However, it is unclear to what degree trees truly avoid drought stress relative to plants that go dormant. ‘Drought resistance’ is the ability to withstand drought exposure, whereas ‘drought resilience’ is a measure of how quickly a tree can resume normal growth when conditions improve .Conifers manage tissue water potential in two main ways: isohydric trees close stomata to maintain water potential, whereas anisohydric species allow water potential to drop . Isohydric trees use increasing abscisic acid concentrations as a signal to keep stomata closed, whereas anisohydric trees use low leaf water potential itself as a signal to close stomata . Anisohydric conifers include many Cupressaceae and some Taxaceae . Xylem architecture affects how changes in stomatal conductivity influence cavitation risk, and anisohydric trees tend to have xylem that is more cavitation resistant . Wider tracheids increase conductivity and the risk of hydraulic failure , whereas those with smaller inter-tracheid pits or more lignified walls are less vulnerable . The reduction of leaf area with branch die-back, reduced needle number or smaller needles can also reduce water loss. Anisohydric species often exhibit branch die-back during drought, whereas isohydric trees typically retain a full canopy until death . Some conifer species can refill xylem following cavitation. This is thought to be an energy-intensive process that depends on carbon reserves . This may explain why droughtstressed trees can exhibit lower refilling capability . Picea abies refills freezing-cavitated xylem before soils have thawed by taking up water through its needles . This could explain why other conifers can refill xylem in the absence of positive root pressures, unlike co-occurring angiosperms . However, refilled xylem may be less resistant to future drought stress, a characteristic known as ‘cavitation fatigue’ .Loss of water potential in cells is associated with cell turgor loss, denaturation of proteins and changes in membrane fluidity. To avoid cellular damage, plants synthesize molecules that act as osmotic balancing agents. These reduce cellular solute potential,gutter berries and may increase turgor at lower water potentials. In addition, hydrophilic compounds can prevent the membranes from leaking .
Other compounds stabilize proteins or detoxify reactive oxygen species. These protective molecules include proteins such as chaperonins and dehydrins , the amino acid proline and various carbohydrates .We hypothesize that protective molecules may be produced earlier during a drought in anisohydric species because leaf water potential drops more quickly .As a result of reduced stomatal conductance , oxidative damage and other factors, photosynthetic rates and chlorophyll concentrations often decline during drought . Therefore, in addition to protecting cells from damage, increased allocation to nonstructural carbohydrates may help to avoid carbon starvation by keeping energy resources in easily mobilizable forms. The patterns of change in overall NSCs and starches seem to differ between species and drought length and severity . Clear evidence of death as a result of carbon depletion is still lacking . However, carbon storage and allocation patterns do vary under drought stress , demonstrating implications of water limitation on carbon availability.The number of branches and leaves affects total transpiration. Rooting depth affects access to deep soil water and is probably crucial for seedlings as well as adult trees in areas with seasonal drought . Deep roots may also redistribute water from deep to shallow soils . More small diameter roots, with high surface area : volume and a lower vulnerability to cavitation, may aid drought resistance . Structural changes can have long-lasting effects. Decreasing soil moisture can induce greater root production, but extended drought reduces root mass , which limits responsiveness to precipitation pulses . Lumen width and cell wall thickness of tracheids are plastic, with those produced in moist seasons and years generally being wider, more numerous and thinner walled than those produced in dry periods . Xylem is often functional for multiple years , and so current drought responses can affect water transport during future drought. The production of protective molecules typically drops soon after normal water potential is restored . However, transcriptional and physiological ‘memory’ in stomatal guard cells has been observed, with stressed plants maintaining smaller stomatal apertures when re-watered . There may also be ‘legacy effects’ on NSC production and traits such as growth and xylem anatomy . Plants that quickly return to normal could gain a growth advantage. In areas in which recurring drought is common, however, we hypothesize that this memory effect reduces mortality risk. There are multiple traits involved at different stages of the drought response .
Stomatal control and patterns of root and shoot growth affect the degree to which a plant avoids drought stress. These traits plus xylem morphology, protective molecule production, changes in carbohydrate metabolism and pathogen defenses influence drought resistance. Finally, the recovery rate of photosynthesis and other processes, the degree of persistent changes in structure and the ability to refill xylem affect drought resilience. In the next two sections, we first review the methods used to date to examine genetic controls on ecologically important traits, and then explore how these methods have been and can be leveraged to test for genetic variation in, and identify the genetic basis of, the traits and processes addressed above.Gene expression or transcriptome studies examine changes in the amount of RNA transcripts to identify genes that are upregulated or down regulated under different conditions. Changes in the amount of a gene product can result in different phenotypic responses, even if all individuals have the same gene sequence. Such changes are responsible for plasticity, and may involve temporary or heritable epigenetic modifications . Gene expression studies may involve a variety of techniques, but most recent studies have used microarray chips – DNA probes to which cDNA or RNA hybridize, resulting in fluorescence – or cDNA sequencing . The latter avoids the need for probe and microarray design and can survey whole novel transcriptomes . Real-time quantitative polymerase chain reaction is highly sensitive, but is most often used to target specific candidate genes or to confirm a subset of expression changes . All techniques are sensitive to which tissues are sampled at what time . Moreover, unless expression responses in different genotypes or populations are explicitly compared, this approach does not address local adaptation.Provenance or common garden studies, where seedlings from many different sources are planted in a common environment, began to reveal heritable differences between tree populations long before the availability of genetic marker data . Provenance studies established in the mid-20th century to identify seed zones for replanting or highly productive genotypes have been re-purposed to investigate potential responses to climate change .
Many recent studies have also used seedling common gardens . Studies conducted across multiple sites, or incorporating multiple treatments, can estimate the plasticity of traits, allowing the fitting of transfer functions that predict performance based on source and planting environments . However, such studies do not reveal which genes are responsible for observed differences unless paired with other techniques. It should be noted that there is usually substantial variation within tree populations . The third set of approaches can be used to investigate the causes of heritable variation between populations and individuals.These approaches aim to identify genes or genomic regions related to a trait or to adaptation along environmental gradients. QTLstudies are a classic way to identify the loci involved in continuous trait variation. However, although QTLs for a number of traits have been identified in trees, this approach has had limited success for a variety of reasons, many of which are reviewed in GonzalezMartınezet al.. For instance, a great deal of time and space is needed to cross parental tree lines and raise a sufficient sample size of progeny. Conifers also have very large genomes with low linkage disequilibrium and, without enough genetic markers available,strawberry gutter system most QTLs are undetectable . In addition, high-resolution genetic/physical maps or positional cloning is needed to identify causal genes/mutations . By contrast, genome scan and association studies make use of large numbers of newly available markers , and are carried out in highly diverse out crossing natural populations . Genome scans identify loci that differ more or less between populations than expected by chance . For instance, outlier Fst values can be used to infer the type of selection: balancing selection results in low Fst and shared alleles, and divergent selection in high Fst with segregated alleles. Genome scans can also identify patterns suggestive of a selective sweep. These studies do not automatically provide information about which, if any, environmental variables are responsible for the pattern. One can test whether patterns of differentiation match an environmental gradient, but this is necessarily a post-hoc interpretation . Association studies use a regression approach to identify loci in which genetic variation is associated with variation in trait values or home environment. Such analyses can be carried out at the individual or population level. Genotype-to-environment association studies identify loci that vary along environmental gradients . An association between an SNP and aridity, for example, suggests that the gene or its regulatory region affects performance in wet vs dry environments. This does not reveal how the locus affects phenotype, and careful interpretation is needed as a result of correlation between climatic variables. Genotype-to-phenotype association studies identify loci correlated with a particular phenotype , but the phenotype may or may not be relevant for fitness in the field. Most association studies in conifers to date have used SNPs in a limited number of candidate genes . This ensures that genes suspected of involvement are surveyed, but limits the ability to identify additional loci. However, with the decreasing cost of sequencing, approaches that generate large numbers of SNPs are increasingly being used for genome-wide association studies . One set of approaches, including RAD-seq and genotyping-by-sequencing , involves the use of restriction enzymes to cut and sequence a small subset of the genome .
This can produce tens of thousands of SNPs with high coverage . Many of these SNPs will be in noncoding regions, which is good for the potential discovery of regulatory regions, but can limit the number of gene associations detected. Another approach involves the creation of a transcriptome or full genome sequence for a species, and the development of probes for all or most of the putative genes to identify SNPs . This approach can also yield useful gene expression data if multiple tissue types or treatments are included in the development of the transcriptome .Most drought gene expression studies in conifers have focused on pine seedlings, with a few investigating other Pinaceae genera . The direction of expression responses to the environment, including dry conditions, is highly conserved between Pinus contorta and Picea glauca 9 engelmannii, even though average expression levels often differ . It is unclear whether this is true across conifer families. No expression studies have focused on adult drought responses. The methods used to induce drought stress vary. Studies have withheld water for a specified period , until soil moisture reached a threshold or needles wilted , or needle water content declined to a certain level . Some have used chemically induced water stress . Caution must therefore be used in interpreting differences across studies, as these could be methodological artifacts . Genes related to signaling and gene transcription are frequently upregulated in drought-stressed seedlings. Changes in signal cascades must precede changes in their targets, and such expression shifts often occur within the first week of drought stress. Those in the ABA pathway are well represented . In addition to being involved in stomatal closure, ABA signaling can affect shoot growth and water uptake . However, there are also ABA-independent pathways in most taxa, which may use leaf water potential as a signal . Upregulation of genes in the ethylene pathway could be related to reduced shoot growth or leaf area . Genes related to protective molecules are also frequently upregulated . Late-embryogenesis-abundant proteins, named for their role in seeds, appear to stabilize proteins and membranes and prevent protein aggregation . Dehydrins, a subgroup of LEAs, often protect against drought stress, although some are induced by other abiotic stresses . Heat shock proteins, detoxifification enzymes and genes in the synthesis and transport pathways of osmoprotective carbohydrates and proline may also be upregulated. Genes involved in pathogen or biotic stress defenses are often upregulated during drought stress, but those involved in growth, including cell division and wall construction, are often downregulated .