similar variations among independent transformants were observed when the castor fatty acid hydroxylase gene was expressed in the a. thaliana fad2/fae double mutant or fatty acid elongase gene from nasturtium was overexpressed in wt a. thaliana. in line with results obtained with the rod mutant, expression of the two flax pdct genes in wt a. thaliana substantially increased pufa production in transgenic lines. on average, the proportion of c18-pufa increased by % and % in lupdct1- or lupdct2-expressing lines, respectively, at the concomitant expense of 18:. these data provide further support that pdct increases pufa accumulation by enhancing the channelling of monounsaturated acyl moieties through desaturation on pc and making available the resulting polyunsaturated acyl moieties for tag synthesis. compositional analysis of flax seed lipids has previously revealed that approximately 95% of its tag is enriched with pufas at any one of the sn-positions, whereas only approximately 40% of its tag contains c18-pufas at all three sn-positions, indicating that there is a vast potential to increase the proportion of pufa-rich tag in this species.  we have identified two novel pdct homologs from flax, which encode enzymes catalyzing increases in c18-pufa levels substantially in metabolically engineered yeast and transgenic a. thaliana seeds. these flax pdcts appear to play an important dual role in the determination of pufa content by efficiently channelling monounsaturated fas into pc for desaturation and moving the resulting pufas out of pc for subsequent use in tag synthesis. these results have distinguished pdct as an ideal candidate gene for future bioengineering of flax and other oil crops to produce improved levels of pufas for widespread use in industrial and nutraceutical applications. 
these flax pdct proteins appear to play an important dual role in the determination of pufa content by efficiently channelling monounsaturated fas into pc for desaturation and moving the resulting pufas out of pc for subsequent use in tag synthesis. in this study, we describe the isolation and characterization of two pdct genes from flax with very high nucleotide sequence identity whose deduced amino acid sequences exhibited approximately 55% identity with that of a. thaliana pdct. these results indicate that flax pdcts would be useful for bioengineering of oil crops to increase pufa levels for applications in human food and nutritional supplements, animal feed and industrial bioproducts. complementation studies with the a. thaliana rod mutant demonstrated that the flax pdcts were capable of restoring pufa levels in planta. furthermore, pufa levels increased in saccharomyces cerevisiae when the flax pdcts were co-expressed with fatty acid desaturases, fad and fad while seed-specific expression of lupdct and lupdct in a. thaliana resulted in % and % increases in c18-pufas, respectively, with a concomitant decrease in the proportion of oleic acid. the two novel pdct homologs from flax are capable of increasing c18-pufa levels substantially in metabolically engineered yeast and transgenic a. thaliana seeds. this polyunsaturated fatty acid is the major determinant for the quality of flax seed oil in food, nutraceuticals and industrial applications. the recently identified enzyme: phosphatidylcholine diacylglycerol cholinephosphotransferase, catalyzes the interconversion between phosphatidylcholine and diacylglycerol, and has been shown to play an important role in pufa accumulation in arabidopsis thaliana seeds. two flax pdct genes were identified using homology-based approach. flax is an agriculturally important crop with seed oil enriched in α-linolenic acid.
 flax is an agriculturally important crop with seed oil enriched in α-linolenic acid. this polyunsaturated fatty acid is the major determinant for the quality of flax seed oil in food, nutraceuticals and industrial applications. the recently identified enzyme: phosphatidylcholine diacylglycerol cholinephosphotransferase, catalyzes the interconversion between phosphatidylcholine and diacylglycerol, and has been shown to play an important role in pufa accumulation in arabidopsis thaliana seeds. methods two flax pdct genes were identified using homology-based approach.  in this study, we describe the isolation and characterization of two pdct genes from flax with very high nucleotide sequence identity whose deduced amino acid sequences exhibited approximately 55% identity with that of a. thaliana pdct. the genes encoded functionally active enzymes that were strongly expressed in developing embryos. complementation studies with the a. thaliana rod mutant demonstrated that the flax pdcts were capable of restoring pufa levels in planta. furthermore, pufa levels increased in saccharomyces cerevisiae when the flax pdcts were co-expressed with fatty acid desaturases, fad and fad while seed-specific expression of lupdct and lupdct in a. thaliana resulted in % and % increases in c18-pufas, respectively, with a concomitant decrease in the proportion of oleic acid.  the two novel pdct homologs from flax are capable of increasing c18-pufa levels substantially in metabolically engineered yeast and transgenic a. thaliana seeds. these flax pdct proteins appear to play an important dual role in the determination of pufa content by efficiently channelling monounsaturated fas into pc for desaturation and moving the resulting pufas out of pc for subsequent use in tag synthesis. these results indicate that flax pdcts would be useful for bioengineering of oil crops to increase pufa levels for applications in human food and nutritional supplements, animal feed and industrial bioproducts. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords α-linolenic acidlinum usitatissimumphosphatidylcholine-diacyglycerol interconversionatrod1pufasaccharomyces cerevisiaeissue-copyright-statement© the author 2015 