BACKGROUND
sucrose is the primary product of photosynthesis and the initial form of transported sugar in most plants.
in higher plants, sucrose is hydrolyzed by either invertases to form glucose and fructose or sucrose synthases  to form uridine diphosphate glucose  and fructose  <cit> .
the carbon in glucose and fructose is further channeled into various primary or secondary metabolic pathways based on the spatiotemporal activity of metabolizing enzymes  <cit> .
sugar metabolizing enzymes have, therefore, been recognized as key gatekeepers of carbon allocation and partitioning pathways in plants  <cit> .
the sugar, udp-glucose, represents an important branch point in carbohydrate metabolism that can potentially be channeled directly for synthesis of starch, sucrose, cellulose or hemicellulose and pectin via synthesis of other nucleotide sugars such as udp-glucuronic acid  <cit> .
udp-glucose pyrophopharylase , also referred to as uglcpp or uridine triphosphate glucose-1-p uridylyltransferase, is a sugar-metabolizing enzyme  that catalyzes the reversible formation of udp-glucose and pyrophosphate from glucose-1-phosphate and uridine triphosphate .
in photosynthetically active mature leaves, ugpase catalyzes the reaction in the direction of udp-glucose and sucrose via coupled action of sucrose phosphate synthase  .
however, in sink organs, which depend on imported sucrose, ugpase is proposed to function in the conversion of udp-glucose, which is produced via susy hydrolysis of sucrose, to glucose-1-phosphate  <cit> .
based on an analysis of the most recent version of the populus trichocarpa genome and previous reports from hybrid aspen , rice  and arabidopsis thaliana, ugpase genes appear to belong to small two-gene containing families  <cit> .
ugpases have been purified from and expressed in several eukaryotic and prokaryotic organisms  <cit> .
in vitro enzyme assays using atugpase <dig> and atugpase <dig> expressed in escherichia coli suggested that both the isoforms have slight preference for the pyrophosphorylation direction of the reaction  <cit> .
atugpase <dig> exhibited higher activity than that of atugpase <dig> in both directions, pyrophosphorolysis and synthesis  <cit> .
the two isoforms showed no activity with other sugars such as adp-glucose/atp or galactose-1-p  <cit> .
antisense suppression of ugpase <dig> in arabidopsis was reported to have no effect on plant growth, however, soluble carbohydrate and starch content was found to be reduced  <cit> .
tobacco  plants overexpressing axugpase, a ugpase gene from acetobacter xylinum, exhibited increased plant height in several lines, similar total dry weight relative to controls and no significant changes in sugars, starch or cellulose levels  <cit> .
however, overexpression of the same bacterial ugpase in hybrid poplar  resulted in significantly reduced growth, increased levels of soluble sugars, starch and cellulose, and reduced lignin  <cit> .
three studies involving antisense downregulation of a native ugpase gene in potato  led to contrasting and inconclusive results.
an early report suggested that a strong 95% reduction in ugpase transcripts and enzyme activity had no apparent effects on plant growth and sugar levels  <cit> .
in two subsequent studies, however, antisense suppression of ugpase in potato resulted in significantly lower sucrose levels  <cit> .
based on microarray analysis of cell types representing the discrete stages of xylem development and wood formation in populus, native ugpase expression was enhanced during late xylem cell expansion and secondary cell wall formation, suggesting a potential functional role for ugpase in udp-glucose substrate provision during secondary cell wall formation  <cit> .
in total, these results imply distinct host-specific-functional activities of ugpase in alternate plant species.
a functional genomics investigation of native populus ugpase gene has not been reported.
given that plant metabolism is tightly regulated for coordinated plant response to various internal and external cues, and carbon flow links primary metabolism to secondary metabolism, it is surprising how little we know about the enzymes interconnecting the primary and secondary metabolic pathways.
it is plausible that manipulation of sugar metabolizing enzymes, such as ugpase, can affect both primary sugar and secondary wall carbohydrate metabolism; however, it is unclear if and how such manipulations may quantitatively and qualitatively affect secondary metabolic pathways of shikimate, phenylpropanoid and lignin biosynthesis.
therefore, we have undertaken a detailed metabolic characterization of populus deltoides plants overexpressing a native pdugpase <dig> gene.
transgenic plants had pronounced growth and metabolic changes relative to control plants.
the changes in cell wall properties were, however, subtle with significantly reduced syringyl-to-guaiacyl ratios as one of the few alterations in transgenic cell walls.
overall, our results support a role for pdugpase <dig> in carbohydrate metabolism, secondary metabolism and plant growth and development.
RESULTS
phylogenetic analysis and expression of ugpase in populus
in several previous reports including populus, the ugpase gene family has been reported to comprise of two genes  <cit> .
a phylogenetic analysis was performed using two previously reported ugpase isoforms from populus  <cit> , orthologs from other plant species and axugpase as an outgroup.
ugpase <dig> and ugpase <dig> cluster together closer than with any other ugpases .
while pdugpase <dig> and pdugpase <dig>  which appear to have arisen during the salicoid duplication event  <cit> , share more than 97% similarity  with each other, they also share 92% similarity with atugpases vs. 90% with a gymnosperm ugpase .
there is a lower, 87%, identity between the rice paralogs, osugpase <dig> and osugpase <dig>  and a 93% identity between the arabidopsis paralogs, atugpase <dig> and atugpase <dig>  the similarity of plant ugpases with the previously characterized axugpase averaged at 21% .
the nucleotide binding loop  and insertion loop , involved in dimer formation and stabilization  <cit> , are conserved in atugpase and pdugpase but absent in axugpase , suggesting a potentially distinct enzymatic mechanism in plants involving dimer- or-oligomerization of proteins.
in support of this hypothesis, it has been shown that the activity of barley ugpase is regulated by di- or oligomerization status of the protein, with monomer status representative of the active form of ugpase enzyme  <cit> .figure  <dig>
phylogenetic analyses of ugpases from
populus
and other species.
analysis was conducted in mega <dig> program using the neighbor-joining method.
the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test  are shown next to the branches.
ugpases from this study are in bold.
atugpase <dig> : at3g03250; atugpase2: at5g <dig> ; axugpase :aaa <dig> ; egugpase : acf <dig> ; osugpase <dig> : abd57308; osugpase2: af249880; ptrugp <dig> : potri.004g074400; ptrugp <dig>  potri.017g144700; rcugpase : xp_ <dig> ; stugpase : aal <dig> ; zmugpase : np_ <dig> .
expression of the two pdugpase genes was quantified in eight populus tissue types including young leaf , mature leaf , young stem , mature stem , petiole of a mature leaf, phloem , xylem  and root by quantitative reverse transcriptase pcr .
both isoforms were expressed in all tissues, with an overall higher expression level of ugpase <dig> relative to ugpase <dig> .
among all the tissues profiled, expression of pdugpase <dig> was relatively higher in mature leaves and xylem, while expression of pdugpase <dig> was higher in young leaves and roots.
our results are consistent with previously published data involving alternate tissues and organs, which showed that ugpase <dig> and ugpase <dig> were abundant in flowers and young leaves  <cit> .
in two additional studies, ugpase activity was also higher in xylem than in leaves  <cit> .
in potato, where only one isoform has been reported, ugpase activity was abundant in sink tissues, such as stems and tubers  <cit> .
in arabidopsis, ugpase <dig> was reported to have enhanced expression in leaves and stems compared to flowers, whereas ugpase <dig> had enhanced expression in flowers  <cit> .figure  <dig>
relative expression of
ugpase
isoforms in different tissues.
expression of ugpase <dig> and ugpase <dig> in yl, young leaf, ml, mature leaf, ys, young stem, ms, mature stem, pet, petiole, phl, phloem, xyl, xylem and rt, root.
data represent means ± se .
relative expression was calculated based on the expression of reference genes ubiquitin-conjugating enzyme e <dig>  and 18s ribosomal rna .
morphological characterization of pdugpase <dig> overexpression plants
transgenic populus deltoides plants overexpressing the pdugpase <dig> gene  under the control of a constitutive polyubiquitin  <dig>  promoter were maintained and characterized under greenhouse conditions.
in an initial transgenic phenotyping study, all pdugpase <dig> overexpression lines were found to be consistently shorter in height  with thinner stems  compared to control plants .
assessment of an independently grown second set of transgenic plants confirmed that plant height and stem diameter were consistently smaller relative to controls .
conversely, leaf area  and petiole length  were significantly greater in transgenics , though total leaf dry weight was reduced as a result of fewer leaves .
additionally, reduced plant height and stem diameter resulted in a significant reduction in stem dry weight .
when compared to controls, leaf-to-stem biomass ratio doubled , whereas root weight and root surface area were reduced  in transgenic plants .
we quantified ugpase <dig> transcript levels in mature leaf petioles of three selected lines and found that pdugpase <dig> expression increased 2-fold .
our results suggest that modest changes in native ugpase <dig> levels can have pronounced morphological effects on plant phenotype in woody perennials such as populus.figure  <dig>
phenotypic characterization of
ugpase2
overexpression lines.
differences in plant height  and stem diameter , leaf area  petiole length  root system  and root surface area  among control and ugpase <dig> transgenic plants.
data represent means ± se .
* indicates statistically significant, p ≤  <dig>  based on student’s t-tests.
dry weight of different plant parts in control and two
ugpase2
transgenic lines.
dry weights corresponding to leaf , stem , and root  and the ratio of leaf to stem dry weight  are presented.
data represent means ± se .
*indicates statistically significant, p ≤  <dig>  based on student’s t-tests.
previous studies of suppression or overexpression of ugpase in other plant species have reported phenotypes ranging from no relative change to severely reduced plant height.
in potato, for example, despite a strong 96% reduction in ugpase enzyme activity as a result of antisense suppression, plant growth or tuber development was found to be unaffected  <cit> .
in arabidopsis, the t-dna mutants of ugpase  <dig>  ugpase  <dig> and double knockouts had no effect on growth rate, biomass production and flowering in mature plants, however, it was noted that young plantlets in agar had reduced hypocotyl and root length  <cit> .
a small increase in plant height without apparent effect on dry weight was reported for tobacco plants overexpressing the acetobacter ugpase under control of a constitutively expressed 35s promoter from cauliflower mosaic virus or a vascular-specific parsley 4-coumarate:coa ligase  promoter  <cit> .
in populus plants overexpressing the same bacterial ugpase under the control of a constitutively expressed 35s promoter, however, was reported to display significant reduction in plant height, stem diameter, leaf area and internode length  <cit> .
contrastingly, we have observed that the leaf area is dramatically greater in pdugpase <dig> plants relative to control.
the distinct aspect of the present work relative to the previous transgenic populus ugpase study  <cit>  is the use of a native gene from populus deltoides here.
given the fact that pdugpase <dig> has only a 20% amino acid similarity with that of acetobacter ugpase , a difference between phenotypic responses of plants overexpressing the two genes is not unexpected.
cell wall composition of pdugpase <dig> overexpressing populus
hplc analysis of stem cell wall sugar composition showed that glucose and xylose were the most abundant sugars in all samples as expected .
among glucose, xylose, galactose, arabinose and mannose, significant changes were observed only in mannose levels, with lower mannose levels in transgenic samples.
while cellulose and lignin content appeared unaffected; the syringyl-to-guaiacyl  ratio of lignin was significantly reduced in transgenic lines .
in order to explore whether overexpression of ugpase <dig> has any effect on the transcription of carbohydrate metabolism or/and cellulose pathway, a subset of sugar metabolism and cellulose synthesis-related genes were studied .
the panel of genes that we studied included, sucrose transporters, sut <dig>  linked previously to enhanced cellulose production  <cit> , and sut <dig>  linked to carbon partitioning  <cit> ; genes that encode sucrose hydrolyzing enzymes  <cit> , vacuolar invertases, vin <dig> and vin3; neutral invertase, nin <dig>  <cit> .
additionally, the secondary cell wall associated cellulose synthase  genes, cesa7b and cesa8b, and korrigan  genes, kor <dig> and kor <dig>  were included based on previously reports of proposed roles in cellulose biosynthesis  <cit> .
susy <dig> and susy <dig> transcripts and corresponding proteins have been shown to be elevated in tissue contexts with enhanced cellulose biosynthesis such as secondary xylem development and tension stress response  <cit> .
furthermore, a functional role for susy in supplying sugars for cellulose synthesis has been proposed  <cit> .
the expression of most sugar metabolism genes was not significantly altered except sut <dig> which was significantly higher in all the three transgenic lines .
whether sut <dig> has a direct role in sucrose transport and carbon partitioning in populus is as yet unanswered.
of the surveyed cellulose pathway genes, cesa and susy isoforms were not significantly altered, but kor <dig> and kor <dig> were significantly increased in young leaves of transgenic lines .
these results suggest that overexpression of pdugpase <dig> may be associated with modest changes in transcript levels of cellulose pathway-associated genes but that does not translate into changes in cellulose levels .
the unchanged cellulose levels in overexpression lines of pdugpase <dig> contradicts with previous reports where cellulose levels were increased in axugpase overexpression lines  <cit> .
while the implication of low sequence identity between the bacterial and populus ugpase isoforms on enzyme activity is yet to be clarified, the functional activity of axugpase has shown to complement cellulose negative mutant phenotype by channeling sugar substrate for cellulose synthesis  <cit> .table  <dig>
structural carbohydrate content  in stems of control and three
ugpase2
transgenic lines
cellobiose
glucose
xylose
galactose
arabinose
mannose
control
ugpase-1
ugpase-2
ugpase-3
data represent means ± se .
*indicates statistically significant, p ≤  <dig>  based on student’s t-tests.
cell wall composition analysis in control and three
ugpase2
overexpression lines.
a. cellulose levels in different tissues, b. lignin and c. syringyl  to guaiacyl  ratio in stems of control and transgenic lines.
yl and ml represent young and mature leaf, respectively.
data represent means ± se .
*indicates statistically significant, p ≤  <dig>  based on student’s t-tests.
expression of selected sugar metabolism  and cellulose pathway genes  in young leaves of control and
ugpase2
overexpression lines.
relative expression was calculated based on the expression of reference genes ubiquitin-conjugating enzyme e <dig> and 18s ribosomal rna.
sut, sucrose transporter; vin, vacuolar invertase; nin, neutral invertase; sps, sucrose phosphate synthase; cesa, cellulose synthase; susy, sucrose synthase; kor, korrigan.
accessions: sut <dig>  potri.019g085800; sut <dig>  potri.002g106900; vin <dig>  potri.003g112600; vin <dig>  potri.015g127100; nin <dig>  potri.019g082000; sps <dig>  potri.018g124700; cesa7b, potri.018g103900; cesa8b, potri.004g059600; kor <dig>  poptri.003g151700; kor <dig>  poptri.001g078900; susy <dig>  potri.018g063500; susy <dig>  potri.006g136700;18s, af206999; ubcc, potri.006g <dig>  data represent means ± se .
*indicates statistically significant, p ≤  <dig>  based on student’s t-tests.
metabolic profiles of xylem, phloem and leaves of pdgpase <dig> overexpressing populus
secondary metabolites such as phenolics and tannins are derived from the carbon in glucose channeled via the shikimate and phenylpropanoid pathway  <cit> .
in order to characterize the broader changes in secondary metabolism, we undertook a detailed metabolomics study.
as expected, the xylem tissue sample, which contain dead xylem cells, had the fewest statistically significant metabolite differences  between pdugpase <dig> overexpression lines and control plants .
still, among the significant differences, shikimic acid and maleic acid were reduced to 55% and 26% of controls, respectively.
several amino acids  and phenolic glycosides  were increased  <dig> x- <dig> x in the transgenics.
additionally, glycerol-1/3-phosphate and α-monopalmitin, two fatty acid-related metabolites, were moderately elevated  <dig> - <dig> x.
we have observed a significant increase  in total phenolic levels in transgenic samples relative to control .
the increase in phenolics may be correlated with the decreased plant height and altered lignin composition.
down-regulation of cinnamoyl-coa reductase , a gene in monolignol pathway in populus was reported to affect plant height, lignin content, s/g ratio, and reduction in several sugars including mannose, however, the levels of phenolics were found to be elevated  <cit> .
antisense down-regulation of 4cl gene in populus has been reported to result in reduced lignin content and plant height but increased levels of phenolics  <cit> .
in the pdugpase plants studies here, we observe a significant reduction in plant height and s/g ratio, and an increase in phenolics.
many more metabolites were affected in the phloem of ugpase <dig> transgenic lines relative to control plants .
similar responses included the reduction of shikimic acid and increases in  <dig>  5-dihydroxybenzoic acid-5-o-glucoside, salicylic acid-2-o-glucoside and salicin.
an example of a contrasting response included the decline in the phenolic acid glycoside  that was elevated in the xylem of ugpase <dig> plants.
additionally, there were declines in soluble sugars in the phloem of ugpase <dig> lines, including decreases in fructose, galactose, glucose and raffinose ranging from 40-68%, compared to that of controls.
many aromatic metabolites were reduced, including catechin, catechol, salicyl alcohol, salicylic acid and higher-order salicylates, including salicortin and α-salicyloylsalicin and five caffeoyl-conjugates, two of which  were not detected in plants overexpressing ugpase <dig> .
these latter declines were coupled with a decline in sinapyl alcohol and its glucoside, syringin, but not coniferyl alcohol nor its glucoside, coniferin.
these observations sit in contrast with the large number of increases in caffeic acid and other caffeoyl-conjugates and feruloyl-conjugates in the phloem of ugpase <dig> transgenic lines.
overall, both the total phenolic metabolites and soluble sugars were reduced in the phloem of ugpase <dig> lines relative to the levels measured in xylem.
udp-glucose acts as a substrate for glycosylation of small secondary metabolites  <cit> .
the effect of ugpase <dig> overexpression on the ratio of aglycones to glycosides of phenolics was measured in phloem samples of control and transgenic plants .
we found that as compared to controls, ugpase <dig> transgenic plants have a greater glycoside to aglycone ratio for the salicylates and a few other aromatic acids, but not for the monolignol glycosides.
the most prominent change was a 5-fold increase in salicin, a possible precursor of higher-order phenolic glycosides.
in general, metabolomic responses in leaves of pdugpase <dig> overexpression lines were similar to those in the phloem .
in contrast with stem phloem responses, caffeoylpopuloside and a caffeoyl-conjugate at rt  <dig>  declined.
additionally, cis- and trans-3-o-caffeoylquinic acids were enhanced in leaves, as were three other caffeoyl-conjugates .
two caffeoyl-glycoside conjugates at rt  <dig>  and  <dig>  were not detectable in leaves of control plants .
other benzoic acid-related and salicylic acid-related metabolites in the transgenics, including  <dig> -dihydroxybenzoic acid-3-o-glucoside, a partially identified dihydroxybenzoic acid-gallic acid glycoside , salicyl alcohol, catechol, and salicyloyl-salicortin, were higher.
reductions were observed in fatty acid-related metabolites, including glyceric acid and, mono- and di-galactosylglycerol.
neither salicylic acid nor its glucoside were significantly different between in leaves of transgenic and control plants.
furthermore, calorimetric assays showed that phenolic content was higher, and tannin, starch, sucrose, and glucose levels were lower in transgenic leaves relative to control .figure  <dig>
estimation of non-structural carbohydrates in control and
ugpase2
transgenic lines.
levels of glucose, sucrose and starch in young leaves , mature leaves  and stems  are presented.
data represent means ± se .
* indicates statistically significant, p ≤  <dig>  based on student’s t-tests.
levels of phenolics and tannins from mature leaves and petiole of control and three
ugpase2
overexpression lines
leaf
petiole
phenolics mg g
−1
tannins od g
−1
phenolics mg g
−1
tannins od g
−1
control
ugpase-1
ugpase-2
ugpase-3
data represent means ± se .
*indicates statistically significant, p ≤  <dig>  based on student’s t-tests.
similar metabolite phenotypes of populus plants misexpressing two distinct candidate cell wall biosynthesis genes
it is interesting to note that many of the metabolic responses reported here were similarly observed in a recent study of p. deltoides kor-like rnai knockdown mutants .
that is, with both overexpression of pdugpase <dig> in the present study and knockdown of kor-like genes, there was an accumulation of caffeoyl-conjugates, including caffeoylpopuloside.
both types of transgenics also had declines in the same types of caffeoyl-conjugates.
the close parallel in the metabolic phenotypes between these different transgenics was also evident in the leaves, where both exhibited declines in syringin and caffeoyl conjugates at rt  <dig>  and  <dig>  min and increases in cis- and trans-3-o-caffeoylquinic acid and caffeoyl-conjugates at rt  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig>  min.
given that these two groups of genes have been proposed to play a role in cell wall biosynthesis and remodeling pathways  <cit> , the premise of observed similarities in metabolite levels in response to perturbations of cell wall formation is interesting.
with s-lignin deposition affected more than g-lignin, there may be a reduced flux of carbon to lignin precursors at the terminus of the lignin pathway.
this hypothesis is supported by accumulation of phenolic acid  conjugates upstream of monolignol synthesis with shikimic acid and other unidentified moieties .
in ccr down-regulated populus, the decrease in lignin and s/g ratio was accompanied by an increase in feruloyl conjugates  <cit> .
a thorough characterization of accumulating caffeoyl-conjugates is merited, likely revealing the nature of lignin or cell wall storage components that have not previously been documented.
the greater number and nature of metabolite responses in phloem versus xylem suggests that metabolite trafficking between these organs plays a major role in shuttling, modifying and storing precursors that are directly not incorporated into the cell wall.
therefore, metabolite trafficking mechanisms in cell wall assembly also merit more attention.
additional detailed study of alterations in cell wall-related properties such as cellulose crystallinity, lignin-carbohydrate cross-linkages and structural features revealed by electron microscopy, as well as stem strength properties are needed to support a possible explanation of the observed similarity in metabolite profiles as a potential consequence of the aberrant cell wall formation in pdugpase overexpression and pdkor knockdown transgenic plants.
overexpression of a bacterial or populus ugpase result in contrasting phenotypes
in tobacco, overexpression of axugpase under the control of a cauliflower mosaic virus 35s promoter or a vascular-specific parsley 4cl promoter resulted in a slight increase in plant height via increased internode length  <cit> .
however, total plant dry weight was unaltered.
although overexpression of the axugpase was evident at the transcript level, in both leaves and xylem, ugpase enzyme activity was not significantly altered.
in contrast, overexpression of the same axugpase under the control of a 35s promoter in hybrid poplar resulted in significant reduction in plant height, stem diameter, leaf area and internode length  <cit> .
while sucrose and glucose levels were greater in hybrid poplar overexpressing axugpase  <cit> , their levels were lower in populus overexpressing the native ugpase <dig> under the control of a ubiquitin promoter.
moreover, leaf area of transgenic hybrid poplar plants overexpressing axugpase were nearly 75% smaller relative to control, while populus plants overexpressing native ugpase <dig> resulted in increased leaf area by a factor of two .
one plausible explanation is that the differential conserved domain composition of axugpase and pdugpase <dig> enzymes  contributes to differential utilization of sugars synthesized in transgenic leaves towards either leaf growth or storage purposes.
at the metabolic level, our results also contrast with those reported for hybrid poplar overexpressing a bacterial ugpase, such that there was a 233x increase in salicylic acid-2-o-glucoside in developing xylem, relatively no change in other phenolic glycosides and elevated soluble sugars using the axugpase construct  <cit> .
in our study, salicylic acid-2-o-glucoside was only modestly elevated  in xylem, along with a number of other phenolic glycosides with a similar magnitude of response of  <dig> - <dig> x. the greatest number and magnitude of metabolite responses were observed in phloem tissue of the stem, where both soluble sugars and total phenolic metabolites declined .
a decline in higher-order salicylates was offset by large accumulations of conjugates of caffeic acid and ferulic acid.
in contrast to the study undertaken by coleman et al.
<cit> , where lignin content was reported to be reduced and the s/g ratio increased, transgenic stem lignin content in the present study was unchanged while s/g ratio decreased.
this change in lignin composition  co-occurred with an increase in the concentrations of monolignols and their glucosides in the phloem of stems.
while coniferyl alcohol and its glucoside, coniferin were unaltered in phloem of ugpase <dig> plants, sinapyl alcohol and its glucoside, syringin, were both greatly reduced.
CONCLUSIONS
the present report addresses a gap in our understanding of the functional role of ugpase <dig> in woody perennials such as populus.
we have presented metabolomics-based evidence in support of a functional role for a plant ugpase <dig> in the tightly coupled primary and secondary metabolic pathways.
our results are in contrast with previously reported results based on overexpression of a low-identity bacterial ugpase in hybrid poplar.
the observed lack of impact on cellulose levels and contrasting plant morphology of populus ugpase <dig> overexpression lines lead to the hypothesis that plant ugpases may have evolved to play a functional role divergent from that reported for bacterial ugpase.
future research aimed at clarifying the precise enzymatic roles and pathway contexts of ugpase <dig> would provide mechanistic insights into the pronounced phenotypic effects resulting from overexpression of a native ugpase <dig> in populus.

 sucrose is the primary product of photosynthesis and the initial form of transported sugar in most plants. in higher plants, sucrose is hydrolyzed by either invertases to form glucose and fructose or sucrose synthases to form uridine diphosphate glucose and fructose. the carbon in glucose and fructose is further channeled into various primary or secondary metabolic pathways based on the spatiotemporal activity of metabolizing enzymes. sugar metabolizing enzymes have, therefore, been recognized as key gatekeepers of carbon allocation and partitioning pathways in plants. the sugar, udp-glucose, represents an important branch point in carbohydrate metabolism that can potentially be channeled directly for synthesis of starch, sucrose, cellulose or hemicellulose and pectin via synthesis of other nucleotide sugars such as udp-glucuronic acid. udp-glucose pyrophopharylase, also referred to as uglcpp or uridine triphosphate glucose-1-p uridylyltransferase, is a sugar-metabolizing enzyme that catalyzes the reversible formation of udp-glucose and pyrophosphate from glucose-1-phosphate and uridine triphosphate. in photosynthetically active mature leaves, ugpase catalyzes the reaction in the direction of udp-glucose and sucrose via coupled action of sucrose phosphate synthase. however, in sink organs, which depend on imported sucrose, ugpase is proposed to function in the conversion of udp-glucose, which is produced via susy hydrolysis of sucrose, to glucose-1-phosphate. based on an analysis of the most recent version of the populus trichocarpa genome and previous reports from hybrid aspen, rice and arabidopsis thaliana, ugpase genes appear to belong to small two-gene containing families. ugpases have been purified from and expressed in several eukaryotic and prokaryotic organisms. in vitro enzyme assays using atugpase and atugpase expressed in escherichia coli suggested that both the isoforms have slight preference for the pyrophosphorylation direction of the reaction. atugpase exhibited higher activity than that of atugpase in both directions, pyrophosphorolysis and synthesis. the two isoforms showed no activity with other sugars such as adp-glucose/atp or galactose-1-p. antisense suppression of ugpase in arabidopsis was reported to have no effect on plant growth, however, soluble carbohydrate and starch content was found to be reduced. tobacco plants overexpressing axugpase, a ugpase gene from acetobacter xylinum, exhibited increased plant height in several lines, similar total dry weight relative to controls and no significant changes in sugars, starch or cellulose levels. however, overexpression of the same bacterial ugpase in hybrid poplar resulted in significantly reduced growth, increased levels of soluble sugars, starch and cellulose, and reduced lignin. three studies involving antisense downregulation of a native ugpase gene in potato led to contrasting and inconclusive results. an early report suggested that a strong 95% reduction in ugpase transcripts and enzyme activity had no apparent effects on plant growth and sugar levels. in two subsequent studies, however, antisense suppression of ugpase in potato resulted in significantly lower sucrose levels. based on microarray analysis of cell types representing the discrete stages of xylem development and wood formation in populus, native ugpase expression was enhanced during late xylem cell expansion and secondary cell wall formation, suggesting a potential functional role for ugpase in udp-glucose substrate provision during secondary cell wall formation. in total, these results imply distinct host-specific-functional activities of ugpase in alternate plant species. a functional genomics investigation of native populus ugpase gene has not been reported. given that plant metabolism is tightly regulated for coordinated plant response to various internal and external cues, and carbon flow links primary metabolism to secondary metabolism, it is surprising how little we know about the enzymes interconnecting the primary and secondary metabolic pathways. it is plausible that manipulation of sugar metabolizing enzymes, such as ugpase, can affect both primary sugar and secondary wall carbohydrate metabolism; however, it is unclear if and how such manipulations may quantitatively and qualitatively affect secondary metabolic pathways of shikimate, phenylpropanoid and lignin biosynthesis. therefore, we have undertaken a detailed metabolic characterization of populus deltoides plants overexpressing a native pdugpase gene. transgenic plants had pronounced growth and metabolic changes relative to control plants. the changes in cell wall properties were, however, subtle with significantly reduced syringyl-to-guaiacyl ratios as one of the few alterations in transgenic cell walls. overall, our results support a role for pdugpase in carbohydrate metabolism, secondary metabolism and plant growth and development.  phylogenetic analysis and expression of ugpase in populus in several previous reports including populus, the ugpase gene family has been reported to comprise of two genes. a phylogenetic analysis was performed using two previously reported ugpase isoforms from populus, orthologs from other plant species and axugpase as an outgroup. ugpase and ugpase cluster together closer than with any other ugpases. while pdugpase and pdugpase which appear to have arisen during the salicoid duplication event, share more than 97% similarity with each other, they also share 92% similarity with atugpases vs. 90% with a gymnosperm ugpase. there is a lower, 87%, identity between the rice paralogs, osugpase and osugpase and a 93% identity between the arabidopsis paralogs, atugpase and atugpase the similarity of plant ugpases with the previously characterized axugpase averaged at 21%. the nucleotide binding loop and insertion loop, involved in dimer formation and stabilization, are conserved in atugpase and pdugpase but absent in axugpase, suggesting a potentially distinct enzymatic mechanism in plants involving dimer- or-oligomerization of proteins. in support of this hypothesis, it has been shown that the activity of barley ugpase is regulated by di- or oligomerization status of the protein, with monomer status representative of the active form of ugpase enzyme.figure phylogenetic analyses of ugpases from populus and other species. analysis was conducted in mega program using the neighbor-joining method. the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. ugpases from this study are in bold. atugpase: at3g03250; atugpase2: at5g; axugpase:aaa; egugpase: acf; osugpase: abd57308; osugpase2: af249880; ptrugp: potri.004g074400; ptrugp potri.017g144700; rcugpase: xp_; stugpase: aal; zmugpase: np_. expression of the two pdugpase genes was quantified in eight populus tissue types including young leaf, mature leaf, young stem, mature stem, petiole of a mature leaf, phloem, xylem and root by quantitative reverse transcriptase pcr. both isoforms were expressed in all tissues, with an overall higher expression level of ugpase relative to ugpase. among all the tissues profiled, expression of pdugpase was relatively higher in mature leaves and xylem, while expression of pdugpase was higher in young leaves and roots. our results are consistent with previously published data involving alternate tissues and organs, which showed that ugpase and ugpase were abundant in flowers and young leaves. in two additional studies, ugpase activity was also higher in xylem than in leaves. in potato, where only one isoform has been reported, ugpase activity was abundant in sink tissues, such as stems and tubers. in arabidopsis, ugpase was reported to have enhanced expression in leaves and stems compared to flowers, whereas ugpase had enhanced expression in flowers.figure relative expression of ugpase isoforms in different tissues. expression of ugpase and ugpase in yl, young leaf, ml, mature leaf, ys, young stem, ms, mature stem, pet, petiole, phl, phloem, xyl, xylem and rt, root. data represent means ± se. relative expression was calculated based on the expression of reference genes ubiquitin-conjugating enzyme e and 18s ribosomal rna. morphological characterization of pdugpase overexpression plants transgenic populus deltoides plants overexpressing the pdugpase gene under the control of a constitutive polyubiquitin promoter were maintained and characterized under greenhouse conditions. in an initial transgenic phenotyping study, all pdugpase overexpression lines were found to be consistently shorter in height with thinner stems compared to control plants. assessment of an independently grown second set of transgenic plants confirmed that plant height and stem diameter were consistently smaller relative to controls. conversely, leaf area and petiole length were significantly greater in transgenics, though total leaf dry weight was reduced as a result of fewer leaves. additionally, reduced plant height and stem diameter resulted in a significant reduction in stem dry weight. when compared to controls, leaf-to-stem biomass ratio doubled, whereas root weight and root surface area were reduced in transgenic plants. we quantified ugpase transcript levels in mature leaf petioles of three selected lines and found that pdugpase expression increased 2-fold. our results suggest that modest changes in native ugpase levels can have pronounced morphological effects on plant phenotype in woody perennials such as populus.figure phenotypic characterization of ugpase2 overexpression lines. differences in plant height and stem diameter, leaf area petiole length root system and root surface area among control and ugpase transgenic plants. data represent means ± se. * indicates statistically significant, p ≤  based on student’s t-tests. dry weight of different plant parts in control and two ugpase2 transgenic lines. dry weights corresponding to leaf, stem, and root and the ratio of leaf to stem dry weight are presented. data represent means ± se. *indicates statistically significant, p ≤ based on student’s t-tests. previous studies of suppression or overexpression of ugpase in other plant species have reported phenotypes ranging from no relative change to severely reduced plant height. in potato, for example, despite a strong 96% reduction in ugpase enzyme activity as a result of antisense suppression, plant growth or tuber development was found to be unaffected. in arabidopsis, the t-dna mutants of ugpase ugpase and double knockouts had no effect on growth rate, biomass production and flowering in mature plants, however, it was noted that young plantlets in agar had reduced hypocotyl and root length. a small increase in plant height without apparent effect on dry weight was reported for tobacco plants overexpressing the acetobacter ugpase under control of a constitutively expressed 35s promoter from cauliflower mosaic virus or a vascular-specific parsley 4-coumarate:coa ligase promoter. in populus plants overexpressing the same bacterial ugpase under the control of a constitutively expressed 35s promoter, however, was reported to display significant reduction in plant height, stem diameter, leaf area and internode length. contrastingly, we have observed that the leaf area is dramatically greater in pdugpase plants relative to control. the distinct aspect of the present work relative to the previous transgenic populus ugpase study is the use of a native gene from populus deltoides here. given the fact that pdugpase has only a 20% amino acid similarity with that of acetobacter ugpase, a difference between phenotypic responses of plants overexpressing the two genes is not unexpected. cell wall composition of pdugpase overexpressing populus hplc analysis of stem cell wall sugar composition showed that glucose and xylose were the most abundant sugars in all samples as expected. among glucose, xylose, galactose, arabinose and mannose, significant changes were observed only in mannose levels, with lower mannose levels in transgenic samples. while cellulose and lignin content appeared unaffected; the syringyl-to-guaiacyl ratio of lignin was significantly reduced in transgenic lines. in order to explore whether overexpression of ugpase has any effect on the transcription of carbohydrate metabolism or/and cellulose pathway, a subset of sugar metabolism and cellulose synthesis-related genes were studied. the panel of genes that we studied included, sucrose transporters, sut linked previously to enhanced cellulose production, and sut linked to carbon partitioning; genes that encode sucrose hydrolyzing enzymes, vacuolar invertases, vin and vin3; neutral invertase, nin. additionally, the secondary cell wall associated cellulose synthase genes, cesa7b and cesa8b, and korrigan genes, kor and kor were included based on previously reports of proposed roles in cellulose biosynthesis. susy and susy transcripts and corresponding proteins have been shown to be elevated in tissue contexts with enhanced cellulose biosynthesis such as secondary xylem development and tension stress response. furthermore, a functional role for susy in supplying sugars for cellulose synthesis has been proposed. the expression of most sugar metabolism genes was not significantly altered except sut which was significantly higher in all the three transgenic lines. whether sut has a direct role in sucrose transport and carbon partitioning in populus is as yet unanswered. of the surveyed cellulose pathway genes, cesa and susy isoforms were not significantly altered, but kor and kor were significantly increased in young leaves of transgenic lines. these results suggest that overexpression of pdugpase may be associated with modest changes in transcript levels of cellulose pathway-associated genes but that does not translate into changes in cellulose levels. the unchanged cellulose levels in overexpression lines of pdugpase contradicts with previous reports where cellulose levels were increased in axugpase overexpression lines. while the implication of low sequence identity between the bacterial and populus ugpase isoforms on enzyme activity is yet to be clarified, the functional activity of axugpase has shown to complement cellulose negative mutant phenotype by channeling sugar substrate for cellulose synthesis.table structural carbohydrate content in stems of control and three ugpase2 transgenic lines cellobiose glucose xylose galactose arabinose mannose control ugpase-1 ugpase-2 ugpase-3 data represent means ± se. *indicates statistically significant, p ≤  based on student’s t-tests. cell wall composition analysis in control and three ugpase2 overexpression lines. a. cellulose levels in different tissues, b. lignin and c. syringyl to guaiacyl ratio in stems of control and transgenic lines. yl and ml represent young and mature leaf, respectively. data represent means ± se. *indicates statistically significant, p ≤  based on student’s t-tests. expression of selected sugar metabolism and cellulose pathway genes in young leaves of control and ugpase2 overexpression lines. relative expression was calculated based on the expression of reference genes ubiquitin-conjugating enzyme e and 18s ribosomal rna. sut, sucrose transporter; vin, vacuolar invertase; nin, neutral invertase; sps, sucrose phosphate synthase; cesa, cellulose synthase; susy, sucrose synthase; kor, korrigan. accessions: sut potri.019g085800; sut potri.002g106900; vin potri.003g112600; vin potri.015g127100; nin potri.019g082000; sps potri.018g124700; cesa7b, potri.018g103900; cesa8b, potri.004g059600; kor poptri.003g151700; kor poptri.001g078900; susy potri.018g063500; susy potri.006g136700;18s, af206999; ubcc, potri.006g data represent means ± se. *indicates statistically significant, p ≤  based on student’s t-tests. metabolic profiles of xylem, phloem and leaves of pdgpase overexpressing populus secondary metabolites such as phenolics and tannins are derived from the carbon in glucose channeled via the shikimate and phenylpropanoid pathway. in order to characterize the broader changes in secondary metabolism, we undertook a detailed metabolomics study. as expected, the xylem tissue sample, which contain dead xylem cells, had the fewest statistically significant metabolite differences between pdugpase overexpression lines and control plants. still, among the significant differences, shikimic acid and maleic acid were reduced to 55% and 26% of controls, respectively. several amino acids and phenolic glycosides were increased x- x in the transgenics. additionally, glycerol-1/3-phosphate and α-monopalmitin, two fatty acid-related metabolites, were moderately elevated - x. we have observed a significant increase in total phenolic levels in transgenic samples relative to control. the increase in phenolics may be correlated with the decreased plant height and altered lignin composition. down-regulation of cinnamoyl-coa reductase, a gene in monolignol pathway in populus was reported to affect plant height, lignin content, s/g ratio, and reduction in several sugars including mannose, however, the levels of phenolics were found to be elevated. antisense down-regulation of 4cl gene in populus has been reported to result in reduced lignin content and plant height but increased levels of phenolics. in the pdugpase plants studies here, we observe a significant reduction in plant height and s/g ratio, and an increase in phenolics. many more metabolites were affected in the phloem of ugpase transgenic lines relative to control plants. similar responses included the reduction of shikimic acid and increases in 5-dihydroxybenzoic acid-5-o-glucoside, salicylic acid-2-o-glucoside and salicin. an example of a contrasting response included the decline in the phenolic acid glycoside that was elevated in the xylem of ugpase plants. additionally, there were declines in soluble sugars in the phloem of ugpase lines, including decreases in fructose, galactose, glucose and raffinose ranging from 40-68%, compared to that of controls. many aromatic metabolites were reduced, including catechin, catechol, salicyl alcohol, salicylic acid and higher-order salicylates, including salicortin and α-salicyloylsalicin and five caffeoyl-conjugates, two of which were not detected in plants overexpressing ugpase. these latter declines were coupled with a decline in sinapyl alcohol and its glucoside, syringin, but not coniferyl alcohol nor its glucoside, coniferin. these observations sit in contrast with the large number of increases in caffeic acid and other caffeoyl-conjugates and feruloyl-conjugates in the phloem of ugpase transgenic lines. overall, both the total phenolic metabolites and soluble sugars were reduced in the phloem of ugpase lines relative to the levels measured in xylem. udp-glucose acts as a substrate for glycosylation of small secondary metabolites. the effect of ugpase overexpression on the ratio of aglycones to glycosides of phenolics was measured in phloem samples of control and transgenic plants. we found that as compared to controls, ugpase transgenic plants have a greater glycoside to aglycone ratio for the salicylates and a few other aromatic acids, but not for the monolignol glycosides. the most prominent change was a 5-fold increase in salicin, a possible precursor of higher-order phenolic glycosides. in general, metabolomic responses in leaves of pdugpase overexpression lines were similar to those in the phloem. in contrast with stem phloem responses, caffeoylpopuloside and a caffeoyl-conjugate at rt declined. additionally, cis- and trans-3-o-caffeoylquinic acids were enhanced in leaves, as were three other caffeoyl-conjugates. two caffeoyl-glycoside conjugates at rt and were not detectable in leaves of control plants. other benzoic acid-related and salicylic acid-related metabolites in the transgenics, including -dihydroxybenzoic acid-3-o-glucoside, a partially identified dihydroxybenzoic acid-gallic acid glycoside, salicyl alcohol, catechol, and salicyloyl-salicortin, were higher. reductions were observed in fatty acid-related metabolites, including glyceric acid and, mono- and di-galactosylglycerol. neither salicylic acid nor its glucoside were significantly different between in leaves of transgenic and control plants. furthermore, calorimetric assays showed that phenolic content was higher, and tannin, starch, sucrose, and glucose levels were lower in transgenic leaves relative to control.figure estimation of non-structural carbohydrates in control and ugpase2 transgenic lines. levels of glucose, sucrose and starch in young leaves, mature leaves and stems are presented. data represent means ± se. * indicates statistically significant, p ≤  based on student’s t-tests. levels of phenolics and tannins from mature leaves and petiole of control and three ugpase2 overexpression lines leaf petiole phenolics mg g −1 tannins od g −1 phenolics mg g −1 tannins od g −1 control ugpase-1 ugpase-2 ugpase-3 data represent means ± se. *indicates statistically significant, p ≤  based on student’s t-tests. similar metabolite phenotypes of populus plants misexpressing two distinct candidate cell wall biosynthesis genes it is interesting to note that many of the metabolic responses reported here were similarly observed in a recent study of p. deltoides kor-like rnai knockdown mutants. that is, with both overexpression of pdugpase in the present study and knockdown of kor-like genes, there was an accumulation of caffeoyl-conjugates, including caffeoylpopuloside. both types of transgenics also had declines in the same types of caffeoyl-conjugates. the close parallel in the metabolic phenotypes between these different transgenics was also evident in the leaves, where both exhibited declines in syringin and caffeoyl conjugates at rt and  min and increases in cis- and trans-3-o-caffeoylquinic acid and caffeoyl-conjugates at rt,,,,,,,  min. given that these two groups of genes have been proposed to play a role in cell wall biosynthesis and remodeling pathways, the premise of observed similarities in metabolite levels in response to perturbations of cell wall formation is interesting. with s-lignin deposition affected more than g-lignin, there may be a reduced flux of carbon to lignin precursors at the terminus of the lignin pathway. this hypothesis is supported by accumulation of phenolic acid conjugates upstream of monolignol synthesis with shikimic acid and other unidentified moieties. in ccr down-regulated populus, the decrease in lignin and s/g ratio was accompanied by an increase in feruloyl conjugates. a thorough characterization of accumulating caffeoyl-conjugates is merited, likely revealing the nature of lignin or cell wall storage components that have not previously been documented. the greater number and nature of metabolite responses in phloem versus xylem suggests that metabolite trafficking between these organs plays a major role in shuttling, modifying and storing precursors that are directly not incorporated into the cell wall. therefore, metabolite trafficking mechanisms in cell wall assembly also merit more attention. additional detailed study of alterations in cell wall-related properties such as cellulose crystallinity, lignin-carbohydrate cross-linkages and structural features revealed by electron microscopy, as well as stem strength properties are needed to support a possible explanation of the observed similarity in metabolite profiles as a potential consequence of the aberrant cell wall formation in pdugpase overexpression and pdkor knockdown transgenic plants. overexpression of a bacterial or populus ugpase result in contrasting phenotypes in tobacco, overexpression of axugpase under the control of a cauliflower mosaic virus 35s promoter or a vascular-specific parsley 4cl promoter resulted in a slight increase in plant height via increased internode length. however, total plant dry weight was unaltered. although overexpression of the axugpase was evident at the transcript level, in both leaves and xylem, ugpase enzyme activity was not significantly altered. in contrast, overexpression of the same axugpase under the control of a 35s promoter in hybrid poplar resulted in significant reduction in plant height, stem diameter, leaf area and internode length. while sucrose and glucose levels were greater in hybrid poplar overexpressing axugpase, their levels were lower in populus overexpressing the native ugpase under the control of a ubiquitin promoter. moreover, leaf area of transgenic hybrid poplar plants overexpressing axugpase were nearly 75% smaller relative to control, while populus plants overexpressing native ugpase resulted in increased leaf area by a factor of two. one plausible explanation is that the differential conserved domain composition of axugpase and pdugpase enzymes contributes to differential utilization of sugars synthesized in transgenic leaves towards either leaf growth or storage purposes. at the metabolic level, our results also contrast with those reported for hybrid poplar overexpressing a bacterial ugpase, such that there was a 233x increase in salicylic acid-2-o-glucoside in developing xylem, relatively no change in other phenolic glycosides and elevated soluble sugars using the axugpase construct. in our study, salicylic acid-2-o-glucoside was only modestly elevated in xylem, along with a number of other phenolic glycosides with a similar magnitude of response of - x. the greatest number and magnitude of metabolite responses were observed in phloem tissue of the stem, where both soluble sugars and total phenolic metabolites declined. a decline in higher-order salicylates was offset by large accumulations of conjugates of caffeic acid and ferulic acid. in contrast to the study undertaken by coleman et al., where lignin content was reported to be reduced and the s/g ratio increased, transgenic stem lignin content in the present study was unchanged while s/g ratio decreased. this change in lignin composition co-occurred with an increase in the concentrations of monolignols and their glucosides in the phloem of stems. while coniferyl alcohol and its glucoside, coniferin were unaltered in phloem of ugpase plants, sinapyl alcohol and its glucoside, syringin, were both greatly reduced.  the present report addresses a gap in our understanding of the functional role of ugpase in woody perennials such as populus. we have presented metabolomics-based evidence in support of a functional role for a plant ugpase in the tightly coupled primary and secondary metabolic pathways. our results are in contrast with previously reported results based on overexpression of a low-identity bacterial ugpase in hybrid poplar. the observed lack of impact on cellulose levels and contrasting plant morphology of populus ugpase overexpression lines lead to the hypothesis that plant ugpases may have evolved to play a functional role divergent from that reported for bacterial ugpase. future research aimed at clarifying the precise enzymatic roles and pathway contexts of ugpase would provide mechanistic insights into the pronounced phenotypic effects resulting from overexpression of a native ugpase in populus. 
while cellulose and lignin levels in the cell walls were not significantly altered, the syringyl-to-guaiacyl ratio was significantly reduced. metabolomic analyses showed that manipulation of pdugpase results in perturbations in primary, as well as secondary metabolism, resulting in reduced sugar and starch levels and increased phenolics, such as caffeoyl and feruloyl conjugates. metabolic profilingprimary and secondary metabolismcell wallugpasepopulusissue-copyright-statement© the author 2014 we characterized the functional role of a ugpase gene in populus deltoides, pdugpase overexpression of the native gene resulted in increased leaf area and leaf-to-shoot biomass ratio but decreased shoot and root growth. these results demonstrate that pdugpase plays a key role in the tightly coupled primary and secondary metabolic pathways and perturbation in its function results in pronounced effects on growth and metabolism beyond cell wall biosynthesis of populus. the functional role of ugpase in perennial woody plants is poorly understood.
 udp-glucose pyrophosphorylase is a sugar-metabolizing enzyme that catalyzes a reversible reaction of udp-glucose and pyrophosphate from glucose-1-phosphate and utp. udp-glucose is a key intermediate sugar that is channeled to multiple metabolic pathways. the functional role of ugpase in perennial woody plants is poorly understood.  we characterized the functional role of a ugpase gene in populus deltoides, pdugpase overexpression of the native gene resulted in increased leaf area and leaf-to-shoot biomass ratio but decreased shoot and root growth. metabolomic analyses showed that manipulation of pdugpase results in perturbations in primary, as well as secondary metabolism, resulting in reduced sugar and starch levels and increased phenolics, such as caffeoyl and feruloyl conjugates. while cellulose and lignin levels in the cell walls were not significantly altered, the syringyl-to-guaiacyl ratio was significantly reduced.  these results demonstrate that pdugpase plays a key role in the tightly coupled primary and secondary metabolic pathways and perturbation in its function results in pronounced effects on growth and metabolism beyond cell wall biosynthesis of populus. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords metabolic profilingprimary and secondary metabolismcell wallugpasepopulusissue-copyright-statement© the author 2014 