BACKGROUND
among different forms of carbon storage, triacylglycerol  is the most energy dense, yielding more than double amount of energy on a per weight basis as compared to starch.
due to the increased global need for plant oil derived products for food but also for non-food applications to decrease our dependence on fossil oil, the interest in mechanisms for regulation of carbon allocation into oil in plant storage tissues has gained a lot of attention and can be of great economic and environmental importance in the process of developing new high-yielding oil crops  <cit> .
to be available for tag synthesis, carbons from sucrose must first be converted to pyruvate through cytosolic or plastidic glycolytic pathways and at some point be imported into the plastid to yield acetyl-coenzyme a  which feeds fatty acid  synthesis with carbon backbones.
fas are subsequently transported out to the endoplasmic reticulum where tag assembly takes place  <cit> .
the seed-specific, loss-of-function mutant wrinkled <dig> of arabidopsis thaliana with 80 % reduction in oil content and increased levels of sucrose but not of starch in mature seeds was by enzymatic analyses shown to be impaired in the allocation of carbon into glycolysis  <cit> .
the corresponding gene, atwrinkled <dig> , encodes a protein with two apetala2-ethylene responsive element-binding protein  motifs that is a signature for plant-specific transcription factors involved in a wide range of developmental processes  <cit> .
gene expression analyses using microarrays have further revealed that some of the down-regulated genes in seeds of the wri <dig> mutant are involved in glycolysis and fa synthesis, rather than in tag assembly  <cit> .
moreover, atwri <dig> has been shown to bind to the aw-box sequence in upstream regions of several genes involved in fa synthesis and glycolysis .
homologs to atwri <dig> have been identified in oil-dense embryonic tissues of several other plant species as rape seed  and maize   <cit> .
a homolog to atwri <dig> was also associated with the large difference in carbon allocation to oil between oil palm  and date palm  mesocarps with the former mainly storing carbon as oil, the latter as sugar  <cit> .
today’s global plant oil production is mainly dependent on tag storage capacity in oil palm fruits and the embryos of soybean, sunflower, and oilseed rape  <cit> .
studies trying to reveal the regulatory mechanisms behind oil accumulation have mainly focused on embryonic tissues of seeds  <cit> , even though fruit mesocarp and seed endosperm tissues have also been studied  <cit> .
however, the potential to produce oil in already high-yielding but not usually oil dense tissues like cereal grain endosperm and underground tuber or root parenchyma, by directing carbon allocation from starch or sugar into oil by genetic engineering, deserves further attention due to the important applied benefits.
among plant species producing underground tubers, only one is known to have genotypes with high amount of oil , i.e.
yellow nutsedge  <cit> .
among the cereals, oat is the only species known to store relatively high amount of oil  in the grain endosperm  <cit> .
in both these tissues, homologs to the atwri <dig> were expressed during tuber parenchyma and grain endosperm development, respectively .
another interesting tissue for potential oil production is from trees due to the vast amount of wood processed in the pulp industry from which fas are already an important byproduct  <cit> .
studies on perennial plants have indicated that lipids can accumulate transiently in the cambium upon transition to dormancy  <cit>  and a homolog to the atwri <dig> has been identified in poplar from available genomic resources  <cit> .
therefore, the role of wri1-like genes for directing carbon flow into oil synthesis and accumulation could be extended to include diverse types of plant tissues.
in oilseed species like arabidopsis, oil accumulation is part of the embryo development and seed maturation process which is governed by master regulators such as lec <dig>  lec <dig>  fus <dig> and abi <dig>  <cit> .
wri <dig> is linked to this upstream regulatory network and has been shown to be a downstream target and specify the action of lec <dig> towards fa synthesis  <cit> .
however, homologs to these upstream regulators were not found in the mesocarp of oil palm, which suggests that the regulatory networks in non-embryo tissues are different to those in developing seed embryos  <cit> .
in this study, we investigated the effect on global gene expression in leaves where oil accumulation was induced by expression of wri <dig> homologs originating from diverse species and tissues  using a transient gene expression system with agroinfiltration of nicotiana benthamiana.
a time study of oil and starch content as well as rt-qpcr and photosynthesis measurements in leaves expressing arabidopsis wri <dig> between one to five days after infiltration was done to further characterize and complement the transcriptional transitions observed at five days after infiltration.
RESULTS
all homologs were wri1-like
the wri <dig> gene from arabidopsis and four additional homologs from different species representing three dicotyledons and two monocotyledons were used in the present study .
these selected homologs are expressed in different types of tissues that all have induction of oil accumulation at certain developmental or temporal stages.
the arabidopsis gene is expressed in the embryo during seed development as is the potato homolog, and the poplar homolog is expressed in the cambium of the tree.
the oat homolog is expressed in grain endosperm and the nutsedge homolog in underground stem tuber.
from a phylogenetic tree based on homology of full-length cdna it was clear that the novel wri <dig> homologs of this study were more similar to arabidopsis wri <dig> than to either arabidopsis wri <dig>  wri <dig>  or wri <dig>  <cit>  which are more similar to each other than to any of the other presented homologs .
it was also evident from this phylogeny analysis that the two homologs of wri <dig> originating from monocot species were more similar to each other than to the dicot wri <dig> homologs.
the wri <dig> orthologs and homologs so far studied have a high degree of identity and similarity on an amino acid level in the region spanning the two ap <dig> domains but with a high degree of divergence in the n-and c-terminus  <cit>  which was also true for the wri <dig> homologs of this study .
however, outside the dna binding domains there was a high divergence among the different homologs although it was evident that species which are more closely related have a higher degree of amino acid homology and identity.
this in particular applies to the derived amino acid sequences of the n. benthamiana and s. tuberosum homologs of wri <dig> with 82 % identity in contrast to the homolog from c. esculentus which had only 45 % identity to that of n. benthamiana.
the different homologs are hereafter named atwri <dig>  stwri1em, ptwri1ca, aswri1es, and cewri1tp abbreviated after the species and tissue of origin .fig.
<dig> phylogeny tree of wri <dig> homologs.
phylogeny analysis based on full-length cdna sequences of wri homologs from arabidopsis , potato embryo , tobacco , poplar stem , oat endosperm , and nutsedge tuber parenchyma
all wri1-like homologs induced oil accumulation in leaves
all wri <dig> homologs in this study were shown to drastically induce oil  accumulation when transiently expressed in leaves of n. benthamiana by agroinfiltration .
leaves expressing the aswri1es showed the highest oil concentration  followed by atwri <dig>  stwri1em, ptwri1ca, and cewri1tp .
this result shows that the function of wri <dig> is highly conserved across species and storage tissues even though not of typical oil-dense embryo type.
also diacylglycerol levels increased in leaves upon expression of the wri <dig> homologs .
polar lipids  were the most abundant in leaves but did not show any difference between leaves expressing the different wri <dig> homologs or to transformed control .
the leaves expressing the wri <dig> homologs appeared stressed with chlorosis, in particular for leaves expressing stwri1em.
dry matter contents of leaves were not significantly different from transformed control , except for leaves expressing the aswri1es with higher dry matter .fig.
<dig> leaf lipid concentrations.
concentration of different lipid classes , dag; diacylglycerol , pl; polar lipids , and other lipids ) given as nmol fatty acids  on dry weight basis of n. benthamiana leaves expressing wri <dig> from arabidopsis embryo , potato embryo , oat endosperm , poplar stem , nutsedge tuber parenchyma , and transformed control  five days after infiltration.
results are the mean from three biological replicates ± standard deviation.
letters distinguish significant different means according to tukey’s test at level p ≤  <dig>
the fa composition of tag changed upon expression of wri <dig> homologs with lower proportions of 18: <dig> and in general higher proportions of 18: <dig>  as compared to transformed control .
in the polar lipid fraction of leaves, the largest observed differences in fa profile upon expression of wri <dig> homologs were in general increased proportions of 18: <dig> and decreased 18: <dig> .fig.
<dig> fatty acid profiles of lipids.
tag; triacylglycerol , dag; diacylglycerol , pl; polar lipids , and other lipids  in n. benthamiana leaves expressing wri <dig> from arabidopsis embryo , potato embryo , oat endosperm , poplar stem , nutsedge tuber parenchyma , and transformed control  five days after infiltration.
results are the mean from three biological replicates ± standard deviation
since oil accumulation was highest in leaves expressing aswri1es and atwri <dig>  the localization and structural properties of oil were only analyzed for those  together with transformed control leaves .
tem analysis indicated that the accumulated oil was localized to the cytosol and was most probably present as oil droplets and not as distinct organized oil-bodies surrounded by oil-body bound proteins.
this is consistent with that no up-regulation of transcripts encoding oil-body proteins was observed in the transcriptome data .
however, differences in oil quantity could not be clearly visualized by this method.
expression of oleosins in arabidopsis leaves already expressing phospholipid:diacylglycerol acyltransferase promoted oil accumulation giving  <dig>  % oil of leaf dw and also changed the structural storage form into organized oil-bodies  <cit> , and combining oleosins with wri <dig> and diacylglyceroltransferase <dig> in n. tabacum leaves gave even higher oil content, 15 % by leaf dw  <cit> .
this demonstrates the potential of combining a transcription factor with genes involved in both tag synthesis and protection against mobilization, for achieving high oil content in leaves.
for clarification, the aim of our study was not to achieve the highest oil content possible but instead to map the transcriptional changes induced by wri <dig> alone in leaf tissue.fig.
<dig> transmission electron microscopy.
photos of n. benthamiana leaves expressing wri <dig> from oat endosperm  and arabidopsis embryo .
transformed control leaf  five days after infiltration.
cw; cell wall, p; plastid, s; starch, o; oil , m; mitochondria, v; vacuole, n; nucleus
transcriptome sequencing data
leaves expressing the different wri <dig> homologs and transformed control tissue were subjected to transcriptome sequencing five days after agro-infiltration, which is generally used as a suitable time for sufficient transient gene expression and impact on metabolism .
upon expression of wri <dig> homologs in the n. benthamiana leaf tissue, notable differences in the number of differentially expressed genes as compared to transformed control were observed, but all five homologs induced a similar pattern in core metabolic pathways.
expression of stwri1em in leaves induced the highest number of differentially expressed genes for both up-regulated and down-regulated transcripts, followed by atwri <dig>  ptwri1ca, aswri1es, and cewri1tp .
however, since the expression levels of the different wri <dig> homologs varied significantly in leaves , no conclusions regarding species-specific differential gene expression induced by those different homologs could be made from this study.
fold-changes of gene expression given here are therefore the averages from leaves expressing the different wri <dig> homologs.
interesting to note though, is that the expression levels of the different wri <dig> homologs did not correlate either to oil content or number of differential expressed genes in leaves.
in fact, the number of differentially expressed genes was instead correlated with phylogenetic similarity of the different wri <dig> homologs to the n. benthamiana wri <dig>  suggesting that the most closely related stwri1em yielded the highest and cewri1tp the lowest number of differentially expressed genes in leaves.
the lists of differentially expressed genes in leaves  upon expression of the different wri <dig> homologs had in general a similar overall pattern for core metabolic pathways .
here, these general trends are described where the functional equivalence of each gene was assigned according to the closest homolog of arabidopsis .
the effect on global gene expression in leaves induced by wri <dig> homologs was in general strong with many genes shown to be highly differentially regulated.
some genes were virtually switched ‘on’, going from almost non-detectable in transformed control tissue to a relatively high expression in wri1-infiltrated tissue.
this illustrates the strong effect of using a constitutively expressed transcription factor to achieve a drastic change of the gene expression program of a certain tissue.
the results also reveal notable similarities in the effect on transcriptional regulation exerted by wri <dig> homologs coming from such diverse plant species and tissues as were included in this study.
it is important to note that it is not possible to distinguish whether the observed effects on gene expression in leaves five days after agroinfiltration were due to direct or indirect effects of wri <dig> expression.
furthermore, this study only discusses induced changes at the transcript level and cannot state whether these changes is also correlated to protein levels.
carbon directed to the plastid
the oil-inducing effect of wri <dig> in arabidopsis seeds has been attributed to its enhancing expression of genes encoding enzymes in glycolysis as well as fa synthesis, rather than in tag assembly  <cit> .
many of the genes, as for biotin carboxylase carrier protein, kasi, enoyl-acp reductase, acp, fatty acid desaturase  <dig>  plastid pyruvate kinase, and plastid pyruvate dehydrogenase e1α, which are down-regulated in the arabidopsis mutant wri <dig> as compared to in wild-type seeds, are within the set of genes involved in fa synthesis and glycolysis shown to have a bell-shaped expression pattern during wild-type seed development  <cit> .
on the contrary, genes involved in tag assembly are not differentially expressed in the wri <dig> seeds and do not belong to the ‘bell-shaped group’ of genes in the wild-type.
studies on arabidopsis seeds also showed that the activities of several glycolytic enzymes are much decreased in the wri <dig> as compared to wild-type seeds  <cit> .
therefore, it was not surprising that leaves expressing wri <dig> homologs in our study indeed had a much higher expression of genes involved in glycolysis and fa synthesis pathways, as compared to in transformed control .
wri <dig> expression also induced a shift at the transcript level in the organelle distribution of glycolysis with carbon flux being directed to the plastid.
genes encoding enzymes of the upper part of cytosolic glycolysis; sucrose synthase , ppi-dependent fructose-6-phosphate  1-phosphotransferase, and frc-bisphosphate aldolase, and the lower part of plastidic glycolysis; phosphoglycerate mutase, enolase, the α and β subunit of pyruvate kinase, and all three subunits  of pyruvate dehydrogenase were highly up-regulated  in leaves expressing wri <dig>  up-regulated were also transcripts of the plastid translocator for phosphoenolpyruvate , thereby probably ensuring cytosolic glycolysis up to phosphoenolpyruvate followed by uptake into the plastid for further plastidic glycolysis.
interestingly, this transcript pattern of organelle shift for glycolysis is very similar to that of arabidopsis seeds during development  <cit> , indicating that this might be a signature of a plant tissue in transformation from a photosynthetic source to heterotrophic oil accumulation.
this pattern is also in agreement with the observed decreased flux of carbon through the lower part of plastidic glycolysis from phosphoenolpyruvate and downwards in developing embryos of the wri <dig> arabidopsis mutant as determined by 13c metabolic flux analyses  <cit> .fig.
<dig> heat map of differential gene expression involved in central carbon metabolism.
values given are log <dig> of the ratios between rpkm of leaves expressing wri <dig> and transformed control five days after infiltration.
the order of leaf samples expressing the five different wri <dig> homologs are given for the box numbered 1: atwri <dig>  stwri1em, ptwri1ca, aswri1es, and cewri1tp.
abbreviations: e4p; erythrose 4-phosphate, sucr; sucrose, glc; glucose, frc; fructose, frc6p; fructose 6-phosphate, glc6p; glucose 6-phosphate, frc <dig> bp; fructose  <dig> -bisphosphate, udp; uridine diphosphate, glnt; gluconate, r5p; ribose 5-phosphate, ru5p; ribulose 5-phosphate, ru <dig> bp; ribulose  <dig> -bisphosphate, pgal; phosphoglyceraldehyde, pga; phosphoglycerate, dpga; diphosphoglycerate, pep; phosphoenolpyruvate, pyr; pyruvate, oaa, oxaloacetate, mal; malate, adp; adenosinediphospho-, s7p; sedoheptulose 7-phosphate, s <dig> bp; sedoheptulose  <dig> - bisphosphate, xu5p; xylulose 5-phosphate.
enzymes:  <dig>  sucrose synthase,  <dig>  ppi-dependent fructose-6-phosphate 1-phosphotransferase,  <dig>  fructose-bisphosphate aldolase,  <dig>  nadp-dependent glyceraldehyde-3-phosphate dehydrogenase,  <dig>  phosphoenolpyruvate carboxykinase,  <dig>  phosphoenolpyruvate carboxylase,  <dig>  triosephosphate/phosphate translocator,  <dig>  phosphoenolpyruvate/phosphate translocator,  <dig>  phosphoglycerate mutase,  <dig>  enolase,  <dig>  pyruvate kinase ,  <dig>  pyruvate dehydrogenase ,  <dig>  malic enzyme,  <dig>  phosphoglucomutase,  <dig>  adp-glucose pyrophosphorylase large subunit,  <dig>  starch synthase  <dig>   <dig>  β-amylase,  <dig>  glucose-6-phosphate dehydrogenase,  <dig>  transaldolase,  <dig>  triose-phosphate isomerase,  <dig>  fructose- <dig> -bisphosphatase,  <dig>  sedoheptulose  <dig> -bisphosphatase,  <dig>  ribose-5-phosphate isomerase,  <dig>  phosphoribulokinase,  <dig>  rubisco,  <dig>  phosphoglycerate kinase,  <dig>  glyceraldehyde-3-phosphate dehydrogenase ,  <dig>  carboxyl transferase α  subunit of acetyl-coa carboxylase ,  <dig>  biotin carboxylase  of acc,  <dig>  biotin carboxyl carrier protein  <dig>  of acc,  <dig>  acyl carrier protein ,  <dig>  malonyl-coa:acp malonyltransferase,  <dig>  3-ketoacyl-acp synthase iii ,  <dig>  3-ketoacyl-acp reductase,  <dig>  enoyl-acp reductase,  <dig>  3-ketoacyl-acp synthase i ,  <dig>  3-ketoacyl-acp synthase ii
increased storage synthesis
pyruvate from glycolysis is converted by pyruvate dehydrogenase into acetyl-coa which is the two-carbon precursor for de novo fa synthesis in the plastid.
many genes encoding enzymes involved in fa synthesis were shown to be up-regulated in leaves expressing the wri <dig> homologs  again well in agreement with other studies .
transcripts encoding all subunits of acetyl-coa carboxylase , acyl carrier protein , malonyl-coa:acp malonyltransferase and 3-ketoacyl-acp synthase iii , 3-ketoacyl-acp synthase i  were all up-regulated  in leaves expressing wri <dig> homologs.
a very high up-regulation was observed for the reductases of 3-ketoacyl-acp and enoyl-acp of the fatty acid synthase complex .
one transcript that was up-regulated 278-fold in leaves expressing the wri <dig> homologs, with very low expression in the transformed control tissue, was clearly annotated as a kas but phylogenetic analyses  did not allow for an obvious designation to kasi or ii in arabidopsis.
however, no other kas genes exist in n. benthamiana and therefore this transcript most probably encodes the enzyme activity corresponding to that of kasii in arabidopsis.
the high up-regulation of this transcript indicates the importance of the elongation of 16:0-acp to 18:0-acp for increased tag synthesis.
genes encoding enzyme activities in tag assembly were previously not usually associated with wri <dig> regulation .
however, a few transcripts related to tag assembly were in this study found to be up-regulated in leaves upon wri <dig> expression .
one transcript annotated to represent phosphatidylcholine diacylglycerol cholinephosphotransferase  was up-regulated  in leaves expressing the wri <dig> homologs.
pdct is involved in the regulation of tag composition by catalyzing the inter-conversion of diacylglycerol and phosphatidylcholine  <cit> , and interestingly the encoding gene was previously shown to be down-regulated in the triple mutant of arabidopsis with reduced expression of wri <dig>  wri <dig>  and wri <dig>  <cit> .
transcripts encoding the enzymes phospholipase c and d were also up-regulated .
both types of lipases are  thought to be important for the conversion of phosphatidylcholine to diacylglycerol  <cit> .
therefore, pdct and/or phospholipases might therefore be the enzymes defining the observed shift in fa composition of tag upon induction of oil accumulation.
furthermore, a transcript encoding glycerol-3p dehydrogenase, catalyzing the synthesis of glycerol-3p  from dihydroxyacetonephosphate was up-regulated 8-fold.
one plastidic isoform of glycerol-3p dehydrogenase in arabidopsis  was previously shown to be important for the metabolism of plastid-localized glycerolipids  <cit>  and is probably not associated with tag synthesis .
however, the glycerol-3p dehydrogenase shown to be up-regulated in our study  was encoding another plastidic isoform  <cit>  which might be important for tag backbone supply since it is associated with gene expression during seed development in arabidopis , and glycerol-3p can pass the plastid envelope through a permease which was recently shown to play an important role for lipid content of arabidopsis embryos  <cit> .
interestingly, the gene encoding the plastidic glycerol-3p dehydrogenase found to be up-regulated in our study was also identified to be one of  <dig> putative target genes of maize  wri <dig>  <cit> .
many genes involved in amino acid metabolism  were differentially expressed upon expression of wri <dig> homologs in our study  which could possibly indicate increased activities involved with protein storage accumulation.
transcripts encoding vacuolar sorting receptor was 2-fold up-regulated while that of vacuolar processing enzyme as much as 1400-fold up-regulated.
other examples of up-regulated transcripts encoded membrane amino acid transporters  and endoplasmic reticulum to golgi vesicle mediated transport of amino acids .
transcripts encoding asparagine synthetase and glutamate synthase was up-regulated , while on the other hand that of chloroplastic glutamine synthetase was down-regulated .
transcripts annotated as amino transferases of aspartate and alanine were up-regulated .
however, it was difficult to completely dissect the meaning of these differentially expressed genes as pathways were up-regulated or down-regulated during seed development in arabidopsis  <cit> , which could fit with wri <dig> expression although changes in amino acid metabolism and nitrogen remobilization is also consistent with other patterns such as stress or senescence.
changes in expression of genes in amino acid metabolism are also indicative as markers for changes in general metabolism in the leaf tissue  <cit> .
it is interesting to note that increased levels of certain amino acids were actually observed in maize kernels overexpressing the zmwri1a  <cit>  which can indicate an association of wri <dig> with storage protein synthesis.
decreased photosynthesis and starch formation
an important finding in this study was that the expression of wri <dig> homologs in leaves induced a severe down-regulation of transcripts encoding functions in the photosynthetic apparatus , both in the light-dependent reactions  oxidoreductase, subunits of atp generation complex,  <dig> to 66-fold changes, see fig.
6) and the carbon fixation reactions of all three phases of the calvin-benson cycle .
thus the whole calvin-benson cycle was down-regulated together with the light harvesting and probably atp generation of the photosynthetic apparatus .
in agreement with this, the transcript encoding the plastid localized triosephosphate/phosphate translocator, with a substrate specificity allowing organelle exchange of dihydroxyacetone-p, glyceraldehyde-3p, and 3-phosphoglycerate  <cit> , was also down-regulated  in wri <dig> expressing tissue which could indicate a decreased flux of the daily photosynthetically fixed carbon out from the plastid for further sucrose synthesis  <cit> .fig.
<dig> heat map of differential gene expression involved in photosynthesis.
values given are log <dig> of the ratios between rpkm of leaves expressing wri <dig> and transformed control five days after infiltration.
the order of leaf samples expressing the five different wri <dig> homologs are given for the box numbered 1: atwri <dig>  stwri1em, ptwri1ca, aswri1es, and cewri1tp.
<dig>  light harvesting complex photosystem ii subunit  <dig>   <dig>  photosystem ii subunit p,  <dig>  psbq subunit of the oxygen evolving complex of photosystem ii,  <dig>  photosystem ii subunit psbx,  <dig>  photosystem ii type i chlorophyll a/b-binding protein,  <dig>  photosystem ii encoding the light-harvesting chlorophyll a/b binding protein cp <dig> of the antenna system of the photosynthetic apparatus,  <dig>  photosystem ii  <dig> kd protein,  <dig>  involved in the light harvesting complex of photosystem ii,  <dig>  photosystem ii by,  <dig>  subunit of light-harvesting complex ii,  <dig>  photosystem ii type i chlorophyll a/b-binding protein,  <dig>  rieske fes center of cytochrome b6f complex,  <dig>  cytochrome b6f complex subunit,  <dig>  plastocyanin,  <dig>  photosystem i subunit e <dig>   <dig>  photosystem i subunit f,  <dig>  subunit g of photosystem i,  <dig>  photosystem i, subunit h <dig>   <dig>  photosystem i subunit k,  <dig>  protein predicted by sequence similarity with spinach psad to be photosystem i reaction center subunit ii,  <dig>  photosystem i reaction center, sub psi-n,  <dig>  photosystem i subunit o,  <dig>  p subunit of photosystem i,  <dig>  leaf-type ferredoxin:nadp oxidoreductase
one example where functional duality of enzymatic activities became obvious was for frc-bisphosphate aldolase with one up-regulated and one down-regulated transcript .
since this enzyme catalyzes the reversible conversion of both frc-bisphosphate  and sedoheptulose-bisphosphate  into dihydroxyacetonephosphate, it can be speculated that the up-and down-regulated transcripts  actually represent genes encoding aldolase with importance for glycolysis and the calvin cycle, respectively.
transcripts encoding enzymes in starch synthesis  were down-regulated  while that of β-amylase, important for starch degradation, was instead up-regulated 6-fold .
increased gene expression and enzyme activity of β-amylase was previously observed during development of oil-seeds of arabidopsis and rape indicating the importance of starch degradation to yield carbon precursors for oil accumulation in these species  <cit> .
starch breakdown and increased glycolysis was also correlated to increased fa synthesis at transcript level in the cambium of aspen upon developmental transition to dormancy  <cit> .
the expression of the different wri <dig> homologs in leaves induced down-regulation of many genes involved in chloroplast fa desaturation and very long chain fa  and cutin biosynthesis .
examples were transcripts encoding fad <dig> , fad <dig> , lacs <dig> , lacs <dig> long-chain acyl-coa synthetases related  <cit> , cer <dig> , and a cer26-like hxxxd acyltransferase .
in addition, genes annotated as coding for modifier functions of cutin were also down-regulated like cer <dig> which is suggested to be an aldehyde decarbonylase  <cit> , and dcr , which is a hxxxd acyl-transferase  <cit> .
furthermore, a gene homologous to glycerol-3-phosphate acyltransferase shown to be critical for acyl transfer in cutin biosynthesis  <cit>  was down-regulated.
possibly genes encoding p <dig> proteins corresponding to functions of importance for cuticle development as cyp86a <dig>  <cit>  were also among down-regulated genes in leaves expressing wri <dig> homologs.
among all those, the transcript encoding fad <dig> was the most severe down-regulated , with other transcripts showing  <dig> to 7-fold changes.
interesting and in agreement with our results, a reduction of gene expression encoding one of the chloroplast desaturases, fad <dig>  was previously observed in leaf tissue expressing atwri <dig>  <cit> .
in the same publication it was noted that in the presence of increased tag synthesis there was a severe reduction in surface lipids.
our results suggests that the reduction in surface lipids they observed would not be due to a competition for substrates but rather a function of reduced gene expression of transcripts related to surface lipid synthesis in response to expression of wri <dig>
all these changes induced by wri <dig> expression imply that many of the transcripts encoding enzymes giving typical characteristics of a source tissue  were highly down-regulated, suggesting a shift into a heterotrophic tissue with low photosynthetic contribution.
this can probably explain the observed chlorosis of leaves expressing wri <dig>  it can be speculated that wri <dig> mediates repression of photosynthetic source functions also during seed development.
carbons from oil recycled to plastid
in oil-accumulating sink tissue such as a seed, the sucrose from photosynthesizing leaves will feed the developing seed allowing for a continuous carbon flow into storage product accumulation.
however, when expressing a transcription factor that, directly or indirectly, not only induce oil synthesis but also reduce photosynthesis, the sucrose available for the increased fa synthesis is likely to become limiting.
in fact, transcripts encoding an α/β hydrolase with tag lipase activity  was highly up-regulated  while glycerol kinase , and several enzymes involved in β-oxidation of fas  were also up-regulated  <dig> to 6-fold .
furthermore, a transcript encoding malate synthase, involved in the closely connected glyoxylate cycle that produces malate from degraded fas, was also highly up-regulated .
to be available for fa synthesis, malate can then either be translocated into the plastid in exchange for phosphate  <cit>  where it can be converted to pyruvate by malic enzyme, or cytosolic conversion of malate by malate dehydrogenase and phosphoenolpyruvate carboxykinase yields phosphoenolpyruvate which can be transported into the plastid by the phosphoenolpyruvate/phosphate translocator.
several transcripts encoding enzyme activities in both these routes  were up-regulated  in wri <dig> expressing leaf tissue as compared to transformed control.
our results therefore suggest that the new sink-like tissue induced in the leaf by expression of wri <dig> is experiencing carbon source limitations.
in the absence of sucrose import this probably triggers the up-regulation of transcripts encoding enzymes in both tag and fa degradation in the leaf in attempt to yield carbons that can be recycled back, either as malate or phosphoenolpyruvate, into glycolysis.
an alternative or additional explanation could be that increased amount of tag in a leaf mesophyll cell, without proper packaging with oil-body proteins, is more prone to mobilization which induces up-regulation of transcripts encoding enzymes of tag degradation.
this will in turn yield free fas that can be toxic to the cell if accumulating  <cit> , which can explain the up-regulation of transcripts encoding functions in fa degradation.
.
as mentioned earlier, the highest total oil content of leaves achieved in our study  was relatively low as compared to in other studies where the stable expression of combination of the transcription factor wri <dig> with other genes involved in tag assembly and storage  showed that much higher oil content is possible to achieve  in n. tabacum leaves  <cit> .
interestingly, the promoter chosen for atwri <dig> expression in that study was that of arabidopsis rubisco, with the rational of achieving a diurnal expression pattern.
however, the down-regulation of photosynthesis and the calvin-benson cycle induced upon wri <dig> expression in this study suggests that wri <dig> itself might be attenuating the expression of wri <dig> from the rubisco promoter.
it is possible that this plausible self-regulating effect of the atwri <dig> expression could be another key to the high levels of tag accumulated without apparent negative effects on plant vigor in that study.
additional differentially expressed genes of potential importance for seed development
a number of genes were differentially expressed in leaves upon wri <dig> expression where the closest homologs in arabidopsis are expressed during seed development or corresponding proteins are of importance for proper seed development.
for example, transcripts annotated as encoding trehalose-6-phosphate synthase were up-regulated 30-fold .
trehalose-6-phosphate is thought to be a signal of sugar status in plant storage tissues and the synthase is of crucial importance for embryo development in arabidopsis .
transcripts encoding proteins of completely unknown function but where the closest arabidopsis homolog is expressed during seed development were found, exemplified by a transcript annotated as closest homolog to at2g <dig>   which was highly up-regulated  in wri <dig> expressing tissue .
for another transcript which was also highly up-regulated , the closest homolog in arabidopsis was at1g <dig> encoding seuss, a transcriptional adaptor of importance for embryonic development  <cit> .
there were additional transcripts annotated as nucleic acid binding or transcription factors although their functions are unknown and gene families so large that closest homologs in arabidopsis were difficult to discern.
only one transcript related to genes encoding oil body proteins was up-regulated by the wri <dig> homologs which closest homolog was at1g <dig> encoding a putative aquaporin  <cit> .
time study of leaves expressing atwri1
some of the transcriptional transitions observed in leaves induced, directly or indirectly, by the transient expression of all the different wri <dig> homologs were unexpected, such as the effect on transcripts encoding photosynthesis, starch metabolism, and fatty acid degradation.
therefore, the effects of the transient expression of atwri <dig> in n. benthamiana leaves were physiologically characterized in more detail in a time study from one to five days after infiltration .
non-transformed control leaves were also analyzed in this time-study to confirm it was not physiologically different to transformed control .
the expression levels of atwri <dig> and a subset of genes encoding enzymes selected as ‘markers’ for core metabolic pathways  were also determined using rt-qpcr .
gene expression levels were determined relative to the gene l <dig> that was recently suggested to be a suitable reference in n. benthamiana leaves  <cit>  and which also from our transcriptome data set was confirmed to be stable between treatments and with an expression level in a suitable range in comparison to target genes.fig.
<dig> rt-qpcr time study of leaves expressing atwri <dig>  expression levels of atwri <dig>  and a subset of genes encoding enzymes selected as ‘markers’ for core metabolic pathways: pdh; pyruvate dehydrogenase for glycolysis , sbp; sedoheptulose-bisphosphatase for calvin cycle , agp; adp-glc pyrophosphorylase for starch synthesis , and mfp; multifunctional protein for fatty acid degradation  from one to five days after infiltration .
gene expression levels  were determined relative to the reference gene l <dig>  trctrl; transformed control.
results are the mean from three biological replicates ± standard deviation.
letters distinguish significant different means according to tukey’s test at level p ≤  <dig>
increased atwri <dig> expression and tag content from two dai
atwri <dig> expression was barely detectable in leaves at one dai but was much increased already from two dai as compared to in transformed control and non-transformed control, and was only slightly further increased  at five dai .
triacylglycerol content of leaves expressing atwri <dig> was significantly increased already by two dai, even though it continued to increase up to five dai .
this gives insight to how fast the metabolic effect generated by a constitutively expressed transcription factor in leaves can actually be observed.
the expression level of the gene encoding pyruvate dehydrogenase  showed a pattern very similar to that of atwri <dig>  which is well in accordance with that this gene is considered to be a direct transcriptional target of atwri <dig>  <cit> .fig.
<dig> time study of triacylglycerol concentrations in leaves expressing atwri <dig>  given as nmol fatty acids  per mg dry weight  from one to five days after infiltration .
trctrl; transformed control, nontrctrl; non-transformed control.
results are the mean from three biological replicates ± standard deviation.
letters distinguish significant different means according to tukey’s test at level p ≤  <dig>
decreased photosynthetic capacity of leaves
fluorescence efficiency measurements clearly showed decreased photosynthetic capacity in leaves expressing atwri <dig> as compared to both non-transformed leaves and transformed control from three dai  which was well in agreement with the observed severe down-regulation of many transcripts encoding different parts of the light-dependent reactions of photosynthesis  as well as down-regulation of the gene used as a marker for the calvin cycle .
this result also verified that transformed control  was physiologically indistinguishable from non-transformed control .
starch content was measured over time  but no significant decrease could be detected.
this implies that the previously described transcriptional changes observed at five dai that were involved in photosynthesis reactions  and starch metabolism  were not detectable at the metabolic level of starch, at least not over the time period studied.
in agreement with this, the level of the transcript encoding adp-glc pyrophosphorylase  was only statistically distinguishable from transformed control as late as by five dai .
the effect on starch levels in leaves expressing wri <dig> could therefore probably not be expected until after the time period studied .fig.
<dig> time study of photosynthetic capacity of leaves expressing atwri <dig>  electron transport rate from chlorophyll fluorescence measurement in leaves two to five days after infiltration .
trctrl; transformed control, nontrctrl; non-transformed control.
results are the mean from three biological replicates ± standard deviation.
letters distinguish significant different means according to tukey’s test at level p ≤  <dig>
no increase in levels of free fas in leaves
the observed up-regulation of transcripts encoding enzymes involved in tag and fa degradation could, as discussed above, have several different explanations.
it was of interest to know if leaves expressing wri <dig> had increased levels of free fas which can have negative physiological effects  <cit> .
this could be a reason to the observed up-regulation of transcripts encoding functions in fa degradation.
from total lipid analysis in our study it was recognized that all lipids except the polar fraction were increased upon expression of wri <dig> homologs in leaves five dai .
the increase of lipids in the ‘rest’ fraction could therefore potentially include increases of free fas, even though not visually detectable on tlc plates .
however, analysis of free fas in leaves expressing atwri <dig> showed no significant increase between one and five dai , implying that free fa levels were either not increased, or degraded in pace with the release of free fas thereby keeping levels low.
the gene expression level of multifunctional protein, used as a marker for β-oxidation of fas, was significantly increased in leaves expressing atwri <dig> already from two dai and onwards, as compared to transformed control  which suggests a relatively fast response to the wri <dig> mediated changes in the leaf.
CONCLUSIONS
this study gives for the first time an overview on the transcriptional transitions in leaf tissue upon expression of the transcription factor wri <dig>  expression of wri <dig> homologs from five diverse species and tissues in leaf all induced oil accumulation and the same general shifts in gene expression programs with down-regulation of transcripts encoding typical source tissue functions and up-regulation of those encoding typical sink tissue functions.
the severe down-regulation observed for transcripts encoding photosynthetic functions was confirmed physiologically with a significant decreased photosynthetic capacity observed of leaves expressing atwri <dig>  altogether this study revealed a wider range of effects caused, directly or indirectly, by wri <dig> than previously suggested.
these changes are reminiscent of the transcriptional and metabolic transitions detected between the vegetative phases of seed development and maturation in arabidopsis; from high photosynthetic contribution, starch accumulation, and cytosolic metabolism of phosphoenolpyruvate, to oil biosynthesis with decreased photosynthesis and plastid uptake and metabolism of phosphoenolpyruvate, during the course of seed development  <cit> .
however, leaves with wri <dig> expression did not only accumulate oil but transcriptional changes indicated that functions encoding tag and fa degradation was up-regulated, probably in attempt to feed the increased need for carbons into fa and oil synthesis thus creating a futile cycle.
this could explain why high oil content in leaf tissue cannot be achieved by high and constitutive transcriptional activation by wri <dig>  most likely this response is due to an incomplete shift from source to sink tissue and a lack of induction of sucrose import.
it is intriguing that expression of wri <dig> alone can induce these large transcriptional and physiological changes in a leaf, and future studies need to functionally dissect the different roles of wri <dig> and upstream transcriptional actors previously suggested to take part in seed development in general  <cit> .
moreover, identification of the regulatory networks present in other types of oil-storing plant tissues in which wri <dig> is known to act in will be of utmost importance for the development of high-yielding oil crops in the future.

 among different forms of carbon storage, triacylglycerol is the most energy dense, yielding more than double amount of energy on a per weight basis as compared to starch. due to the increased global need for plant oil derived products for food but also for non-food applications to decrease our dependence on fossil oil, the interest in mechanisms for regulation of carbon allocation into oil in plant storage tissues has gained a lot of attention and can be of great economic and environmental importance in the process of developing new high-yielding oil crops. to be available for tag synthesis, carbons from sucrose must first be converted to pyruvate through cytosolic or plastidic glycolytic pathways and at some point be imported into the plastid to yield acetyl-coenzyme a which feeds fatty acid synthesis with carbon backbones. fas are subsequently transported out to the endoplasmic reticulum where tag assembly takes place. the seed-specific, loss-of-function mutant wrinkled of arabidopsis thaliana with 80 % reduction in oil content and increased levels of sucrose but not of starch in mature seeds was by enzymatic analyses shown to be impaired in the allocation of carbon into glycolysis. the corresponding gene, atwrinkled, encodes a protein with two apetala2-ethylene responsive element-binding protein motifs that is a signature for plant-specific transcription factors involved in a wide range of developmental processes. gene expression analyses using microarrays have further revealed that some of the down-regulated genes in seeds of the wri mutant are involved in glycolysis and fa synthesis, rather than in tag assembly. moreover, atwri has been shown to bind to the aw-box sequence in upstream regions of several genes involved in fa synthesis and glycolysis. homologs to atwri have been identified in oil-dense embryonic tissues of several other plant species as rape seed and maize. a homolog to atwri was also associated with the large difference in carbon allocation to oil between oil palm and date palm mesocarps with the former mainly storing carbon as oil, the latter as sugar. today’s global plant oil production is mainly dependent on tag storage capacity in oil palm fruits and the embryos of soybean, sunflower, and oilseed rape. studies trying to reveal the regulatory mechanisms behind oil accumulation have mainly focused on embryonic tissues of seeds, even though fruit mesocarp and seed endosperm tissues have also been studied. however, the potential to produce oil in already high-yielding but not usually oil dense tissues like cereal grain endosperm and underground tuber or root parenchyma, by directing carbon allocation from starch or sugar into oil by genetic engineering, deserves further attention due to the important applied benefits. among plant species producing underground tubers, only one is known to have genotypes with high amount of oil, i.e. yellow nutsedge. among the cereals, oat is the only species known to store relatively high amount of oil in the grain endosperm. in both these tissues, homologs to the atwri were expressed during tuber parenchyma and grain endosperm development, respectively. another interesting tissue for potential oil production is from trees due to the vast amount of wood processed in the pulp industry from which fas are already an important byproduct. studies on perennial plants have indicated that lipids can accumulate transiently in the cambium upon transition to dormancy and a homolog to the atwri has been identified in poplar from available genomic resources. therefore, the role of wri1-like genes for directing carbon flow into oil synthesis and accumulation could be extended to include diverse types of plant tissues. in oilseed species like arabidopsis, oil accumulation is part of the embryo development and seed maturation process which is governed by master regulators such as lec lec fus and abi. wri is linked to this upstream regulatory network and has been shown to be a downstream target and specify the action of lec towards fa synthesis. however, homologs to these upstream regulators were not found in the mesocarp of oil palm, which suggests that the regulatory networks in non-embryo tissues are different to those in developing seed embryos. in this study, we investigated the effect on global gene expression in leaves where oil accumulation was induced by expression of wri homologs originating from diverse species and tissues using a transient gene expression system with agroinfiltration of nicotiana benthamiana. a time study of oil and starch content as well as rt-qpcr and photosynthesis measurements in leaves expressing arabidopsis wri between one to five days after infiltration was done to further characterize and complement the transcriptional transitions observed at five days after infiltration.  all homologs were wri1-like the wri gene from arabidopsis and four additional homologs from different species representing three dicotyledons and two monocotyledons were used in the present study. these selected homologs are expressed in different types of tissues that all have induction of oil accumulation at certain developmental or temporal stages. the arabidopsis gene is expressed in the embryo during seed development as is the potato homolog, and the poplar homolog is expressed in the cambium of the tree. the oat homolog is expressed in grain endosperm and the nutsedge homolog in underground stem tuber. from a phylogenetic tree based on homology of full-length cdna it was clear that the novel wri homologs of this study were more similar to arabidopsis wri than to either arabidopsis wri wri or wri which are more similar to each other than to any of the other presented homologs. it was also evident from this phylogeny analysis that the two homologs of wri originating from monocot species were more similar to each other than to the dicot wri homologs. the wri orthologs and homologs so far studied have a high degree of identity and similarity on an amino acid level in the region spanning the two ap domains but with a high degree of divergence in the n-and c-terminus which was also true for the wri homologs of this study. however, outside the dna binding domains there was a high divergence among the different homologs although it was evident that species which are more closely related have a higher degree of amino acid homology and identity. this in particular applies to the derived amino acid sequences of the n. benthamiana and s. tuberosum homologs of wri with 82 % identity in contrast to the homolog from c. esculentus which had only 45 % identity to that of n. benthamiana. the different homologs are hereafter named atwri stwri1em, ptwri1ca, aswri1es, and cewri1tp abbreviated after the species and tissue of origin.fig. phylogeny tree of wri homologs. phylogeny analysis based on full-length cdna sequences of wri homologs from arabidopsis, potato embryo, tobacco, poplar stem, oat endosperm, and nutsedge tuber parenchyma all wri1-like homologs induced oil accumulation in leaves all wri homologs in this study were shown to drastically induce oil accumulation when transiently expressed in leaves of n. benthamiana by agroinfiltration. leaves expressing the aswri1es showed the highest oil concentration followed by atwri stwri1em, ptwri1ca, and cewri1tp. this result shows that the function of wri is highly conserved across species and storage tissues even though not of typical oil-dense embryo type. also diacylglycerol levels increased in leaves upon expression of the wri homologs. polar lipids were the most abundant in leaves but did not show any difference between leaves expressing the different wri homologs or to transformed control. the leaves expressing the wri homologs appeared stressed with chlorosis, in particular for leaves expressing stwri1em. dry matter contents of leaves were not significantly different from transformed control, except for leaves expressing the aswri1es with higher dry matter.fig. leaf lipid concentrations. concentration of different lipid classes, dag; diacylglycerol, pl; polar lipids, and other lipids ) given as nmol fatty acids on dry weight basis of n. benthamiana leaves expressing wri from arabidopsis embryo, potato embryo, oat endosperm, poplar stem, nutsedge tuber parenchyma, and transformed control five days after infiltration. results are the mean from three biological replicates ± standard deviation. letters distinguish significant different means according to tukey’s test at level p ≤  the fa composition of tag changed upon expression of wri homologs with lower proportions of 18: and in general higher proportions of 18: as compared to transformed control. in the polar lipid fraction of leaves, the largest observed differences in fa profile upon expression of wri homologs were in general increased proportions of 18: and decreased 18:.fig. fatty acid profiles of lipids. tag; triacylglycerol, dag; diacylglycerol, pl; polar lipids, and other lipids in n. benthamiana leaves expressing wri from arabidopsis embryo, potato embryo, oat endosperm, poplar stem, nutsedge tuber parenchyma, and transformed control five days after infiltration. results are the mean from three biological replicates ± standard deviation since oil accumulation was highest in leaves expressing aswri1es and atwri the localization and structural properties of oil were only analyzed for those together with transformed control leaves. tem analysis indicated that the accumulated oil was localized to the cytosol and was most probably present as oil droplets and not as distinct organized oil-bodies surrounded by oil-body bound proteins. this is consistent with that no up-regulation of transcripts encoding oil-body proteins was observed in the transcriptome data. however, differences in oil quantity could not be clearly visualized by this method. expression of oleosins in arabidopsis leaves already expressing phospholipid:diacylglycerol acyltransferase promoted oil accumulation giving  % oil of leaf dw and also changed the structural storage form into organized oil-bodies, and combining oleosins with wri and diacylglyceroltransferase in n. tabacum leaves gave even higher oil content, 15 % by leaf dw. this demonstrates the potential of combining a transcription factor with genes involved in both tag synthesis and protection against mobilization, for achieving high oil content in leaves. for clarification, the aim of our study was not to achieve the highest oil content possible but instead to map the transcriptional changes induced by wri alone in leaf tissue.fig. transmission electron microscopy. photos of n. benthamiana leaves expressing wri from oat endosperm and arabidopsis embryo. transformed control leaf five days after infiltration. cw; cell wall, p; plastid, s; starch, o; oil, m; mitochondria, v; vacuole, n; nucleus transcriptome sequencing data leaves expressing the different wri homologs and transformed control tissue were subjected to transcriptome sequencing five days after agro-infiltration, which is generally used as a suitable time for sufficient transient gene expression and impact on metabolism. upon expression of wri homologs in the n. benthamiana leaf tissue, notable differences in the number of differentially expressed genes as compared to transformed control were observed, but all five homologs induced a similar pattern in core metabolic pathways. expression of stwri1em in leaves induced the highest number of differentially expressed genes for both up-regulated and down-regulated transcripts, followed by atwri ptwri1ca, aswri1es, and cewri1tp. however, since the expression levels of the different wri homologs varied significantly in leaves, no conclusions regarding species-specific differential gene expression induced by those different homologs could be made from this study. fold-changes of gene expression given here are therefore the averages from leaves expressing the different wri homologs. interesting to note though, is that the expression levels of the different wri homologs did not correlate either to oil content or number of differential expressed genes in leaves. in fact, the number of differentially expressed genes was instead correlated with phylogenetic similarity of the different wri homologs to the n. benthamiana wri suggesting that the most closely related stwri1em yielded the highest and cewri1tp the lowest number of differentially expressed genes in leaves. the lists of differentially expressed genes in leaves upon expression of the different wri homologs had in general a similar overall pattern for core metabolic pathways. here, these general trends are described where the functional equivalence of each gene was assigned according to the closest homolog of arabidopsis. the effect on global gene expression in leaves induced by wri homologs was in general strong with many genes shown to be highly differentially regulated. some genes were virtually switched ‘on’, going from almost non-detectable in transformed control tissue to a relatively high expression in wri1-infiltrated tissue. this illustrates the strong effect of using a constitutively expressed transcription factor to achieve a drastic change of the gene expression program of a certain tissue. the results also reveal notable similarities in the effect on transcriptional regulation exerted by wri homologs coming from such diverse plant species and tissues as were included in this study. it is important to note that it is not possible to distinguish whether the observed effects on gene expression in leaves five days after agroinfiltration were due to direct or indirect effects of wri expression. furthermore, this study only discusses induced changes at the transcript level and cannot state whether these changes is also correlated to protein levels. carbon directed to the plastid the oil-inducing effect of wri in arabidopsis seeds has been attributed to its enhancing expression of genes encoding enzymes in glycolysis as well as fa synthesis, rather than in tag assembly. many of the genes, as for biotin carboxylase carrier protein, kasi, enoyl-acp reductase, acp, fatty acid desaturase plastid pyruvate kinase, and plastid pyruvate dehydrogenase e1α, which are down-regulated in the arabidopsis mutant wri as compared to in wild-type seeds, are within the set of genes involved in fa synthesis and glycolysis shown to have a bell-shaped expression pattern during wild-type seed development. on the contrary, genes involved in tag assembly are not differentially expressed in the wri seeds and do not belong to the ‘bell-shaped group’ of genes in the wild-type. studies on arabidopsis seeds also showed that the activities of several glycolytic enzymes are much decreased in the wri as compared to wild-type seeds. therefore, it was not surprising that leaves expressing wri homologs in our study indeed had a much higher expression of genes involved in glycolysis and fa synthesis pathways, as compared to in transformed control. wri expression also induced a shift at the transcript level in the organelle distribution of glycolysis with carbon flux being directed to the plastid. genes encoding enzymes of the upper part of cytosolic glycolysis; sucrose synthase, ppi-dependent fructose-6-phosphate 1-phosphotransferase, and frc-bisphosphate aldolase, and the lower part of plastidic glycolysis; phosphoglycerate mutase, enolase, the α and β subunit of pyruvate kinase, and all three subunits of pyruvate dehydrogenase were highly up-regulated in leaves expressing wri up-regulated were also transcripts of the plastid translocator for phosphoenolpyruvate, thereby probably ensuring cytosolic glycolysis up to phosphoenolpyruvate followed by uptake into the plastid for further plastidic glycolysis. interestingly, this transcript pattern of organelle shift for glycolysis is very similar to that of arabidopsis seeds during development, indicating that this might be a signature of a plant tissue in transformation from a photosynthetic source to heterotrophic oil accumulation. this pattern is also in agreement with the observed decreased flux of carbon through the lower part of plastidic glycolysis from phosphoenolpyruvate and downwards in developing embryos of the wri arabidopsis mutant as determined by 13c metabolic flux analyses.fig. heat map of differential gene expression involved in central carbon metabolism. values given are log of the ratios between rpkm of leaves expressing wri and transformed control five days after infiltration. the order of leaf samples expressing the five different wri homologs are given for the box numbered 1: atwri stwri1em, ptwri1ca, aswri1es, and cewri1tp. abbreviations: e4p; erythrose 4-phosphate, sucr; sucrose, glc; glucose, frc; fructose, frc6p; fructose 6-phosphate, glc6p; glucose 6-phosphate, frc bp; fructose -bisphosphate, udp; uridine diphosphate, glnt; gluconate, r5p; ribose 5-phosphate, ru5p; ribulose 5-phosphate, ru bp; ribulose -bisphosphate, pgal; phosphoglyceraldehyde, pga; phosphoglycerate, dpga; diphosphoglycerate, pep; phosphoenolpyruvate, pyr; pyruvate, oaa, oxaloacetate, mal; malate, adp; adenosinediphospho-, s7p; sedoheptulose 7-phosphate, s bp; sedoheptulose - bisphosphate, xu5p; xylulose 5-phosphate. enzymes: sucrose synthase, ppi-dependent fructose-6-phosphate 1-phosphotransferase, fructose-bisphosphate aldolase, nadp-dependent glyceraldehyde-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, phosphoenolpyruvate carboxylase, triosephosphate/phosphate translocator, phosphoenolpyruvate/phosphate translocator, phosphoglycerate mutase, enolase, pyruvate kinase, pyruvate dehydrogenase, malic enzyme, phosphoglucomutase, adp-glucose pyrophosphorylase large subunit, starch synthase β-amylase, glucose-6-phosphate dehydrogenase, transaldolase, triose-phosphate isomerase, fructose- -bisphosphatase, sedoheptulose -bisphosphatase, ribose-5-phosphate isomerase, phosphoribulokinase, rubisco, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, carboxyl transferase α subunit of acetyl-coa carboxylase, biotin carboxylase of acc, biotin carboxyl carrier protein of acc, acyl carrier protein, malonyl-coa:acp malonyltransferase, 3-ketoacyl-acp synthase iii, 3-ketoacyl-acp reductase, enoyl-acp reductase, 3-ketoacyl-acp synthase i, 3-ketoacyl-acp synthase ii increased storage synthesis pyruvate from glycolysis is converted by pyruvate dehydrogenase into acetyl-coa which is the two-carbon precursor for de novo fa synthesis in the plastid. many genes encoding enzymes involved in fa synthesis were shown to be up-regulated in leaves expressing the wri homologs again well in agreement with other studies. transcripts encoding all subunits of acetyl-coa carboxylase, acyl carrier protein, malonyl-coa:acp malonyltransferase and 3-ketoacyl-acp synthase iii, 3-ketoacyl-acp synthase i were all up-regulated in leaves expressing wri homologs. a very high up-regulation was observed for the reductases of 3-ketoacyl-acp and enoyl-acp of the fatty acid synthase complex. one transcript that was up-regulated 278-fold in leaves expressing the wri homologs, with very low expression in the transformed control tissue, was clearly annotated as a kas but phylogenetic analyses did not allow for an obvious designation to kasi or ii in arabidopsis. however, no other kas genes exist in n. benthamiana and therefore this transcript most probably encodes the enzyme activity corresponding to that of kasii in arabidopsis. the high up-regulation of this transcript indicates the importance of the elongation of 16:0-acp to 18:0-acp for increased tag synthesis. genes encoding enzyme activities in tag assembly were previously not usually associated with wri regulation. however, a few transcripts related to tag assembly were in this study found to be up-regulated in leaves upon wri expression. one transcript annotated to represent phosphatidylcholine diacylglycerol cholinephosphotransferase was up-regulated in leaves expressing the wri homologs. pdct is involved in the regulation of tag composition by catalyzing the inter-conversion of diacylglycerol and phosphatidylcholine, and interestingly the encoding gene was previously shown to be down-regulated in the triple mutant of arabidopsis with reduced expression of wri wri and wri. transcripts encoding the enzymes phospholipase c and d were also up-regulated. both types of lipases are thought to be important for the conversion of phosphatidylcholine to diacylglycerol. therefore, pdct and/or phospholipases might therefore be the enzymes defining the observed shift in fa composition of tag upon induction of oil accumulation. furthermore, a transcript encoding glycerol-3p dehydrogenase, catalyzing the synthesis of glycerol-3p from dihydroxyacetonephosphate was up-regulated 8-fold. one plastidic isoform of glycerol-3p dehydrogenase in arabidopsis was previously shown to be important for the metabolism of plastid-localized glycerolipids and is probably not associated with tag synthesis. however, the glycerol-3p dehydrogenase shown to be up-regulated in our study was encoding another plastidic isoform which might be important for tag backbone supply since it is associated with gene expression during seed development in arabidopis, and glycerol-3p can pass the plastid envelope through a permease which was recently shown to play an important role for lipid content of arabidopsis embryos. interestingly, the gene encoding the plastidic glycerol-3p dehydrogenase found to be up-regulated in our study was also identified to be one of putative target genes of maize wri. many genes involved in amino acid metabolism were differentially expressed upon expression of wri homologs in our study which could possibly indicate increased activities involved with protein storage accumulation. transcripts encoding vacuolar sorting receptor was 2-fold up-regulated while that of vacuolar processing enzyme as much as 1400-fold up-regulated. other examples of up-regulated transcripts encoded membrane amino acid transporters and endoplasmic reticulum to golgi vesicle mediated transport of amino acids. transcripts encoding asparagine synthetase and glutamate synthase was up-regulated, while on the other hand that of chloroplastic glutamine synthetase was down-regulated. transcripts annotated as amino transferases of aspartate and alanine were up-regulated. however, it was difficult to completely dissect the meaning of these differentially expressed genes as pathways were up-regulated or down-regulated during seed development in arabidopsis, which could fit with wri expression although changes in amino acid metabolism and nitrogen remobilization is also consistent with other patterns such as stress or senescence. changes in expression of genes in amino acid metabolism are also indicative as markers for changes in general metabolism in the leaf tissue. it is interesting to note that increased levels of certain amino acids were actually observed in maize kernels overexpressing the zmwri1a which can indicate an association of wri with storage protein synthesis. decreased photosynthesis and starch formation an important finding in this study was that the expression of wri homologs in leaves induced a severe down-regulation of transcripts encoding functions in the photosynthetic apparatus, both in the light-dependent reactions oxidoreductase, subunits of atp generation complex, to 66-fold changes, see fig. 6) and the carbon fixation reactions of all three phases of the calvin-benson cycle. thus the whole calvin-benson cycle was down-regulated together with the light harvesting and probably atp generation of the photosynthetic apparatus. in agreement with this, the transcript encoding the plastid localized triosephosphate/phosphate translocator, with a substrate specificity allowing organelle exchange of dihydroxyacetone-p, glyceraldehyde-3p, and 3-phosphoglycerate, was also down-regulated in wri expressing tissue which could indicate a decreased flux of the daily photosynthetically fixed carbon out from the plastid for further sucrose synthesis.fig. heat map of differential gene expression involved in photosynthesis. values given are log of the ratios between rpkm of leaves expressing wri and transformed control five days after infiltration. the order of leaf samples expressing the five different wri homologs are given for the box numbered 1: atwri stwri1em, ptwri1ca, aswri1es, and cewri1tp. light harvesting complex photosystem ii subunit photosystem ii subunit p, psbq subunit of the oxygen evolving complex of photosystem ii, photosystem ii subunit psbx, photosystem ii type i chlorophyll a/b-binding protein, photosystem ii encoding the light-harvesting chlorophyll a/b binding protein cp of the antenna system of the photosynthetic apparatus, photosystem ii kd protein, involved in the light harvesting complex of photosystem ii, photosystem ii by, subunit of light-harvesting complex ii, photosystem ii type i chlorophyll a/b-binding protein, rieske fes center of cytochrome b6f complex, cytochrome b6f complex subunit, plastocyanin, photosystem i subunit e photosystem i subunit f, subunit g of photosystem i, photosystem i, subunit h photosystem i subunit k, protein predicted by sequence similarity with spinach psad to be photosystem i reaction center subunit ii, photosystem i reaction center, sub psi-n, photosystem i subunit o, p subunit of photosystem i, leaf-type ferredoxin:nadp oxidoreductase one example where functional duality of enzymatic activities became obvious was for frc-bisphosphate aldolase with one up-regulated and one down-regulated transcript. since this enzyme catalyzes the reversible conversion of both frc-bisphosphate and sedoheptulose-bisphosphate into dihydroxyacetonephosphate, it can be speculated that the up-and down-regulated transcripts actually represent genes encoding aldolase with importance for glycolysis and the calvin cycle, respectively. transcripts encoding enzymes in starch synthesis were down-regulated while that of β-amylase, important for starch degradation, was instead up-regulated 6-fold. increased gene expression and enzyme activity of β-amylase was previously observed during development of oil-seeds of arabidopsis and rape indicating the importance of starch degradation to yield carbon precursors for oil accumulation in these species. starch breakdown and increased glycolysis was also correlated to increased fa synthesis at transcript level in the cambium of aspen upon developmental transition to dormancy. the expression of the different wri homologs in leaves induced down-regulation of many genes involved in chloroplast fa desaturation and very long chain fa and cutin biosynthesis. examples were transcripts encoding fad, fad, lacs, lacs long-chain acyl-coa synthetases related, cer, and a cer26-like hxxxd acyltransferase. in addition, genes annotated as coding for modifier functions of cutin were also down-regulated like cer which is suggested to be an aldehyde decarbonylase, and dcr, which is a hxxxd acyl-transferase. furthermore, a gene homologous to glycerol-3-phosphate acyltransferase shown to be critical for acyl transfer in cutin biosynthesis was down-regulated. possibly genes encoding p proteins corresponding to functions of importance for cuticle development as cyp86a were also among down-regulated genes in leaves expressing wri homologs. among all those, the transcript encoding fad was the most severe down-regulated, with other transcripts showing to 7-fold changes. interesting and in agreement with our results, a reduction of gene expression encoding one of the chloroplast desaturases, fad was previously observed in leaf tissue expressing atwri. in the same publication it was noted that in the presence of increased tag synthesis there was a severe reduction in surface lipids. our results suggests that the reduction in surface lipids they observed would not be due to a competition for substrates but rather a function of reduced gene expression of transcripts related to surface lipid synthesis in response to expression of wri all these changes induced by wri expression imply that many of the transcripts encoding enzymes giving typical characteristics of a source tissue were highly down-regulated, suggesting a shift into a heterotrophic tissue with low photosynthetic contribution. this can probably explain the observed chlorosis of leaves expressing wri it can be speculated that wri mediates repression of photosynthetic source functions also during seed development. carbons from oil recycled to plastid in oil-accumulating sink tissue such as a seed, the sucrose from photosynthesizing leaves will feed the developing seed allowing for a continuous carbon flow into storage product accumulation. however, when expressing a transcription factor that, directly or indirectly, not only induce oil synthesis but also reduce photosynthesis, the sucrose available for the increased fa synthesis is likely to become limiting. in fact, transcripts encoding an α/β hydrolase with tag lipase activity was highly up-regulated while glycerol kinase, and several enzymes involved in β-oxidation of fas were also up-regulated to 6-fold. furthermore, a transcript encoding malate synthase, involved in the closely connected glyoxylate cycle that produces malate from degraded fas, was also highly up-regulated. to be available for fa synthesis, malate can then either be translocated into the plastid in exchange for phosphate where it can be converted to pyruvate by malic enzyme, or cytosolic conversion of malate by malate dehydrogenase and phosphoenolpyruvate carboxykinase yields phosphoenolpyruvate which can be transported into the plastid by the phosphoenolpyruvate/phosphate translocator. several transcripts encoding enzyme activities in both these routes were up-regulated in wri expressing leaf tissue as compared to transformed control. our results therefore suggest that the new sink-like tissue induced in the leaf by expression of wri is experiencing carbon source limitations. in the absence of sucrose import this probably triggers the up-regulation of transcripts encoding enzymes in both tag and fa degradation in the leaf in attempt to yield carbons that can be recycled back, either as malate or phosphoenolpyruvate, into glycolysis. an alternative or additional explanation could be that increased amount of tag in a leaf mesophyll cell, without proper packaging with oil-body proteins, is more prone to mobilization which induces up-regulation of transcripts encoding enzymes of tag degradation. this will in turn yield free fas that can be toxic to the cell if accumulating, which can explain the up-regulation of transcripts encoding functions in fa degradation.. as mentioned earlier, the highest total oil content of leaves achieved in our study was relatively low as compared to in other studies where the stable expression of combination of the transcription factor wri with other genes involved in tag assembly and storage showed that much higher oil content is possible to achieve in n. tabacum leaves. interestingly, the promoter chosen for atwri expression in that study was that of arabidopsis rubisco, with the rational of achieving a diurnal expression pattern. however, the down-regulation of photosynthesis and the calvin-benson cycle induced upon wri expression in this study suggests that wri itself might be attenuating the expression of wri from the rubisco promoter. it is possible that this plausible self-regulating effect of the atwri expression could be another key to the high levels of tag accumulated without apparent negative effects on plant vigor in that study. additional differentially expressed genes of potential importance for seed development a number of genes were differentially expressed in leaves upon wri expression where the closest homologs in arabidopsis are expressed during seed development or corresponding proteins are of importance for proper seed development. for example, transcripts annotated as encoding trehalose-6-phosphate synthase were up-regulated 30-fold. trehalose-6-phosphate is thought to be a signal of sugar status in plant storage tissues and the synthase is of crucial importance for embryo development in arabidopsis. transcripts encoding proteins of completely unknown function but where the closest arabidopsis homolog is expressed during seed development were found, exemplified by a transcript annotated as closest homolog to at2g which was highly up-regulated in wri expressing tissue. for another transcript which was also highly up-regulated, the closest homolog in arabidopsis was at1g encoding seuss, a transcriptional adaptor of importance for embryonic development. there were additional transcripts annotated as nucleic acid binding or transcription factors although their functions are unknown and gene families so large that closest homologs in arabidopsis were difficult to discern. only one transcript related to genes encoding oil body proteins was up-regulated by the wri homologs which closest homolog was at1g encoding a putative aquaporin. time study of leaves expressing atwri1 some of the transcriptional transitions observed in leaves induced, directly or indirectly, by the transient expression of all the different wri homologs were unexpected, such as the effect on transcripts encoding photosynthesis, starch metabolism, and fatty acid degradation. therefore, the effects of the transient expression of atwri in n. benthamiana leaves were physiologically characterized in more detail in a time study from one to five days after infiltration. non-transformed control leaves were also analyzed in this time-study to confirm it was not physiologically different to transformed control. the expression levels of atwri and a subset of genes encoding enzymes selected as ‘markers’ for core metabolic pathways were also determined using rt-qpcr. gene expression levels were determined relative to the gene l that was recently suggested to be a suitable reference in n. benthamiana leaves and which also from our transcriptome data set was confirmed to be stable between treatments and with an expression level in a suitable range in comparison to target genes.fig. rt-qpcr time study of leaves expressing atwri expression levels of atwri and a subset of genes encoding enzymes selected as ‘markers’ for core metabolic pathways: pdh; pyruvate dehydrogenase for glycolysis, sbp; sedoheptulose-bisphosphatase for calvin cycle, agp; adp-glc pyrophosphorylase for starch synthesis, and mfp; multifunctional protein for fatty acid degradation  from one to five days after infiltration. gene expression levels were determined relative to the reference gene l trctrl; transformed control. results are the mean from three biological replicates ± standard deviation. letters distinguish significant different means according to tukey’s test at level p ≤  increased atwri expression and tag content from two dai atwri expression was barely detectable in leaves at one dai but was much increased already from two dai as compared to in transformed control and non-transformed control, and was only slightly further increased at five dai. triacylglycerol content of leaves expressing atwri was significantly increased already by two dai, even though it continued to increase up to five dai. this gives insight to how fast the metabolic effect generated by a constitutively expressed transcription factor in leaves can actually be observed. the expression level of the gene encoding pyruvate dehydrogenase showed a pattern very similar to that of atwri which is well in accordance with that this gene is considered to be a direct transcriptional target of atwri.fig. time study of triacylglycerol concentrations in leaves expressing atwri given as nmol fatty acids per mg dry weight from one to five days after infiltration. trctrl; transformed control, nontrctrl; non-transformed control. results are the mean from three biological replicates ± standard deviation. letters distinguish significant different means according to tukey’s test at level p ≤  decreased photosynthetic capacity of leaves fluorescence efficiency measurements clearly showed decreased photosynthetic capacity in leaves expressing atwri as compared to both non-transformed leaves and transformed control from three dai which was well in agreement with the observed severe down-regulation of many transcripts encoding different parts of the light-dependent reactions of photosynthesis as well as down-regulation of the gene used as a marker for the calvin cycle. this result also verified that transformed control was physiologically indistinguishable from non-transformed control. starch content was measured over time but no significant decrease could be detected. this implies that the previously described transcriptional changes observed at five dai that were involved in photosynthesis reactions and starch metabolism were not detectable at the metabolic level of starch, at least not over the time period studied. in agreement with this, the level of the transcript encoding adp-glc pyrophosphorylase was only statistically distinguishable from transformed control as late as by five dai. the effect on starch levels in leaves expressing wri could therefore probably not be expected until after the time period studied.fig. time study of photosynthetic capacity of leaves expressing atwri electron transport rate from chlorophyll fluorescence measurement in leaves two to five days after infiltration. trctrl; transformed control, nontrctrl; non-transformed control. results are the mean from three biological replicates ± standard deviation. letters distinguish significant different means according to tukey’s test at level p ≤  no increase in levels of free fas in leaves the observed up-regulation of transcripts encoding enzymes involved in tag and fa degradation could, as discussed above, have several different explanations. it was of interest to know if leaves expressing wri had increased levels of free fas which can have negative physiological effects. this could be a reason to the observed up-regulation of transcripts encoding functions in fa degradation. from total lipid analysis in our study it was recognized that all lipids except the polar fraction were increased upon expression of wri homologs in leaves five dai. the increase of lipids in the ‘rest’ fraction could therefore potentially include increases of free fas, even though not visually detectable on tlc plates. however, analysis of free fas in leaves expressing atwri showed no significant increase between one and five dai, implying that free fa levels were either not increased, or degraded in pace with the release of free fas thereby keeping levels low. the gene expression level of multifunctional protein, used as a marker for β-oxidation of fas, was significantly increased in leaves expressing atwri already from two dai and onwards, as compared to transformed control which suggests a relatively fast response to the wri mediated changes in the leaf.  this study gives for the first time an overview on the transcriptional transitions in leaf tissue upon expression of the transcription factor wri expression of wri homologs from five diverse species and tissues in leaf all induced oil accumulation and the same general shifts in gene expression programs with down-regulation of transcripts encoding typical source tissue functions and up-regulation of those encoding typical sink tissue functions. the severe down-regulation observed for transcripts encoding photosynthetic functions was confirmed physiologically with a significant decreased photosynthetic capacity observed of leaves expressing atwri altogether this study revealed a wider range of effects caused, directly or indirectly, by wri than previously suggested. these changes are reminiscent of the transcriptional and metabolic transitions detected between the vegetative phases of seed development and maturation in arabidopsis; from high photosynthetic contribution, starch accumulation, and cytosolic metabolism of phosphoenolpyruvate, to oil biosynthesis with decreased photosynthesis and plastid uptake and metabolism of phosphoenolpyruvate, during the course of seed development. however, leaves with wri expression did not only accumulate oil but transcriptional changes indicated that functions encoding tag and fa degradation was up-regulated, probably in attempt to feed the increased need for carbons into fa and oil synthesis thus creating a futile cycle. this could explain why high oil content in leaf tissue cannot be achieved by high and constitutive transcriptional activation by wri most likely this response is due to an incomplete shift from source to sink tissue and a lack of induction of sucrose import. it is intriguing that expression of wri alone can induce these large transcriptional and physiological changes in a leaf, and future studies need to functionally dissect the different roles of wri and upstream transcriptional actors previously suggested to take part in seed development in general. moreover, identification of the regulatory networks present in other types of oil-storing plant tissues in which wri is known to act in will be of utmost importance for the development of high-yielding oil crops in the future. 
here, the effect on global gene expression as well as on physiological changes in leaves transiently expressing five homologs of the transcription factor wrinkled originating from diverse species and tissues; arabidopsis thaliana and potato seed embryo, poplar stem cambium, oat grain endosperm, and nutsedge tuber parenchyma, were studied by agroinfiltration in nicotiana benthamiana. this data gives for the first time a general view on the transcriptional transitions in leaf tissue upon induction of oil synthesis by wri this yields important information about what effects wri may exert on global gene expression during seed and embryo development. the results suggest why high oil content in leaf tissue cannot be achieved by solely transcriptional activation by wri which can be essential knowledge in the development of new high-yielding oil crops. knowledge about the regulation of carbon allocation into oil in plant storage tissue can be of great economic and environmental importance for developing new high-yielding oil crops. transcriptome sequencing revealed that all homologs induced the same general patterns with a drastic shift in gene expression profiles of leaves from that of a typical source tissue to a source-limited sink-like tissue: transcripts encoding enzymes for plastid uptake and metabolism of phosphoenolpyruvate, fatty acid and oil biosynthesis were up-regulated, as were also transcripts encoding starch degradation. carbon accumulation and remobilization are essential mechanisms in plants to ensure energy transfer between plant tissues with different functions or metabolic needs and to support new generations. all wri homologs induced oil accumulation when expressed in leaf tissue. chlorophyll fluorescence measurements of leaves expressing arabidopsis wri showed a significant decrease in photosynthesis, even though effect on starch content could not be observed. transcripts encoding enzymes in photosynthesis and starch synthesis were instead down-regulated. moreover, transcripts representing fatty acid degradation were up-regulated indicating that fatty acids might be degraded to feed the increased need to channel carbons into fatty acid synthesis creating a futile cycle.
 carbon accumulation and remobilization are essential mechanisms in plants to ensure energy transfer between plant tissues with different functions or metabolic needs and to support new generations. knowledge about the regulation of carbon allocation into oil in plant storage tissue can be of great economic and environmental importance for developing new high-yielding oil crops. here, the effect on global gene expression as well as on physiological changes in leaves transiently expressing five homologs of the transcription factor wrinkled originating from diverse species and tissues; arabidopsis thaliana and potato seed embryo, poplar stem cambium, oat grain endosperm, and nutsedge tuber parenchyma, were studied by agroinfiltration in nicotiana benthamiana.  all wri homologs induced oil accumulation when expressed in leaf tissue. transcriptome sequencing revealed that all homologs induced the same general patterns with a drastic shift in gene expression profiles of leaves from that of a typical source tissue to a source-limited sink-like tissue: transcripts encoding enzymes for plastid uptake and metabolism of phosphoenolpyruvate, fatty acid and oil biosynthesis were up-regulated, as were also transcripts encoding starch degradation. transcripts encoding enzymes in photosynthesis and starch synthesis were instead down-regulated. moreover, transcripts representing fatty acid degradation were up-regulated indicating that fatty acids might be degraded to feed the increased need to channel carbons into fatty acid synthesis creating a futile cycle. rt-qpcr analysis of leaves expressing arabidopsis wri showed the temporal trends of transcripts selected as ‘markers’ for key metabolic pathways one to five days after agroinfiltration. chlorophyll fluorescence measurements of leaves expressing arabidopsis wri showed a significant decrease in photosynthesis, even though effect on starch content could not be observed.  this data gives for the first time a general view on the transcriptional transitions in leaf tissue upon induction of oil synthesis by wri this yields important information about what effects wri may exert on global gene expression during seed and embryo development. the results suggest why high oil content in leaf tissue cannot be achieved by solely transcriptional activation by wri which can be essential knowledge in the development of new high-yielding oil crops. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2015 