BACKGROUND
it has been postulated that genes of co-ordinated function that offer an organism some selective advantage  might be identifiable in studies that examine differential gene expression  across species ranges and heterogeneous environments  <cit> .
similarly, dge studies that compare expression profiles of closely related species, such as those that have formed during late tertiary radiation of new zealand's alpine genera, might also help to identify candidate genes that have played a role in species diversification.
to investigate this approach, in an earlier study we compared microarray expression profiles for natural populations of two closely related alpine plant species from the largely endemic new zealand genus pachycladon .
analyses of logfold ratios and ontology analyses in this experiment suggested that secondary metabolite profiles differed among and between populations of these two species.
hplc-uv/gc-ms analyses confirmed these predictions, leading to the hypothesis that herbivory was a major driver of diversification of p. fastigiatum and p. enysii  <cit> .
these results provided encouragement that transcriptome profiling used in a predictive sense provided a promising approach for helping to identify putative adaptations between closely related species.
however, a potential shortcoming with this approach is that gene expression differences between species may also result from genetic drift  <cit> .
moreover, due to posttranslational modifications, transcript abundances may not correlate with protein abundances  <cit> .
profiling of proteomes, which have different underlying evolutionary dynamics from transcriptomes  <cit> , provides an alternative approach for the prediction of downstream physiological processes.
in the present study we have compared findings from gene expression profiles obtained using heterologous microarrays  <cit>  with findings from protein expression profiles generated by shotgun proteomics.
during shotgun proteomics, proteins levels are calculated by determining the number of spectral counts that identify a protein  <cit>  and proteins are identified by searching ms-ms spectra of peptides against reference protein data bases.
our aim was to determine to what extent gene expression profiles were indicative of protein expression profiles and vice versa, and to evaluate both methods with respect to their potential for predicting physiological differences among new zealand pachycladon species.
for this purpose, we also characterized glucosinolate chemotypes and evaluated the extent to which these were predicted from the protein and transcript expression data.
the genus pachycladon is of allopolyploid origin  and has diversified into ten morphologically and ecologically diverse species within the last million years.
nine of the species are endemic to the southern alps of new zealand and one is native to tasmania.
the nine new zealand species form three distinct groups distinguished by their genetic characteristics, morphological features and habitat preferences  <cit> .
species differ markedly in their preference for certain soil types.
for example, the group consisting of p. novae-zelandiae and p. wallii are restricted to soils derived from schist whereas another group consisting of p. fastigiatum, p. enysii and p. stellatum are confined to greywacke soils.
there are also the limestone specialist p. fasciarium and the basicole p. exile.
the remaining two species, p. cheesemanii and p. latisiliquum are geological generalists.
these preferences have led to the hypothesis that adaptation to different soils has to a large part driven diversification and evolution of distinct pachycladon species.
since species also differ in their altitudinal ranges  <cit> , adaptation to different altitudes has been suggested as another possible driver of diversification.
in this study, we compared three pachycladon species with distinct ecological attributes.
these species were p. cheesemanii , p. exile  and p. novae-zelandiae .
the former two are sister species  <cit>  and resemble close overseas pachycladon relatives transberingia and crucihimalaya  <cit> .
p. cheesemanii and p. exile are both polycarpic perennials with a woody caudex, exhibit leaf heterophylly with ovate to broadly elliptic early  leaves with branched hairs on the lamina and petiole and serrate to lobed later  leaves; they have slender and terminal inflorescences and narrow petals and terete  siliques with uniseriate wingless seeds.
p. exile is much smaller in all parts than p. cheesemanii.
p. cheesemanii occurs along a wide altitudinal range  on greywacke, semi-schist, schist and plutonics substrates, it is a true generalist.
p. exile is a lowland species  that grows on base-rich soils.
it was previously recorded from a few sites in central otago but is now known with certainty from only one limestone site and has recently been classified as nationally critical in new zealand  <cit> .
p. novae-zelandiae is also a polycarpic perennial with a semi-woody caudex but is morphologically distinct from p. cheesemanii and p. exile.
it does not produce heteroblastic leaves.
rather it produces oblong to elliptic leaves that are either lobed or crenate and are either hairy  or glabrous; it has lateral inflorescences, and laterally compressed siliques with biseriate wingless seeds.
p. novae-zelandiae grows at much higher altitudes  and is confined to schist soils.
in summary, p. cheesemanii and p. exile are morphologically similar, lower altitude species that are very closely related but which differ in their soil substrate specificities .
in contrast, p. novae-zelandiae is a more distantly related alpine species restricted to schist.
given their different ecologies and degrees of relatedness, the three species are expected to differ in their gene and protein expression profiles.
in the instances that protein profiles corroborate inferences from comparative transcript analyses, these observations have been used to predict physiological differences between species as well as candidate genes for adaptive diversification.
RESULTS
transcriptome - proteome correlations
to investigate the extent to which transcriptional patterns were congruent with protein expression patterns, we calculated correlation coefficients for  <dig> genes surveyed by both transcript and protein profiling .
given the non-normal distributions of both the log <dig> spectral abundance factors and the log <dig> fluorescence intensities, we used spearman's rank correlation coefficient.
each species' proteome correlated most strongly with its own transcriptome with the highest correlation observed in p. cheesemanni  and the lowest in p. novae-zelandiae .
the transcript profile  for each species correlated most strongly with the proteome  of p. cheesemanii.
this was largely due to the fact that the three transcriptomes correlated very strongly with one another  and the p. cheesemanii transcriptome had the highest correlation with its own proteome.
the three proteomes correlated much more weakly with each other  than did the three transcriptomes, indicating greater differences between the protein profiles than between the transcript profiles.
correlations between both transcriptomes and proteomes mirrored phylogenetic relationships with the profiles of sister species p. cheesemanii and p. exile being most similar.
interestingly, both the transcriptome and proteome of p. novae-zelandiae were found to be more similar to those of p. exile than to those of p. cheesemanii.
pairwise spearman rank correlation coefficients between a) protein  and transcript  expression statistics , b) transcript expression statistics and c) protein expression statistics for  <dig> genes surveyed by both microarrays and shotgun proteomics of three pachycladon species.
species abbreviations: ch: p. cheesemanii, ex: p. exile, nz: p. novae-zelandiae.
the highest correlation coefficients for each species are depicted in bold.
note that the proteome of each species correlates most strongly with its own transcriptome .
furthermore, transcriptomes and proteomes of ch correlate more strongly with those of ex than with those of nz , reflecting the closer phylogenetic relationship between ch and ex as opposed to ch and nz.
overlap in transcript and protein up-regulation
for each of the three pachycladon species, we identified up- and down-regulated transcripts and proteins using linear model analysis and wilcoxon rank and t-tests, respectively.
the number of up- and down-regulated transcripts and proteins are summarized in table  <dig> and gene identifiers, descriptions, transcript and protein expression statistics are provided in additional file  <dig>  table s <dig>  we compared expression in each species with expression in the other two species combined  and separately .
for example, expression in ch was compared to expression in ex+nz  but also to ex and nz separately .
for each of the twelve comparisons, we intersected the lists of up-regulated transcripts with lists of up-regulated proteins and found significant overlap  in seven cases .
the extent of overlap between transcript and protein profiles is limited partially as a result of relative sizes of the different data sets .
for example, of the  <dig> transcripts up-regulated in p. cheesemanii when compared with nz+ex combined, only  <dig> were present in the protein data set.
this means the  <dig> genes found up-regulated by both transcript and protein profiling in p. cheesemanii represent an overlap of 23% .
conversely, of the  <dig> proteins up-regulated in p. cheesemanii only  <dig> were present in the transcript data set.
in this case we have found 32%  of the transcripts maximally identifiable by transcript profiling.
interestingly, across the three pachycladon species, an average of  <dig> % of the transcript patterns were confirmed by protein patterns  whereas on average only  <dig> % of the protein patterns were predicted by transcript patterns .
these percentages did differ greatly between species .
transcript patterns were best confirmed by proteins patterns in p. novae-zelandiae  and protein patterns were best confirmed by transcript patterns in p. cheesemanii .
the disagreement of transcript and protein profiling remaining after correcting for different numbers of genes captured by both analyses presumably is explained by the different kinetics of protein and transcript accumulation and degradation.
expression of transcripts  and proteins  in each species  was compared with expression in the remaining two species combined  and separately .
for example,  <dig> transcripts and  <dig> proteins were up-regulated in ch when compared with ex and nz combined and  <dig> transcripts and  <dig> proteins were down-regulated in ch when compared with ex and nz combined.
sixty nine transcripts and  <dig> proteins were up-regulated in ch when compared only to ex and  <dig> transcripts and  <dig> proteins were up-regulated in ch when compared only to nz.
the total numbers of transcripts and proteins investigated by microarray analysis and shotgun proteomics were  <dig> and  <dig>  respectively.
locus ids, gene descriptions, logarithmized expression ratios and adjusted p-values of differentially expressed transcripts and proteins for each of the  <dig> comparison are compiled in additional file  <dig>  table s <dig>
our goal was to identify genes specifically up- and down-regulated in each species with respect to the other two  in the set of  <dig> genes that were covered by both transcript and protein profiling.
in these comparisons the greatest overlap between transcript and protein profiles was found for p. cheesemanii , followed by p. exile  and p. novae-zelandiae .
gene identifiers, descriptions, and expression statistics for these are compiled in table  <dig>  from the genes equally identified by both approaches, some interesting observations and predictions can be derived.
for example, three isoforms of carbonic anhydrases were differentially expressed in p. cheesemanii  and p. novae-zelandiae  .
these genes catalyze the interconversion of carbon dioxide and bicarbonate and have been shown to be involved in photosynthesis, respiration, ph regulation, inorganic carbon transport, ion transport, and water and electrolyte balance  <cit> .
other genes found up-regulated by both approaches in p. cheesemanii are genes involved in translation, photosynthesis light reactions, the calvin cycle, pentose phosphate pathway, fatty acid beta oxidation, serine metabolism, oxidative stress, protein folding and others .
the four commonly up-regulated genes in p. exile are associated with very different processes such as calvin cycle, s-adenosylmethione biosynthetic process, carbohydrate metabolism and others .
in p. novae-zelandiae, two genes encoding vegetative storage proteins are up-regulated which have been implicated in responses to insects and other stress factors.
also up-regulated in p. novae-zelandiae are genes involved in transport, toxin catabolism and response to oxidative stress .
two and four genes were found to be down-regulated by transcript and protein profiling in p. cheesemanii and p. exile, respectively.
the former two are involved in cell wall modification  and carbohydrate metabolism  and the latter four represent the two vegetative storage proteins up-regulated in p. novae-zelandiae, a universal stress protein and a phosphoglucomutase .
since none of the down-regulated transcripts in p. novae-zelandiae  were captured by the common set of  <dig> genes, no overlap in down-regulated transcripts and proteins was observed in p. novae-zelandiae.
locus, description, log fold change , and gene ontology terms  for genes found specifically up- and down-regulated in p. cheesemanii, p. exile and p. novae-zelandiae when compared to the two remaining species combined.
only genes for which both transcripts  and proteins  have been found to be differentially expressed are listed .
glucosinolate profiles and hydrolysis products
to test how faithfully both transcript and protein expression patterns would predict physiological differences between species, we examined a trait, which we had previously shown to vary greatly in pachycladon, namely, glucosinolate biosynthesis and hydrolysis  <cit> .
glucosinolate biosynthesis and hydrolysis genes found to be differentially regulated in the twelve comparisons by either transcript or protein profiling are summarized in table  <dig>
loci involved in glucosinolate biosynthesis and hydrolysis  found up-regulated in each of the twelve comparisons by either transcript or protein profiling.
glucosinolate biosynthesis: at4g <dig>  and at4g <dig>  catalyze the accumulation of hydroxyalkyl and alkenyl glucosinolates, respectively.
at5g <dig> , at2g <dig> , at5g <dig> , and at1g <dig>  are involved in the chain elongation of methionine and thus can be considered indicators for the accumulation of c <dig> glucosinolates as opposed to c <dig> glucosinolates.
glucosinolate hydrolysis: at3g <dig>  and at1g <dig>  determine the outcome of glucosinolate hydrolysis in that esp is necessary for nitrile formation whereas esm <dig> promotes the formation of isothiocyanates.
other myrosinase-binding/associated proteins  whose biological function remains to be determined are also found up-regulated in some of the species.
loci whose expression is in agreement with profiles of glucosinolates  and glucosinolate hydrolysis products  are highlighted in bold.
expectations for glucosinolate profiles were that p. cheesemanii would have higher levels of alkenyl and hydroxyalkyl glucosinolates  than p. exile and p. novae-zelandiae and p. cheesemanii would have higher levels of four carbon  glucosinolates than p. exile  .
conflicting predictions were made for p. novae-zelandiae since the up-regulation of methionine chain elongation proteins, such as mam-i  and two proteins of methylthioalkylmalate dehydrogenase  suggests higher levels of c <dig> glucosinolates in p. novae-zelandiae than in the other two species.
however, up-regulation of mam-i and methylthioalkylmalate synthase  transcript levels in p. cheesemanii when compared to p. novae-zelandiae suggests higher levels of c <dig> glucosinolates in the former .
contradictory predictions were also derived for p. exile, as up-regulation of mam <dig> transcripts indicated higher c <dig> levels in p. exile than in p. novae-zelandiae and down-regulation of mam-i proteins suggested higher levels of c <dig> glucosinolates in p. novae-zelandiae and p. cheesemanii when compared to  p. exile .
to verify these predictions we analyzed glucosinolate profiles in the same specimens that had been used for transcript and protein profiling.
fourteen compounds were identified across the three species .
interestingly, p. cheesemanii and p. exile had very distinct glucosinolate profiles whereas p. cheesemanii and p. novae-zelandiae, despite being less closely related than p. cheesemanii and p. exile, shared their two major compounds allyl and s -2-hydroxy-3-butenyl glucosinolate .
this high similarity in glucosinolate profiles between p. cheesemanii and p. novae-zelandiae was also surprising given that p. cheesemanii and p. exile had been found to have more similar transcript and protein profiles than p. cheesemanii and p. novae-zelandiae .
p. exile did produce almost no alkenyl glucosinolates  and p. cheesemanii and p. novae-zelandiae did not differ significantly in their production of alkenyl glucosinolates  .
thus the prediction from aop <dig> gene expression suggesting higher levels of alkenyl glucosinolates in p. cheesemanii when compared to p. exile and p. novae-zelandiae was only fulfilled for p. exile.
hydroxy alkenyl glucosinolates were not detected in any of the three species contrary to the up-regulation of aop <dig> transcripts in p. cheesemanii.
as previously discussed  <cit> , aop <dig> expression might be an artefact of cross-hybridization of aop <dig> transcripts with the aop <dig> probe and as such constitute a drawback of using a heterologous microarray.
p. novae-zelandiae however produced slightly more c <dig> glucosinolates than p. cheesenmanii  and also accumulated three additional c <dig> glucosinolates  compared to p. cheesemanii which only accumulated the c <dig> compound s-2-hydroxy- <dig> butenyl glucosinolate .
p. exile did not produce any c <dig> glucosinolates.
thus the differential expression of mam-i and the two mam-d proteins  proved to be a reliable indicator of metabolite profiles whereas up-regulation of mam-i transcripts in p. cheesemanii and mam <dig> transcripts in p. exile did not.
neither array nor protein profiling captured the expression of 2-oxoacid-dependent dioxygenase which has recently been shown to catalyze the production of 2-hydroxy-3-butenyl glucosinolates from 3-butenyl glucosinolates  <cit>  and which we predict to be up-regulated in p. cheesemanii and p. novae-zelandiae when compared with p. exile.
expectations for glucosinolate hydrolysis were that during glucosinolate hydrolysis p. exile would produce isothiocyanates  and p. novae-zelandiae would produce nitriles  .
there were no particular expectations for p. cheesemanii although in the absence of myrosinase-associated enzymes, glucosinolates will most likely convert into isothiocyanates.
interestingly, transcript and protein profiling revealed other myrosinase-associated genes to be up-regulated in p. exile  and p. novae-zelandiae  and down-regulated in p. cheesemanii , p. exile  and p. novae-zelandiae  .
all of these additional loci are located in close proximity to esp  or esm <dig>  in a. thaliana.
due to being recently classified as nationally critical in new zealand, no plants of p. exile were available for glucosinolate hydrolysis analysis.
however, the comparison of glucosinolate hydrolysis products of p. cheesemanii and p. novae-zelandiae, confirmed our predictions.
both species have the same major glucosinolates but whereas in p. cheesemanii all compounds hydrolyzed into isothiocyanates, in p. novae-zelandiae, the two major compounds were converted into their corresponding nitriles and epithionitriles .
this esp-associated polymorphism in glucosinolate hydrolysis products had been found earlier in two other closely related pachycladon species, namely p. enysii  and p. fastigiatum   <cit> .
however, p. enysii and p. fastigiatum's glucosinolate profiles were different from those of p. cheesemanii and p. novae-zelandiae in that both of the latter produced s-2-hydroxy-3-butenyl, a compound neither present in p. enysii nor p. fastigiatum.
the strong differential expression of myrosinase-associated transcripts and proteins is consistent with the hypothesis that glucosinolate hydrolysis has been under selection during the divergence of pachycladon species.
note that p. novae-zelandiae is capable of converting its main glucosinoalates  into its corresponding nitriles -3-hydroxy-pent-4-enenitril) and epithionitriles -3-hydroxy-3--thiiran-2-yl)propanenitril, 2--thiiran-2-yl)ethanenitril) whereas p. cheesemanii is not.
despite minor glucosinolates being converted to isothiocyanates in p. novae-zelandiae, 65% of its glucosinolate breakdown products are nitriles/epithionitriles.
in p. cheesemanii,  <dig> % of glucosinolates are converted to isothiocyanates indicating nitrile/epithionitrile forming activity in p. novae-zelandiae but not p. cheesemanii.
abbreviations: goitrin, goitrin; allyl-itc, 2-propenylisothiocyanate; 3-but-itc, 3-butenylisothiocyanate; 3mtp-itc, 3-methylthiopropylisothiocyanate; 3msop-itc, 3-methylsulfinylpropylisothiocyanate; 4mtb-itc, 4-methylthiopropylisothiocynate; 4msob-itc, 4-methylsulfinylbutylisothiocyanate; 8mto-itc, 8-methylthiooctylisothiocyanate; 9mtn-itc, 9-methylthiononyl-isothiocyanate; 2oh3but-cn, s-3-hydroxy-pent-4-enenitrile; epithio-2oh3b-i, diastereomer  <dig> of -3-hydroxy-3--thiiran-2-yl)propanenitril; epithio-2oh3b-ii, diastereomer  <dig> of -3-hydroxy-3--thiiran-2-yl)propanenitril; epithio-allyl, 2--thiiran-2-yl)ethanenitril; 3mtp-cn, 4-methylthiobutanenitrile; 4mtb-cn, 5-methylthiopentanenitrile.
in summary, predictions of glucosinolate metabolism from protein profiling were more accurate than predictions from transcript profiling.
based on protein profiles we predicted that p. novae-zelandiae would accumulate more c <dig> glucosinolates than the other two species and would produce nitriles in hydrolysis instead of isothiocyanates.
both predictions were confirmed.
in contrast, the prediction from transcript profiles that p. cheesemanii would produce more alkenyl glucosinolates than p. novae-zelandiae and p. exile was only validated for p. exile.
discussion
in this study we compared protein profiles with transcript profiles for three species of new zealand alpine pachycladon.
we employed a heterologous approach to obtain both transcript and protein profiles using arabidopsis microarrays for transcript quantification and the arabidopsis genome as a reference for the quantification of peptide spectra.
such a heterologous approach may limit the number of transcripts and proteins that can be unambiguously identified and whose expression can be reliably quantified.
thus we cannot rule out that these limitations contributed to some of the incongruence we found between transcript and protein patterns.
however, the fact that we obtained both transcript and protein data for a set of  <dig> genes and that we found significant correlations and overlap in differential transcript and protein expression demonstrates that the benefits of using heterologous approaches outweigh their drawbacks.
heterologous microarray studies have long been perceived as a valuable tool for the study of species diversity  <cit> .
our study is one of the first that combines the use of heterologous microarrays with heterologous shotgun proteomics in the context of ecological prediction.
we adopted two approaches to estimate congruence between transcript and protein profiles.
first, we determined 'overall congruence' through correlations of transcript and protein abundance for a set of  <dig> genes surveyed by both microarray analyses and shotgun proteomics.
these correlations differed between species and the highest correlation was found in p. cheesemanii .
the coefficients we observed were consistent with those found in other, non-plant systems .
we then determined congruence in differential expression by investigating down- stream prediction  and up-stream prediction .
we found that transcript patterns predicted protein patterns best in p. novae-zelandiae , followed by p. exile  and p. cheesemanii .
up-stream prediction was on average much weaker  than down-stream prediction , and the results differed markedly between the three species .
given considerable incongruence of transcript and protein patterns, we suggest that for the prediction of physiologies consideration needs to be given to both transcript and protein profiles.
although more minor in scope than transcript profiling, protein profiling not only proved to be a valuable tool for validation of transcript profiling but, in this study, was also a more accurate predictor of metabolite patterns than transcript profiling.
interestingly, proteomes were more dissimilar between species than transcriptomes.
this may be due to different protein turnover rates in these species, or it may be explained as a consequence of a greater divergence of the species' proteomes.
in other words, depending on down-stream processes such as translation, post-transcriptional modification and protein degradation, relatively similar transcription profiles may give rise to relatively dissimilar protein profiles.
thus small differences in transcript levels may manifest themselves in large differences in protein levels and vice versa, which may also be an explanation for the relatively large degree of discordance between transcript and protein profiles.
transcript and protein profiles largely mirrored inferred phylogenetic relatedness among species  <cit>  with the profiles of p. cheesemanii and p. exile being more similar to each other than each was to the profiles of p. novae-zelandiae.
interestingly, p. cheesemanii and p. novae-zelandiae had more similar glucosinolate profiles than did p. cheesemanii and p. exile.
the former species shared their main two compounds allyl and s-2-hydroxy-3-butenyl.
p. exile neither produced alkenyl nor c <dig> glucosinolates, thus its profile is most similar to that of the p. fastigiatum chemotype  <dig>  <cit> .
in contrast to p. cheesemanii and p. exile, p. novae-zelandiae produced a complex blend of glucosinolates.
the finding that similarity patterns in glucosinolate profiles do not correspond to phylogenetic relationships might be explained by selection for glucosinolate metabolism during diversification of pachycladon species, and is an hypothesis that requires further investigation.
in the present study, a number of candidate genes have been identified as having been potentially important during diversification of pachycladon species.
these include myrosinase-binding proteins two of which, namely, esp  and esm <dig>  have been shown to direct glucosinolate hydrolysis towards nitriles and isothiocyanates and thus directly affect the toxicity and palatability of plant tissue to herbivores  <cit> .
we found the esp protein up-regulated in p. novae-zelandiae  along with genes that in a. thaliana are physically linked to esp, such as at1g <dig> and at1g <dig>  as well as at3g <dig> which is physically linked to esm <dig>  in p. exile, there was evidence for up-regulation of at1g <dig>  at1g <dig>  at1g <dig> and esm <dig>  given previous observations  <cit>  of up-regulation of esm <dig> in p. fastigiatum , at1g <dig> and at1g <dig> in p. fastigiatum and esp in p. enysii , we hypothesize that at least two gene clusters have been under selection during pleistocene species radiation of pachycladon  <cit> .
these two clusters correspond to five loci on a. thaliana chromosome one  and two loci on chromosome three  which are all annotated as myrosinase-associated proteins.
we have begun to characterize both clusters in several pachycladon species regarding their gene structure and nucleotide diversity patterns.
other genes of potential adaptive significance in pachycladon are genes which mediate differences in the glucosinolate profiles of arabidopsis.
these include methylthioalkylmalate synthase , methylthioalkylmalate isomerases  and methylthioalkylmalate dehydrogenases , which specify side chain lengths of aliphatic glucosinolates and oxo-acid-dependent-dioxygenases which catalyze the synthesis of alkenyl  and hydroxyalkenyl  glucosinolates.
their potential importance in divergence of pachycladon species is suggested in the present study by the up-regulation of at2g <dig>  at1g <dig> and at5g <dig> in p. novae-zelandiae and of at4g <dig> and at2g <dig> in p. cheesemanii.
previous findings of up-regulation of at5g <dig>  at2g <dig>  at3g <dig>  at5g <dig>  at1g <dig>  at4g <dig> in p. enysii  <cit> , as well as the presence of strikingly different glucosinolate profiles among pachycladon species  are also consistent with an hypothesis of adaptive diversification of glucosinolate metabolism in pachycladon.
in addition to the potential significance of glucosinolate biosynthesis and hydrolysis genes during pachycladon diversification, we observed expression patterns that may be indicative of ecophysiological differences among pachycladon species.
for example, the up-regulation of different beta carbonic anhydrase genes in p. cheesemanii  and p. novae-zelandiae  suggests differential recruitment of carbonic anhydrases during pachycladon diversification and regulation of physiological processes requiring the interconversion of carbon dioxide and bicarbonate.
chloroplast isoforms may contribute to maintenance of adequate carbon dioxide concentration for rubisco whereas cytoplasmic isoforms may provide bicarbonate to phosphoenolpyruvate decarboxylase  <cit> .
this is the primary carboxylating enzyme in c <dig> and cam plants and is also an enzyme replenishing tricarboxylic acid cycle intermediates in leaves of c <dig> plants  <cit> .
reports of co-localization of carbonic anhydrases with phosphoenolpyruvate decarboxylase and pyruvate kinase in roots indicate a role of carbonic anhydrases in re-assimilation of carbon dioxide into metabolic intermediates also in non-photosynthetic tissues  <cit> .
thus the differential expression of three beta carbonic anhydrases suggests that p. novae-zelandiae and p. cheesemanii may regulate carbon dioxide and bicarbonate supply to rubisco and phosphoenolpyruvate decarboxylase differently.
interestingly, a suite of genes up-regulated in p. cheesemanii have recently been found important in the drought responses of populus genotypes that exhibit contrasting leaf carbon isotope discrimination measurements .
these are genes involved in oxidative stress  and protein folding .
other genes involved in drought response in populus were genes involved in carbon fixation , photosynthesis , atp synthesis  and protein folding .
up-regulated in p. cheesemanii was a similar set of genes: for carbon fixation , photosynthesis , atp synthesis  and protein folding .
in populus, abundances of some proteins, amongst them a mitochondrial succinyl coa ligase protein, was found to be correlated with leaf carbon isotope discrimination  <cit> .
the same gene is among the few genes specifically up-regulated in p. exile .
oxidative stress genes and chloroplast-localized molecular chaperones have also been found up-regulated or constitutively higher expressed in drought resistant genotypes of potato  <cit>  and barley  <cit> .
among these were homologues of glutathione-s-transferase and glutathione transporters which were also up-regulated in p. novae-zealandiae as well as a homologue of thioredoxin which was up-regulated in p. cheesemanii.
given that many elements of the transcriptomic and proteomic signatures of drought response in populus, solanum and hordeum are found in differential transcript and protein patterns of pachycladon, we hypothesize differences in drought resistance and water use efficiency between pachcyladon species.
these differences may have evolved in response to differences in soil water availability between species' habitats.
based on our findings we have initiated comparisons of carbon dioxide assimilation rates and carbon isotope discrimination to characterize photosynthesis and water use efficiency in pachycladon.
furthermore, efforts are underway to measure soil moisture levels and other environmental parameters at native pachycladon sites.
other genes potentially interesting for species diversification in pachycladon are serine racemase  and two vegetative storage proteins .
however, their biological roles in plant metabolism are not fully understood, hindering the interpretation of their differential expression in pachyladon in the context of diversification.
serine racemase is primarily expressed in elongating and developing cells such as root tips, developing leaves and shoot meristems in a. thaliana  <cit> .
the enzyme has been demonstrated to have a higher dehydration than racemisation activity leading to the hypothesis that it has a role in providing pyruvate from serine in highly dividing cells.
pyruvate is a key metabolic intermediate of many pathways, such as glycolysis and the biosynthesis of amino acids, organic acids and fatty acids.
thus, similarly to carbonic anydrase, serine racemase may act in the regeneration of metabolic intermediates.
one of the vegetative storage proteins  has been shown to have an 'anti-insect' activity that is associated with its phosphatase activity, and is primarily expressed in arabidopsis flowers.
it is induced by wounding, methyl jasmonate, phosphate deprivation and insect feeding and assumed to also play a role in temporary amino acid storage  <cit> .
given these multiple roles in abiotic and biotic stress responses, the preferential expression in p. novae-zelandiae may confer this species an advantage in coping with stress compared to p. exile and p. cheesemanii.
CONCLUSIONS
in summary, although shotgun proteomics provided information on fewer genes than transcript profiling, it provided a valuable means for validating high throughput gene expression data and predicting physiological attributes of plants.
the combined application of transcript and protein profiling has allowed us to identify candidate genes potentially important in adaptive diversification.
in pachycladon we identified genes with confirmed or predicted roles in glucosinolate biosynthesis and hydrolysis, carbon dioxide bicarbonate conversion, water use efficiency and general stress-related processes.
from these gene sets we predict that adaptation to herbivores and pathogens and soil moisture have been important in driving diversification of pachycladon species.
differences in photosynthesis and carbon fixation may indicate selection for different growth rates.
characterization of gene clusters, physiological and habitat differences in pachycladon are underway to follow up predictions from this comparative profiling study.
microarray analyses of pachycladon roots are also underway to help test the hypothesis that adaptation to different soil types has been a major driver of diversification in pachycladon.

 it has been postulated that genes of co-ordinated function that offer an organism some selective advantage might be identifiable in studies that examine differential gene expression across species ranges and heterogeneous environments. similarly, dge studies that compare expression profiles of closely related species, such as those that have formed during late tertiary radiation of new zealand's alpine genera, might also help to identify candidate genes that have played a role in species diversification. to investigate this approach, in an earlier study we compared microarray expression profiles for natural populations of two closely related alpine plant species from the largely endemic new zealand genus pachycladon. analyses of logfold ratios and ontology analyses in this experiment suggested that secondary metabolite profiles differed among and between populations of these two species. hplc-uv/gc-ms analyses confirmed these predictions, leading to the hypothesis that herbivory was a major driver of diversification of p. fastigiatum and p. enysii. these results provided encouragement that transcriptome profiling used in a predictive sense provided a promising approach for helping to identify putative adaptations between closely related species. however, a potential shortcoming with this approach is that gene expression differences between species may also result from genetic drift. moreover, due to posttranslational modifications, transcript abundances may not correlate with protein abundances. profiling of proteomes, which have different underlying evolutionary dynamics from transcriptomes, provides an alternative approach for the prediction of downstream physiological processes. in the present study we have compared findings from gene expression profiles obtained using heterologous microarrays with findings from protein expression profiles generated by shotgun proteomics. during shotgun proteomics, proteins levels are calculated by determining the number of spectral counts that identify a protein and proteins are identified by searching ms-ms spectra of peptides against reference protein data bases. our aim was to determine to what extent gene expression profiles were indicative of protein expression profiles and vice versa, and to evaluate both methods with respect to their potential for predicting physiological differences among new zealand pachycladon species. for this purpose, we also characterized glucosinolate chemotypes and evaluated the extent to which these were predicted from the protein and transcript expression data. the genus pachycladon is of allopolyploid origin and has diversified into ten morphologically and ecologically diverse species within the last million years. nine of the species are endemic to the southern alps of new zealand and one is native to tasmania. the nine new zealand species form three distinct groups distinguished by their genetic characteristics, morphological features and habitat preferences. species differ markedly in their preference for certain soil types. for example, the group consisting of p. novae-zelandiae and p. wallii are restricted to soils derived from schist whereas another group consisting of p. fastigiatum, p. enysii and p. stellatum are confined to greywacke soils. there are also the limestone specialist p. fasciarium and the basicole p. exile. the remaining two species, p. cheesemanii and p. latisiliquum are geological generalists. these preferences have led to the hypothesis that adaptation to different soils has to a large part driven diversification and evolution of distinct pachycladon species. since species also differ in their altitudinal ranges, adaptation to different altitudes has been suggested as another possible driver of diversification. in this study, we compared three pachycladon species with distinct ecological attributes. these species were p. cheesemanii, p. exile and p. novae-zelandiae. the former two are sister species and resemble close overseas pachycladon relatives transberingia and crucihimalaya. p. cheesemanii and p. exile are both polycarpic perennials with a woody caudex, exhibit leaf heterophylly with ovate to broadly elliptic early leaves with branched hairs on the lamina and petiole and serrate to lobed later leaves; they have slender and terminal inflorescences and narrow petals and terete siliques with uniseriate wingless seeds. p. exile is much smaller in all parts than p. cheesemanii. p. cheesemanii occurs along a wide altitudinal range on greywacke, semi-schist, schist and plutonics substrates, it is a true generalist. p. exile is a lowland species that grows on base-rich soils. it was previously recorded from a few sites in central otago but is now known with certainty from only one limestone site and has recently been classified as nationally critical in new zealand. p. novae-zelandiae is also a polycarpic perennial with a semi-woody caudex but is morphologically distinct from p. cheesemanii and p. exile. it does not produce heteroblastic leaves. rather it produces oblong to elliptic leaves that are either lobed or crenate and are either hairy or glabrous; it has lateral inflorescences, and laterally compressed siliques with biseriate wingless seeds. p. novae-zelandiae grows at much higher altitudes and is confined to schist soils. in summary, p. cheesemanii and p. exile are morphologically similar, lower altitude species that are very closely related but which differ in their soil substrate specificities. in contrast, p. novae-zelandiae is a more distantly related alpine species restricted to schist. given their different ecologies and degrees of relatedness, the three species are expected to differ in their gene and protein expression profiles. in the instances that protein profiles corroborate inferences from comparative transcript analyses, these observations have been used to predict physiological differences between species as well as candidate genes for adaptive diversification.  transcriptome - proteome correlations to investigate the extent to which transcriptional patterns were congruent with protein expression patterns, we calculated correlation coefficients for genes surveyed by both transcript and protein profiling. given the non-normal distributions of both the log spectral abundance factors and the log fluorescence intensities, we used spearman's rank correlation coefficient. each species' proteome correlated most strongly with its own transcriptome with the highest correlation observed in p. cheesemanni and the lowest in p. novae-zelandiae. the transcript profile for each species correlated most strongly with the proteome of p. cheesemanii. this was largely due to the fact that the three transcriptomes correlated very strongly with one another and the p. cheesemanii transcriptome had the highest correlation with its own proteome. the three proteomes correlated much more weakly with each other than did the three transcriptomes, indicating greater differences between the protein profiles than between the transcript profiles. correlations between both transcriptomes and proteomes mirrored phylogenetic relationships with the profiles of sister species p. cheesemanii and p. exile being most similar. interestingly, both the transcriptome and proteome of p. novae-zelandiae were found to be more similar to those of p. exile than to those of p. cheesemanii. pairwise spearman rank correlation coefficients between a) protein and transcript expression statistics, b) transcript expression statistics and c) protein expression statistics for genes surveyed by both microarrays and shotgun proteomics of three pachycladon species. species abbreviations: ch: p. cheesemanii, ex: p. exile, nz: p. novae-zelandiae. the highest correlation coefficients for each species are depicted in bold. note that the proteome of each species correlates most strongly with its own transcriptome. furthermore, transcriptomes and proteomes of ch correlate more strongly with those of ex than with those of nz, reflecting the closer phylogenetic relationship between ch and ex as opposed to ch and nz. overlap in transcript and protein up-regulation for each of the three pachycladon species, we identified up- and down-regulated transcripts and proteins using linear model analysis and wilcoxon rank and t-tests, respectively. the number of up- and down-regulated transcripts and proteins are summarized in table and gene identifiers, descriptions, transcript and protein expression statistics are provided in additional file table s we compared expression in each species with expression in the other two species combined and separately. for example, expression in ch was compared to expression in ex+nz but also to ex and nz separately. for each of the twelve comparisons, we intersected the lists of up-regulated transcripts with lists of up-regulated proteins and found significant overlap in seven cases. the extent of overlap between transcript and protein profiles is limited partially as a result of relative sizes of the different data sets. for example, of the transcripts up-regulated in p. cheesemanii when compared with nz+ex combined, only were present in the protein data set. this means the genes found up-regulated by both transcript and protein profiling in p. cheesemanii represent an overlap of 23%. conversely, of the proteins up-regulated in p. cheesemanii only were present in the transcript data set. in this case we have found 32% of the transcripts maximally identifiable by transcript profiling. interestingly, across the three pachycladon species, an average of % of the transcript patterns were confirmed by protein patterns whereas on average only % of the protein patterns were predicted by transcript patterns. these percentages did differ greatly between species. transcript patterns were best confirmed by proteins patterns in p. novae-zelandiae and protein patterns were best confirmed by transcript patterns in p. cheesemanii. the disagreement of transcript and protein profiling remaining after correcting for different numbers of genes captured by both analyses presumably is explained by the different kinetics of protein and transcript accumulation and degradation. expression of transcripts and proteins in each species was compared with expression in the remaining two species combined and separately. for example, transcripts and proteins were up-regulated in ch when compared with ex and nz combined and transcripts and proteins were down-regulated in ch when compared with ex and nz combined. sixty nine transcripts and proteins were up-regulated in ch when compared only to ex and transcripts and proteins were up-regulated in ch when compared only to nz. the total numbers of transcripts and proteins investigated by microarray analysis and shotgun proteomics were and respectively. locus ids, gene descriptions, logarithmized expression ratios and adjusted p-values of differentially expressed transcripts and proteins for each of the comparison are compiled in additional file table s our goal was to identify genes specifically up- and down-regulated in each species with respect to the other two in the set of genes that were covered by both transcript and protein profiling. in these comparisons the greatest overlap between transcript and protein profiles was found for p. cheesemanii, followed by p. exile and p. novae-zelandiae. gene identifiers, descriptions, and expression statistics for these are compiled in table from the genes equally identified by both approaches, some interesting observations and predictions can be derived. for example, three isoforms of carbonic anhydrases were differentially expressed in p. cheesemanii and p. novae-zelandiae. these genes catalyze the interconversion of carbon dioxide and bicarbonate and have been shown to be involved in photosynthesis, respiration, ph regulation, inorganic carbon transport, ion transport, and water and electrolyte balance. other genes found up-regulated by both approaches in p. cheesemanii are genes involved in translation, photosynthesis light reactions, the calvin cycle, pentose phosphate pathway, fatty acid beta oxidation, serine metabolism, oxidative stress, protein folding and others. the four commonly up-regulated genes in p. exile are associated with very different processes such as calvin cycle, s-adenosylmethione biosynthetic process, carbohydrate metabolism and others. in p. novae-zelandiae, two genes encoding vegetative storage proteins are up-regulated which have been implicated in responses to insects and other stress factors. also up-regulated in p. novae-zelandiae are genes involved in transport, toxin catabolism and response to oxidative stress. two and four genes were found to be down-regulated by transcript and protein profiling in p. cheesemanii and p. exile, respectively. the former two are involved in cell wall modification and carbohydrate metabolism and the latter four represent the two vegetative storage proteins up-regulated in p. novae-zelandiae, a universal stress protein and a phosphoglucomutase. since none of the down-regulated transcripts in p. novae-zelandiae were captured by the common set of genes, no overlap in down-regulated transcripts and proteins was observed in p. novae-zelandiae. locus, description, log fold change, and gene ontology terms for genes found specifically up- and down-regulated in p. cheesemanii, p. exile and p. novae-zelandiae when compared to the two remaining species combined. only genes for which both transcripts and proteins have been found to be differentially expressed are listed. glucosinolate profiles and hydrolysis products to test how faithfully both transcript and protein expression patterns would predict physiological differences between species, we examined a trait, which we had previously shown to vary greatly in pachycladon, namely, glucosinolate biosynthesis and hydrolysis. glucosinolate biosynthesis and hydrolysis genes found to be differentially regulated in the twelve comparisons by either transcript or protein profiling are summarized in table loci involved in glucosinolate biosynthesis and hydrolysis found up-regulated in each of the twelve comparisons by either transcript or protein profiling. glucosinolate biosynthesis: at4g and at4g catalyze the accumulation of hydroxyalkyl and alkenyl glucosinolates, respectively. at5g, at2g, at5g, and at1g are involved in the chain elongation of methionine and thus can be considered indicators for the accumulation of c glucosinolates as opposed to c glucosinolates. glucosinolate hydrolysis: at3g and at1g determine the outcome of glucosinolate hydrolysis in that esp is necessary for nitrile formation whereas esm promotes the formation of isothiocyanates. other myrosinase-binding/associated proteins whose biological function remains to be determined are also found up-regulated in some of the species. loci whose expression is in agreement with profiles of glucosinolates and glucosinolate hydrolysis products are highlighted in bold. expectations for glucosinolate profiles were that p. cheesemanii would have higher levels of alkenyl and hydroxyalkyl glucosinolates than p. exile and p. novae-zelandiae and p. cheesemanii would have higher levels of four carbon glucosinolates than p. exile. conflicting predictions were made for p. novae-zelandiae since the up-regulation of methionine chain elongation proteins, such as mam-i and two proteins of methylthioalkylmalate dehydrogenase suggests higher levels of c glucosinolates in p. novae-zelandiae than in the other two species. however, up-regulation of mam-i and methylthioalkylmalate synthase transcript levels in p. cheesemanii when compared to p. novae-zelandiae suggests higher levels of c glucosinolates in the former. contradictory predictions were also derived for p. exile, as up-regulation of mam transcripts indicated higher c levels in p. exile than in p. novae-zelandiae and down-regulation of mam-i proteins suggested higher levels of c glucosinolates in p. novae-zelandiae and p. cheesemanii when compared to p. exile. to verify these predictions we analyzed glucosinolate profiles in the same specimens that had been used for transcript and protein profiling. fourteen compounds were identified across the three species. interestingly, p. cheesemanii and p. exile had very distinct glucosinolate profiles whereas p. cheesemanii and p. novae-zelandiae, despite being less closely related than p. cheesemanii and p. exile, shared their two major compounds allyl and s -2-hydroxy-3-butenyl glucosinolate. this high similarity in glucosinolate profiles between p. cheesemanii and p. novae-zelandiae was also surprising given that p. cheesemanii and p. exile had been found to have more similar transcript and protein profiles than p. cheesemanii and p. novae-zelandiae. p. exile did produce almost no alkenyl glucosinolates and p. cheesemanii and p. novae-zelandiae did not differ significantly in their production of alkenyl glucosinolates. thus the prediction from aop gene expression suggesting higher levels of alkenyl glucosinolates in p. cheesemanii when compared to p. exile and p. novae-zelandiae was only fulfilled for p. exile. hydroxy alkenyl glucosinolates were not detected in any of the three species contrary to the up-regulation of aop transcripts in p. cheesemanii. as previously discussed, aop expression might be an artefact of cross-hybridization of aop transcripts with the aop probe and as such constitute a drawback of using a heterologous microarray. p. novae-zelandiae however produced slightly more c glucosinolates than p. cheesenmanii and also accumulated three additional c glucosinolates compared to p. cheesemanii which only accumulated the c compound s-2-hydroxy- butenyl glucosinolate. p. exile did not produce any c glucosinolates. thus the differential expression of mam-i and the two mam-d proteins proved to be a reliable indicator of metabolite profiles whereas up-regulation of mam-i transcripts in p. cheesemanii and mam transcripts in p. exile did not. neither array nor protein profiling captured the expression of 2-oxoacid-dependent dioxygenase which has recently been shown to catalyze the production of 2-hydroxy-3-butenyl glucosinolates from 3-butenyl glucosinolates and which we predict to be up-regulated in p. cheesemanii and p. novae-zelandiae when compared with p. exile. expectations for glucosinolate hydrolysis were that during glucosinolate hydrolysis p. exile would produce isothiocyanates and p. novae-zelandiae would produce nitriles. there were no particular expectations for p. cheesemanii although in the absence of myrosinase-associated enzymes, glucosinolates will most likely convert into isothiocyanates. interestingly, transcript and protein profiling revealed other myrosinase-associated genes to be up-regulated in p. exile and p. novae-zelandiae and down-regulated in p. cheesemanii, p. exile and p. novae-zelandiae. all of these additional loci are located in close proximity to esp or esm in a. thaliana. due to being recently classified as nationally critical in new zealand, no plants of p. exile were available for glucosinolate hydrolysis analysis. however, the comparison of glucosinolate hydrolysis products of p. cheesemanii and p. novae-zelandiae, confirmed our predictions. both species have the same major glucosinolates but whereas in p. cheesemanii all compounds hydrolyzed into isothiocyanates, in p. novae-zelandiae, the two major compounds were converted into their corresponding nitriles and epithionitriles. this esp-associated polymorphism in glucosinolate hydrolysis products had been found earlier in two other closely related pachycladon species, namely p. enysii and p. fastigiatum. however, p. enysii and p. fastigiatum's glucosinolate profiles were different from those of p. cheesemanii and p. novae-zelandiae in that both of the latter produced s-2-hydroxy-3-butenyl, a compound neither present in p. enysii nor p. fastigiatum. the strong differential expression of myrosinase-associated transcripts and proteins is consistent with the hypothesis that glucosinolate hydrolysis has been under selection during the divergence of pachycladon species. note that p. novae-zelandiae is capable of converting its main glucosinoalates into its corresponding nitriles -3-hydroxy-pent-4-enenitril) and epithionitriles -3-hydroxy-3--thiiran-2-yl)propanenitril, 2--thiiran-2-yl)ethanenitril) whereas p. cheesemanii is not. despite minor glucosinolates being converted to isothiocyanates in p. novae-zelandiae, 65% of its glucosinolate breakdown products are nitriles/epithionitriles. in p. cheesemanii, % of glucosinolates are converted to isothiocyanates indicating nitrile/epithionitrile forming activity in p. novae-zelandiae but not p. cheesemanii. abbreviations: goitrin, goitrin; allyl-itc, 2-propenylisothiocyanate; 3-but-itc, 3-butenylisothiocyanate; 3mtp-itc, 3-methylthiopropylisothiocyanate; 3msop-itc, 3-methylsulfinylpropylisothiocyanate; 4mtb-itc, 4-methylthiopropylisothiocynate; 4msob-itc, 4-methylsulfinylbutylisothiocyanate; 8mto-itc, 8-methylthiooctylisothiocyanate; 9mtn-itc, 9-methylthiononyl-isothiocyanate; 2oh3but-cn, s-3-hydroxy-pent-4-enenitrile; epithio-2oh3b-i, diastereomer of -3-hydroxy-3--thiiran-2-yl)propanenitril; epithio-2oh3b-ii, diastereomer of -3-hydroxy-3--thiiran-2-yl)propanenitril; epithio-allyl, 2--thiiran-2-yl)ethanenitril; 3mtp-cn, 4-methylthiobutanenitrile; 4mtb-cn, 5-methylthiopentanenitrile. in summary, predictions of glucosinolate metabolism from protein profiling were more accurate than predictions from transcript profiling. based on protein profiles we predicted that p. novae-zelandiae would accumulate more c glucosinolates than the other two species and would produce nitriles in hydrolysis instead of isothiocyanates. both predictions were confirmed. in contrast, the prediction from transcript profiles that p. cheesemanii would produce more alkenyl glucosinolates than p. novae-zelandiae and p. exile was only validated for p. exile. discussion in this study we compared protein profiles with transcript profiles for three species of new zealand alpine pachycladon. we employed a heterologous approach to obtain both transcript and protein profiles using arabidopsis microarrays for transcript quantification and the arabidopsis genome as a reference for the quantification of peptide spectra. such a heterologous approach may limit the number of transcripts and proteins that can be unambiguously identified and whose expression can be reliably quantified. thus we cannot rule out that these limitations contributed to some of the incongruence we found between transcript and protein patterns. however, the fact that we obtained both transcript and protein data for a set of genes and that we found significant correlations and overlap in differential transcript and protein expression demonstrates that the benefits of using heterologous approaches outweigh their drawbacks. heterologous microarray studies have long been perceived as a valuable tool for the study of species diversity. our study is one of the first that combines the use of heterologous microarrays with heterologous shotgun proteomics in the context of ecological prediction. we adopted two approaches to estimate congruence between transcript and protein profiles. first, we determined'overall congruence' through correlations of transcript and protein abundance for a set of genes surveyed by both microarray analyses and shotgun proteomics. these correlations differed between species and the highest correlation was found in p. cheesemanii. the coefficients we observed were consistent with those found in other, non-plant systems. we then determined congruence in differential expression by investigating down- stream prediction and up-stream prediction. we found that transcript patterns predicted protein patterns best in p. novae-zelandiae, followed by p. exile and p. cheesemanii. up-stream prediction was on average much weaker than down-stream prediction, and the results differed markedly between the three species. given considerable incongruence of transcript and protein patterns, we suggest that for the prediction of physiologies consideration needs to be given to both transcript and protein profiles. although more minor in scope than transcript profiling, protein profiling not only proved to be a valuable tool for validation of transcript profiling but, in this study, was also a more accurate predictor of metabolite patterns than transcript profiling. interestingly, proteomes were more dissimilar between species than transcriptomes. this may be due to different protein turnover rates in these species, or it may be explained as a consequence of a greater divergence of the species' proteomes. in other words, depending on down-stream processes such as translation, post-transcriptional modification and protein degradation, relatively similar transcription profiles may give rise to relatively dissimilar protein profiles. thus small differences in transcript levels may manifest themselves in large differences in protein levels and vice versa, which may also be an explanation for the relatively large degree of discordance between transcript and protein profiles. transcript and protein profiles largely mirrored inferred phylogenetic relatedness among species with the profiles of p. cheesemanii and p. exile being more similar to each other than each was to the profiles of p. novae-zelandiae. interestingly, p. cheesemanii and p. novae-zelandiae had more similar glucosinolate profiles than did p. cheesemanii and p. exile. the former species shared their main two compounds allyl and s-2-hydroxy-3-butenyl. p. exile neither produced alkenyl nor c glucosinolates, thus its profile is most similar to that of the p. fastigiatum chemotype. in contrast to p. cheesemanii and p. exile, p. novae-zelandiae produced a complex blend of glucosinolates. the finding that similarity patterns in glucosinolate profiles do not correspond to phylogenetic relationships might be explained by selection for glucosinolate metabolism during diversification of pachycladon species, and is an hypothesis that requires further investigation. in the present study, a number of candidate genes have been identified as having been potentially important during diversification of pachycladon species. these include myrosinase-binding proteins two of which, namely, esp and esm have been shown to direct glucosinolate hydrolysis towards nitriles and isothiocyanates and thus directly affect the toxicity and palatability of plant tissue to herbivores. we found the esp protein up-regulated in p. novae-zelandiae along with genes that in a. thaliana are physically linked to esp, such as at1g and at1g as well as at3g which is physically linked to esm in p. exile, there was evidence for up-regulation of at1g at1g at1g and esm given previous observations of up-regulation of esm in p. fastigiatum, at1g and at1g in p. fastigiatum and esp in p. enysii, we hypothesize that at least two gene clusters have been under selection during pleistocene species radiation of pachycladon. these two clusters correspond to five loci on a. thaliana chromosome one and two loci on chromosome three which are all annotated as myrosinase-associated proteins. we have begun to characterize both clusters in several pachycladon species regarding their gene structure and nucleotide diversity patterns. other genes of potential adaptive significance in pachycladon are genes which mediate differences in the glucosinolate profiles of arabidopsis. these include methylthioalkylmalate synthase, methylthioalkylmalate isomerases and methylthioalkylmalate dehydrogenases, which specify side chain lengths of aliphatic glucosinolates and oxo-acid-dependent-dioxygenases which catalyze the synthesis of alkenyl and hydroxyalkenyl glucosinolates. their potential importance in divergence of pachycladon species is suggested in the present study by the up-regulation of at2g at1g and at5g in p. novae-zelandiae and of at4g and at2g in p. cheesemanii. previous findings of up-regulation of at5g at2g at3g at5g at1g at4g in p. enysii, as well as the presence of strikingly different glucosinolate profiles among pachycladon species are also consistent with an hypothesis of adaptive diversification of glucosinolate metabolism in pachycladon. in addition to the potential significance of glucosinolate biosynthesis and hydrolysis genes during pachycladon diversification, we observed expression patterns that may be indicative of ecophysiological differences among pachycladon species. for example, the up-regulation of different beta carbonic anhydrase genes in p. cheesemanii and p. novae-zelandiae suggests differential recruitment of carbonic anhydrases during pachycladon diversification and regulation of physiological processes requiring the interconversion of carbon dioxide and bicarbonate. chloroplast isoforms may contribute to maintenance of adequate carbon dioxide concentration for rubisco whereas cytoplasmic isoforms may provide bicarbonate to phosphoenolpyruvate decarboxylase. this is the primary carboxylating enzyme in c and cam plants and is also an enzyme replenishing tricarboxylic acid cycle intermediates in leaves of c plants. reports of co-localization of carbonic anhydrases with phosphoenolpyruvate decarboxylase and pyruvate kinase in roots indicate a role of carbonic anhydrases in re-assimilation of carbon dioxide into metabolic intermediates also in non-photosynthetic tissues. thus the differential expression of three beta carbonic anhydrases suggests that p. novae-zelandiae and p. cheesemanii may regulate carbon dioxide and bicarbonate supply to rubisco and phosphoenolpyruvate decarboxylase differently. interestingly, a suite of genes up-regulated in p. cheesemanii have recently been found important in the drought responses of populus genotypes that exhibit contrasting leaf carbon isotope discrimination measurements. these are genes involved in oxidative stress and protein folding. other genes involved in drought response in populus were genes involved in carbon fixation, photosynthesis, atp synthesis and protein folding. up-regulated in p. cheesemanii was a similar set of genes: for carbon fixation, photosynthesis, atp synthesis and protein folding. in populus, abundances of some proteins, amongst them a mitochondrial succinyl coa ligase protein, was found to be correlated with leaf carbon isotope discrimination. the same gene is among the few genes specifically up-regulated in p. exile. oxidative stress genes and chloroplast-localized molecular chaperones have also been found up-regulated or constitutively higher expressed in drought resistant genotypes of potato and barley. among these were homologues of glutathione-s-transferase and glutathione transporters which were also up-regulated in p. novae-zealandiae as well as a homologue of thioredoxin which was up-regulated in p. cheesemanii. given that many elements of the transcriptomic and proteomic signatures of drought response in populus, solanum and hordeum are found in differential transcript and protein patterns of pachycladon, we hypothesize differences in drought resistance and water use efficiency between pachcyladon species. these differences may have evolved in response to differences in soil water availability between species' habitats. based on our findings we have initiated comparisons of carbon dioxide assimilation rates and carbon isotope discrimination to characterize photosynthesis and water use efficiency in pachycladon. furthermore, efforts are underway to measure soil moisture levels and other environmental parameters at native pachycladon sites. other genes potentially interesting for species diversification in pachycladon are serine racemase and two vegetative storage proteins. however, their biological roles in plant metabolism are not fully understood, hindering the interpretation of their differential expression in pachyladon in the context of diversification. serine racemase is primarily expressed in elongating and developing cells such as root tips, developing leaves and shoot meristems in a. thaliana. the enzyme has been demonstrated to have a higher dehydration than racemisation activity leading to the hypothesis that it has a role in providing pyruvate from serine in highly dividing cells. pyruvate is a key metabolic intermediate of many pathways, such as glycolysis and the biosynthesis of amino acids, organic acids and fatty acids. thus, similarly to carbonic anydrase, serine racemase may act in the regeneration of metabolic intermediates. one of the vegetative storage proteins has been shown to have an'anti-insect' activity that is associated with its phosphatase activity, and is primarily expressed in arabidopsis flowers. it is induced by wounding, methyl jasmonate, phosphate deprivation and insect feeding and assumed to also play a role in temporary amino acid storage. given these multiple roles in abiotic and biotic stress responses, the preferential expression in p. novae-zelandiae may confer this species an advantage in coping with stress compared to p. exile and p. cheesemanii.  in summary, although shotgun proteomics provided information on fewer genes than transcript profiling, it provided a valuable means for validating high throughput gene expression data and predicting physiological attributes of plants. the combined application of transcript and protein profiling has allowed us to identify candidate genes potentially important in adaptive diversification. in pachycladon we identified genes with confirmed or predicted roles in glucosinolate biosynthesis and hydrolysis, carbon dioxide bicarbonate conversion, water use efficiency and general stress-related processes. from these gene sets we predict that adaptation to herbivores and pathogens and soil moisture have been important in driving diversification of pachycladon species. differences in photosynthesis and carbon fixation may indicate selection for different growth rates. characterization of gene clusters, physiological and habitat differences in pachycladon are underway to follow up predictions from this comparative profiling study. microarray analyses of pachycladon roots are also underway to help test the hypothesis that adaptation to different soil types has been a major driver of diversification in pachycladon. 
in the present study we use shotgun proteomics to validate inferences from microarray studies regarding physiological differences in three pachycladon species. evidence from heterologous microarrays and shotgun proteomics revealed differential expression of genes involved in glucosinolate hydrolysis and biosynthesis, the interconversion of carbon dioxide and bicarbonate, water use efficiency and others. our results indicate that protein profiling provides a valuable tool for validating transcript profiles in studies of adaptive divergence. we have used transcript and protein profiling to predict physiological processes that evolved differently during diversification of three pachycladon species. overall, prediction of protein abundances from transcript profiles was stronger than prediction of transcript abundance from protein profiles. protein profiles also proved to be more accurate predictors of glucosinolate profiles than transcript profiles. the similarity of species profiles for both transcripts and proteins reflected previously inferred phylogenetic relationships while glucosinolate chemotypes did not. transcript profiling of closely related species provides a means for identifying genes potentially important in species diversification. we compare transcript and protein profiling and evaluate their predictive value for inferring glucosinolate chemotypes characteristic of these species. differences in glucosinolate hydrolysis products were directly confirmed. this approach has also identified candidate genes potentially important in adaptation, which are now the focus of ongoing study. however, the predictive value of transcript profiling for inferring downstream-physiological processes has been unclear.
 transcript profiling of closely related species provides a means for identifying genes potentially important in species diversification. however, the predictive value of transcript profiling for inferring downstream-physiological processes has been unclear. in the present study we use shotgun proteomics to validate inferences from microarray studies regarding physiological differences in three pachycladon species. we compare transcript and protein profiling and evaluate their predictive value for inferring glucosinolate chemotypes characteristic of these species.  evidence from heterologous microarrays and shotgun proteomics revealed differential expression of genes involved in glucosinolate hydrolysis and biosynthesis, the interconversion of carbon dioxide and bicarbonate, water use efficiency and others. differences in glucosinolate hydrolysis products were directly confirmed. overall, prediction of protein abundances from transcript profiles was stronger than prediction of transcript abundance from protein profiles. protein profiles also proved to be more accurate predictors of glucosinolate profiles than transcript profiles. the similarity of species profiles for both transcripts and proteins reflected previously inferred phylogenetic relationships while glucosinolate chemotypes did not.  we have used transcript and protein profiling to predict physiological processes that evolved differently during diversification of three pachycladon species. this approach has also identified candidate genes potentially important in adaptation, which are now the focus of ongoing study. our results indicate that protein profiling provides a valuable tool for validating transcript profiles in studies of adaptive divergence. 