Prompted by the publication of the complete genome sequence of Populus trichocarpa in 2006 and taking advantage from a set of modern genomic and phenotyping tools, research studies are shedding light on the molecular mechanisms underlying poplar responses to drought stress. Exciting information is accumulating on tree-specific processes including embolism formation and repair, the impact of drought stress on woody biomass yield and quality, and the long-term effects of drought events. This mounting knowledge can be exploited to select more tolerant genotypes and can be translated to other tree species, improving our understanding of forest dynamics under rapidly changing environmental conditions.
Poplar (Populus spp.) is a high-value crop for woody biomass production. Global poplar plantations cover approximately 31.4 million hectares. The largest cultivated areas are located in Canada and China, followed by Europe with France and Italy being the main countries. In Italy, plantations are mostly found in the Po’ valley and cover about 43,400 hectares . Intensive genetic improvement programs have been implemented from the early years of the 20th century, with three main objectives: yield, pest and pathogen resistance, and wood quality . However, abiotic stresses, especially drought, are rising in importance, posing new challenges to ecosystems and agricultural activities . The impact of drought stress can be particularly severe on forest trees due to intrinsic vulnerabilities related to their size, which requires a complex vascular water transport system from soil to canopy, and their long generation time, which slows genetic adaptation .
Poplar copes with water deficit deploying drought avoidance and drought tolerance strategies . Avoidance mechanisms include the control of transpiration through the regulation of stomatal conductance, deposition of cuticular waxes to limit non-stomatal transpiration, increased root growth, and reduced leaf area and leaf shedding . Drought avoidance strategies are particularly effective in trees as they can generally rely on high degree of phenotypic plasticity to adjust their morphological traits . Tolerance mechanisms are aimed at maintaining the biological functions under stress conditions; these encompass the accumulation of osmolytes (glycine betaine, proline, sugars) that help maintain water fluxes and cell turgor , the synthesis of protective molecules as late embryogenesis abundant (LEA) proteins and proline, the expression of genes encoding ROS scavenging enzymes and aquaporins . The deployment of avoidance and tolerance mechanisms is energetically costly, thus it involves a trade-off between stress resilience and growth.
The most productive poplar genotypes under non-limiting water conditions display the greatest yield reduction yield under drought conditions . This variability has been found in and between poplar species and is correlated to water availability in the natural habitat . This partially untapped diversity is explored by linkage mapping and genome-wide association scan (GWAS).
As example, an interspecific F1 population of 144 individuals derived from a crossing between P. deltoides and P. simonii was used to analyse five drought-related traits at seedling stage. This analysis led to the identification of 63 quantitative trait loci (QTLs) associated to drought tolerance traits . The functional annotation of genes identified within the QTLs pointed out a large variety of gene functions.
Using a F2 mapping population derived from the cross between one P. deltoides and one P. trichocarpa showing contrasting physiological traits, 54 QTLs specific for drought response were mapped . Low leaf osmotic potential at full turgor was used as a proxy for measuring the degree of dehydration in poplar.
An F2 population derived from a P. trichocarpa × P. deltoides cross was extensively phenotyped for low osmotic potential at full turgor in presence of soil limiting and non-limiting water conditions. The screening allowed to identify seven significant QTLs accounting for 5.5 to 19.1% of the total phenotypic variation observed .
Transcriptomics has been widely applied to investigate the molecular bases of poplar responses to drought. Analysis of the transcriptome of root apices and leaves of the P. deltoides × P. nigra hybrids Carpaccio and Soligo allowed the identification of genes and processes differentially regulated in roots and leaves during rapid and long-term responses to drought . Mining the response of P. euphratica, a poplar species adapted to arid environments, exposed to water deficit for 7 weeks at 4 different intensities revealed that specific regulatory pathways are activated according to the severity of the imposed stress. Candidate marker genes were detected encoding fo transcription factors and heat shock proteins (HSPs) .
In P. nigra drought-stressed roots, genes responding to oxidative stress, including some encoding for reactive oxygen species (ROS) scavenging enzymes, were downregulated . Downregulation of genes involved in protection from oxidative stress was also detected in P. trichocarpa xylem parenchyma cells after the induction of embolism . The transcriptome of P. euphratica plants exposed to drought stress showed that, under moderate drought, stomatal closure was inhibited concomitantly to transcriptional remodelling of genes involved in photoprotection and ROS detoxification. The results allowed the discovery of candidate genes implicated in the inhibition of stomatal closure and in the modulation of the ascorbate-glutathione and ubiquitin-proteasome pathways . P. trichocarpa plants overexpressing WRKY75, a member of a transcription factor family characterized by the conserved amino acid sequence WRKYGQK and implicated in stress responses, displayed higher photosynthetic and growth rate under drought conditions. PtrWRKY75 activated the expression of PHENYLALANINE AMMONIA LYASE 1 (PAL1), which resulted in increased salicylic acid (SA) and ROS accumulation that acted as signals to induce stomatal closure .
Three P. nigra clones with different behaviour under limiting water conditions were analyzed. The tolerant clone displayed enrichment of genes encoding bark storage proteins and HSPs, while the avoidant and the drought-escape clones activated genes implicated in secondary metabolism, programmed cell death, and leaf senescence . Intraspecific variation was also found in P. balsamifera and P. simonii genotypes exposed to water withdrawal treatments, evidencing differential accumulation of citric acid and oligosaccharides and differential expression of genes implicated in phytohormone metabolism, osmoregulation, oxidative stress, carbohydrate metabolism, and amino acid transport .
A microarray analysis of the response to combined drought and salt stress in leaves of a P. alba × P. glandulosa hybrid allowed the identification of genes modulated by both stresses, evidencing a crosstalk between the two response patterns . In plants of P. simonii exposed to a combination of heat and drought, genes modulated by single or combined conditions were identified in roots and leaves. High temperature and/or drought activated abscisic acid (ABA) accumulation but repressed the metabolism of auxin and other phytohormones . In addition, 197 miRNAs conserved between P. euphratica and P. trichocarpa and 58 miRNAs specific for P. euphratica were shown to be differentially expressed in P. euphratica plants exposed to drought . A similar analysis was applied to P. trichocarpa . In these studies, degradome analysis led to the identification of target genes putatively involved in drought responses.
Genes controlling stomatal development have been identified in poplar by sequence homology with their Arabidopsis homologs . These include STOMAGEN, encoding a signal peptide that acts as a positive regulator of stomatal development, ERECTA (ER), which encodes a leucine rich repeat (LRR) receptor-like kinases, STOMATA DENSITY AND DISTRIBUTION 1 (SDD1), encoding a protease that acts as negative regulator of stomatal development, FAMA, encoding a basic helix-loop-helix (bHLH) transcription factor, YODA (YDA) encoding a MAP kinase kinase kinase that activates a signal cascade which negatively regulates stomatal development, and TOO MANY MOUTHS (TMM), encoding an LRR receptor-like protein that modulates stomatal patterning. Environmental cues modulate the expression of these regulators. In P. balsamifera plants exposed to drought, newly formed leaves were characterized by reduced stomatal index (the ration between stomatal density and the sum of stomatal density and epidermal cell density). This reduction was correlated with the expression of STOMAGEN and FAMA . Wang et al.  isolated, from the hybrid poplar clone NE-19, PdEPF1, a homolog of Arabidopsis EPIDERMAL PATTERNING FACTOR 1 which encodes a signal peptide that inhibits the progression towards guard cell differentiation acting antagonistically to STOMAGEN. PdEF1 was induced by drought stress and ABA and P. tomentosa plants overexpressing PdEPF1 displayed reduced stomatal density on the abaxial leaf surface, higher water use efficiency (WUE) and increased drought tolerance. WUE, the amount of biomass assimilated per unit of water used, is a key concept used to evaluate plant productive potential under different levels of water availabilty . Interestingly, in well-watered conditions the growth rate and the photosynthetic rate of wild type and PdEPF1 overexpressing plants was similar, indicating that no yield penalty was conferred by the transgene. Based on these results, PdEPF1 can be regarded as an interesting gene for breeding and biotechnology programmes. Also the overexpression of PdERECTA and PdEPFL6, both negative regulators of stomata differentiation, resulted in decreased stomatal density and increased drought tolerance without a significant reduction in biomass accumulation under non-limiting water conditions .
However, when STOMAGEN was overexpressed in the poplar hybrid clone 84K, plants displayed increased stomatal density and conductance, higher net photosynthetic rate, and higher vegetative growth , suggesting a direct correlation between stomatal density and yield. Furthermore, several reports have highlighted that increased, rather than decreased, stomatal density can be regarded as an evolutionary adaptation to dry environments . Denser stomata tend to be smaller and faster in closing or opening in response to changing environmental conditions, allowing more efficient gas exchanges that result in reduced risks of hydraulic dysfunction. Natural variation in stomatal traits has been explored in P. trichocarpa by GWAS using genotypes collected from the species natural range in the north-western America and Canada region. Genotypes originated from northern latitudes or higher altitude areas were characterized by increased adaxial stomatal density, which may compensate with faster growth the limitations posed by a shorter growing season . GWAS allowed to associate several genes and alleles with stomatal traits, including PtSPCH1, a P. trichocarpa homologue of Arabidopsis SPEECHLESS 1 (SPCH1) which encodes a bHLH transcriptional activator of stomata differentiation . Furthermore, a multitrait GWAS approach integrated with co-expression network analysis led to the identification of potential regulatory networks underlying three key traits related to yield and responses to abiotic stresses: carbon isotope composition (used as an estimate for WUE), stomatal density, and leaf area .
Stomatal closure under water stress conditions is mainly induced by ABA perception in guard cells, which is mediated by the PYR1/PYL (Pyrabactin Resistance 1/PYR1-like)/Regulatory Component of ABA Receptors (RCARs) proteins . Transgenic P. davidiana × P. bolleana poplars overexpressing two ABA receptors (PtPYRL1 and PtPYRL5) from P. trichocarpa displayed increased biomass accumulation and reduced oxidative stress under drought condition, while they did not differ significantly from the wild type under non-limiting water conditions . However, in a field experiment, P. tremula × P. tremuloides transgenic lines overexpressing the RCAR1/PYL9 ABA receptor displayed, surprisingly, increased biomass accumulation under non-limiting water conditions, while drought tolerance was not improved . Furthermore, overexpression of several components of the ABA signalling pathway, including members of type 2C protein phosphatases (PP2C) gene family, genes encoding ABA-Responsive Element Binding (AREB) transcription factors, and genes encoding basic region/leucine zipper motif (bZIP) transcription factors evidenced that the activation of ABA signalling exerts a repressive action on leaf and shoot growth while promoting root growth, which can result in reduced biomass accumulation in conditions of non-limiting water availability . However, the positive role played by ABA signalling was confirmed by P. tomentosa plants overexpressing PtrMYB94, a drought- and ABA-induced transcription factor from P. trichocarpa, which displayed increased ABA accumulation, expression of ABA- and drought-responsive genes, and drought tolerance .
PeSHN1, an APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) homologue of Arabidopsis WAX INDUCER1/SHINE1 (WIN1/SHN1), which is a positive regulator of cuticular waxes biosynthesis , was isolated from of the elite P. × euramericana clone ‘Neva’ . P. alba × P. glandulosa 84K plants overexpressing PeSHN1 displayed increased wax accumulation, decreased transpiration and increased photosynthetic activity and WUE under drought conditions. PeSHN1 overexpressing plants also displayed altered wax composition, with a shift towards longer-chain fatty acids, aldehydes, and alkanes . Subsequent studies pointed out that the molecular composition of cuticular waxes was of pre-eminent importance for the control of cuticular transpiration in poplar species and hybrids .
In poplar root traits are highly plastic; the ability of the root system to access deep or fluctuating water tables is considered as a key adaptive trait of poplar species evolved in arid regions as P. euphratica .
Root growth dynamics under limiting water availability is controlled by ABA and auxin . P. tremula × P. tremuloides transgenic lines overexpressing AREB3, a transcription factor involved in ABA signaling, display increased drought tolerance and biomass re-allocation with increased root/shoot ratio under water limiting conditions, but also severely reduced productivity under well-irrigated conditions . Poplars overexpressing NUCLEAR FACTOR-YB21 (PdNF-YB21), another transcription factor involved in drought responses and ABA signalling, displayed increased root growth and drought tolerance, while knock-out mutants showed an opposite phenotype . PdNF-YB21 was shown to interact with PdFUSCA3 (PdFUS3), which directly activated the transcription of PdNCED3, a gene encoding a rate-limiting enzyme of ABA biosynthesis. The resulting increase in ABA concentration promoted auxin transport to the root tips, which in turn stimulated root growth .
Root functional adaptations to drought include the upregulation of PalERF2, an AP2/ERF transcription factor from P. alba var. pyramidalis that promotes the expression of Pi transporters improving Pi uptake . Furthermore, the basic region/leucine zipper 1-like (PtabZIP1L) transcription factor from P. tremula × P. alba was shown to promote lateral root growth under osmotic and drought stress . Transcriptional network analyses in P. tremula × P. alba identified PtaJAZ3 and PtaRAP2.6, homologs of Arabidopsis JAZ3 (Jasmonate-Zim_Domain protein 3) and RAP2.6 (Related to Apetala 2.6), which are are involved in jasmonate and ABA signalling and promote root elongation and lateral root proliferation during drought stress .
Molecular players controlling root plasticity under limited water availability and adventitious root development are partially overlapping. WUSCHEL-related homeobox (WOX) transcription factors were characterized as regulators of cell division and root formation in several plant species, and as determinants of rooting ability of poplar cuttings . Recently, poplar plants overexpressing PagWOX11/12a have been shown displaying increased root biomass and drought tolerance while opposite phenotypes were displayed by PagWOX11/12a downregulated plants .
Soil microorganisms with different degrees of associations with plant roots can promote growth and drought tolerance by a variety of mechanisms, including: extension of the root absorbing surface, facilitation of ion mobility and uptake, modulation of plant gene expression patterns and hormonal pathways, stimulation of antioxidant capability, modulation of water transport and stomatal movement .
Plants of P. tremula × P. tremuloides inoculated with an ectomycorrhizal fungus and exposed to drought stress displayed increased expression of aquaporin genes which contribute to water uptake and transport . Poplar establishes both ecto- and endomycorrhizal symbiosis, but ectomycorrhizal fungi have been reported to be more effective in increasing root hydraulic conductivity of P. balsamifera plants . The composition of poplar root-associated microbiome is modified by soil water content and by the genotype of the host plant . Root-associated microbiome of P. trichocarpa drought-adapted genotypes displays bigger changes then that of drought-sensitive genotypes, suggesting that root environment of drought-tolerant genotypes exposed to limited water can be enriched for microorganisms playing beneficial roles in stress conditions .
Physiological and molecular responses to drought stress of poplar hybrids propagated by cuttings differ according to the climatic characteristics of the sites where were grown the plants from which the cuttings were collected . Differences in transcriptome responses are paralleled by differences in genome-wide DNA methylation patterns. The global DNA methylation rate in the shoot apex of P. deltoides × P. nigra hybrids is differentially affected by water stress and correlates with yield under both liming- and nonlimiting-water conditions . Genes involved in ethylene, jasmonate, and SA pathways are differentially methylated and expressed in shoot apical meristems of poplar plants during drought stress and recovery . Repression of jasmonate- and SA-responsive genes after rewatering was associated with gene body hypomethylation . A decrease in global DNA methylation rate was also observed in the shoot apex of field grown P. nigra plants originated from different natural populations in response to drought . Gene body methylation in P. trichocarpa was reported to both promote and repress transcription .
ABA signalling genes encoding PP2C phosphatases, HOMEOBOX7 (HB7)-related proteins and LEAs, show expression patterns shaped by stress memory in different tissues and organs of P. × canescens plants exposed to cycles of water stress and recovery , suggesting that also ABA signalling may be regulated epigenetically.
Recently, it has been reported that in drought conditions the growth rate of P. tremula × P. alba plants downregulated for DECREASED IN DNA METHYLATION 1 (DDM1) is substantially unaffected while the height and diameter growth rates of WT plants is severely reduced . This drought-tolerant phenotype is associated with increased accumulation of SA and reduced accumulation of cytokinins in the shoot apex.
During drought and/or high evaporative demand, the water column within the lumen of tree xylem vessels can be subjected to high tension, resulting in embolism formation  after entry and expansion of air bubbles in xylem . Embolism decreases water transport across the stem, inducing plant desiccation, tissue damage, and decline of plant productivity . Massive embolized vessels are the main drivers of tree mortality . Embolism formation depends on several factors: tension of the water column, water chemical properties, surface tension in the xylem sap, and wood anatomical and physicochemical properties such as diameter, length and connectivity of conduits, density, and pit characteristics , as well as to history of previous embolism activity .
The vulnerability to embolism is species-specific and tissue-specific, and is generally quantified by P50, i.e., the value of water potential at which 50% of hydraulic conductivity is lost : the more negative the P50 value, the higher is tree tolerance to water stress. Poplars are among temperate woody plants most vulnerable to drought-induced embolism, having an average P50 close to 1.44 MPa , although with remarkable differences between species: P. tremuloides is relatively resistant (P50 = 2.13 MPa), whereas P. euphratica is the most vulnerable (P50 = 0.70 MPa) . Different environmental conditions and physiological unbalances affect hydraulic functionality in poplar: prolonged shading, for instance, increases xylem vulnerability to embolism in P. nigra, possibly due to lower sugar accumulated in stem tissues ; in the same species, sugars derived from photosynthesis in woody tissue reduce xylem vulnerability to drought .
Poplars are often able to regain or mitigate the loss of transport capacity in xylem, even in presence of diffuse leaf desiccation . Embolism repair involves vessel-associated cells (VACs) that supply water and energy, in connection with an accumulation of fructose and glucose in vessel apoplast . The solutes accumulated in apoplast generate an osmotic gradient that triggers the entry of water into the embolized conduits, allowing for recovery. In addition, water entry is facilitated by aquaporins, plasma-membrane water channels upregulated in response to embolism .
Drought induces a general reorganization of xylem architecture regulated, in poplars, mainly by ABA, the most abundant hormone in xylem sap . ABA accumulation leads to increased vessel number and decreased vessel cross-sectional area, which affects both water conduction and embolism vulnerability .
The effects of drought-induced alterations on wood quality, however, are still poorly understood. Fiber length can be influenced by drought as well, but to vary degrees depending on the time of growing season when the stress occurs: i.e., P. nigra × P. maximowiczii plants exposed to drought in early summer displayed a significant reduction of fiber length, while with drought occurring in late summer such effect was minimized .
Except for foliar parasites (i.e., the Marssonina leaf spot agent), many other poplar pathogens, inhabiting bark or root tissues, generally are enhanced by water stress. Over a given threshold, these weak pathogens are able to pass from a latency phase to an actively pathogenic phase, in a perspective in which pathogenicity is a consequence of fine-tuned interactions between host, environment, and microbiome, i.e., microbes priming host defences or debilitating host physiology . Some fungal parasites induce symptoms in absence of stress, although less aggressively than in water-stress conditions, while others require a stressed host to start the disease, such as Cytospora chrysosperma (Pers.) Fr. on quaking aspen  and other poplar species.
A water-stressed tree results disadvantaged versus a weak pathogen because of several factors : increased bark glucose content like in Armillaria root rot ; increased bark amino-acid content like in Hypoxylon canker of aspen ; inhibited synthesis of antifungal compounds and phytoalexins, once again in Hypoxylon canker ; xylem embolisms associated with low water potential that can provide preferential routes for the spread of internal pathogens .
As regards water stress versus insect pests, different feeding guilds (suckers, borers, miners, etc.) may show different responses to a water-stressed host. Generally speaking, however, a moderate drought induces an increase of defence compounds whereas a strong drought ends up inhibiting their metabolism so that, in this last case, polyphagous insects may be favoured . In addition, water-stressed trees may synthetize chemicals – such as ethanol, monoterpenes, and others – attractive for infestations . Lastly, high temperatures, often simultaneous with drought occurrence, may enhance insect voltinism independently from the altered conditions of target plants.
This is not always true for host-specific pests: for example, Chrysomela populi L., the poplar leaf chewer, showed decreased survival and feeding after induced water stress in hybrid poplars , and the woolly poplar aphid, Phloeomyzus passerinii (Signoret), is reported as being impaired by water stress: its optimal development occurs at moderate temperature (20–25 ◦C) and air humidity of more than 70%, under the shady microclimate provided by trees near maturity . However, according to the so called “plant vigour hypothesis” formulated in connection with sap-feeding insects (including aphids) , the reduced vigour of trees exposed to drought is per se enough to explain reduced fitness of these insects and, consequently, abundance. In conclusion, the effect of a drought episode on the occurrence of certain insect pests on poplars must be evaluated on case-to-case basis considering all interacting factors.
The phenotypic traits related to drought tolerance include rooting power, growth habit, resistance to biotic and abiotic stresses, growth rate, and wood physical characteristics such as basal density, colour, and hue.
In poplar breeding programmes, field measurements are beginning to be integrated with standardized high-throughput field phenotyping (HTFP) technologies. Unmanned Aerial Vehicles (UAVs), equipped with cameras and remote sensors, allow the detection of high-resolution spectral responses: thermal infrared-equipped UAVs have been used to assess drought response of P. nigra populations taking advantage from the strict correlation between leaf temperature and stomatal conductance .
Spectral images from satellites are very useful for rapid evaluations of large areas, allowing analyses of typology of vegetation cover and tree health conditions by direct and indirect measures of temperature, evapotranspiration, and other factors. However, integration with proximal sensing data is still necessary to provide adequate resolution and the flexibility to collect accurate phenotypic data at multiple timepoints. Since ground-level surveys are time-consuming, require numerous personnel and are not always possible, UAVs equipped with cameras and sensors can be used to bridge the gap between time-consuming ground-based measurements and satellite/airborne observations .
Research on drought responses in poplar has provided thus far a remarkable amount of published data. These data demonstrate that the basic response pathways elucidated in herbaceous model plants, such as Arabidopsis, are largely conserved in poplar. In addition, however, research efforts have shed light on processes and mechanisms that are specific for woody plants. Transcriptomics applied to drought-adapted poplar species as P. euphratica and P simonii has disclosed finely regulated responses to different levels of drought stress and to combined drought and heat stress. Genetics and genomics studies have led to the identification of differentially expressed genes associated to drought. In the stomata differentiation pathway, PtSPCH1 was identified by GWAS as a key factor correlated with the distribution range of P. trichocarpa, while allelic variation of PtSPCH1 was correlated with adaptation to different environmental conditions. Studies on the role of ABA signalling in biomass trade-off and resource re-allocation between roots and canopy enriched the knowledge of long-term responses to drought. Furthermore, long-term responses have been linked with phenotypic plasticity, molecular memory and epigenetic modifications, which are emerging processes in the stress biology of trees. Finally, the intensive research on the physiological and molecular mechanisms involved in xylem embolism has allowed to draw models for embolism repair. Future studies may be aimed to assess to what extent these mechanisms are conserved among forest trees and to evaluate their roles in shaping forest dynamics under changing environmental conditions. Results from poplar research also provide a guide for breeding programs aimed to obtain drought-resilient clones with high productivity.