BACKGROUND
flax lignans are reported for a wide range of health benefits .
various plant species have been shown to produce different types of lignans including secoisolariciresinol diglucoside  known as the main lignan in flax  seed  <cit> .
lignans in flax seed are usually found in a glycosylated form, sdg, and ester-lined within oligomeric chains  <cit>  making a macromolecule  <cit> ; its monomeric aglycone  and intermediate monoglucoside  forms not being accumulated in the seed.
glycosylation leads to the structural complexity and diversity of phytochemicals  <cit> .
it ensures the stability and water solubility of plant natural products, reduces their reactivity or toxicity  <cit> , and eases the transport and storage in the cells  <cit> .
glycosylation is achieved by cazymes which include the glycosyltransferases  superfamily.
gts have been classified into  <dig> families, family  <dig> being referred to as uridine glycosyltransferases   <cit> .
in plants, ugts have a  <dig> amino acid characteristic signature box commonly known as plant secondary product glycosyltransferase  box  <cit>  and which role in ligand binding and catalysis has been documented in plants  <cit> .
ugts transfer udp-activated sugars such as udp-glucose to acceptor subtracts to form glucosides  <cit> .
in arabidopsis, more than  <dig> ugts have been reported and grouped into  <dig> sub-families based on sequence homology  <cit> .
in flax, barvkar et al.
<cit>  recently reported  <dig> ugts from the flax draft genome  <cit>  and organized them into  <dig> groups .
however, no functional characterization was provided.
concurrently to barvkar’s flax genome data mining study  <cit> , we cloned and characterized five family  <dig> ugt genes  and demonstrated that ugt74s <dig>  was the only one able to glucosylate seco by sequentially forming smg and sdg  <cit> .
further, by performing 3d modeling of the ugt74s <dig> protein followed by ligand docking, targeted site-directed mutagenesis, heterologous expression and enzyme assays, gln <dig> and ser <dig> were found to be essential for the glucosylation of smg into sdg, whereas trp <dig> and his <dig> appeared to be critical for ugt74s <dig> glucosylation activity toward seco in vitro  <cit> .
however, whether ugt74s <dig> was the only ugt achieving the seco glucosylation into sdg in the flax genome remained unknown.
gene duplication is a common feature of many plant genomes.
evidence for ancient whole genome duplication events have been reported for flax  <cit> , a finding supported by in-depth studies of duplication of fatty acid biosynthetic genes .
similar studies on the ugt family have not yet been conducted in flax, despite the reported ugt diversity  <cit> .
more specifically, the fate of ugt74s <dig> through the flax genome evolution and its relation to other ugts is unknown, as are the functional roles of its close relatives.
the objectives of this study were to further study the diversity and evolution of ugt genes in flax, identify ugts closely related to ugt74s <dig>  and assess their functionality through glucosylation activity assays towards seco for sdg formation.
methods
plant materials
flax  seeds of cv ac mcduff  <cit>  were planted in growing seasons 2008– <dig> at aafc harrington farm  and tissues were sampled as previously described  <cit> .
rna and dna isolation
total rna was extracted from developing flax seed, leaf and stem tissues using the trizol rna kit  as previously described  <cit> .
the extracted rna samples were further purified using the purelink™ rna mini kit , quantified using the qubit rna hs assay kit , and the quality was checked by agarose gel electrophoresis and experion rna analyzer  as previously described with slight modifications  <cit> .
genomic dna was extracted from flax leaves using the qiagen dneasy plant mini kit , quantified using the qubit dsdna br assay kit , and the quality was verified by agarose gel electrophoresis.
genome-wide mining and phylogenetic analysis of ugts
ugt74s <dig> , ugt74t <dig> , ugt89b <dig> , ugt94h <dig> , ugt712b <dig>   <cit>  and  <dig> previously identified flax ugts  <cit>  were used as queries to identify putative ugts from the  <dig>  annotated genes of the flax genome assembly  <cit>  using blast with an e-value of 1e- <dig>  the identified ugt candidates were further annotated by analyzing their gene structure and sequence similarity.
phylogenetic analysis of genome-wide ugts was performed using mega  <dig>   <cit> .
the protein sequences for  <dig> out of  <dig> ugts were found to be very short compared to others, probably truncated genes, and were excluded from the tree construction.
to get a general overview of the tree topology from as many ugts as possible, protein sequences of the remaining  <dig> ugt sequences were aligned in a first step using clustalw  <cit>  and a phylogenetic tree was constructed using the neighbor-joining  algorithm with  <dig> bootstrap replicates.
because ugts are characterized by a  <dig> amino acid pspg motif signature box  <cit>  known to be involved in substrate recognition and catalysis  <cit> , ugt candidate gene list was narrowed down to only those carrying a pspg motif.
the  <dig> conserved amino acids of the pspg motif and their 50 bp upstream and downstream sequences from  <dig> ugts were extracted.
these amino acid sequences were aligned with muscle  <cit>  and a phylogenetic tree was constructed using the maximum parsimony analysis method with  <dig> bootstrap replicates.
gene duplication analysis and ugt divergence
gene duplication analysis was conducted by self-blast of the entire putative ugt database using a threshold e-value of 1e- <dig>  <cit> .
pairs of ugt genes returning reciprocal top hits of each other and having identical or highly similar gene structure were declared duplicate copies.
the identified ugt genes were assigned to the  <dig> flax linkage groups, each representing a chromosome, based on the sorted flax genome sequence  <cit> .
duplication and divergence times were determined from the ratio of calculated non-synonymous substitution  over the calculated synonymous substitution  values  <cit> .
briefly,  <dig> full-length cdna sequences  were aligned in mega v <dig>   <cit>  and the ka/ks ratio was calculated  <cit> .
ks values lower than  <dig>  were removed from the dataset as suggested by sveinson et al.
<cit> .
furthermore, ks values larger than  <dig> were excluded to minimize the saturation effects  <cit> .
the evolutionary distance between pairs of genes was determined based on the ks corrected with the nei-gojobori model of nucleotide evolution which accounts for multiple substitutions per site 26].
the divergence  of a pair of duplicated genes was converted into duplication or divergence time  in million years  following the equation t = k// <dig>  where r is the substitution rate of  <dig>  × 10− <dig> substitutions per synonymous site per year  <cit> .
functional analysis of ugt74s <dig> and its closely related ugts lus <dig> and lus10014148
to assess the functionality of the two ugts most closely related to ugt74s <dig>  gene splicing of genomic dna was performed to recover the full length coding sequence  of lus <dig> and lus <dig> which have short single introns  <cit> .
tissue-specific and heterologous gene expression studies of these two genes were undertaken alongside ugt74s <dig>
lus <dig> and lus <dig> gene splicing from genomic dna
extension of overlapping gene segments by pcr is a simple technique for gene splicing  .
to delete the intron from the genomic dna through joining the two exons together, genomic sequences corresponding to lus <dig>  and lus <dig>  were used to design gene specific primers .
for each sequence, an external flanking primer pair was designed from the 5′ and 3′ end of the cds, referred to as exonic forward and reverse primers.
additionally, two 30-nucleotide internal primers were designed.
the first internal primer called exon i-r consisted of the reverse complement to the first  <dig> nucleotides in the sense strand of exon ii  and the last  <dig> nucleotides of the reverse strand of the exon i .
the second internal primer called exon ii-f consisted of the last  <dig> nucleotides in the sense strand of exon i  and the first  <dig> nucleotides of exon ii sense strand  as previously described  .
the gene splicing was conducted in three steps.
the initial step was the amplification of the two exons using the 5′ exonic i forward and exon i-r in a first reaction, and exon ii-f and 3′ exonic ii reverse in a second reaction.
dna was diluted and 3 μl aliquots  were used as template in subsequent pcr reactions.
pcr cycles consisted of an initial denaturation at 94 °c for 2 min followed by 30 cycles of 94 °c for 30 s, 55 °c for 45 s and 72 °c for 90 s prior to a final extension at 72 °c for 10 min.
aliquots of 6 μl of the pcr products were resolved on 1% agarose gels stained with ethidium bromide.
the amplified exon fragments i and ii were purified using the qiaquick pcr purification kit , diluted  <dig> folds and 4 μl aliquots of each purified product were pooled and used as template in the second gene splicing step where full length templates for each gene were generated.
during pcr, overlapping strands of the two intermediate products form a duplex, providing taq with a free 3′end for extension and a single strand for polymerization, thereby generating a single full-length product.
this pcr reaction consisted of 10 cycles at 95 °c for 30 s, 25 °c for 45 s, and 72 °c for 90 s. the resulting pcr product was diluted  <dig> folds and 3 μl aliquots were used as a template in a third and final pcr reaction conducted at 94 °c for 3 min followed by 30 cycles of 94 °c for 30 s, 55 °c for 45 s and 72 °c for 90 s, followed by a final extension at 72 °c for 10 min.
the fused full length fragments were purified with the qiaquick gel extraction kit , cloned into topo ta vector and transformed in e. coli  similar to previous descriptions  <cit> .
ugt74s <dig>  lus <dig> and lus <dig> gene expression in different flax tissue
to quantify the gene expression levels of lus <dig> and lus <dig> in different flax tissues , real-time pcr primers were designed from the 3′ end of lus <dig>  lus <dig>  ugt74s <dig>  and a ribosomal rna , the latter being used for data normalization  as performed in a previous report  <cit> .
first strand cdna synthesis and real-time pcr conditions were as previously described  <cit> .
the output gene expression data were generated using the 2-∆∆ct method  <cit> , and the results were presented as fold changes expression relative to that of 0 day after anthesis  for all tissues  <cit> .
cloning and heterologous expression
the topo ta cloned full length fragments for lus <dig> and lus <dig> were sequenced for confirmation.
flanking primers carrying restriction enzyme sites  were designed to shuttle the full length lus <dig> and lus <dig> into the yeast  expression vectors pyes2/nt c and b , respectively.
the pyes2/nt c construct carrying the full-length cdna for ugt74s <dig> was previously described  <cit> .
all three constructs were transformed in the yeast  strain invsc <dig> following manufacturer’s instructions  and single transformant colonies were cultured, induced, harvested and lysed as previously reported  <cit> .
protein expression for each of the three ugts, including ugt74s <dig> as control, was monitored by western blot using equal amount of proteins and diluted antibodies raised against the xpresstm epitope present between the 6× histidine tag and the multiple cloning site as previously described  <cit> .
enzyme assays and reaction products determination and quantitation
to determine whether the two ugts  could glucosylate seco into sdg as reported before for ugt74s <dig>  <cit> , the purified native proteins obtained from the yeast cultures expressing ugt74s <dig>  lus <dig>  or lus <dig> were reacted with seco , the only substrate relevant to this study, in the presence of udp-glucose  <cit>  or udp-galactose.
the 100 μl reaction mixture composition and reaction incubation conditions were exactly as described in our previous report  <cit> .
for separation, identification, and quantitation of the reactants and their products, a waters h-class acquity uplc system  equipped with a tqd tandem mass spectrometer  and a waters csh c <dig> column  were used as previously described  <cit> , albeit with slight modifications.
in addition to ms <dig> scanning mode, selected ion recording  spectra were collected to improve the detection sensitivity of seco, smg, sdg and seco monogalactoside .
the capillary voltage was set at 3 kv, the extractor at 3 v, and rf lens at  <dig>  v. the chromatographic parameters followed a binary gradient system composed of 3% formic acid in water  and acetonitrile , varying according to the following program: t <dig>  a = 98%; t1 =  <dig>  min, a = 0%; t2 = 6 min, a = 0% isocratic; t3 = 7 min, a = 98%; t4 = 8 min, a = 98% isocratic.
peaks detected at 280 nm, indicative of phenolic compounds, and were validated using authentic standards  purchased from chromadex  as described in  <cit> .
a standard curve for sdg was created using the sdg standard described above.
purified smg standard was prepared in-house as previously described  <cit> .
all reactions were carried out in triplicates and the data are presented as the means ± standard deviations.
a one-tailed student’s t-test was performed to test the statistical significance of metabolite production levels by ugt74s <dig>  lus <dig> and lus <dig>  <cit> .
RESULTS
ugt gene duplication and diversity in the flax genome
genome-wide mining for ugt genes identified  <dig> non-redundant sequences dispersed on all  <dig> flax chromosomes .
with a mere ten sequences, chromosomes  <dig> had the fewest ugt genes whereas chromosome  <dig> harbored the most with  <dig> .
a total of  <dig>  of the identified ugt genes were duplicated, of which  <dig>  corresponded to intra-chromosomal duplications .
fourteen  of those occurred on chromosome  <dig> whereas no intra-chromosomal duplications were observed on chromosomes  <dig>   <dig>   <dig>   <dig> and  <dig> .
ugt74s <dig> was located on chromosome  <dig> and displayed no evidence of gene duplication.
phylogenetic analysis from the  <dig> out of the  <dig> ugts, excluding  <dig> truncated sequences, revealed four main clusters  and  <dig> sub-clusters.
cluster  <dig> was the most diverse, accounted for 53%  of the ugt genes, and included eight  of the  <dig> groups previously reported  <cit> .
the fifteenth new group identified in this study was hereafter named “o” .
ugt74s <dig> clustered within group l as previously reported and belonged to a small gene family of five ugts including lus <dig> , lus <dig> , lus <dig> , lus <dig>  and lus <dig> , supported by a 53% bootstrap value and hereafter called the lus <dig> subclade.
two members of this subclade, lus <dig> and lus <dig>  were shown to be duplicated genes located on chromosomes  <dig> and  <dig>  respectively, and were the most closely related to ugt74s <dig> .
this small gene family clustered with a sister subclade, herein called lus10006352/lus <dig> subclade, that was supported with a 51% bootstrap value and which included lus <dig> , lus <dig> , lus <dig> and lus <dig> .
to further assess the consistency of the observed groupings, a new tree was constructed using a conserved region from  <dig> ugt sequences.
consistent with the first large phylogenetic tree, this reduced tree showed, with relatively high bootstrap values , the same configuration and groupings as previously reported  <cit> .
however, the a, b, c, e and m groups were not as well resolved as in the previous tree.
in this second phylogenetic tree, group “l” was well supported by an 89% bootstrap value and the lus <dig> subclade comprised lus <dig> , lus <dig> , lus <dig> , lus <dig> and lus <dig> while the lus10006352/lus <dig> subclade included lus <dig> , lus <dig>  and lus <dig> , thus providing more confidence in our phylogeny for inferring the evolutionary history of the ugts.
following udp-glucosyltransferase nomenclature, lus <dig>  lus <dig>  lus <dig>  lus <dig>  lus100lus <dig>  lus <dig> and lus <dig> were annotated as members of ugt74s <dig>  ugt74s <dig>  ugt74s3- <dig>  ugt74s <dig>  ugt74s <dig>  ugt74s <dig>  ugt74s5- <dig> sub-families, respectively.
therefore, lus <dig> and lus <dig> will be hereafter designated by their ugt nomenclature: ugt74s <dig> and ugt74s <dig>  alongside with ugt74s <dig> .fig.
<dig> phylogenetic relationship among  <dig> flax ugts based on the conserved pspg motif and its surrounding regions.
the different groups are indicated by alphabetic letters a-o around and inside the circular tree.
ugt74s <dig> belongs to a small gene family highlighted in red within group l and is marked with an asterisk
ugt gene divergence
we examined ugt sequence divergence by determining the ka/ks ratio among  <dig> ugt sequences carrying the pspg motif.
after ks calculation and data filtration,  <dig>   ugt gene pairs with ks ranging between  <dig>  and  <dig>  were retained.
overall, 82% of the gene pairs had ks values < <dig>  including 45% with ks values < <dig> .
the ks mean value was  <dig>  and the distribution peaked at  <dig>  .
similarly,  <dig> and 71% of the ugt gene pairs had ka/ks <  <dig>  and ka/ks <  <dig>  respectively.
less than 1% of the gene pairs had ka/ks =  <dig> .
the distribution frequency of the individual ka/ks values peaked at  <dig>  .fig.
<dig> frequency distribution of ks values and ka/ks ratios among  <dig>  ugt gene pairs obtained from  <dig> ugt sequences
the gene pairs involving the sdg lignan-forming gene ugt74s <dig>  had ks <  <dig> and ka/ks <  <dig> in a proportion of  <dig> and 56%, respectively.
within the lus <dig> and lus10006352/lus <dig> subclades, all ks derived from gene pairs involving ugt74s <dig> ranged from  <dig>  to  <dig> .
in contrast, only two of the eight gene pairs  had ka/ks <  <dig>  the remaining 75% of the gene pairs displayed ka/ks > <dig>
the divergence time between pairs of the  <dig> ugt genes ranged from  <dig>  to  <dig>  mya .
the divergence time between the  <dig> genes clustered in group l spread from  <dig>  to  <dig> mya .
within the lus <dig> and lus10006352/lus <dig> subclades, lus <dig>  and lus <dig>  appeared to be the most ancient, diverging from one another approximately  <dig> mya.
ugt74s <dig>  may have diverged from the other seven ugts clustered in these two subclades around 16– <dig> mya.
based on our inferences, ugt74s <dig>  probably duplicated from ugt74s <dig>  ~ <dig> mya.
the evolutionary relationships between ugt74s <dig> , lus <dig>  ugt74s <dig>  and ugt74s5- <dig>  were found to be complex.
the duplication study identified both ugt74s <dig>  and ugt74s5- <dig>  as duplicates of ugt74s <dig>  on one hand, and also ugt74s <dig>  as a duplicate of lus <dig>  on the other hand, with duplication/divergence times of about  <dig> and  <dig>  mya for ugt74s5/ugt74s5- <dig> and lus10006720/ugt74s <dig>  respectively.
however, the divergence time for the ugt74s5/ugt74s <dig> pair could not be determined using the filtration criteria set .table  <dig> pairwise matrix of flax ugt divergence time in mya for the  <dig> ugt genes clustered lus <dig> and lus10006351/lus <dig> subclades within the group “l” of the phylogenetic tree.
the most recent and ancient divergence times are highlighted in blue and green, respectively
n/a, denotes that the gene pairs did not meet the filtration criteria set in methods
ugt74s <dig> , ugt74s <dig> , and ugt74s <dig>  are indicated by an asterisk
structural gene organization of ugt74s <dig> and its closest relatives
ugt74s <dig> is a 1407 bp gene with a single intron of 739 bp  <cit> .
to assess the functionality of the two ugts that were the most closely related to ugt74s <dig>  i.e., ugt74s <dig> and ugt74s <dig>  we obtained their full coding regions by gene splicing.
ugt74s <dig> generated a spliced fused cds of 1416 bp by joining the exons of  <dig> and 738 bp from a 1492 bp genomic dna fragment.
similarly, a spliced fused cds of 1371 bp was obtained by joining the exons of  <dig> and 728 bp from a 1464 bp genomic dna product for ugt74s <dig> .
the amplified products were of expected sizes as estimated from the original sequences in the ncbi database  and in phytozome .
in this study, ugt74s <dig> and ugt74s <dig> had  <dig> and 93 bp introns, respectively and their fused spliced coding sequences were confirmed as 100% identical to those already reported .
it is noteworthy to mention that ugt74s <dig>  corresponding to ncbi accession jn <dig> , was reported by barvkar et al.
<cit>  with a 27 bp truncation at its 5′ end .
this truncation was detected by comparing the amplified genomic and spliced fused sequences of ugt74s <dig> with that of jn <dig>   <cit>  and of lus <dig> g from the flax draft genome .
in ugt74s <dig>  the intron is located between nucleotide positions  <dig> and  <dig>  whereas it is located between nucleotide position  <dig> and  <dig> of ugt74s <dig> .
thus, the full genomic sequence size for ugt74s <dig> is 1464 bp and not 1437 bp as previously reported  <cit> .
the spliced full length cdnas for ugt74s <dig>  and ugt74s <dig>  were predicted to code for  <dig> and  <dig> amino acids, respectively, and as also observed in this study .
both showed nearly 62% identities to ugt74s <dig> and displayed 80% identity to one other at the amino acid levels.
the conserved  <dig> amino acids pspg motif  was located between amino acids  <dig> and  <dig> in ugt74s <dig> and between  <dig> and  <dig> in ugt74s <dig>  the pspg motif of ugt74s <dig> and ugt74s <dig> shared  <dig> and 80% identity with ugt74s <dig>  respectively .
four amino acids found to be conserved within the pspg motif of ugt74s <dig> and ugt74s <dig> were substituted in ugt74s1: e339q, a345k, m362l and w374c.
ugt74s <dig> had three unique amino acids changes  and ugt74s <dig> had two .
ugt74s <dig>  ugt74s <dig> and ugt74s <dig> gene expression in different flax tissues
real time gene expression levels of ugt74s <dig> and ugt74s <dig> were similar in developing seeds and leaves but were markedly contrasting in stems and roots.
moreover, their expression patterns differed from that of ugt74s <dig> in all tissues .
in developing seeds, ugt74s <dig> expression mimic a bell curve pattern, peaking at  <dig> daa  as previously observed  <cit> .
at eight and  <dig> daa, ugt74s <dig> and ugt74s <dig> expression levels were 3– <dig> times lower than ugt74s <dig> whereas their expression levels were higher  than that of ugt74s <dig> at maturity .
in contrast, ugt74s <dig> was barely expressed in root and stem tissues where ugt74s <dig> and ugt74s <dig> were highly expressed.
expression of all three ugt genes was low in leaves .fig.
<dig> expression profile for ugt74s <dig>  ugt74s <dig>  and ugt74s <dig>  in developing seeds sampled at  <dig>   <dig> and 16 days after anthesis  and at maturity , as well as leaf, root and stem tissues of flax cultivar ac mcduff.
a, ugt74s1; b, ugt74s <dig> ; and c, ugt74s <dig>
heterologous protein expression and enzyme activity
in this study, we wanted to assess whether the ugt74s <dig> and ugt74s <dig> enzymes had the ability to glycosylate seco as reported for ugt74s <dig>  <cit> .
thus, protein expression and glycosylation activity toward seco were assessed for ugt74s <dig> and ugt74s <dig> in comparison with ugt74s <dig>  the full-length cdnas for ugt74s <dig> and ugt74s <dig> were expressed in yeast as previously described for ugt74s <dig>  the western blot analysis showed that all three proteins highly expressed after 8 h of induction with 2% galactose .
ugt74s <dig> and ugt74s <dig> showed strong intensity signals comparable to the  <dig>  kda of ugt74s <dig> previously reported  <cit>  and produced  <dig>  and  <dig>  kda proteins, respectively, in agreement with their predicted sequences .fig.
<dig> western blot of ugt74s <dig>  ugt <dig> 4s <dig>  and ugt74s <dig>  and reaction products observed from enzyme assays using purified native enzymes.
a, western blot of the histag-purified ugt74s <dig>  ugt74s <dig> and ugt74s <dig> probed using antixpresstm antibody.
the  <dig> and 50 kda protein molecular marker sizes corresponding to the western c precision plus protein marker mixed with conjugant  are pinpointed.
b, reaction products formed by each of ugt74s <dig>  ugt74s <dig> and ugt74s <dig> after enzyme reaction with seco as substrate and udp-glucose or udp-galactose as sugar donor.
sdg secoisolarciresinol diglucoside, smg secoisolariciresinol monoglucoside, smgal secoisolariciresinol monogalactoside.
c. uplc chromatograms  showing the reaction products of 50 μg of purified proteins for the three ugts using seco as substrate and udp-glucose as sugar donor.
insets show 10× magnification to visualize trace peaks of smg in ugt74s <dig> and ugt74s <dig> reaction products.
appreciable accumulation of sdg  and smg  was only observed for ugt74s <dig>  the unreacted seco  is present in all three chromatograms
to determine the glucosylation activity of ugt74s <dig> and ugt74s <dig>  enzyme assays were performed using the purified proteins, with ugt74s <dig> as a positive control .
although ugt74s <dig> and ugt74s <dig> protein expression was similar to ugt74s <dig> , their glucosylation activity towards seco was seven fold less and both failed to produce sdg under our experimental conditions .
no significant difference  in glycosylation activity was observed between ugt74s <dig> and ugt74s <dig> towards seco.
by using udp-galactose as an alternative sugar donor for the production of seco glycosides, all three proteins formed trace amount of seco monogalactosides .
but no seco digalactosides were detected with any of the three enzymes tested .
no difference was observed among all three proteins with regards to the smgal levels when udp-galactose was used as alternate sugar donor.
discussion
ugts are a large and complex family of enzymes that catalyze glycosidic bond formation  <cit> .
flax ugt74s <dig> has recently been reported as an enzyme sequentially glucosylating seco into its monoglucoside smg and diglucoside sdg when expressed in yeast  <cit> .
to get more insights into the exclusivity of ugt74s <dig> to achieve seco glucosylation into sdg in flax, genome-wide mining, tissue-specific and heterologous gene expression, and enzyme activity assays were conducted.
the study confirmed our previous findings  <cit>  and further shows that ugt74s <dig> may be the key enzyme controlling seco glucosylation in flax, although ugt74s <dig> and ugt74s <dig> may also contribute to a minor extent in supplying smg as substrate to ugt74s <dig> for the second glucosylation step.
a total of  <dig> ugts were found dispersed throughout the ~373 mb flax genome  <cit> , corresponding to a gene density of  <dig>  ugt/mb, which is similar to arabidopsis  but higher than rice , apple , grapevine , poplar , soybean  and sorghum .
the percentage of ugts among the total predicted genes in flax is  <dig> %, higher than previously reported in dicots  and falls in the same range as monocots   <cit> .
gene duplication is a common phenomenon in eukaryotes, such as plants  <cit> .
it leads to genetic novelty and diversity, evolution, and functional diversification , phenomena that are required by vascular plant species for adaptation and plasticity  <cit> .
paleoploidy events including whole genome duplications have been described in the genus linum  <cit>  and are considered key drivers of gene duplication .
previous studies predicted  <dig>  genes in the flax genome, of which  <dig> genes were predicted to be duplicated  <cit> .
of the  <dig> ugts identified in this study,  <dig>  were found to be duplicated and accounted for  <dig> % of all duplicated genes of the flax genome.
ks has been previously used as a time indicator for duplication events, and its frequency distribution was reported to reflect the relative time of genome duplication  <cit> .
accordingly, the ks calculated from the ugt gene pairs peaked at  <dig> , and ranged from  <dig>  to  <dig> , indicating ancient duplications  <cit>  during ugt evolution.
using the ks values, the divergence among the pairs of  <dig> genes was estimated to have occurred  <dig> – <dig>  mya.
likewise, the divergence time between the  <dig> genes clustered in the l group spans from  <dig>  to  <dig> mya.
these results suggest that some flax ugt duplication events may have taken place prior to the most recent whole genome duplication estimated at 5– <dig> mya  <cit> .
our ka/ks analyses showed that 71% of the ugt gene pairs had ka/ks <  <dig>  indicating that most of the flax ugts have evolved under a purifying  selection  <cit> .
about a quarter  of the gene pairs had ka/ks >  <dig>  thus being under positive selection pressure whereas  <dig> % of the ugt genes found to be under neutral selection  <cit> .
in our study, full protein sequences carrying pspg motif were used to calculate ka and ks.
similarly, full length gene sequences have been used to determine the ks/ka values for the wheat and rice homologues  <cit> .
whether potential biases may be associated with the conserved domain’s own divergence rate, it is reasonable to assume that such bias, if any, should be evenly distributed and minimal because all the considered sequences had the pspg motif and, thus, would not affect to the drawn conclusions.
despite the high rate of ugt duplication in flax, ugt74s <dig> gene appeared as a single copy gene located on linkage group  <dig> .
this gene was found to be involved in  <dig> gene pairs based on the criteria set in this study, of which 56% had ka/ks <  <dig> and 44% with ka/ks >  <dig>  almost a 50/ <dig> ratio for negative and positive selection.
when a single member of a gene family is undergoing positive selection, it shows high ka/ks ratios when paired with all members of its family  <cit> .
this fact was substantiated within the lus <dig> and lus10006352/lus <dig> subclades where 75% of the gene pairs involving ugt74s <dig> had ka/ks > <dig>  indicating that ugt74s <dig> is under positive selection and, as such, it is experiencing reduced selective constraints  <cit>  to remain a singleton  <cit> .
positive selection has been reported for ugt73c <dig> and ugt73c <dig>  playing a role in saponin-mediated insect resistance  <cit> , and lignans, which have also been reported to take part in plant defense against insect  <cit> .
it is possible that ugt74s <dig> was duplicated during one or both of the proposed ancient duplication events and, that its paralog were subsequently deleted through gene loss as previously reported  <cit> .
single copy genes have been evoked to play key roles in maintaining essential metabolic processes in plants through macromolecular complex formation  <cit> .
ugt74s <dig> may have been maintained as a single copy gene in flax for seco glucosylation into sdg lignans during adaptive divergence as was reported for udp-glucose pyrophosphorylase in sorghum  <cit> .
phylogenetic studies of ugts have previously described  <dig> and  <dig> distinct groups using  <dig> ugt amino acid sequences from arabidopsis  <cit>  and  <dig> ugts from nine higher plants  <cit> , respectively.
in flax, barvkar et al.
<cit>  described  <dig> groups based on  <dig> ugts.
the  <dig> sub-clusters observed in the current study included the  <dig> groups previously reported in flax  <cit>  and identified a new group named “o”.
the grouping observed here is comparable to the 14– <dig> groups previously reported  <cit>  and highlights the diversity of ugts in flax, possibly through concerted evolution, a phenomenon also known in other higher plants  <cit> .
duplicated genes may face three different fates: nonfunctionalization in which one copy becomes silenced; neofunctionalization where one copy acquires a novel, beneficial function while the other copy retains the original function; or subfunctionalization where both copies become partially compromised by the accumulation of mutations  <cit> .
ugt74s <dig> and ugt74s <dig>  which originated from a duplication event, were the closest to ugt74s <dig> that glucosylates seco into sdg  <cit> .
because high sequence similarity does not necessarily translate into similarity in expression and function, ugt74s <dig> and ugt74s <dig> tissue-specific expression and protein functionality towards seco were investigated.
contrary to ugt74s <dig>  ugt74s <dig> and ugt74s <dig> were barely expressed in the seeds at 8– <dig> daa developmental stages, when sdg lignan synthesis increases rapidly  <cit> .
but, these  <dig> duplicated genes were more expressed in vegetative tissues and in mature seeds  than ugt74s <dig>  and showed different expression patterns, perhaps because of their tissue-specific promoters or their neofunctionalization after the duplication events  <cit> .
the reported data suggest that these  <dig> genes may play major roles in other biological processes yet to be elucidated, as opposed to seco glucosylation into sdg.
high protein expression levels were achieved in the heterologous yeast expression system but under the conditions described in this study, ugt74s <dig> and ugt74s <dig> were unable to convert seco into sdg.
however, they were able to produce small amounts of smg with an overall glucosylation activity towards seco seven times lower than ugt74s <dig> suggesting that ugt74s <dig> and ugt74s <dig> proteins may prefer a different substrate.
in contrast, ugt74s <dig> consistently produced higher amounts of sdg along with small amounts of smg and, its gene was more highly expressed in developing seeds where sdg lignans accumulate as previously reported  <cit> .
these observations suggest that ugt74s <dig> may be the key functional player in seco glucosylation into sdg, due to its ability to achieve both required glucosylation steps; first glucosylating seco into smg and then smg into sdg .
additionally, mutations in ugt74s <dig> lead to reduced or abolished sdg production in vitro  <cit> , further demonstrating its essential function.
while not all the  <dig> ugts reported in this study were biochemically evaluated for their ability to glucosylate seco, the finding that ugt74s <dig> and ugt74s <dig>  produced smg from seco at a very low rates and failed to form any sdg, indicates that these two genes are not as essential as ugt74s <dig>  which performs both required steps  <cit> .
although involvement of other cryptic ugts in the seco glucosylation process leading to sdg has not been exhaustively ruled out, one may question the evolutionary advantages of maintaining redundant genes and duplicated pathways for the same biochemical function.
moreover, recent evidence coming from a follow-up study conducted in our laboratory and focusing on tilling of ethyl methane sulfonate  flax mutant lines support the idea that ugt74s <dig> is unique in controlling seco glucosylation into sdg .
in the current study, ugt74s <dig> and ugt74s <dig> were able to glycosylate seco with udp-glucose or udp-galactose at low efficiency, forming smg or smgal respectively, under the described conditions, but failed to do so for sdg as did ugt74s <dig>  and therefore, no further biochemical characterizations or optimizations were performed for these two enzymes.
indeed, if ugt74s <dig> and ugt74s <dig> were able to glycosylate smg into sdg, even at a low rate, one would expect to observe trace sdg peak using the very sensitive ms detection methods, even under standard conditions, which would have warranted further biochemical optimization of the enzyme reactions.
however, this was not the case under our experimental conditions, suggesting that these two proteins may not have the ability to perform the second glycosylation step.
these enzyme activity data are in line with the conclusions drawn from the differential temporal gene expression data in the developing seeds, and reinforce the idea that ugt74s <dig> and ugt74s <dig> may play other roles in different metabolic processes apart from sdg lignan glucosylation.
the role played by the pspg motif in ligand binding and catalysis has been well-documented in plants  <cit> .
the variations within the pspg observed between ugt74s <dig> and its two closely related ugt genes may be responsible for the differences in their substrate specificity as previously reported  <cit>  and may account for the observed differential functionality.
CONCLUSIONS
ugts form a large gene family where the majority of the members are duplicated in the flax genome and subject to negative or purifying selection pressures whereas ugt74s <dig> is a singleton that has evolved under positive selection pressure.
flax ugt74s <dig>  and ugt74s <dig>  were the most closely related to ugt74s <dig> but their encoded proteins were unable to glycosylate smg into sdg.
whereas not all ugts in the genome were biochemically tested in this study, it is unlikely that other redundant ugt genes having the same roles in smg glucosylation as ugt74s <dig> would be maintained without a specific evolutionary advantage.
however, the possibility of cryptic ugt functionality cannot be totally ruled out.
nonetheless, the current study provides additional insights into ugt gene duplication events in flax and could be of interest to plant evolutionary biologists, breeders and plant natural product chemists.
additional files
additional file 1: figure s <dig>  schematic representation of gene splicing pcr .
additional file 2: table s <dig>  primers used for pcr mediated splicing, cloning and real-time gene expression of lus <dig> and lus <dig>
additional file 3: figure s <dig>  circos map of  <dig> ugts on the  <dig> flax chromosomes.
lines in the center of the map join duplicates.
scale for each chromosome is indicated in mb.
ugt74s <dig>  ugt74s <dig>  and ugt74s <dig>  are marked with asterisks.
additional file 4: table s <dig>  list, location and coordinates of the  <dig> ugts found in the flax genome.
ugt locus corresponding to ugt74s <dig> is highlighted in bold red.
additional file 5: figure s <dig>  distribution of ugt genes on the  <dig> chromosomes in the flax genome.
the term “first and second copy” is arbitrary and was used only for graph presentation purpose.
the fifteen chromosomes in the flax genome are indicated by lu <dig> to lu <dig>
additional file 6: figure s <dig>  phylogenetic relationship among  <dig> flax ugts and depicting  <dig> groups.
sixty-eight ugts with relatively short sequences were excluded.
the four main clusters are indicated by alpha numeric numbers 1– <dig> and the different groups are indicated by alphabetic letters a-o around and inside the circular tree.
ugt74s <dig> belongs to a small gene family highlighted in red within group l and is marked with an asterisk.
additional file 7: table s <dig>
ka, ks, ka/ks and divergence time for  <dig>  ugt genes pairs after filtration.
additional file 8: table s <dig>  pairwise matrix of flax ugt divergence time in mya for  <dig> genes clustered in group “l” of the phylogenetic tree.
the ugts found in lus <dig> and lus10006351/lus <dig> subclades are highlighted in green.
the most recent and ancient divergence times are highlighted in blue and yellow, respectively.
additional file 9: figure s <dig>  comparison of genomic dna and full length coding regions for lus <dig>  and lus <dig>  as obtained by gene fusion pcr.
a, amplified exon i  and exon ii  of lus <dig> were fused to make the gene fusion product and compared to the genomic dna amplicon; b, amplified exon i  and exon ii  of lus <dig> were fused and similarly compared to its genomic dna amplicon.
molecular ladder sizes are indicated by arrows.
additional file 10: figure s <dig>  sequence alignment between the fused spliced cds from lus <dig>  and lus <dig>  and their respective corresponding genomic and transcript sequences reported in ncbi and phytozome.
a, sequence alignment of the fused spliced lus <dig>  cds with its amplified genomic region before splicing , its corresponding genomic sequences reported in ncbi and phytozome , and its transcript sequence from phytozome .
the genomic sequences show the 76 bp intron located between nucleotide positions  <dig> and  <dig> whereas the intron is missing in the published transcript sequence and in the fused spliced cds reported in the current study; b, sequence alignment of the fused spliced lus <dig>  cds with its amplified region before splicing , its corresponding genomic sequences reported in ncbi and phytozome , and its transcript sequence from phytozome .
the genomic sequences show the 93 bp intron located between nucleotide position  <dig> and  <dig> whereas the intron is missing in the published transcript sequence and in the fused spliced cds reported in the current study.
additional file 11: figure s <dig>  protein sequence alignment between ugt74s <dig>  lus <dig>  and lus <dig> .
the pspg region is indicated by a red box and where amino acid variations between the three proteins appear as a white indentation in the black box representing the consensus sequence.
all amino acid positions were numbered according to their location in ugt74s <dig>
abbreviations
cdscoding sequence
daaday after anthesis
emsethyl methane sulfonate
kanon-synonymous substitution
kssynonymous substitution
msmass spectrometry
myamillion year ago
ncbinational center for biotechnology information
pspgplant secondary product glycosyltransferase
sdgsecoisolariciresinol diglucoside
secosecoisolariciresinol
smgsecoisolariciresinol monoglucoside
smgalsecoisolariciresinol monogalactoside
tillingtargeted induced local lesion in genome
udpuridine diphosphate
ugturidine glycosyltransferases
uplcultra performance liquid chromatography

 flax lignans are reported for a wide range of health benefits. various plant species have been shown to produce different types of lignans including secoisolariciresinol diglucoside known as the main lignan in flax seed. lignans in flax seed are usually found in a glycosylated form, sdg, and ester-lined within oligomeric chains making a macromolecule; its monomeric aglycone and intermediate monoglucoside forms not being accumulated in the seed. glycosylation leads to the structural complexity and diversity of phytochemicals. it ensures the stability and water solubility of plant natural products, reduces their reactivity or toxicity, and eases the transport and storage in the cells. glycosylation is achieved by cazymes which include the glycosyltransferases superfamily. gts have been classified into families, family being referred to as uridine glycosyltransferases. in plants, ugts have a amino acid characteristic signature box commonly known as plant secondary product glycosyltransferase box and which role in ligand binding and catalysis has been documented in plants. ugts transfer udp-activated sugars such as udp-glucose to acceptor subtracts to form glucosides. in arabidopsis, more than ugts have been reported and grouped into sub-families based on sequence homology. in flax, barvkar et al. recently reported ugts from the flax draft genome and organized them into groups. however, no functional characterization was provided. concurrently to barvkar’s flax genome data mining study, we cloned and characterized five family ugt genes and demonstrated that ugt74s was the only one able to glucosylate seco by sequentially forming smg and sdg. further, by performing 3d modeling of the ugt74s protein followed by ligand docking, targeted site-directed mutagenesis, heterologous expression and enzyme assays, gln and ser were found to be essential for the glucosylation of smg into sdg, whereas trp and his appeared to be critical for ugt74s glucosylation activity toward seco in vitro. however, whether ugt74s was the only ugt achieving the seco glucosylation into sdg in the flax genome remained unknown. gene duplication is a common feature of many plant genomes. evidence for ancient whole genome duplication events have been reported for flax, a finding supported by in-depth studies of duplication of fatty acid biosynthetic genes. similar studies on the ugt family have not yet been conducted in flax, despite the reported ugt diversity. more specifically, the fate of ugt74s through the flax genome evolution and its relation to other ugts is unknown, as are the functional roles of its close relatives. the objectives of this study were to further study the diversity and evolution of ugt genes in flax, identify ugts closely related to ugt74s and assess their functionality through glucosylation activity assays towards seco for sdg formation. methods plant materials flax seeds of cv ac mcduff were planted in growing seasons 2008– at aafc harrington farm and tissues were sampled as previously described. rna and dna isolation total rna was extracted from developing flax seed, leaf and stem tissues using the trizol rna kit as previously described. the extracted rna samples were further purified using the purelink™ rna mini kit, quantified using the qubit rna hs assay kit, and the quality was checked by agarose gel electrophoresis and experion rna analyzer as previously described with slight modifications. genomic dna was extracted from flax leaves using the qiagen dneasy plant mini kit, quantified using the qubit dsdna br assay kit, and the quality was verified by agarose gel electrophoresis. genome-wide mining and phylogenetic analysis of ugts ugt74s, ugt74t, ugt89b, ugt94h, ugt712b and previously identified flax ugts were used as queries to identify putative ugts from the annotated genes of the flax genome assembly using blast with an e-value of 1e- the identified ugt candidates were further annotated by analyzing their gene structure and sequence similarity. phylogenetic analysis of genome-wide ugts was performed using mega. the protein sequences for out of ugts were found to be very short compared to others, probably truncated genes, and were excluded from the tree construction. to get a general overview of the tree topology from as many ugts as possible, protein sequences of the remaining ugt sequences were aligned in a first step using clustalw and a phylogenetic tree was constructed using the neighbor-joining algorithm with bootstrap replicates. because ugts are characterized by a amino acid pspg motif signature box known to be involved in substrate recognition and catalysis, ugt candidate gene list was narrowed down to only those carrying a pspg motif. the conserved amino acids of the pspg motif and their 50 bp upstream and downstream sequences from ugts were extracted. these amino acid sequences were aligned with muscle and a phylogenetic tree was constructed using the maximum parsimony analysis method with bootstrap replicates. gene duplication analysis and ugt divergence gene duplication analysis was conducted by self-blast of the entire putative ugt database using a threshold e-value of 1e-. pairs of ugt genes returning reciprocal top hits of each other and having identical or highly similar gene structure were declared duplicate copies. the identified ugt genes were assigned to the flax linkage groups, each representing a chromosome, based on the sorted flax genome sequence. duplication and divergence times were determined from the ratio of calculated non-synonymous substitution over the calculated synonymous substitution values. briefly, full-length cdna sequences were aligned in mega v and the ka/ks ratio was calculated. ks values lower than were removed from the dataset as suggested by sveinson et al.. furthermore, ks values larger than were excluded to minimize the saturation effects. the evolutionary distance between pairs of genes was determined based on the ks corrected with the nei-gojobori model of nucleotide evolution which accounts for multiple substitutions per site 26]. the divergence of a pair of duplicated genes was converted into duplication or divergence time in million years following the equation t = k// where r is the substitution rate of  × 10− substitutions per synonymous site per year. functional analysis of ugt74s and its closely related ugts lus and lus10014148 to assess the functionality of the two ugts most closely related to ugt74s gene splicing of genomic dna was performed to recover the full length coding sequence of lus and lus which have short single introns. tissue-specific and heterologous gene expression studies of these two genes were undertaken alongside ugt74s lus and lus gene splicing from genomic dna extension of overlapping gene segments by pcr is a simple technique for gene splicing. to delete the intron from the genomic dna through joining the two exons together, genomic sequences corresponding to lus and lus were used to design gene specific primers. for each sequence, an external flanking primer pair was designed from the 5′ and 3′ end of the cds, referred to as exonic forward and reverse primers. additionally, two 30-nucleotide internal primers were designed. the first internal primer called exon i-r consisted of the reverse complement to the first nucleotides in the sense strand of exon ii and the last nucleotides of the reverse strand of the exon i. the second internal primer called exon ii-f consisted of the last nucleotides in the sense strand of exon i and the first nucleotides of exon ii sense strand as previously described. the gene splicing was conducted in three steps. the initial step was the amplification of the two exons using the 5′ exonic i forward and exon i-r in a first reaction, and exon ii-f and 3′ exonic ii reverse in a second reaction. dna was diluted and 3 μl aliquots were used as template in subsequent pcr reactions. pcr cycles consisted of an initial denaturation at 94 °c for 2 min followed by 30 cycles of 94 °c for 30 s, 55 °c for 45 s and 72 °c for 90 s prior to a final extension at 72 °c for 10 min. aliquots of 6 μl of the pcr products were resolved on 1% agarose gels stained with ethidium bromide. the amplified exon fragments i and ii were purified using the qiaquick pcr purification kit, diluted folds and 4 μl aliquots of each purified product were pooled and used as template in the second gene splicing step where full length templates for each gene were generated. during pcr, overlapping strands of the two intermediate products form a duplex, providing taq with a free 3′end for extension and a single strand for polymerization, thereby generating a single full-length product. this pcr reaction consisted of 10 cycles at 95 °c for 30 s, 25 °c for 45 s, and 72 °c for 90 s. the resulting pcr product was diluted folds and 3 μl aliquots were used as a template in a third and final pcr reaction conducted at 94 °c for 3 min followed by 30 cycles of 94 °c for 30 s, 55 °c for 45 s and 72 °c for 90 s, followed by a final extension at 72 °c for 10 min. the fused full length fragments were purified with the qiaquick gel extraction kit, cloned into topo ta vector and transformed in e. coli similar to previous descriptions. ugt74s lus and lus gene expression in different flax tissue to quantify the gene expression levels of lus and lus in different flax tissues, real-time pcr primers were designed from the 3′ end of lus lus ugt74s and a ribosomal rna, the latter being used for data normalization as performed in a previous report. first strand cdna synthesis and real-time pcr conditions were as previously described. the output gene expression data were generated using the 2-∆∆ct method, and the results were presented as fold changes expression relative to that of 0 day after anthesis for all tissues. cloning and heterologous expression the topo ta cloned full length fragments for lus and lus were sequenced for confirmation. flanking primers carrying restriction enzyme sites were designed to shuttle the full length lus and lus into the yeast expression vectors pyes2/nt c and b, respectively. the pyes2/nt c construct carrying the full-length cdna for ugt74s was previously described. all three constructs were transformed in the yeast strain invsc following manufacturer’s instructions and single transformant colonies were cultured, induced, harvested and lysed as previously reported. protein expression for each of the three ugts, including ugt74s as control, was monitored by western blot using equal amount of proteins and diluted antibodies raised against the xpresstm epitope present between the 6× histidine tag and the multiple cloning site as previously described. enzyme assays and reaction products determination and quantitation to determine whether the two ugts could glucosylate seco into sdg as reported before for ugt74s, the purified native proteins obtained from the yeast cultures expressing ugt74s lus or lus were reacted with seco, the only substrate relevant to this study, in the presence of udp-glucose or udp-galactose. the 100 μl reaction mixture composition and reaction incubation conditions were exactly as described in our previous report. for separation, identification, and quantitation of the reactants and their products, a waters h-class acquity uplc system equipped with a tqd tandem mass spectrometer and a waters csh c column were used as previously described, albeit with slight modifications. in addition to ms scanning mode, selected ion recording spectra were collected to improve the detection sensitivity of seco, smg, sdg and seco monogalactoside. the capillary voltage was set at 3 kv, the extractor at 3 v, and rf lens at  v. the chromatographic parameters followed a binary gradient system composed of 3% formic acid in water and acetonitrile, varying according to the following program: t a = 98%; t1 =   min, a = 0%; t2 = 6 min, a = 0% isocratic; t3 = 7 min, a = 98%; t4 = 8 min, a = 98% isocratic. peaks detected at 280 nm, indicative of phenolic compounds, and were validated using authentic standards purchased from chromadex as described in. a standard curve for sdg was created using the sdg standard described above. purified smg standard was prepared in-house as previously described. all reactions were carried out in triplicates and the data are presented as the means ± standard deviations. a one-tailed student’s t-test was performed to test the statistical significance of metabolite production levels by ugt74s lus and lus.  ugt gene duplication and diversity in the flax genome genome-wide mining for ugt genes identified non-redundant sequences dispersed on all flax chromosomes. with a mere ten sequences, chromosomes had the fewest ugt genes whereas chromosome harbored the most with. a total of of the identified ugt genes were duplicated, of which corresponded to intra-chromosomal duplications. fourteen of those occurred on chromosome whereas no intra-chromosomal duplications were observed on chromosomes and. ugt74s was located on chromosome and displayed no evidence of gene duplication. phylogenetic analysis from the out of the ugts, excluding truncated sequences, revealed four main clusters and sub-clusters. cluster was the most diverse, accounted for 53% of the ugt genes, and included eight of the groups previously reported. the fifteenth new group identified in this study was hereafter named “o”. ugt74s clustered within group l as previously reported and belonged to a small gene family of five ugts including lus, lus, lus, lus and lus, supported by a 53% bootstrap value and hereafter called the lus subclade. two members of this subclade, lus and lus were shown to be duplicated genes located on chromosomes and respectively, and were the most closely related to ugt74s. this small gene family clustered with a sister subclade, herein called lus10006352/lus subclade, that was supported with a 51% bootstrap value and which included lus, lus, lus and lus. to further assess the consistency of the observed groupings, a new tree was constructed using a conserved region from ugt sequences. consistent with the first large phylogenetic tree, this reduced tree showed, with relatively high bootstrap values, the same configuration and groupings as previously reported. however, the a, b, c, e and m groups were not as well resolved as in the previous tree. in this second phylogenetic tree, group “l” was well supported by an 89% bootstrap value and the lus subclade comprised lus, lus, lus, lus and lus while the lus10006352/lus subclade included lus, lus and lus, thus providing more confidence in our phylogeny for inferring the evolutionary history of the ugts. following udp-glucosyltransferase nomenclature, lus lus lus lus lus100lus lus and lus were annotated as members of ugt74s ugt74s ugt74s3- ugt74s ugt74s ugt74s ugt74s5- sub-families, respectively. therefore, lus and lus will be hereafter designated by their ugt nomenclature: ugt74s and ugt74s alongside with ugt74s.fig. phylogenetic relationship among flax ugts based on the conserved pspg motif and its surrounding regions. the different groups are indicated by alphabetic letters a-o around and inside the circular tree. ugt74s belongs to a small gene family highlighted in red within group l and is marked with an asterisk ugt gene divergence we examined ugt sequence divergence by determining the ka/ks ratio among ugt sequences carrying the pspg motif. after ks calculation and data filtration, ugt gene pairs with ks ranging between and were retained. overall, 82% of the gene pairs had ks values < including 45% with ks values <. the ks mean value was and the distribution peaked at. similarly, and 71% of the ugt gene pairs had ka/ks <  and ka/ks <  respectively. less than 1% of the gene pairs had ka/ks = . the distribution frequency of the individual ka/ks values peaked at.fig. frequency distribution of ks values and ka/ks ratios among ugt gene pairs obtained from ugt sequences the gene pairs involving the sdg lignan-forming gene ugt74s had ks <  and ka/ks <  in a proportion of and 56%, respectively. within the lus and lus10006352/lus subclades, all ks derived from gene pairs involving ugt74s ranged from to. in contrast, only two of the eight gene pairs had ka/ks <  the remaining 75% of the gene pairs displayed ka/ks > the divergence time between pairs of the ugt genes ranged from to mya. the divergence time between the genes clustered in group l spread from to mya. within the lus and lus10006352/lus subclades, lus and lus appeared to be the most ancient, diverging from one another approximately mya. ugt74s may have diverged from the other seven ugts clustered in these two subclades around 16– mya. based on our inferences, ugt74s probably duplicated from ugt74s ~ mya. the evolutionary relationships between ugt74s, lus ugt74s and ugt74s5- were found to be complex. the duplication study identified both ugt74s and ugt74s5- as duplicates of ugt74s on one hand, and also ugt74s as a duplicate of lus on the other hand, with duplication/divergence times of about and mya for ugt74s5/ugt74s5- and lus10006720/ugt74s respectively. however, the divergence time for the ugt74s5/ugt74s pair could not be determined using the filtration criteria set.table pairwise matrix of flax ugt divergence time in mya for the ugt genes clustered lus and lus10006351/lus subclades within the group “l” of the phylogenetic tree. the most recent and ancient divergence times are highlighted in blue and green, respectively n/a, denotes that the gene pairs did not meet the filtration criteria set in methods ugt74s, ugt74s, and ugt74s are indicated by an asterisk structural gene organization of ugt74s and its closest relatives ugt74s is a 1407 bp gene with a single intron of 739 bp. to assess the functionality of the two ugts that were the most closely related to ugt74s i.e., ugt74s and ugt74s we obtained their full coding regions by gene splicing. ugt74s generated a spliced fused cds of 1416 bp by joining the exons of and 738 bp from a 1492 bp genomic dna fragment. similarly, a spliced fused cds of 1371 bp was obtained by joining the exons of and 728 bp from a 1464 bp genomic dna product for ugt74s. the amplified products were of expected sizes as estimated from the original sequences in the ncbi database and in phytozome. in this study, ugt74s and ugt74s had and 93 bp introns, respectively and their fused spliced coding sequences were confirmed as 100% identical to those already reported. it is noteworthy to mention that ugt74s corresponding to ncbi accession jn, was reported by barvkar et al. with a 27 bp truncation at its 5′ end. this truncation was detected by comparing the amplified genomic and spliced fused sequences of ugt74s with that of jn and of lus g from the flax draft genome. in ugt74s the intron is located between nucleotide positions and whereas it is located between nucleotide position and of ugt74s. thus, the full genomic sequence size for ugt74s is 1464 bp and not 1437 bp as previously reported. the spliced full length cdnas for ugt74s and ugt74s were predicted to code for and amino acids, respectively, and as also observed in this study. both showed nearly 62% identities to ugt74s and displayed 80% identity to one other at the amino acid levels. the conserved amino acids pspg motif was located between amino acids and in ugt74s and between and in ugt74s the pspg motif of ugt74s and ugt74s shared and 80% identity with ugt74s respectively. four amino acids found to be conserved within the pspg motif of ugt74s and ugt74s were substituted in ugt74s1: e339q, a345k, m362l and w374c. ugt74s had three unique amino acids changes and ugt74s had two. ugt74s ugt74s and ugt74s gene expression in different flax tissues real time gene expression levels of ugt74s and ugt74s were similar in developing seeds and leaves but were markedly contrasting in stems and roots. moreover, their expression patterns differed from that of ugt74s in all tissues. in developing seeds, ugt74s expression mimic a bell curve pattern, peaking at daa as previously observed. at eight and daa, ugt74s and ugt74s expression levels were 3– times lower than ugt74s whereas their expression levels were higher than that of ugt74s at maturity. in contrast, ugt74s was barely expressed in root and stem tissues where ugt74s and ugt74s were highly expressed. expression of all three ugt genes was low in leaves.fig. expression profile for ugt74s ugt74s and ugt74s in developing seeds sampled at and 16 days after anthesis and at maturity, as well as leaf, root and stem tissues of flax cultivar ac mcduff. a, ugt74s1; b, ugt74s; and c, ugt74s heterologous protein expression and enzyme activity in this study, we wanted to assess whether the ugt74s and ugt74s enzymes had the ability to glycosylate seco as reported for ugt74s. thus, protein expression and glycosylation activity toward seco were assessed for ugt74s and ugt74s in comparison with ugt74s the full-length cdnas for ugt74s and ugt74s were expressed in yeast as previously described for ugt74s the western blot analysis showed that all three proteins highly expressed after 8 h of induction with 2% galactose. ugt74s and ugt74s showed strong intensity signals comparable to the  kda of ugt74s previously reported and produced and  kda proteins, respectively, in agreement with their predicted sequences.fig. western blot of ugt74s ugt 4s and ugt74s and reaction products observed from enzyme assays using purified native enzymes. a, western blot of the histag-purified ugt74s ugt74s and ugt74s probed using antixpresstm antibody. the and 50 kda protein molecular marker sizes corresponding to the western c precision plus protein marker mixed with conjugant are pinpointed. b, reaction products formed by each of ugt74s ugt74s and ugt74s after enzyme reaction with seco as substrate and udp-glucose or udp-galactose as sugar donor. sdg secoisolarciresinol diglucoside, smg secoisolariciresinol monoglucoside, smgal secoisolariciresinol monogalactoside. c. uplc chromatograms showing the reaction products of 50 μg of purified proteins for the three ugts using seco as substrate and udp-glucose as sugar donor. insets show 10× magnification to visualize trace peaks of smg in ugt74s and ugt74s reaction products. appreciable accumulation of sdg and smg was only observed for ugt74s the unreacted seco is present in all three chromatograms to determine the glucosylation activity of ugt74s and ugt74s enzyme assays were performed using the purified proteins, with ugt74s as a positive control. although ugt74s and ugt74s protein expression was similar to ugt74s, their glucosylation activity towards seco was seven fold less and both failed to produce sdg under our experimental conditions. no significant difference in glycosylation activity was observed between ugt74s and ugt74s towards seco. by using udp-galactose as an alternative sugar donor for the production of seco glycosides, all three proteins formed trace amount of seco monogalactosides. but no seco digalactosides were detected with any of the three enzymes tested. no difference was observed among all three proteins with regards to the smgal levels when udp-galactose was used as alternate sugar donor. discussion ugts are a large and complex family of enzymes that catalyze glycosidic bond formation. flax ugt74s has recently been reported as an enzyme sequentially glucosylating seco into its monoglucoside smg and diglucoside sdg when expressed in yeast. to get more insights into the exclusivity of ugt74s to achieve seco glucosylation into sdg in flax, genome-wide mining, tissue-specific and heterologous gene expression, and enzyme activity assays were conducted. the study confirmed our previous findings and further shows that ugt74s may be the key enzyme controlling seco glucosylation in flax, although ugt74s and ugt74s may also contribute to a minor extent in supplying smg as substrate to ugt74s for the second glucosylation step. a total of ugts were found dispersed throughout the ~373 mb flax genome, corresponding to a gene density of ugt/mb, which is similar to arabidopsis but higher than rice, apple, grapevine, poplar, soybean and sorghum. the percentage of ugts among the total predicted genes in flax is %, higher than previously reported in dicots and falls in the same range as monocots. gene duplication is a common phenomenon in eukaryotes, such as plants. it leads to genetic novelty and diversity, evolution, and functional diversification, phenomena that are required by vascular plant species for adaptation and plasticity. paleoploidy events including whole genome duplications have been described in the genus linum and are considered key drivers of gene duplication. previous studies predicted genes in the flax genome, of which genes were predicted to be duplicated. of the ugts identified in this study, were found to be duplicated and accounted for % of all duplicated genes of the flax genome. ks has been previously used as a time indicator for duplication events, and its frequency distribution was reported to reflect the relative time of genome duplication. accordingly, the ks calculated from the ugt gene pairs peaked at, and ranged from to, indicating ancient duplications during ugt evolution. using the ks values, the divergence among the pairs of genes was estimated to have occurred – mya. likewise, the divergence time between the genes clustered in the l group spans from to mya. these results suggest that some flax ugt duplication events may have taken place prior to the most recent whole genome duplication estimated at 5– mya. our ka/ks analyses showed that 71% of the ugt gene pairs had ka/ks <  indicating that most of the flax ugts have evolved under a purifying selection. about a quarter of the gene pairs had ka/ks >  thus being under positive selection pressure whereas % of the ugt genes found to be under neutral selection. in our study, full protein sequences carrying pspg motif were used to calculate ka and ks. similarly, full length gene sequences have been used to determine the ks/ka values for the wheat and rice homologues. whether potential biases may be associated with the conserved domain’s own divergence rate, it is reasonable to assume that such bias, if any, should be evenly distributed and minimal because all the considered sequences had the pspg motif and, thus, would not affect to the drawn conclusions. despite the high rate of ugt duplication in flax, ugt74s gene appeared as a single copy gene located on linkage group. this gene was found to be involved in gene pairs based on the criteria set in this study, of which 56% had ka/ks <  and 44% with ka/ks >  almost a 50/ ratio for negative and positive selection. when a single member of a gene family is undergoing positive selection, it shows high ka/ks ratios when paired with all members of its family. this fact was substantiated within the lus and lus10006352/lus subclades where 75% of the gene pairs involving ugt74s had ka/ks > indicating that ugt74s is under positive selection and, as such, it is experiencing reduced selective constraints to remain a singleton. positive selection has been reported for ugt73c and ugt73c playing a role in saponin-mediated insect resistance, and lignans, which have also been reported to take part in plant defense against insect. it is possible that ugt74s was duplicated during one or both of the proposed ancient duplication events and, that its paralog were subsequently deleted through gene loss as previously reported. single copy genes have been evoked to play key roles in maintaining essential metabolic processes in plants through macromolecular complex formation. ugt74s may have been maintained as a single copy gene in flax for seco glucosylation into sdg lignans during adaptive divergence as was reported for udp-glucose pyrophosphorylase in sorghum. phylogenetic studies of ugts have previously described and distinct groups using ugt amino acid sequences from arabidopsis and ugts from nine higher plants, respectively. in flax, barvkar et al. described groups based on ugts. the sub-clusters observed in the current study included the groups previously reported in flax and identified a new group named “o”. the grouping observed here is comparable to the 14– groups previously reported and highlights the diversity of ugts in flax, possibly through concerted evolution, a phenomenon also known in other higher plants. duplicated genes may face three different fates: nonfunctionalization in which one copy becomes silenced; neofunctionalization where one copy acquires a novel, beneficial function while the other copy retains the original function; or subfunctionalization where both copies become partially compromised by the accumulation of mutations. ugt74s and ugt74s which originated from a duplication event, were the closest to ugt74s that glucosylates seco into sdg. because high sequence similarity does not necessarily translate into similarity in expression and function, ugt74s and ugt74s tissue-specific expression and protein functionality towards seco were investigated. contrary to ugt74s ugt74s and ugt74s were barely expressed in the seeds at 8– daa developmental stages, when sdg lignan synthesis increases rapidly. but, these duplicated genes were more expressed in vegetative tissues and in mature seeds than ugt74s and showed different expression patterns, perhaps because of their tissue-specific promoters or their neofunctionalization after the duplication events. the reported data suggest that these genes may play major roles in other biological processes yet to be elucidated, as opposed to seco glucosylation into sdg. high protein expression levels were achieved in the heterologous yeast expression system but under the conditions described in this study, ugt74s and ugt74s were unable to convert seco into sdg. however, they were able to produce small amounts of smg with an overall glucosylation activity towards seco seven times lower than ugt74s suggesting that ugt74s and ugt74s proteins may prefer a different substrate. in contrast, ugt74s consistently produced higher amounts of sdg along with small amounts of smg and, its gene was more highly expressed in developing seeds where sdg lignans accumulate as previously reported. these observations suggest that ugt74s may be the key functional player in seco glucosylation into sdg, due to its ability to achieve both required glucosylation steps; first glucosylating seco into smg and then smg into sdg. additionally, mutations in ugt74s lead to reduced or abolished sdg production in vitro, further demonstrating its essential function. while not all the ugts reported in this study were biochemically evaluated for their ability to glucosylate seco, the finding that ugt74s and ugt74s produced smg from seco at a very low rates and failed to form any sdg, indicates that these two genes are not as essential as ugt74s which performs both required steps. although involvement of other cryptic ugts in the seco glucosylation process leading to sdg has not been exhaustively ruled out, one may question the evolutionary advantages of maintaining redundant genes and duplicated pathways for the same biochemical function. moreover, recent evidence coming from a follow-up study conducted in our laboratory and focusing on tilling of ethyl methane sulfonate flax mutant lines support the idea that ugt74s is unique in controlling seco glucosylation into sdg. in the current study, ugt74s and ugt74s were able to glycosylate seco with udp-glucose or udp-galactose at low efficiency, forming smg or smgal respectively, under the described conditions, but failed to do so for sdg as did ugt74s and therefore, no further biochemical characterizations or optimizations were performed for these two enzymes. indeed, if ugt74s and ugt74s were able to glycosylate smg into sdg, even at a low rate, one would expect to observe trace sdg peak using the very sensitive ms detection methods, even under standard conditions, which would have warranted further biochemical optimization of the enzyme reactions. however, this was not the case under our experimental conditions, suggesting that these two proteins may not have the ability to perform the second glycosylation step. these enzyme activity data are in line with the conclusions drawn from the differential temporal gene expression data in the developing seeds, and reinforce the idea that ugt74s and ugt74s may play other roles in different metabolic processes apart from sdg lignan glucosylation. the role played by the pspg motif in ligand binding and catalysis has been well-documented in plants. the variations within the pspg observed between ugt74s and its two closely related ugt genes may be responsible for the differences in their substrate specificity as previously reported and may account for the observed differential functionality.  ugts form a large gene family where the majority of the members are duplicated in the flax genome and subject to negative or purifying selection pressures whereas ugt74s is a singleton that has evolved under positive selection pressure. flax ugt74s and ugt74s were the most closely related to ugt74s but their encoded proteins were unable to glycosylate smg into sdg. whereas not all ugts in the genome were biochemically tested in this study, it is unlikely that other redundant ugt genes having the same roles in smg glucosylation as ugt74s would be maintained without a specific evolutionary advantage. however, the possibility of cryptic ugt functionality cannot be totally ruled out. nonetheless, the current study provides additional insights into ugt gene duplication events in flax and could be of interest to plant evolutionary biologists, breeders and plant natural product chemists. additional files additional file 1: figure s schematic representation of gene splicing pcr. additional file 2: table s primers used for pcr mediated splicing, cloning and real-time gene expression of lus and lus additional file 3: figure s circos map of ugts on the flax chromosomes. lines in the center of the map join duplicates. scale for each chromosome is indicated in mb. ugt74s ugt74s and ugt74s are marked with asterisks. additional file 4: table s list, location and coordinates of the ugts found in the flax genome. ugt locus corresponding to ugt74s is highlighted in bold red. additional file 5: figure s distribution of ugt genes on the chromosomes in the flax genome. the term “first and second copy” is arbitrary and was used only for graph presentation purpose. the fifteen chromosomes in the flax genome are indicated by lu to lu additional file 6: figure s phylogenetic relationship among flax ugts and depicting groups. sixty-eight ugts with relatively short sequences were excluded. the four main clusters are indicated by alpha numeric numbers 1– and the different groups are indicated by alphabetic letters a-o around and inside the circular tree. ugt74s belongs to a small gene family highlighted in red within group l and is marked with an asterisk. additional file 7: table s ka, ks, ka/ks and divergence time for ugt genes pairs after filtration. additional file 8: table s pairwise matrix of flax ugt divergence time in mya for genes clustered in group “l” of the phylogenetic tree. the ugts found in lus and lus10006351/lus subclades are highlighted in green. the most recent and ancient divergence times are highlighted in blue and yellow, respectively. additional file 9: figure s comparison of genomic dna and full length coding regions for lus and lus as obtained by gene fusion pcr. a, amplified exon i and exon ii of lus were fused to make the gene fusion product and compared to the genomic dna amplicon; b, amplified exon i and exon ii of lus were fused and similarly compared to its genomic dna amplicon. molecular ladder sizes are indicated by arrows. additional file 10: figure s sequence alignment between the fused spliced cds from lus and lus and their respective corresponding genomic and transcript sequences reported in ncbi and phytozome. a, sequence alignment of the fused spliced lus cds with its amplified genomic region before splicing, its corresponding genomic sequences reported in ncbi and phytozome, and its transcript sequence from phytozome. the genomic sequences show the 76 bp intron located between nucleotide positions and whereas the intron is missing in the published transcript sequence and in the fused spliced cds reported in the current study; b, sequence alignment of the fused spliced lus cds with its amplified region before splicing, its corresponding genomic sequences reported in ncbi and phytozome, and its transcript sequence from phytozome. the genomic sequences show the 93 bp intron located between nucleotide position and whereas the intron is missing in the published transcript sequence and in the fused spliced cds reported in the current study. additional file 11: figure s protein sequence alignment between ugt74s lus and lus. the pspg region is indicated by a red box and where amino acid variations between the three proteins appear as a white indentation in the black box representing the consensus sequence. all amino acid positions were numbered according to their location in ugt74s abbreviations cdscoding sequence daaday after anthesis emsethyl methane sulfonate kanon-synonymous substitution kssynonymous substitution msmass spectrometry myamillion year ago ncbinational center for biotechnology information pspgplant secondary product glycosyltransferase sdgsecoisolariciresinol diglucoside secosecoisolariciresinol smgsecoisolariciresinol monoglucoside smgalsecoisolariciresinol monogalactoside tillingtargeted induced local lesion in genome udpuridine diphosphate ugturidine glycosyltransferases uplcultra performance liquid chromatography 
ugt74s a single copy gene located on chromosome displayed no evidence of duplication and was deemed to be under positive selection pressure. the study suggests that ugt74s 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 ugt74s phylogenetic and gene duplication analyses, heterologous gene expression and enzyme assays were conducted to identify family members closely related to ugt74s and to establish their roles in seco glucosylation. the phylogenetic analysis identified four main clusters where cluster which included ugt74s was the most diverse. flax ugts diverged – mya and 71% were found to be under purifying selection pressure. however, whether this gene is the only ugt achieving seco glucosylation in flax was not known. flax ugt74s was recently reported to sequentially glucosylate seco into smg and sdg in vitro.
 flax lignan, commonly known as secoisolariciresinol diglucoside, 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 until lately. flax ugt74s 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.  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 ugt74s and to establish their roles in seco glucosylation. a total of different ugts were identified, of which were duplicated. flax ugts diverged – mya and 71% were found to be under purifying selection pressure. ugt74s a single copy gene located on chromosome displayed no evidence of duplication and was deemed to be under positive selection pressure. the phylogenetic analysis identified four main clusters where cluster which included ugt74s was the most diverse. the duplicated ugt74s and ugt74s located on chromosomes and respectively, were the most closely related to ugt74s and were differentially expressed in different tissues. heterologous expression levels of ugt74s ugt74s and ugt74s proteins were similar but ugt74s and ugt74s glucosylation activity towards seco was seven fold less than ugt74s in addition, they both failed to produce sdg, suggesting neofunctionalization following their divergence from ugt74s  we showed that ugt74s is closely related to two duplicated genes, ugt74s and ugt74s which, unlike ugt74s failed to glucosylate smg into sdg. the study suggests that ugt74s 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 ugt74s electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords flaxsecoisolariciresinolsdg lignangenome mininggene duplicationugtsneofunctionalizationissue-copyright-statement© the author 2017 