BACKGROUND
changes in climate over geological timescales have played a major role in the evolution of the australian vegetation.
northward continental drift, beginning in the late oligocence , led to aridity cycles of increasing severity and duration during the miocene  which have promoted a range of adaptive traits characteristic of the australian vegetation.
isolation of gene pools via developmental, climatic and edaphic factors, coupled with high degrees of outcrossing has promoted speciation among many australian plant families, most notably the genus eucalyptus l’hér genus giving rise to > <dig> species.
the genus euclayptus is widely distributed across australia in tropical, arid, temperate and mediterranean climatic zones occupying an extensive ecological range from regions with annual rainfall as low as 250 mm  <cit> , to the wet sclerophyll forests of eastern and south-western australia where rainfall exceeds 1500 mm year− <dig>
among a range of edaphic and climatic factors governing species distributions, water availability is a significant determinant of eucalypt growth, survival and productivity .
a range of adaptive and acclimation traits to enhance growth and survival under the effects of water deficit are prevalent including anatomical changes in leaf shape and area  <cit> , changes in biomass allocation  <cit> , variable stomatal control  <cit> , differences in cell wall reinforcement  <cit> , cell wall water storage  <cit>  and cellular osmolarity  <cit> .
these properties act in concert in many eucalypt species, undoubtedly combining to produce high levels of site-specific variation in performance and survival.
the primary function of physiological and chemical adaptations, either by tolerance or avoidance, ultimately serves to maintain cellular function.
the scope of environmental conditions tolerated by the genus eucalyptus, and the diversity of physiological responses to the availability of water detected among contrasting taxonomic groups  <cit>  suggests both isohydric and anisohydric approaches to plant growth and survival are prevalent within the genus.
the concomitant effects of water deficit, such as redox imbalances within photosynthetic reactions or the capacity to dissipate excessive leaf temperatures require the enhancement of homeostatic mechanisms to maintain cellular function.
the complexity and interrelatedness of biochemical and physiological pathways of plants presents a major challenge to our ability to study plant scale responses.
more recent technological advances to collect network scale molecular data  <cit>  have led to progress in the adoption of a ‘systems biology’ approach  to the study of plants that is still being defined  <cit> .
in addition to the scope of challenges faced by ‘omic’ approaches to comprehensively encompass spatio-temporal variation, interpretation of such datasets is challenging as the development of suitable bioinformatic tools lags behind our ability to acquire data, especially for non-model species.
emerging technologies in bioinformatics to enable a systems approach to multidisciplinary investigations will undoubtedly prove vital in the development of ‘plant systems biology’ and the investigation of dynamic systems from the gene to the landscape scale.
for eucalyptus, understanding physiological and chemical responses of species is presently based on stochastic investigations of pathways that are part of a larger dynamic network.
the molecular basis of acclimation offers a logical reductionist approach to investigating network complexity and is now receiving significant attention .
in plant systems, transcriptional regulation of pathways governing a cascade of chemical and physiological response mechanisms underpinning homeostasis are typically considered in isolation with little context to more general adjustments in metabolism and growth.
investigations collecting ‘omic’ data , such as the ‘transcriptome’, therefore offers considerable advantages to guide physiological investigations and provide upstream information on regulatory processes in a broader context of plant scale homeostasis.
whilst it is well known that quantitative and qualitative regulation of plant metabolism exists throughout the framework, transcript libraries offer a logical ‘first step’ in developing higher-level understanding of whole plant responses to environmental change.
despite a broad understanding of physiological and chemical traits in eucalyptus species conferring acclimation to the effects of aridity, less is known regarding the molecular basis of such traits.
more recent investigations at both the genomic  and transcriptome level  <cit>  are proving highly informative for our understanding of ‘omic’ responses by eucalypts in adapting to and mitigating against changing growth conditions.
studies into water stress response in model and crop species, which are generally short lived annuals with low tolerance to water deficit, tend to focus on identifying early gene expression changes that trigger the initial responses to a change in water status .
in contrast, eucalypt species are long lived perennials thus subjected to seasonal changes in water availability that can vary in severity from year to year depending on climatic zones and prevailing weather conditions  <cit> .
tolerating such temporal variability and amplitude of osmotic potentials  <cit>  is crucial for survival and reproduction.
adopting a comparative transcriptomics approach , though a combination of experimental design, next generation sequencing and novel bioinformatics approaches, we investigate the gene expression profiles of two eucalyptus species of contrasting taxonomy under controlled and quantified medium term water deficit treatment in order to understand the gene sets that become stably established in responses to prolonged water stress in each species.
the species used in this study, eucalyptus globulus labill.
and eucalyptus cladocalyx f.
muell., have contrasting properties for tolerating water deficit with e. globulus occupying more ‘mesic’ and e. cladocalyx more ‘xeric’ environments  <cit>  with differences in isohydric/anisohydric behaviour observed in traits such as stomatal control  <cit>  and both qualitative and quantitative chemical traits for homeostatic regulation of the cellular environment  <cit> .
despite these differences, both species are anatomically similar in leaf and plant structure.
on this basis, this comparative transcriptome study facilitates an investigation of transcriptome wide differential gene expression on a background of eucalyptus responses to the effects of medium term water deficit to provide insight into the molecular mechanisms that differentiate the ‘mesic’ and ‘xeric’ ecotypes.
RESULTS
leaf level physiological measurements indicate acute water deficit responses
leaf gas exchange in control  plants was consistent for both species throughout the study compared to severe stress  treated plants, with average net carbon assimilation values higher for e. globulus than for e. cladocalyx .
both net carbon assimilation and stomatal conductance reduced with treatment intensity.
sub-stomatal carbon concentration , similarly reduced with treatment indicating that limitations to carbon assimilation are largely attributable to stomatal limitations to diffusion rather than reductions in carboxylation arising to biochemical factors .
from these results it is clear that the main limitation is water availability in line with the treatments applied.table  <dig> leaf level physiological measurements indicate acute water deficit responses due to treatment
e. globulus
e. cladocalyx
net co <dig> assimilation , stomatal conductance  and assimilation weighted sub stomatal carbon concentration  for e. globulus and e. cladocalyx seedlings subjected to 8 weeks water deficit  treatment and well watered  control conditions.
post hoc column indicates whether values measured were statistical different  or not
sequencing data
of the  <dig> , <dig> raw reads  <dig> , <dig> passed filtering as high quality .
of the hq reads  <dig> , <dig> aligned to  <dig>  of the  <dig>  annotated e. grandis v <dig>  genes and these were used in the differential expression analysis.
most of the remaining reads either aligned to the plastid genomes  or to ribosomal genes  with on average  <dig> %  of hq reads explained per sample .
investigation of the unmapped reads indicated these largely derived from unannotated ribosomal genes with ~ <dig> % of reads explained when mapped to the genome sequence as opposed to annotated genes.
a measure of the inter-library variation between replicates  is the common coefficient of biological variation  <cit>  and was estimated as  <dig>  .
a multi-dimensional scaling analysis of the transcriptome data  shows clear discrimination between the two species and between ww and ss treatments with the tissues clustering by type.
this high-level view clearly shows that the physiological differences between the species and treatments, as described above, are reflected in the transcriptome and that the isohydric strategy of e. globulus is reflected in a lesser transcriptome response in all tissues.fig.
<dig> multi-dimensional scaling plot of transcriptome data distinguishes samples by tissue and species.
multi-dimensional scaling plot showing the main separation to be between stem derived tissues  and leaf like tissues  on the x-axis and species  on the y-axis
transcripts show differential ecotypic responses to water deficit
differential expression analysis  aimed to identify genes showing a general response to treatment, and secondly, to identify genes with ecotype specific responses to treatment.
in all cases a stringent fdr of  <dig>  was used to determine significance.
in total  <dig> genes showed a significant differential expression response to treatment, with  <dig> having a higher expression in the ss treatment and  <dig> having higher expression in the ww control.
of the  <dig> genes,  <dig> displayed a significant tissue specific response with 69% in e. cladocalyx and 71% in e. globulus exhibiting a stem tissue response.
by way of validation we compared the set of differentially expressed genes discovered in this study to those discovered in an independent water deficit experiment where similar stress and well watered treatments were applied to biological replicate plants from the same two species.
as only leaf tissues were assayed in the validation study we would only expect to see validation of genes that showed a leaf tissue specific response and a global tissue response, approximately half the genes detected.
also, as the two studies differed in the level of replication we would expect a difference in power to detect differential expression.
however, if the genes reported in this study were largely false discoveries it would be extremely unlikely for us to observe a significant overlap between the detected gene sets and further, the direction and magnitude of changes would also be expected to be uncorrelated.
of the  <dig> genes detected as significantly differentially expressed in stem and/or leaf tissue in this study   <dig> genes in the validation study  showed a significant differential expression and this change is in the same direction and of a similar magnitude in more than 99% of genes .
this validation result strongly supports that the signals reported in this study are true signals and not false discoveries.
gene ontology  analysis of biological function using panther functional classification on genes identified to have an fdr <  <dig>  indicated that the majority of the genes more highly expressed in the ss treatment where involved in metabolic processes  or unknown/unclassified , while for the ww control, genes related to metabolic processes  and cellular processes  or where unknown/unclassified  were more highly represented .
agrigo singular enrichment analysis  indicated that  <dig> gene ontologies relating to biological function were significantly enriched  in the ss treatment and included; response to stress, stimulus, abiotic stimulus and water , whereas  <dig> gene ontologies relating to biological function were significantly enriched  in the ww control and included; flavonoid and phenylpropanoid biosynthetic and metabolic processes .fig.
<dig> gene ontology functional classification of differentially expressed genes by treatment and species.
gene ontology functional classifications undertaken using panther for genes significantly more highly expressed in response to the water deficit  treatment  and control   as well as for genes showing a significant species specific response to water deficit  treatment  and control
highly represented gene families  showing higher expression in the ss treatment included; major facilitator superfamily protein , nad-binding rossmann-fold superfamily protein  and expansin-like b <dig> , whereas gene families highly represented in the ww control included; nb-arc domain-containing disease resistance proteins , disease resistance protein , putative , ankyrin repeat family protein , leucine-rich repeat protein kinase family protein , pathogenesis-related thaumatin superfamily protein , basic helix-loop-helix  dna-binding superfamily protein , bifunctional inhibitor/lipid-transfer protein/seed storage 2s albumin superfamily protein , subtilase family protein , rad-like  <dig> , p-loop containing nucleoside triphosphate hydrolases superfamily protein  and germin .
regarding the ecotype specific response to the treatment,  <dig> genes showed a significant effect and included;  <dig> genes that showed significantly higher expression in e. cladocalyx in the ss treatment,  <dig> a significantly higher expression in this species in the ww control, and six, a significantly higher expression in e. globulus in the ww control .
no genes showed significantly higher expression in e. globulus due to the ss treatment.
of these genes,  <dig> displayed a significant tissue specific response with 80% of these genes in both e. cladocalyx and e. globulus had a stem tissue response to treatment.
go analysis of biological function indicated that the majority of the more highly expressed genes  in the ss treatment where either unknown/unclassified  or involved in metabolic processes  while for the ww control, genes related to metabolic processes , cellular processes  and biological regulation  or unknown/unclassified  were more highly represented .
sea indicated that  <dig> gene ontologies relating to biological function were significantly enriched  in the ss treatment and included; response to stress, stimulus and abiotic stimulus , whereas  <dig> gene ontologies relating to biological function were significantly enriched  in the ww control and included; defence response, programmed cell death, death and cell death .table  <dig> transcripts showing a significant differential ecotypic response to water deficit treatment
genes showing significantly higher species specific expression  in either the water deficit  treatment or control  in both e. cladocalyx and e. globulus from most to least significant
of the  <dig> genes that showed a significant response to the ss treatment,  <dig> showed an opposing response by species and  <dig> showed a similar but enhanced response relative to e. globulus with  <dig> of these genes, including the top  <dig>  being unknown.
the expansin-like b <dig>  was the only gene family to be represented more than once.
in the case of the  <dig> genes that showed a significant response in e. cladocalyx to the ww control,  <dig> showed an opposite response and  <dig> showed a similar but enhanced response.
ten genes in this group where unknown while other highly represented gene families included; disease resistance protein  putative , nb-arc domain-containing disease resistance protein  and soybean gene regulated by cold- <dig> .
for the genes in e. globulus that showed a significant response to the ww control, all six exhibited an opposite response when compared to e. cladocalyx and comprised of only one unknown gene.
the 20s proteasome beta subunit g <dig> and nb-arc domain-containing disease resistance protein  gene families were represented more than once.
network analysis reveals key pathways linked to differential responses to water deficit between ecotypes
a network analysis of treatment responsive genes  and of known pathways using cystoscape™ revealed two main linked clusters; cluster a, comprising  <dig> genes linked to  <dig> pathways, and cluster b, comprising of  <dig> genes linked to  <dig> pathways, as well as approximately  <dig> small unlinked clusters containing between  <dig> and  <dig> genes  with links to between  <dig> and  <dig> pathways  .
due to the complexity of these networks, a brief description of the key observations for the main clusters is detailed below with the low resolution figures provided  being for demonstrative purposes as it is not possible to present the extent of detail in this format.
those wanting to view additional details and/or undertake further analysis of the network presented here are encouraged to download the cytoscape v <dig> .
<dig>   and view the cytoscape file  and interpretation instructions .fig.
<dig> metabolic pathway network analysis of differentially expressed genes in response to water deficit treatment.
cytoscape graph depicting genes with an fdr of < <dig>  with known links to pathways in plant metabolic networks.
genes and pathway interactions are found in two main clusters, cluster ﻿a , and cluster b  as well as numerous small ‘unlinked’ clusters .
sub-clusters are also identified within cluster a  and cluster b
cluster a
cluster a  contained  <dig> genes from  <dig> families that showed a response  to the ss treatment e.g.
more highly expressed, compared to  <dig> genes from  <dig> gene families that showed a response in the ww control.
eighteen of the  <dig> pathways showed differential gene responses to treatment, e.g.
at least one linked gene showing higher expression in the ss treatment and ww control, of which  <dig> genes showed a higher response to the ss treatment and  <dig> genes in response to the ww control.
four pathways showed an exclusive response to the ss treatment only and were linked to one gene, whereas  <dig> pathways showed an exclusive response to the ww control and were linked to  <dig> genes.
pathways represented in cluster a included sub-clusters relating to; aromatic polyketides, brassinosteroid, flavonoid, flavonol, flavone, leucodelphinidin, leucopelargonidin, leucocyanidin, luteolin, phloridzin, pinobanksin and resveratrol biosynthesis , luteolin, kaempferol, quercitin, apigenin glycoside as well as anthocyanin, pelargonidin, rutin, salvianin and shisonin biosynthesis , beta alanine, phenylpropanoid acid, methylquercetin, quercetinsulphate, coumarins, and suberin biosynthethesis as well as leucine and valine degradation  and phenylpropanoid, spermidine hydroxycinnamic acid, chlorogenic acid and scopoletin biosynthesis .
genes represented more than once in these clusters in are listed in table  <dig> table  <dig> highly represented gene families identified in metabolic pathway network analysis
gene families more highly represented in sub-clusters within cluster a and b identified as part of the network pathway analysis in cytoscape
within this cluster, nine genes showed a different response to treatment at a species level,  .
four of these genes showed a differential response, e.g.
a greater relative change in expression between treatments in one species, in the ss treatment and five in the ww control where in all cases the change was observed in e. cladocalyx.
two genes showed a significant differential response to the treatment  in the leaf tissue of e. cladocalyx  which were linked to nine different pathways .table  <dig> gene and associated metabolic pathways showing significant response to water deficit treatment in e. cladocalyx
genes showing a significantly higher species specific expression  linked to metabolic pathway networks in e. cladocalyx seedlings subject to either a water deficit  treatment or control  over an 8 week period.
table notes in which treatment  and tissue  the significant response was observed in
cluster b
cluster b  contained  <dig> genes from  <dig> gene families that showed highest expression in the ss treatment and  <dig> genes from  <dig> gene families that showed a highest expression in the ww control.
of the  <dig> pathways present,  <dig> showed differential responses to the treatment, e.g.
at least one linked gene showing higher expression in the ww and ss treatment, where  <dig> genes showed the response in the ss treatment and  <dig> genes in the ww control.
<dig> pathways showed a response only in the ss treatment and were linked to  <dig> genes, whereas  <dig> pathways linked to  <dig> genes showed a response in the ww control only.
pathways represented in cluster b included sub-clusters relating to; colonic acid, udp-galactose, udp-galactoronate, gdp & udp-glucose, udp-glucuronate, starch, udp-rhamnose and udp-xylose biosynthesis, galactose, melibiose, stachyose, sucrose, trehalose and xylose degradation as well as sucrose and starch metabolism, udp-sugars inter-conversion and pentose phosphate pathways , malate-oxaloacetate shuttle, pyruvate decarboxylation, glycolysis, gluconeogenesis, glyoxylate cycle, rubisco shunt, tca cycle, as well as glutamine and acetyl-coa biosynthesis and aspartate degradation pathways , linoleate and ricinoleate biosynthesis as well as phospholipases, phosphatidylcholine acyl editing, phospholipid desaturation and fatty acid activation, degradation and oxidation pathways , jasmonic acid, traumatin and -3-hexen-1-yl acetate as well as fatty acid beta-oxidation pathways , benzoate, ferulate, glycine, monolignal glucoside and sinapate biosynthesis as well as benzoate, ethanol and lysine degradation, acetate conversion to acetylco-a and alkane and fatty acid oxidation , and 4-hydroxyphenylpyruvate, glucosinolate, plastoquinol, rosmarinic acid and vitamin e biosynthesis as well as tyrosine degradation pathways .
genes represented more than once in these clusters in are listed in table  <dig>
within this cluster,  <dig> genes showed a differential response to treatment at a species level  .
eight genes showed a differential response, e.g.
greater relative change in expression between treatments in one species, to the ss treatment and ten to the ww control where, in all but three cases, this response was observed in e. cladocalyx.
nine genes showed a significant differential response to the treatment  in mostly the stem tissue of e. cladocalyx  which were linked to  <dig> different pathways .
minor clusters
of the  <dig> small clusters identified   <dig> showed a significant response e.g.
higher expression, as a result of the ss treatment only,  <dig> as a result of the ww control only and  <dig> showed responses in both ss treatment and ww control.
these clusters comprised  <dig> genes from  <dig> gene families where  <dig>  representing a total of  <dig> gene families, showed a significant response to the ss treatment and  <dig>  representing  <dig> gene families, showed a significant response to the ww control.
gene families responsive to the ss treatment that were more highly represented  included; alpha/beta-hydrolases , basic chitinases , zinc-binding dehydrogenases , aicarft/impchase bienzymes , indole-3-acetate beta-d-glucosyltransferases .
on the other hand, gene families responsive to the ww control included; cytochrome p <dig> , glycosyl hydrolases , pectin lyases , alpha carbonic anhydrase , alpha/beta-hydrolases , beta-galactosidases , carboxyesterases , cellulose synthase , peroxidases  and udp-glucosyltransferases .
within these  <dig> clusters,  <dig> genes showed a notable differential response to treatment at a species level  .
nineteen of these genes showed a differential response, e.g.
greater relative change in expression between treatments in one species, in response to the ss treatment and  <dig> in response to the ww control where in all but five cases, this response was observed in e. cladocalyx.
thirteen genes showed a significant differential response to the treatment  in often both the leaf and stem tissue of e. cladocalyx  which were linked to  <dig> different pathways .
discussion
this comparative expression profile for eucalyptus species of contrasting ecotype under the effects of medium term water deficit provides insight into the molecular mechanisms that underpin adaptive and acclimation responses.
plant responses to treatment induced a stomatal response with concurrent physiological changes indicating that the main limitation for water deficit treated plants compared to the well watered was water availability.
the treatments triggered a cascade of gene expression responses associated with the concomitant effects of water deficit with several patterns in gene expression emerging and offering insight into the comparative stress response mechanisms employed by eucalyptus species.
notably, this study highlights the enhanced transcriptional responses observed in key pathways of species adapted to more water limiting environments.
this investigation demonstrates the importance of a plant scale transcriptomic approach encompassing multiple tissue types.
over 78% of genes identified as responding to the stress treatment occurred in the stem, demonstrating the potential for significant regulatory processes being modulated from this tissue.
previous and current approaches investigating plant responses to resource availability are focused on leaves potentially overlooking significant molecular, chemical and physiological regulation of processes governing stress responses.
such results clearly demonstrate the importance of ‘whole plant’ studies adhering to the literal interpretation of the term.
encompassing multiple tissue types exacerbates a major challenge to ‘omic approaches – the complexity of interpreting such large volumes of data.
the bioinformatic approach outlined here, has successfully highlighted patterns in transcript data and produced interpretable representations of network structure.
it is important to recall that our intention for this study is not to comprehensively summarise the entire transcriptome, nor the pathway analysis constructed, but to offer insight into the transcriptional response mechanisms to applied water deficit and provide a framework for further investigation into the molecular, chemical and physiological mechanisms underpinning tolerance to water deficit in the genus.
the known unknowns
among the most highly induced transcripts observed in this study, many were of unknown function.
whilst repositories of eucalyptus sequence data are improving rapidly in scope  much work remains to complete the functional annotation of the genome, especially given the broad ecotypic scope, chemical composition and diversity of physiological and growth responses within the genus.
the targeted experimental and bioinformatic approaches employed here prioritises a small set of genes as candidates for further investigation.
this use of contrasting ecotypes has enabled us to distinguish common mechanisms and species-specific responses from the ‘background’ of gene expression necessary for general cellular function.
this approach offers great potential for more functional, mechanistic approaches to characterising transcriptome wide responses to the onset of water deficit with implications for both upstream  and downstream  responses.
photoprotection and redox balance
amongst the most immediate challenges to photosynthetic tissues during resource imbalances is avoiding the consequences of excessive photon flux.
in this study, several patterns emerge at the network scale pertaining to the maintenance of redox balance.
for example, in cluster a, a range of photoprotective response mechanisms from the phosphoenolpyruvate pathways are observed to respond including transcripts governing redox balance, most notably those of several cytochrome p <dig> enzymes which play a central role in the oxidation of organic compounds  <cit> .
the role of p450s in oxidising organic compounds, or as the terminal of electron transport chains  represents significant homeostatic control over redox balance.
such control aids in the preservation of photosynthetic infrastructure and the reactions of primary photosynthesis  thus mitigating excess photochemical energy experienced during times of stress or alternatively in times of excessive photoassimilate production.
similarly, antioxidant systems such as the glutathione and ascorbate pathways appear regulated in cluster a and in the unclustered networks supporting previous studies on the role of antioxidant pathways and redox balance in eucalyptus leaves .
sequencing and subsequent annotation of these pathways provides solid evidence for their inducible responses in eucalyptus tissues and together with the induction of p <dig> enzymes provide a useful basis for further characterisation in maintaining redox balance under conditions requiring quenching of excess photochemical energy or as an indicator of photoassimilate supply to cellular metabolism.
phytohormones and signalling molecules
rapid elicitation of signalling molecules represents upstream regulation of plant processes governing plant scale resource allocation and utilization.
a range of genes involved in the synthesis of phytohormones is observed to respond in the present network.
genes encoding phytohormones in the gibberellin, auxin and cytokinin groups are observed, most notably among cluster a and the associated gene analogues encoding photoprotective properties.
the wide distribution of phytohormonal responses across the network likely reflects the broad chemical and physiological processes that rely on hormonal regulation in higher plants.
of particular note is the up regulation of genes governing the synthesis of quercitin in e. cladocalyx.
quercitin is a strong antioxidant compound  <cit>  that is antagonistic to the functions of the auxin class of phytohormone  <cit> .
inhibiting the role of auxin may lead to broad changes in resource allocation to the scale of plant growth habit.
such influence may lead to changes in plant scale resource allocation and concomitant traits associated with resource limitation.
it is tempting to suggest that the expression of quercitin and its antagonistic effects on a major hormonal signalling pathway may serve to explain at least some of the variation in growth habit and water use observed across the genus  <cit> .
further investigations of this gene expression pattern and spatio-temporal assessments of transcript and compound abundance may serve to unravel this fundamental process associated with ecotypic variation within eucalyptus.
primary photosynthesis and cellular metabolism
a range of pathways and gene analogues governing the cycling of primary photosynthates and respiratory substrates were observed across the network.
combined, these indicate a broad reconfiguration of primary photosynthetic reactions in response to the water deficit treatment.
for example, udp-d galactose-4-epimerase- <dig> governs the inter-conversion of galactose to glucose primarily for input into glycolysis.
this gene shows differential regulation in response to the treatment .
udp-d galactose-4-epimerase- <dig>  producing glucose-1-phosphate, may also provide substrate for alternative pathways, most notably to the synthesis of inositols via inositol  <dig> phosphate synthase and downstream pathways previously linked to stress tolerance – including that within the eucalyptus genus  <cit> .
cycling of photoassimilates and subsequent removal from the primary photosynthetic reactions of the cell is thought to alleviate sugar-mediated repression of photosynthesis in plants  thus avoiding, at least in part, photo-oxidative damage.
a major component of this is the galactosylation of sugars for symplastic loading mechanisms characteristic of many tree species  <cit>  including that of eucalyptus  <cit> .
gene analogues governing the galactosylation of carbohydrates to form di-, tri- and tetra-saccharides such as melibiose, raffinose and stachyose is observed  with potential implications for rates of carbon export from leaves.
the interconnectedness of pathways within primary metabolism is a major contributor to the capacity for homeostatic regulation of the system to balance inputs such as water and light.
it is therefore not surprising that response mechanisms such as redox balance, photoprotection and changes in the cycling of primary photosynthates dominate the response to the water deficit treatment and share commonalities with previous investigations in response to alternative stress types .
an example of this is the expression of phosphogluconolactonase  closely linked with the pentose phosphate pathway.
the pentose phosphate pathway may replace glycolysis under suboptimal conditions producing nadph and a range of  <dig> and  <dig> carbon sugars.
products of the pentose phosphate pathway may provide substrate for several biochemical processes such as input back into glycolysis or the shikimic acid pathways for subsequent synthesis of aromatic compounds.
within primary metabolism, the necessity for tight regulation of the network means that subtle differences in transcript abundance may have large consequences on the observed phenotype.
it is therefore important to remember that transcript abundance – even that of differentially expressed genes among contrasting ecotypes – is not a sole indicator of the significance of the response.
secondary metabolism
previous investigations into secondary metabolites in eucalyptus have illustrated a broad diversity and range of concentrations among leaf oils  <cit> , waxes  <cit> , phenolics  <cit>  and tannins  <cit>  with several efforts to characterise genetic associations that underpin this diversity  <cit> .
in this study, both common and differential response in the expression of candidate genes governing the synthesis of secondary metabolites was observed between the two species.
for example, tyrosine transaminase  is differentially expressed in response to treatment.
tyrosine is a phenolic amino acid thus may act as a precursor to the synthesis of other phenolic compounds or alkaloids  <cit> .
linkages between the synthesis of tyrosine and the synthesis of compounds known to have homeostatic redox functions such as the vitamin e in close proximity to gene analogues for plastiquinol biosynthesis indicate that redox balance is a major factor in plant acclimation strategies to the effects of imbalances between the availability of water and excessive photon flux.
the diversity of secondary metabolism in the genus is reflected within the network compiled and serves as a valuable sequence resource to investigate differentially expressed chemical traits within the genus.
this study reports a novel experimental approach that achieves a comparative investigation of transcriptome responses two eucalyptus species of contrasting ecotype to medium term water deficit.
the range of transcript responses among the species highlights the mechanisms employed to maintain cellular function under a contrasting environmental conditions.
these patterns, in several cases having the potential to influence broader structural and chemical processes governing adaptation to water deficit, are likely to be reflected in gene expression among a broader range of species within the genus and thus are candidates underpinning inter-species variation.
this study highlights the strength of a transcriptomic approach to characterise plant responses in the context of metabolic network scale expression profiles and follow up experiments on more species and a range of time points will enable discovery of the genes that regulate species specific responses to water deficit as well as to the gene sets that define those responses.
compilation of such repositories for transcriptome data will undoubtedly serve as a valuable resource for further investigation at a range of scales into the responses employed by eucalyptus to tolerate such a wide scope of environmental conditions.
CONCLUSIONS
this study describes and annotates an enhanced transcriptional response to water deficit in key pathways and associated genes in a ‘dry’  climate eucalypt species adapted to water limited environments compared to a species originating from less arid conditions .
the observed transcriptional response was of a greater magnitude and diversity in stem tissue.
of the differentially expressed genes, a significant proportion were of unknown function, indicating both unique and poorly understood mechanisms underlying the success of members in this genus in water limiting environments.
genes with known function were found in pathways linked to photoprotection and redox balance, phytohormone and long-distance signalling, primary photosynthesis and cellular metabolism.
this suggests that the ‘dry’ climate eucalypt species has tightly regulated mechanisms across the metabolic network for broad scale modification of their biochemistry to reduce water loss while coping with the resultant increases in heat and light energy associated with the inability regulate this via modified physiology.
identification of these pathways and associated genes provides the opportunity to further investigate and understand the mechanisms and genetic variation linked to this important environmental response and assist in genomic efforts linked to managing native populations or tree improvement programs.

 changes in climate over geological timescales have played a major role in the evolution of the australian vegetation. northward continental drift, beginning in the late oligocence, led to aridity cycles of increasing severity and duration during the miocene which have promoted a range of adaptive traits characteristic of the australian vegetation. isolation of gene pools via developmental, climatic and edaphic factors, coupled with high degrees of outcrossing has promoted speciation among many australian plant families, most notably the genus eucalyptus l’hér genus giving rise to > species. the genus euclayptus is widely distributed across australia in tropical, arid, temperate and mediterranean climatic zones occupying an extensive ecological range from regions with annual rainfall as low as 250 mm, to the wet sclerophyll forests of eastern and south-western australia where rainfall exceeds 1500 mm year− among a range of edaphic and climatic factors governing species distributions, water availability is a significant determinant of eucalypt growth, survival and productivity. a range of adaptive and acclimation traits to enhance growth and survival under the effects of water deficit are prevalent including anatomical changes in leaf shape and area, changes in biomass allocation, variable stomatal control, differences in cell wall reinforcement, cell wall water storage and cellular osmolarity. these properties act in concert in many eucalypt species, undoubtedly combining to produce high levels of site-specific variation in performance and survival. the primary function of physiological and chemical adaptations, either by tolerance or avoidance, ultimately serves to maintain cellular function. the scope of environmental conditions tolerated by the genus eucalyptus, and the diversity of physiological responses to the availability of water detected among contrasting taxonomic groups suggests both isohydric and anisohydric approaches to plant growth and survival are prevalent within the genus. the concomitant effects of water deficit, such as redox imbalances within photosynthetic reactions or the capacity to dissipate excessive leaf temperatures require the enhancement of homeostatic mechanisms to maintain cellular function. the complexity and interrelatedness of biochemical and physiological pathways of plants presents a major challenge to our ability to study plant scale responses. more recent technological advances to collect network scale molecular data have led to progress in the adoption of a ‘systems biology’ approach to the study of plants that is still being defined. in addition to the scope of challenges faced by ‘omic’ approaches to comprehensively encompass spatio-temporal variation, interpretation of such datasets is challenging as the development of suitable bioinformatic tools lags behind our ability to acquire data, especially for non-model species. emerging technologies in bioinformatics to enable a systems approach to multidisciplinary investigations will undoubtedly prove vital in the development of ‘plant systems biology’ and the investigation of dynamic systems from the gene to the landscape scale. for eucalyptus, understanding physiological and chemical responses of species is presently based on stochastic investigations of pathways that are part of a larger dynamic network. the molecular basis of acclimation offers a logical reductionist approach to investigating network complexity and is now receiving significant attention. in plant systems, transcriptional regulation of pathways governing a cascade of chemical and physiological response mechanisms underpinning homeostasis are typically considered in isolation with little context to more general adjustments in metabolism and growth. investigations collecting ‘omic’ data, such as the ‘transcriptome’, therefore offers considerable advantages to guide physiological investigations and provide upstream information on regulatory processes in a broader context of plant scale homeostasis. whilst it is well known that quantitative and qualitative regulation of plant metabolism exists throughout the framework, transcript libraries offer a logical ‘first step’ in developing higher-level understanding of whole plant responses to environmental change. despite a broad understanding of physiological and chemical traits in eucalyptus species conferring acclimation to the effects of aridity, less is known regarding the molecular basis of such traits. more recent investigations at both the genomic and transcriptome level are proving highly informative for our understanding of ‘omic’ responses by eucalypts in adapting to and mitigating against changing growth conditions. studies into water stress response in model and crop species, which are generally short lived annuals with low tolerance to water deficit, tend to focus on identifying early gene expression changes that trigger the initial responses to a change in water status. in contrast, eucalypt species are long lived perennials thus subjected to seasonal changes in water availability that can vary in severity from year to year depending on climatic zones and prevailing weather conditions. tolerating such temporal variability and amplitude of osmotic potentials is crucial for survival and reproduction. adopting a comparative transcriptomics approach, though a combination of experimental design, next generation sequencing and novel bioinformatics approaches, we investigate the gene expression profiles of two eucalyptus species of contrasting taxonomy under controlled and quantified medium term water deficit treatment in order to understand the gene sets that become stably established in responses to prolonged water stress in each species. the species used in this study, eucalyptus globulus labill. and eucalyptus cladocalyx f. muell., have contrasting properties for tolerating water deficit with e. globulus occupying more ‘mesic’ and e. cladocalyx more ‘xeric’ environments with differences in isohydric/anisohydric behaviour observed in traits such as stomatal control and both qualitative and quantitative chemical traits for homeostatic regulation of the cellular environment. despite these differences, both species are anatomically similar in leaf and plant structure. on this basis, this comparative transcriptome study facilitates an investigation of transcriptome wide differential gene expression on a background of eucalyptus responses to the effects of medium term water deficit to provide insight into the molecular mechanisms that differentiate the ‘mesic’ and ‘xeric’ ecotypes.  leaf level physiological measurements indicate acute water deficit responses leaf gas exchange in control plants was consistent for both species throughout the study compared to severe stress treated plants, with average net carbon assimilation values higher for e. globulus than for e. cladocalyx. both net carbon assimilation and stomatal conductance reduced with treatment intensity. sub-stomatal carbon concentration, similarly reduced with treatment indicating that limitations to carbon assimilation are largely attributable to stomatal limitations to diffusion rather than reductions in carboxylation arising to biochemical factors. from these results it is clear that the main limitation is water availability in line with the treatments applied.table leaf level physiological measurements indicate acute water deficit responses due to treatment e. globulus e. cladocalyx net co assimilation, stomatal conductance and assimilation weighted sub stomatal carbon concentration for e. globulus and e. cladocalyx seedlings subjected to 8 weeks water deficit treatment and well watered control conditions. post hoc column indicates whether values measured were statistical different or not sequencing data of the, raw reads, passed filtering as high quality. of the hq reads, aligned to of the annotated e. grandis v genes and these were used in the differential expression analysis. most of the remaining reads either aligned to the plastid genomes or to ribosomal genes with on average % of hq reads explained per sample. investigation of the unmapped reads indicated these largely derived from unannotated ribosomal genes with ~ % of reads explained when mapped to the genome sequence as opposed to annotated genes. a measure of the inter-library variation between replicates is the common coefficient of biological variation and was estimated as. a multi-dimensional scaling analysis of the transcriptome data shows clear discrimination between the two species and between ww and ss treatments with the tissues clustering by type. this high-level view clearly shows that the physiological differences between the species and treatments, as described above, are reflected in the transcriptome and that the isohydric strategy of e. globulus is reflected in a lesser transcriptome response in all tissues.fig. multi-dimensional scaling plot of transcriptome data distinguishes samples by tissue and species. multi-dimensional scaling plot showing the main separation to be between stem derived tissues and leaf like tissues on the x-axis and species on the y-axis transcripts show differential ecotypic responses to water deficit differential expression analysis aimed to identify genes showing a general response to treatment, and secondly, to identify genes with ecotype specific responses to treatment. in all cases a stringent fdr of was used to determine significance. in total genes showed a significant differential expression response to treatment, with having a higher expression in the ss treatment and having higher expression in the ww control. of the genes, displayed a significant tissue specific response with 69% in e. cladocalyx and 71% in e. globulus exhibiting a stem tissue response. by way of validation we compared the set of differentially expressed genes discovered in this study to those discovered in an independent water deficit experiment where similar stress and well watered treatments were applied to biological replicate plants from the same two species. as only leaf tissues were assayed in the validation study we would only expect to see validation of genes that showed a leaf tissue specific response and a global tissue response, approximately half the genes detected. also, as the two studies differed in the level of replication we would expect a difference in power to detect differential expression. however, if the genes reported in this study were largely false discoveries it would be extremely unlikely for us to observe a significant overlap between the detected gene sets and further, the direction and magnitude of changes would also be expected to be uncorrelated. of the genes detected as significantly differentially expressed in stem and/or leaf tissue in this study genes in the validation study showed a significant differential expression and this change is in the same direction and of a similar magnitude in more than 99% of genes. this validation result strongly supports that the signals reported in this study are true signals and not false discoveries. gene ontology analysis of biological function using panther functional classification on genes identified to have an fdr <  indicated that the majority of the genes more highly expressed in the ss treatment where involved in metabolic processes or unknown/unclassified, while for the ww control, genes related to metabolic processes and cellular processes or where unknown/unclassified were more highly represented. agrigo singular enrichment analysis indicated that gene ontologies relating to biological function were significantly enriched in the ss treatment and included; response to stress, stimulus, abiotic stimulus and water, whereas gene ontologies relating to biological function were significantly enriched in the ww control and included; flavonoid and phenylpropanoid biosynthetic and metabolic processes.fig. gene ontology functional classification of differentially expressed genes by treatment and species. gene ontology functional classifications undertaken using panther for genes significantly more highly expressed in response to the water deficit treatment and control as well as for genes showing a significant species specific response to water deficit treatment and control highly represented gene families showing higher expression in the ss treatment included; major facilitator superfamily protein, nad-binding rossmann-fold superfamily protein and expansin-like b, whereas gene families highly represented in the ww control included; nb-arc domain-containing disease resistance proteins, disease resistance protein, putative, ankyrin repeat family protein, leucine-rich repeat protein kinase family protein, pathogenesis-related thaumatin superfamily protein, basic helix-loop-helix dna-binding superfamily protein, bifunctional inhibitor/lipid-transfer protein/seed storage 2s albumin superfamily protein, subtilase family protein, rad-like, p-loop containing nucleoside triphosphate hydrolases superfamily protein and germin. regarding the ecotype specific response to the treatment, genes showed a significant effect and included; genes that showed significantly higher expression in e. cladocalyx in the ss treatment, a significantly higher expression in this species in the ww control, and six, a significantly higher expression in e. globulus in the ww control. no genes showed significantly higher expression in e. globulus due to the ss treatment. of these genes, displayed a significant tissue specific response with 80% of these genes in both e. cladocalyx and e. globulus had a stem tissue response to treatment. go analysis of biological function indicated that the majority of the more highly expressed genes in the ss treatment where either unknown/unclassified or involved in metabolic processes while for the ww control, genes related to metabolic processes, cellular processes and biological regulation or unknown/unclassified were more highly represented. sea indicated that gene ontologies relating to biological function were significantly enriched in the ss treatment and included; response to stress, stimulus and abiotic stimulus, whereas gene ontologies relating to biological function were significantly enriched in the ww control and included; defence response, programmed cell death, death and cell death.table transcripts showing a significant differential ecotypic response to water deficit treatment genes showing significantly higher species specific expression in either the water deficit treatment or control in both e. cladocalyx and e. globulus from most to least significant of the genes that showed a significant response to the ss treatment, showed an opposing response by species and showed a similar but enhanced response relative to e. globulus with of these genes, including the top being unknown. the expansin-like b was the only gene family to be represented more than once. in the case of the genes that showed a significant response in e. cladocalyx to the ww control, showed an opposite response and showed a similar but enhanced response. ten genes in this group where unknown while other highly represented gene families included; disease resistance protein putative, nb-arc domain-containing disease resistance protein and soybean gene regulated by cold-. for the genes in e. globulus that showed a significant response to the ww control, all six exhibited an opposite response when compared to e. cladocalyx and comprised of only one unknown gene. the 20s proteasome beta subunit g and nb-arc domain-containing disease resistance protein gene families were represented more than once. network analysis reveals key pathways linked to differential responses to water deficit between ecotypes a network analysis of treatment responsive genes and of known pathways using cystoscape™ revealed two main linked clusters; cluster a, comprising genes linked to pathways, and cluster b, comprising of genes linked to pathways, as well as approximately small unlinked clusters containing between and genes with links to between and pathways. due to the complexity of these networks, a brief description of the key observations for the main clusters is detailed below with the low resolution figures provided being for demonstrative purposes as it is not possible to present the extent of detail in this format. those wanting to view additional details and/or undertake further analysis of the network presented here are encouraged to download the cytoscape v. and view the cytoscape file and interpretation instructions.fig. metabolic pathway network analysis of differentially expressed genes in response to water deficit treatment. cytoscape graph depicting genes with an fdr of < with known links to pathways in plant metabolic networks. genes and pathway interactions are found in two main clusters, cluster ﻿a, and cluster b as well as numerous small ‘unlinked’ clusters. sub-clusters are also identified within cluster a and cluster b cluster a cluster a contained genes from families that showed a response to the ss treatment e.g. more highly expressed, compared to genes from gene families that showed a response in the ww control. eighteen of the pathways showed differential gene responses to treatment, e.g. at least one linked gene showing higher expression in the ss treatment and ww control, of which genes showed a higher response to the ss treatment and genes in response to the ww control. four pathways showed an exclusive response to the ss treatment only and were linked to one gene, whereas pathways showed an exclusive response to the ww control and were linked to genes. pathways represented in cluster a included sub-clusters relating to; aromatic polyketides, brassinosteroid, flavonoid, flavonol, flavone, leucodelphinidin, leucopelargonidin, leucocyanidin, luteolin, phloridzin, pinobanksin and resveratrol biosynthesis, luteolin, kaempferol, quercitin, apigenin glycoside as well as anthocyanin, pelargonidin, rutin, salvianin and shisonin biosynthesis, beta alanine, phenylpropanoid acid, methylquercetin, quercetinsulphate, coumarins, and suberin biosynthethesis as well as leucine and valine degradation and phenylpropanoid, spermidine hydroxycinnamic acid, chlorogenic acid and scopoletin biosynthesis. genes represented more than once in these clusters in are listed in table  table highly represented gene families identified in metabolic pathway network analysis gene families more highly represented in sub-clusters within cluster a and b identified as part of the network pathway analysis in cytoscape within this cluster, nine genes showed a different response to treatment at a species level,. four of these genes showed a differential response, e.g. a greater relative change in expression between treatments in one species, in the ss treatment and five in the ww control where in all cases the change was observed in e. cladocalyx. two genes showed a significant differential response to the treatment in the leaf tissue of e. cladocalyx which were linked to nine different pathways.table gene and associated metabolic pathways showing significant response to water deficit treatment in e. cladocalyx genes showing a significantly higher species specific expression linked to metabolic pathway networks in e. cladocalyx seedlings subject to either a water deficit treatment or control over an 8 week period. table notes in which treatment and tissue the significant response was observed in cluster b cluster b contained genes from gene families that showed highest expression in the ss treatment and genes from gene families that showed a highest expression in the ww control. of the pathways present, showed differential responses to the treatment, e.g. at least one linked gene showing higher expression in the ww and ss treatment, where genes showed the response in the ss treatment and genes in the ww control. pathways showed a response only in the ss treatment and were linked to genes, whereas pathways linked to genes showed a response in the ww control only. pathways represented in cluster b included sub-clusters relating to; colonic acid, udp-galactose, udp-galactoronate, gdp & udp-glucose, udp-glucuronate, starch, udp-rhamnose and udp-xylose biosynthesis, galactose, melibiose, stachyose, sucrose, trehalose and xylose degradation as well as sucrose and starch metabolism, udp-sugars inter-conversion and pentose phosphate pathways, malate-oxaloacetate shuttle, pyruvate decarboxylation, glycolysis, gluconeogenesis, glyoxylate cycle, rubisco shunt, tca cycle, as well as glutamine and acetyl-coa biosynthesis and aspartate degradation pathways, linoleate and ricinoleate biosynthesis as well as phospholipases, phosphatidylcholine acyl editing, phospholipid desaturation and fatty acid activation, degradation and oxidation pathways, jasmonic acid, traumatin and -3-hexen-1-yl acetate as well as fatty acid beta-oxidation pathways, benzoate, ferulate, glycine, monolignal glucoside and sinapate biosynthesis as well as benzoate, ethanol and lysine degradation, acetate conversion to acetylco-a and alkane and fatty acid oxidation, and 4-hydroxyphenylpyruvate, glucosinolate, plastoquinol, rosmarinic acid and vitamin e biosynthesis as well as tyrosine degradation pathways. genes represented more than once in these clusters in are listed in table  within this cluster, genes showed a differential response to treatment at a species level. eight genes showed a differential response, e.g. greater relative change in expression between treatments in one species, to the ss treatment and ten to the ww control where, in all but three cases, this response was observed in e. cladocalyx. nine genes showed a significant differential response to the treatment in mostly the stem tissue of e. cladocalyx which were linked to different pathways. minor clusters of the small clusters identified showed a significant response e.g. higher expression, as a result of the ss treatment only, as a result of the ww control only and showed responses in both ss treatment and ww control. these clusters comprised genes from gene families where representing a total of gene families, showed a significant response to the ss treatment and representing gene families, showed a significant response to the ww control. gene families responsive to the ss treatment that were more highly represented included; alpha/beta-hydrolases, basic chitinases, zinc-binding dehydrogenases, aicarft/impchase bienzymes, indole-3-acetate beta-d-glucosyltransferases. on the other hand, gene families responsive to the ww control included; cytochrome p, glycosyl hydrolases, pectin lyases, alpha carbonic anhydrase, alpha/beta-hydrolases, beta-galactosidases, carboxyesterases, cellulose synthase, peroxidases and udp-glucosyltransferases. within these clusters, genes showed a notable differential response to treatment at a species level. nineteen of these genes showed a differential response, e.g. greater relative change in expression between treatments in one species, in response to the ss treatment and in response to the ww control where in all but five cases, this response was observed in e. cladocalyx. thirteen genes showed a significant differential response to the treatment in often both the leaf and stem tissue of e. cladocalyx which were linked to different pathways. discussion this comparative expression profile for eucalyptus species of contrasting ecotype under the effects of medium term water deficit provides insight into the molecular mechanisms that underpin adaptive and acclimation responses. plant responses to treatment induced a stomatal response with concurrent physiological changes indicating that the main limitation for water deficit treated plants compared to the well watered was water availability. the treatments triggered a cascade of gene expression responses associated with the concomitant effects of water deficit with several patterns in gene expression emerging and offering insight into the comparative stress response mechanisms employed by eucalyptus species. notably, this study highlights the enhanced transcriptional responses observed in key pathways of species adapted to more water limiting environments. this investigation demonstrates the importance of a plant scale transcriptomic approach encompassing multiple tissue types. over 78% of genes identified as responding to the stress treatment occurred in the stem, demonstrating the potential for significant regulatory processes being modulated from this tissue. previous and current approaches investigating plant responses to resource availability are focused on leaves potentially overlooking significant molecular, chemical and physiological regulation of processes governing stress responses. such results clearly demonstrate the importance of ‘whole plant’ studies adhering to the literal interpretation of the term. encompassing multiple tissue types exacerbates a major challenge to ‘omic approaches – the complexity of interpreting such large volumes of data. the bioinformatic approach outlined here, has successfully highlighted patterns in transcript data and produced interpretable representations of network structure. it is important to recall that our intention for this study is not to comprehensively summarise the entire transcriptome, nor the pathway analysis constructed, but to offer insight into the transcriptional response mechanisms to applied water deficit and provide a framework for further investigation into the molecular, chemical and physiological mechanisms underpinning tolerance to water deficit in the genus. the known unknowns among the most highly induced transcripts observed in this study, many were of unknown function. whilst repositories of eucalyptus sequence data are improving rapidly in scope much work remains to complete the functional annotation of the genome, especially given the broad ecotypic scope, chemical composition and diversity of physiological and growth responses within the genus. the targeted experimental and bioinformatic approaches employed here prioritises a small set of genes as candidates for further investigation. this use of contrasting ecotypes has enabled us to distinguish common mechanisms and species-specific responses from the ‘background’ of gene expression necessary for general cellular function. this approach offers great potential for more functional, mechanistic approaches to characterising transcriptome wide responses to the onset of water deficit with implications for both upstream and downstream responses. photoprotection and redox balance amongst the most immediate challenges to photosynthetic tissues during resource imbalances is avoiding the consequences of excessive photon flux. in this study, several patterns emerge at the network scale pertaining to the maintenance of redox balance. for example, in cluster a, a range of photoprotective response mechanisms from the phosphoenolpyruvate pathways are observed to respond including transcripts governing redox balance, most notably those of several cytochrome p enzymes which play a central role in the oxidation of organic compounds. the role of p450s in oxidising organic compounds, or as the terminal of electron transport chains represents significant homeostatic control over redox balance. such control aids in the preservation of photosynthetic infrastructure and the reactions of primary photosynthesis thus mitigating excess photochemical energy experienced during times of stress or alternatively in times of excessive photoassimilate production. similarly, antioxidant systems such as the glutathione and ascorbate pathways appear regulated in cluster a and in the unclustered networks supporting previous studies on the role of antioxidant pathways and redox balance in eucalyptus leaves. sequencing and subsequent annotation of these pathways provides solid evidence for their inducible responses in eucalyptus tissues and together with the induction of p enzymes provide a useful basis for further characterisation in maintaining redox balance under conditions requiring quenching of excess photochemical energy or as an indicator of photoassimilate supply to cellular metabolism. phytohormones and signalling molecules rapid elicitation of signalling molecules represents upstream regulation of plant processes governing plant scale resource allocation and utilization. a range of genes involved in the synthesis of phytohormones is observed to respond in the present network. genes encoding phytohormones in the gibberellin, auxin and cytokinin groups are observed, most notably among cluster a and the associated gene analogues encoding photoprotective properties. the wide distribution of phytohormonal responses across the network likely reflects the broad chemical and physiological processes that rely on hormonal regulation in higher plants. of particular note is the up regulation of genes governing the synthesis of quercitin in e. cladocalyx. quercitin is a strong antioxidant compound that is antagonistic to the functions of the auxin class of phytohormone. inhibiting the role of auxin may lead to broad changes in resource allocation to the scale of plant growth habit. such influence may lead to changes in plant scale resource allocation and concomitant traits associated with resource limitation. it is tempting to suggest that the expression of quercitin and its antagonistic effects on a major hormonal signalling pathway may serve to explain at least some of the variation in growth habit and water use observed across the genus. further investigations of this gene expression pattern and spatio-temporal assessments of transcript and compound abundance may serve to unravel this fundamental process associated with ecotypic variation within eucalyptus. primary photosynthesis and cellular metabolism a range of pathways and gene analogues governing the cycling of primary photosynthates and respiratory substrates were observed across the network. combined, these indicate a broad reconfiguration of primary photosynthetic reactions in response to the water deficit treatment. for example, udp-d galactose-4-epimerase- governs the inter-conversion of galactose to glucose primarily for input into glycolysis. this gene shows differential regulation in response to the treatment. udp-d galactose-4-epimerase- producing glucose-1-phosphate, may also provide substrate for alternative pathways, most notably to the synthesis of inositols via inositol phosphate synthase and downstream pathways previously linked to stress tolerance – including that within the eucalyptus genus. cycling of photoassimilates and subsequent removal from the primary photosynthetic reactions of the cell is thought to alleviate sugar-mediated repression of photosynthesis in plants thus avoiding, at least in part, photo-oxidative damage. a major component of this is the galactosylation of sugars for symplastic loading mechanisms characteristic of many tree species including that of eucalyptus. gene analogues governing the galactosylation of carbohydrates to form di-, tri- and tetra-saccharides such as melibiose, raffinose and stachyose is observed with potential implications for rates of carbon export from leaves. the interconnectedness of pathways within primary metabolism is a major contributor to the capacity for homeostatic regulation of the system to balance inputs such as water and light. it is therefore not surprising that response mechanisms such as redox balance, photoprotection and changes in the cycling of primary photosynthates dominate the response to the water deficit treatment and share commonalities with previous investigations in response to alternative stress types. an example of this is the expression of phosphogluconolactonase closely linked with the pentose phosphate pathway. the pentose phosphate pathway may replace glycolysis under suboptimal conditions producing nadph and a range of and carbon sugars. products of the pentose phosphate pathway may provide substrate for several biochemical processes such as input back into glycolysis or the shikimic acid pathways for subsequent synthesis of aromatic compounds. within primary metabolism, the necessity for tight regulation of the network means that subtle differences in transcript abundance may have large consequences on the observed phenotype. it is therefore important to remember that transcript abundance – even that of differentially expressed genes among contrasting ecotypes – is not a sole indicator of the significance of the response. secondary metabolism previous investigations into secondary metabolites in eucalyptus have illustrated a broad diversity and range of concentrations among leaf oils, waxes, phenolics and tannins with several efforts to characterise genetic associations that underpin this diversity. in this study, both common and differential response in the expression of candidate genes governing the synthesis of secondary metabolites was observed between the two species. for example, tyrosine transaminase is differentially expressed in response to treatment. tyrosine is a phenolic amino acid thus may act as a precursor to the synthesis of other phenolic compounds or alkaloids. linkages between the synthesis of tyrosine and the synthesis of compounds known to have homeostatic redox functions such as the vitamin e in close proximity to gene analogues for plastiquinol biosynthesis indicate that redox balance is a major factor in plant acclimation strategies to the effects of imbalances between the availability of water and excessive photon flux. the diversity of secondary metabolism in the genus is reflected within the network compiled and serves as a valuable sequence resource to investigate differentially expressed chemical traits within the genus. this study reports a novel experimental approach that achieves a comparative investigation of transcriptome responses two eucalyptus species of contrasting ecotype to medium term water deficit. the range of transcript responses among the species highlights the mechanisms employed to maintain cellular function under a contrasting environmental conditions. these patterns, in several cases having the potential to influence broader structural and chemical processes governing adaptation to water deficit, are likely to be reflected in gene expression among a broader range of species within the genus and thus are candidates underpinning inter-species variation. this study highlights the strength of a transcriptomic approach to characterise plant responses in the context of metabolic network scale expression profiles and follow up experiments on more species and a range of time points will enable discovery of the genes that regulate species specific responses to water deficit as well as to the gene sets that define those responses. compilation of such repositories for transcriptome data will undoubtedly serve as a valuable resource for further investigation at a range of scales into the responses employed by eucalyptus to tolerate such a wide scope of environmental conditions.  this study describes and annotates an enhanced transcriptional response to water deficit in key pathways and associated genes in a ‘dry’ climate eucalypt species adapted to water limited environments compared to a species originating from less arid conditions. the observed transcriptional response was of a greater magnitude and diversity in stem tissue. of the differentially expressed genes, a significant proportion were of unknown function, indicating both unique and poorly understood mechanisms underlying the success of members in this genus in water limiting environments. genes with known function were found in pathways linked to photoprotection and redox balance, phytohormone and long-distance signalling, primary photosynthesis and cellular metabolism. this suggests that the ‘dry’ climate eucalypt species has tightly regulated mechanisms across the metabolic network for broad scale modification of their biochemistry to reduce water loss while coping with the resultant increases in heat and light energy associated with the inability regulate this via modified physiology. identification of these pathways and associated genes provides the opportunity to further investigate and understand the mechanisms and genetic variation linked to this important environmental response and assist in genomic efforts linked to managing native populations or tree improvement programs. 
key genes that differentiated species responses were linked to photoprotection/redox balance, phytohormone/signalling, primary photosynthesis/cellular metabolism and secondary metabolism based on plant metabolic pathway network analysis. this knowledge provides the opportunity to further investigate and understand the mechanisms and genetic variation linked to this important environmental response that will assist with genomic efforts in managing native populations as well as in tree improvement programs under future climate scenarios. climatic and edaphic conditions over geological timescales have generated enormous diversity of adaptive traits and high speciation within the genus eucalyptus. eucalypt species occur from high rainfall to semi-arid zones and from the tropics to latitudes as high as 43°s. these results highlight a more definitive response to water deficit by a ‘dry’ climate eucalypt, particularly in stem tissue, identifying key pathways and associated genes that are responsible for the differences between ‘wet’ and ‘dry’ climate eucalypts. the majority of these differentially expressed genes, were expressed in stem tissue. one hundred twenty million high-quality hiseq reads were created from tissue samples in plants that had been successfully subjected to a water deficit treatment or a well-watered control. eucalyptustranscriptomicwater deficithomeostasisecotypehttp://dx.doi.org/ /501100001782university of melbourneissue-copyright-statement© the author 2017
 climatic and edaphic conditions over geological timescales have generated enormous diversity of adaptive traits and high speciation within the genus eucalyptus. eucalypt species occur from high rainfall to semi-arid zones and from the tropics to latitudes as high as 43°s. despite several morphological and metabolomic characterizations, little is known regarding gene expression differences that underpin differences in tolerance to environmental change. using species of contrasting taxonomy, morphology and physiology, this study combines physiological characterizations with ‘second-generation’ sequencing to identify key genes involved in eucalypt responses to medium-term water limitation.  one hundred twenty million high-quality hiseq reads were created from tissue samples in plants that had been successfully subjected to a water deficit treatment or a well-watered control. alignment to the e. grandis genome saw genes of which exhibited differential expression in one or both ecotypes in response to the treatment. further analysis identified genes that demonstrated a significant species-specific response of which were linked to the ‘dry’ species e. cladocalyx where of these genes were uncharacterised. the majority of these differentially expressed genes, were expressed in stem tissue. key genes that differentiated species responses were linked to photoprotection/redox balance, phytohormone/signalling, primary photosynthesis/cellular metabolism and secondary metabolism based on plant metabolic pathway network analysis.  these results highlight a more definitive response to water deficit by a ‘dry’ climate eucalypt, particularly in stem tissue, identifying key pathways and associated genes that are responsible for the differences between ‘wet’ and ‘dry’ climate eucalypts. this knowledge provides the opportunity to further investigate and understand the mechanisms and genetic variation linked to this important environmental response that will assist with genomic efforts in managing native populations as well as in tree improvement programs under future climate scenarios. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords eucalyptustranscriptomicwater deficithomeostasisecotypehttp://dx.doi.org/ /501100001782university of melbourneissue-copyright-statement© the author 2017 