Abstract

Drought is a significant threat to forest health and the establishment of productive tree plantations. There is therefore great interest in understanding the mechanisms underpinning drought responses in forest trees. This review considers the means by which plants in general, and forest trees specifically, both detect and respond to water limitation. The review focuses on molecular-level responses to a drought stimulus, with an emphasis on responses that involve genome-wide reconfigurations in transcript abundance and protein complement in forest trees. A historical view of the molecular analysis of such responses shows a remarkable transition from understanding the impact of drought on individual genes to a more comprehensive picture of the suites of genes and proteins that constitute a drought response. Attention is paid to how this understanding might further the aims of preserving forest health and improve plantation productivity. The review suggests that genome-wide analysis of forest tree drought responses can be leveraged to provide new tools for conservation of adaptive variation and targets for selective breeding or directed modification of forest tree genotypes that can better contend with future drought scenarios.

Introduction

Alterations in global climate and precipitation regimes strongly influence forest distribution and survival (Allen and Breshears, 1998; Engelbrecht et al., 2007; Shaw et al., 2005). While forest trees are sessile organisms, they possess many attributes that allow them to contend with variable water availability, within limits (Chaves et al., 2003; Ingram and Bartels, 2003). Nevertheless, expected rates of global climate change are unprecedented, with longer and more severe periods of drought predicted (Intergovernmental Panel on Climate Change, 2007). This may have a profound effect on forest health, as water limitation is one of the leading contributors to forest declines globally (Bigler et al., 2006; van Mantgem et al., 2009; Allen et al., 2010). For example, in western Canadian aspen forests, drought negatively impacted growth and survival after a severe regional drought during the 2001–2002 growing season (Hogg et al., 2008), with similar reductions in forest productivity observed in Europe (Ciais et al., 2005).

Drought is a multidimensional environmental factor, affecting tree responses from the molecular level to the forest stand level. Interpretation of the drought response at the stand and tree level is complex because it involves consideration of the stress effects and responses (Yordanov et al., 2000). Nevertheless, negative impacts of drought are observed in many facets of forest health, including seedling recruitment, productivity, susceptibility to pathogen or insect attack and fire susceptibility (Hogg and Wein, 2005; van Mantgem et al., 2009; Zhao and Running, 2010). Consequently, there is considerable incentive to better understand the means by which forest trees respond to drought, so as to develop strategies for preservation of forest tree growth and survival against this particular environmental threat.

Given recent advances in genome biology, there is great scope to develop a more fulsome mechanistic understanding of forest tree drought responses. In keeping with this, over the past decades, advances made in genetics, molecular biology, genomics, proteomics and bioinformatics have provided ever-growing insights into how forest trees respond to drought. This review considers the emergence of those insights and how they might shape the protection of forest trees from drought in the future. Readers interested in a broader consideration of the genomics of forest tree responses to abiotic stress or the modification of specific genes as a means by which to achieve drought tolerance are directed to excellent reviews elsewhere (for review, see Polle et al. (2006) and Fischer and Polle (2010)).

Responses of forest trees to drought

A reduction of available water impinges on trees’ ability to grow and transpire by affecting the soil–plant water continuum. Often, in response to declines in plant water potential, in order to reduce water loss under drought stress, forest trees reduce transpiration by closing their stomatal pores, at the expense of CO2 assimilation (Cowan, 1977; Jarvis and Jarvis, 1963). Additionally, water fluxes in a tree can further be disrupted through cavitation or xylem embolism at high xylem tensions induced by water stress. Cavitation, or xylem embolism, is the filling of xylem with air or water vapour instead of water, leading to a reduction in the water conductivity of the plant. The reduction of water conductance within a tree can, in turn, limit growth (Tyree et al., 1994; Rice et al., 2004).

Forest trees utilize a range of strategies to contend with drought. A series of molecular, biochemical, physiological and morphological changes underpin plant response to water deprivation, and the extents of these responses are highly variable and complex (for review see Chaves et al. (2003) and Ingram and Bartels (2003)). The variability in drought responses is a function of the severity and duration of the drought stress (Chaves et al., 2003), superimposed upon genetic variation at the individual, population and species levels (Hamanishi et al., 2010; Wilkins et al., 2009b; Zhang et al., 2004). Foresters have known for many years that different tree species have variable responses to drought. Almost 50 years ago, in a cross-species examination of tree seedlings under drought conditions, Jarvis and Jarvis (1963) concluded that Pinus spp. were the most drought-tolerant species, whereas Populus tremuloides was the most susceptible.

Variation in the drought response is not only seen between forest tree species but also seen intraspecifically. For example, Pseudotsuga menziesii (Douglas fir) seedlings, from distinct geographic regions, exhibited variable drought resistance when grown under water-deficit conditions in a greenhouse (Ferrell and Woodard, 1966). Similarly, variation in response to water availability has been observed in progeny and provenance testing in Pinus taeda (loblolly pine) (Teskey et al., 1987). These early genetic studies, aimed at examining drought tolerance, focused on the relationship between genotype and environment and established the importance of genetic variation in drought tolerance both inter- and intraspecifically among forest trees. Nevertheless, the mechanisms that underpinned drought tolerance and resistance in forest trees and their molecular basis were much less well understood.

Forest trees posses a wide array of traits that confer drought tolerance. The ability to avoid drought stress is dependent on the trees’ ability to minimize water loss and maximize water uptake (Chaves et al., 2003). For example, some forest trees can increase water uptake through more extensive and deeper roots (Nguyen and Lamant, 1989). In order to minimize water loss under drought conditions, forest trees can utilize a variety of traits including altered leaf morphology (e.g. cuticular wax (Hadley and Smith, 1990)), reduction in leaf area (e.g. increased leaf abscission (Munné-Bosch and Alegre, 2004)) and reduction in stomatal conductance. Stomatal control plays a particularly important role in regulating water balance within a plant and, in turn, CO2 assimilation. Although stomatal closure is an efficient mechanism to contend with water shortages by limiting water loss through the stomatal pores (Froux et al., 2005), it cannot solely prevent water balance decline in trees. Consequently, forest tree growth and survival under drought stress is frequently dependent on many of the aforementioned strategies acting in concert. Although inroads have been made in dissecting the physiological responses to drought in forest trees, understanding the depths of the molecular underpinnings is scant in forest trees relative to herbaceous annual plant species.

Plant perception of water status and downstream signalling pathways

Some of the means by which plants sense water-deficit conditions and subsequently induce downstream molecular signalling response cascades have been elucidated (for review see Shinozaki and Yamaguchi-Shinozaki (2007)). In order to mount any response to drought, plants must first sense water-deficit conditions. Although the precise mechanism underlying drought perception in plants is not well understood, there are multiple hypotheses related to how roots sense drought conditions in the soil. Under conditions of decreased soil water, the plant phytohormone abscisic acid (ABA) accumulates in the soil solution. The increase in soil ABA concentration may act as a mechanism by which roots sense reduced soil water (Slovik et al., 1995; Hartung et al., 1996). Drought may also be perceived through a reduction in turgor by osmosensors. Urao et al. (1999) identified an Arabidopsis thaliana transmembrane histidine kinase, AtHK1, which has putative function as an osmosensor. AtHK1 senses osmotic changes and transmits a stress signal to downstream mitogen-activated protein kinase signalling cascades, which in turn induce drought-responsive gene expression (Urao et al., 1999). In red river gum (Eucalyptus camaldulensis), Liu et al. (2001) identified two HKT1 homologues that can sense changes in solute concentration, similar to AtHK1. Eucalyptus HKT1 homologues altered sodium and potassium transport in Xenopus oocytes, suggesting a role in osmoperception and osmoregulation. These homologues are strong candidates for tree proteins that play a key role in the perception of water limitation leading to drought response signalling.

Downstream of water-deficit sensing, the steps in the drought-responsive pathway proceed via one of two signalling pathways: the ABA-dependent and the ABA-independent pathways (Shinozaki and Yamaguchi-Shinozaki, 1996). Under drought conditions, increasing levels of ABA are observed in the roots and shoots; ABA is thought to play an important role in root to shoot signalling (Davies and Zhang, 1991; Walton et al., 1976; Zeevaart and Creelman, 1988). The ABA signal is modified through changes to xylem or apoplastic pH, influencing the signalling process by moderating sensitivity and availability of ABA in planta (Wilkinson et al., 1998; Bahrun et al., 2002; Sobeih et al., 2004). ABA not only acts in the signalling of drought stress but also plays a central role in regulating drought response in plants.

One of the most prominent roles of ABA is in the regulation of stomatal movement in response to drought (for review, see Belin et al. (2010), Wilkinson and Davies (2002) and Popko et al. (2010)). Mediating stomatal aperture under drought conditions allows plants to limit water loss and regulate water balance during periods of water deficit. Another important role of ABA-mediated drought response in plants is the maintenance of root growth under mild or moderate drought stress, whereas leaf growth under drought conditions is restricted (Hsiao and Xu, 2000). Under drought conditions, growth allocation patterns in plants are altered and the variable growth rates observed in roots and shoots are correlated with ABA levels; however, the regulation of growth rates may be mediated through another plant hormone, ethylene (Sharp et al., 2004).

Despite what is known about the role of ABA in mediating plant response to drought stress, little was known about ABA perception by the plant cell until recently. Initial reports of putative ABA receptors were considered controversial because of limited evidence of central roles in ABA perception and response (McCourt and Creelman, 2008). More recently, using a chemical screen technique, Park et al. (2009) identified a protein, pyrabactin resistant 1 (PYR1), which is involved in ABA signalling. PYR1 and PYR1-like receptors are necessary for many plant responses to ABA. Members of the PYRABACTIN RESISTANT / PYRABACTIN RESISTANT-LIKE (PYR/PRL) family of receptor proteins interact downstream with HAB1, a 2C protein phosphatase (PP2C). PP2Cs negatively regulate ABA signalling (Saez et al., 2004). Through proteomic approaches, another group simultaneously identified the ABA receptor, RACR1, which belongs to the same PYR/PYL family of receptors (Ma et al., 2009). This family of receptor proteins appears to be highly conserved across crop plants, and recent work is aimed at elucidating members in tree species (for review, see Klingler et al. (2010)). Saavedra et al. (2010) identified a PP2C homologue from beech tree (Fagus sylvatica) that is a negative regulator of ABA signalling and showed that FsPP2C interacts with Arabidopsis PYR7 and PYR8. Identification of ABA receptor protein homologues is important for understanding the perception of ABA and its involvement in the drought response in trees.

Molecular outputs in response to water-deficit signalling

Whole-plant responses to ABA are underpinned by ABA-dependent changes in gene expression that are mediated through the action of ABA-inducible transcription factors controlling the expression of genes containing cis-acting ABA response elements (ABREs) (for review, see Ingram and Bartels (2003)). The ABRE is stereotypically found in the upstream regulatory regions of drought-responsive genes (Giuliano et al., 1988; Bray, 1994). ABRE-like sequences have also been identified in the upstream regulatory regions of drought-inducible genes, including the G-box sequence (Williams et al., 1992; Shen et al., 1996). Members of the bZIP protein family are known to bind to ABRE and ABRE-like sequences and, in turn, activate ABA-dependent gene expression (Guiltinan et al., 1990; Choi et al., 2000; Uno et al., 2000).

Among the best-characterized drought-induced genes, RESPONSIVE TO DESSICATION 22 (RD22) has ABA-regulated transcription. ABA-mediated regulation of RD22 transcription requires the synthesis of an MYC (rd22BP1/AtMYC2) and an MYB (AtMYB2) transcription factor, both of which are induced by ABA. AtMYC2 and AtMYB2 act as transcriptional activators and bind cis-elements in the promoter of RD22 (Abe et al., 1997; Abe et al., 2003). AtMYC2 and AtMYB2 also are involved in ABA-dependent gene expression of other ABA-inducible genes (Abe et al., 2003).

While many drought responses are mediated by ABA, plants also have ABA-independent responses to drought conditions. Several genes are induced under drought conditions that are not dependent on ABA (Shinozaki and Yamaguchi-Shinozaki, 1996). Often these genes contain a conserved dehydration-responsive element (DRE) in their upstream gene regulatory region, which functions to recruit transcription factors that are not regulated by ABA. Many of the non-ABA abiotic stress signalling pathways are complex, and it is hypothesized that the DRE cis-acting element plays a role mediating different stress-signalling cascades, resulting in an overall plant response to abiotic stress (Knight and Knight, 2001).

Integration of the ABA-dependent and ABA-independent signalling cascades also occurs through downstream gene regulation. For example, the gene RD29A contains both an ABRE and a DRE within its cis-acting upstream gene regulatory region. In the initial stages of drought stress, expression of RD29A is independent of ABA but later is dependent on ABA for gene expression (Shinozaki and Yamaguchi-Shinozaki, 2000).

Through molecular analysis of multiple plant species, insights have been gained into a range of proteins that are induced under water-deficit conditions (for review see Ramanjulu and Bartels (2002)). For example, the hydrophobic late-embryogenesis-abundant (LEA) proteins accumulate under drought stress and are commonly associated with tolerance to water-deficit conditions (Welin et al., 1994). Recent evidence suggests that LEA proteins may have an important role in the stabilization of other proteins and membranes, as well as the prevention of protein aggregation during periods of water deficit (Close, 1996; Goyal et al., 2005). In a poplar clone (P. euramericana cv Dorskamp), the rapid induction of a LEA family protein, dehydrins, gene expression was observed after osmotic stress was imposed on the clones (Caruso et al., 2002). Similar increases in transcript or protein levels of LEA family proteins in other forest trees, such as spruce, have been observed (Blodner et al., 2007).

Aquaporins are another major class of proteins that play a key role in the water-deficit response. Aquaporins are channel proteins that are found in cellular membranes and are responsible for water flux and are crucial for maintaining proper water balance (for review, see Maurel et al. (2008)). There are two major groups of aquaporins: those found specifically in plasma membranes are plasma membrane intrinsic proteins (PIPs), whereas those found in the tonoplast are known as tonoplast membrane intrinsic proteins. Both classes of water transport proteins are important for maintaining water status in the plant, which is vital for photosynthesis and subsequently growth. In Eucalyptus, PIPs are essential for normal growth; a reduction in PIPs resulted in a suppression of growth (Tsuchihira et al., 2010). The expression of aquaporins is dynamic in response to plant water status. Under drought stress conditions, the expression of Plasma-membrane-Intrinsic-Protein (PIP)-type aquaporins was reduced in tobacco plants, hypothesized to decrease water transport (Mahdieh et al., 2008). In white poplar (Populus alba L.), Berta et al. (2010) identified five transcripts for aquaporin proteins that were up-regulated in following re-watering in trees that experienced drought stress. Accumulation of aquaporins following re-watering may be integral to restoration of water transport of plants under well-watered conditions. In poplar trees, members of the PIP1 family of aquaporins are important for recovery from xylem embolism Populus (Secchi and Zwieniecki, 2010). The functional variability and importance of aquaporins in a trees’ response to drought is reflected in the distinct responses of aquaporins in trees with different drought response strategies. Under drought conditions, the drought responsiveness of specific aquaporin family members varied between two poplar clones (P. balsamifera and P. simonii× P. balsamifera) that had contrasting drought response strategies (Almeida-Rodriguez et al., 2010). The variability in aquaporin response may reflect the different roles of aquaporins, with respect to water transport, in trees.

Over the past decades, examination of the drought response in herbaceous annual plant species has revealed many details about the molecular pathways involved in the drought response. This had led to the identification of many important proteins that accumulate under drought conditions, including transporter proteins, messenger RNA-binding proteins, proteases and many others involved in regulation and signal transduction (for review, see Ingram and Bartels (2003)). More recently, progress has been made uncovering the molecular mechanisms underpinning these pathways and, in turn, drought tolerance and resistance in forest trees. Currently, genomic approaches are being brought to bear the drought responses that enable the integration of knowledge of tree-level responses with gene expression and function.

Early identification of drought-responsive genes in forest trees

Prior to the genomic era, foresters and plant biologists alike were limited to studying the function of one or a few genes at a time. Early studies in trees revealed that drought-responsive genes initially identified in herbaceous annual plants, such as dehydrins and heat-shock proteins, and had homologues expressed in the bark tissue of various woody plants (Wisniewski et al., 1996). Insights into the molecular response of trees to drought began to improve through the identification of genes induced by drought in trees. For example, a number of drought-induced genes were first identified in Pinus taeda through comparative analysis of complementary DNA (cDNA) clones whose expression was induced under water-deficit conditions (Chang et al., 1996). Chang et al. (1996) were able to further characterize four water-deficit-induced cDNAs, providing insight into their sequences and patterns of expression. Based on sequence similarity to characterized genes in other plant species, the majority of these genes were thought to function in cell wall reinforcement and hypothesized to participate in the adaptation of the cells to water-deficit stress (Chang et al., 1996). To identify larger numbers of drought-responsive genes without basing discovery on a priori knowledge, Dubos and Plomion (2003) pioneered the use of cDNA-Amplified Fragment Length Polymorphism (AFLP) to identify drought-responsive genes in roots and then the needles of Pinus pinaster (maritime pine) (Dubos et al., 2003). Dubos et al. (2003) identified 48 putative genes that were drought responsive in Pinus pinaster seedlings. Of these 48 genes, many corresponded to proteins of known function with roles in photosynthesis, carbohydrate metabolism, cell wall synthesis and plant defence; however, a relatively high proportion corresponded to genes of unknown function (Dubos et al., 2003). Similar experiments using a cDNA-AFLP technique with almond (Prunus amygdalus) identified drought-responsive genes in young leaves of different cultivars with variable drought response (Campalans et al., 2001).

The breadth and depth of gene discovery in forest trees was expanded through partial sequencing of transcribed cDNA libraries of loblolly pine (Allona et al., 1998) and poplar (Sterky et al., 1998) to generate compendia of expressed sequence tags (ESTs). These pioneering EST efforts focused almost exclusively on genes involved in wood formation but generated information about gene expression and coding sequences in what were, at that time, almost completely uncharacterized genomes. These initial efforts in gene discovery, although modest by today’s standards, were ground breaking and provided important foundations for future studies.

Following the initial efforts in pine and poplar, the number of reported ESTs from forest tree species, including birch, pine and eucalyptus, increased year by year (Strabala, 2004; Li et al., 2009; Wang et al., 2010). Nevertheless, many of these efforts continued to focus ESTs that were related to wood formation. With an increasing desire to gain insights into stress responses in forest trees, biologists carried out EST analysis on other tissues under various treatment regimes. Ujino-Ihara et al. (2000) identified ∼1400 ESTs from the inner bark from a sugi tree (Cryptomeria japonica), which was felled 2 days prior to EST analysis in order to enrich the EST library in drought, wounding and other stress-related genes. In order to directly identify water-responsive genes in loblolly pine, Lorenz et al. (2006) subjected seedlings to various watering regimes and generated an EST library from the root tissues. In these studies, some of the ESTs identified were homologous to genes previously identified as drought responsive in herbaceous plants, such as LEAs and dehydrins. Although there was some degree of similarity between ESTs identified in trees and previous attempts in other plant species, many of the transcripts identified in trees were of unknown function. Although the identification of drought-induced ESTs in forest trees was important for early gene identification and transcriptome studies, the EST libraries generated from these studies played key roles in founding broader analyses of tree transcriptome activity (Nagaraj et al., 2007).

Genome-wide dissection of forest tree drought responses—whole transcriptome analyses

During the course of EST discovery efforts, it was clear that the identification of subsets of drought-responsive genes was insufficient to fully understand the complexities of the drought response in forest trees. cDNA-AFLP techniques and EST frequencies revealed basic data with respect to gene expression; however, genome-wide analysis techniques, such as microarray analysis, had the potential to reveal global gene expression patterns. Early microarray platform experiments investigating the gene expression patterns in hybrid aspen, based on a small set of ESTs identified in wood formation (Sterky et al., 1998), revealed unique tissue-specific transcript profiles in differentiating xylem (Hertzberg et al., 2001). Some of the earliest insights into transcriptome responses to drought in forest trees was determined using cDNA microarray based on ESTs libraries from specific tissues, such as xylem, shoot tips or pollen. Heath et al. (2002) used a 384 pine cDNA microarray to investigate the adaptation to mild drought in pine seedlings. Although the number of genes being investigated was limited, the importance of molecular chaperones and membrane transport proteins was revealed. These particular proteins are postulated to be vital in cell maintenance and repair and therefore necessary for forest trees to cope with mild drought stress (Heath et al., 2002).

Early microarray experiments, aimed at investigating the molecular basis of a given trait or response, had potential; however, they were constrained by the number of genes under investigation. As development of genetic resources for forest trees continued, more comprehensive microarrays were established that enabled relationships between physiological responses and genome-wide gene expression profiles to be investigated. Watkinson et al. (2003) used a microarray consisting of ∼2100 cDNA clones to examine the gene expression in drought-stressed loblolly pine that revealed that alterations in gene expression patterns in response to drought in loblolly pine were not only qualitative but also quantitative. The increased number of transcripts examined, representing 15 functional categories, allowed the authors to correlate patterns of expression with acclimation to mild or severe drought and define roles for specific groups of genes (Watkinson et al., 2003).

Although EST sequencing and early microarray experiments provided significant insights into groups of drought-responsive genes, the sequencing of the complete black cottonwood (P. trichocarpa Torr. & Gray) genome represented a significant milestone in the ability to explore the drought transcriptome in its entirety in a tree (Tuskan et al., 2006). The Populus genome sequence was not only integral in the development of more comprehensive whole-genome microarray platforms but also important for comparative analyses with other plant genomes, such as Arabidopsis. In the ‘post-genomic’ era, a number of microarray resources were developed for poplar species using the available sequence data. POP2, a spotted cDNA array representing more than 100 000 ESTs and ∼40 per cent of predicted gene models from the Populus genome, was used to investigate the global drought response in black cottonwood (P. trichocarpa) and eastern cottonwood (P. deltoides Bart.; Sterky et al., 2004; Street et al., 2006). Street et al. (2006) identified genes with contrasting responses to drought in the two Populus species and hypothesized that the control of gene expression may be an important process in species divergence.

In addition to cDNA microarrays, two short oligonucleotide microarrays were developed for Populus: the Affymetrix GeneChip Poplar Genome Array (www.affymetrix.com) and the Nimblegen Populus whole-genome array (http://www.nimblegen.com/products/exp/eukaryotic.html). Both of these microarrays were designed based on the gene model sequences from the poplar genome sequence (Tuskan et al., 2006), as well as available publicly available EST sequences. Using Affymetrix GeneChip Poplar Genome Arrays, Wilkins et al. (2009b) were able to identify divergent responses in gene expression profiles in response to drought between two poplar hybrids, suggesting that it is difficult to capture a genome-wide drought response with one or a few Populus genotypes. They also showed that transcriptional responses to drought are time of day dependent in hybrid poplars, indicating that any investigation into the molecular-level responses to drought should factor time of day in order to fully grasp the molecular basis of such a response.

The ability to identify a Populus-specific drought transcriptome is becoming increasingly more difficult as whole-genome arrays uncover variation in the transcriptomes within a given species, P. balsamifera (balsam poplar) (Hamanishi et al., 2010). Hamanishi et al. (2010) compared phenotypic traits with gene expression profiles, showing that balsam poplar genotypes that exhibited increased magnitude change in gene expression were also able to sustain growth under drought conditions. Differences in the drought-responsive transcriptomes among balsam poplar genotypes was related to the extent of intra-specific DNA sequence variation, suggesting that genetic relatedness is likely an indicator of a shared drought response.

Until recently, many gene expression studies on forest trees focused on leaves or roots provided insights into transcriptome response in a given organ; however, the subtleties of the drought response at the cellular level could not be identified from these experiments. Berta et al. (2010) examined the transcriptome response to water deficit in the wood-forming tissue in white poplar (Populus alba). This study investigated the transcriptome response to drought, as well as the interplay between wood formation and drought response in trees. Many drought-responsive gene networks were shared between different tissues (e.g. leaves and roots), but some transcripts were identified that may have had specific roles in modulating wood formation under drought stress (Berta et al., 2010). Together, these studies on Populus reveal the complexities in the genome-wide drought response. The variability in the drought transcriptomes of various forest trees on a temporal and spatial scale, as well as the variability that is present among various individuals, can be exploited for breeding and selection of drought-resistant stock.

Genome-wide dissection of forest tree drought responses—quantitative trait locus mapping and association studies

The response of forest trees to drought stress at the morphological and transcriptome level is complex and highly variable, both intra- and inter-specifically. In order to dissect such a complex trait, biologists have often employed quantitative genetic techniques to reveal genetic intervals to which variability in the trait can be ascribed. Quantitative trait locus (QTL) analysis is advantageous for analysing traits, where a priori knowledge of molecular underpinnings or genes is elusive. Genetic maps of many forest trees have been generated for forest trees, and many QTL have been identified for drought-related traits. In rapidly growing willow hybrids (Salix dasyclados × Salix viminalis), Rönnberg-Wästljung et al. (2005) identified a few QTLs that had a significant effect on water use efficiency (WUE), and the authors noted that the analysis revealed the complex nature of drought tolerance in willow. Similarly, in pedunculate oak (Quercus robur L.), Brendel et al. (2008) identified 10 QTL for WUE, where only a few QTL were responsible for the larger proportion of the clonal variation. Generally, in forest tree species, the number of QTL identified, and the amount of variation that is explained by any given QTL reveals the complexity of drought tolerance or WUE. The knowledge gained from QTL analysis in tree species is useful for tree breeding; however, these gains have been restricted by difficulties and time-consuming nature of identifying genes or genes located at a given QTL for species with limited genomic sequence availability.

In order to overcome the obstacles associated with QTL mapping experiments, biologists have used association or linkage disequilibrium (LD) mapping in order to determine the genetics underpinning complex traits, as it is proposed to be more efficient than QTL mapping (Hall et al., 2010). Association mapping relies on the association of genomic regions containing genetic markers with complex traits. With time, the availability of genetic markers has improved allowing the use of association or LD mapping to become more prevalent. Using a candidate gene loci approach, Gonzalez-Martinez et al. (2006) estimated the LD estimate for 18 drought-tolerance candidate genes in loblolly pine (P taeda). A majority of the drought-tolerance candidate genes showed neutral selection, with the exception of ccoaomt-1 and early response to drought 3 (erd3) (Gonzalez-Martinez et al., 2006).

An alternative association mapping approach is a whole-genome scan. In white spruce (Picea glauca), single-nucleotide polymorphisms (SNPs) were identified in expressed genes and used as genetic markers for mapping purposes (Namroud et al., 2008). Although the authors noted limitations in their methods, Namroud et al. (2008) found potential associations between local adaptation of candidate genes and phenotypic attributes of populations. The benefits of identifying genes under potential selection for drought tolerance in non-model tree species through association mapping has the potential to be very useful for tree breeding strategies in the future.

From drought transcriptome to drought proteome

Studies unveiling the drought-responsive transcriptome in trees have provided a wealth of information regarding the molecular underpinnings of the drought response; however, analysis of the proteome reveals the abundance of final gene products that may be important in understanding the down stream drought response. The drought proteome has been examined in several tree species, including poplar (Plomion et al., 2006) and spruce (Blodner et al., 2007). Initial drought studies examining both gene and protein expression in poplar revealed limited overlap between drought-related transcripts and proteins, suggesting the need for complementary approaches to unveil the mechanisms and molecular plasticity that control drought responses in trees (Plomion et al., 2006).

Many proteome studies focused on drought response in forest trees assess the role of genotype in shaping the response. Proteome analysis of eight P. x euramericana genotypes with varying intrinsic water use efficiencies revealed a number of proteins with significant genotype by treatment interaction. A large majority of these proteins were found to be chloroplastic in nature and involved in control of carbon fixation (Bonhomme et al., 2009). While comparing two native poplar species from China, Yang et al. (2010) examined the combined effect of physiological and proteome response to drought stress. Although the two species of poplar differed in their responses, to drought stress, it is evident that physiological and proteomic processes are important for maintenance of cellular homeostasis under drought conditions. Responses to drought stress are not limited to the species level; Zhang et al. (2010) identified sex-specific variation in the expression of drought-responsive proteins in P. cathayana. Many photosynthetic-related and stress-responses proteins have a significant sex by drought interaction effect (Zhang et al., 2010). The differences in the proteomic response to drought observed between the sexes may provide insight into the variability in their productivity as well as their response to drought stress.

Investigations into proteomic variation among genotypes provide excellent insights into the variability in drought-stress response among trees; however, investigations within a given individual will shed light on the dynamic nature of plant stress responses. Pechanova et al. (2010) examined the proteome within apoplastic continuum of P. deletoides. Using a systems approach, the dynamic nature of the proteome between leaves and stems was investigated and many stress-responsive proteins were identified. Interestingly, a large constituent of diverse peroxides thought to play a role in cell wall modifications were identified. Complexities in the proteome among and within individual trees highlight the diversity in the drought response. Using system approaches, combining genomic and proteomic methods of investigation will increase our ability to understand the complex and dynamic responses of forest trees to drought stress.

Recent advances in genome analysis

In the field of genomics, many milestones have been passed, including the sequencing of whole genomes, which have opened many doors to our understanding of tree molecular biology (for review, see Deschamps and Campbell (2009)). The next-generation high-throughput (HTP) sequencing technologies offer more opportunities to ask other, more complex, biological questions (Mardis, 2008). While the use of poplar as a model species has been highly beneficial to the understanding of biology and molecular underpinnings of trees to stress, technology is moving at such a pace that forest biologists can now investigate different tree species with many of the genomic and technological advantages of a model species. The ability to rapidly sequence genomes at increased depth and speed allows for the rapid increase in available genomic resources, including sequence data, physical maps and molecular genetic markers. All these advantages will improve marker-aided tree breeding and tree improvement methods.

Next-generation HTP sequencing technology not only provides better insight into sequence variation but also gives us the ability to investigate epigenetic modifications. Epigenetic modifications, such as DNA or histone modifications, play key roles in regulating gene expression and, therefore, plant growth and development. Under stress conditions, epigenetic modifications play important roles regulating the expression of stress-induced genes (Boyko and Kovalchuk, 2008; Chinnusamy and Zhu, 2009). Some epigenetic modifications are heritable and may provide a sort of ‘stress-memory’ to plants, allowing them to better cope with future stress conditions; however, the benefit of being better able to cope with these conditions may be at the expense of growth (Chinnusamy and Zhu, 2009). While genotypic variation for DNA methylation is observed among poplar hybrids under drought stress and is correlated to productivity under non-stress conditions (Gourcilleau et al., 2010), understanding the role and the mechanisms by which epigenetic modifications regulate gene expression under stress conditions.

Synergistic approaches, including the integration of whole-genome expression data with genotyping data, such as SNP marker analysis, similarly offer the opportunity to link sometimes apparently disparate biological mechanisms to derive a more holistic description of tree responses to a drought stimulus. Advances in the next-generation sequencing technology opens doors for the ability to examine genomic sequences for non-model tree species, as well as many individuals of the same species to gain insight into sequence variation, as well as epigenetic modifications. These new technologies create opportunities to increase our wealth of information about stress adaptation and to delineate the relationships among phenotypic, genetic and epigenetic variation in forest trees.

Perspectives

As climate and precipitation regimes change and impinge on forest productivity, it is becoming increasingly clear that understating how trees adapt and survive under adverse conditions is important for many reasons. Increased periods of drought stress may limit the ability for trees to survive; however, with new found knowledge of molecular responses to drought in forest trees, we may be able to equip ourselves with the ability to plant more resilient stock that is best suited for future conditions.

Over the past few decades, we have accelerated from the initial discovery of individual genes involved in a drought response to variations observed at whole transcriptome level. Microarray studies and other HTP transcriptome analyses have revealed many complexities in the drought response among forest trees. Links between phenotypic observations and transcriptome responses reveal potential mechanisms for adaptation to drought. With further efforts in other “-omics’ platforms, investigators had the ability to also examine proteomic and metabolic responses to drought in trees. Although the response at any given molecular level reveal much information about how trees respond to drought, the integration of the many various high-throughput platforms may uncover many complex molecular mechanisms and pathways that underpin the drought response. A more holistic or systems biology approach will be important in order to understand the relative importance of various pathways and mechanisms. For example, transcriptome studies reveal many genes involved in the synthesis of raffinose and galactinol sugars are found with higher transcript abundance in drought-treated trees (Shinozaki and Yamaguchi-Shinozaki, 2007; Hamanishi et al., 2010). These metabolites are thought to have an osmoprotectant role under drought conditions. Understanding the mechanisms and molecular plasticity of each level of this pathway could be important to exploit this innate drought protection mechanism in trees.

The ability to capitalize on the new genomics technologies has the potential to lead to strategies to better preserve existing tree populations, as well as improve the productivity of new stands and plantations under changing climates. One might make use of these technologies, based on whole-genome approaches or multi-pronged systems biology approaches in order to identify genes and gene products related to drought responses. The identified genes, or gene products, can be used for strategies for selection or directed modification of trees with enhanced capacity to tolerate drought. For example, identification of drought-resistant QTLs in rice (Bernier et al., 2009) has played an important role in the marker-aided selection of drought-tolerant rice varieties (Steele, 2009). The identification of genes, such as homologues of the AtMYB61 gene in Arabidopsis involved in the closure of stomata, and therefore regulation of water loss (Liang et al., 2005), can be used for the future modification of tree stock with enhanced drought tolerance. Using bioinformatic methods, the relationship of genes from the herbaceous Arabidopsis can be transferred to forest trees, such as Populus (Wilkins et al., 2009a), and the roles of genes, such as MYB61, can be inferred. Using these tools, we can engineer trees with enhanced drought tolerance through combination of genomic, bioinformatics and prior knowledge of drought responses in plants. Improved planting stock helps improve or maintain productivity in areas influenced by increasing levels of drought. As well, knowledge of the molecular responses to drought will facilitate in the identification of naturally occurring variation in the drought response. This variation can be selected for or used as a focus for conservation in forests. In this period of uncertainty about our climatic future, genomic approaches that enable us to enhance and increase precision and improve rates of identification of resilient individuals will be of paramount importance.

Conflict of Interest Statement

None declared.

We are most grateful for very useful comments on the draft manuscript provided by two anonymous reviewers. Research in the Campbell laboratory is generously supported by the Natural Science and Engineering Research Council of Canada (NSERC), the Canada Foundation for Innovation (CFI), the Ontario Research Fund (ORF), Genome Canada and the University of Toronto.

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