Genes up-regulated in water deficit-treated apple roots vs other plants responding to water deficit1
Water availability is the single most important factor determining plant survival. Many environments experience water-limited periods of various degrees and duration. The issue of water availability for agricultural crops where maintenance of high yields over variable growing seasons is desirable is particular critical. For plants, the strategy choices for survival can be summarized as dehydration avoidance (e.g., deep rooting), dehydration tolerance (e.g., accumulation of osmoprotectants) and drought escape (e.g., reproductive completion before the dry season). Drought adaptation in most plants is controlled by complicated interactions between anatomy, physiology and biochemistry, all of which are directly or indirectly under genetic control [1-3].
2. Water use efficiency and drought resistance
All living organisms have evolved mechanisms for adapting to changes in their environment, whether biotic such as pest related, or abiotic such as physically effected. For plants this is especially challenging, since they are unable to relocate to avoid adverse conditions. As a result, numerous strategies employed by plants leading to successful acclimation have been identified. Broadly speaking, these adaptive mechanisms can be divided into two major categories, namely morphological modifications and physiological adjustments. Through combinations of these basic strategies, plants can respond quickly to environmental cues, often maintaining the response for relatively long periods [4,5].
The term drought resistance is sometimes confused with water use efficiency (WUE). Drought resistance is determined primarily by ‘drought avoidance’ (high plant water status maintained under water deficit) and/or ‘drought tolerance’ (capacity to sustain plant function in a dehydrated state) [6,7]. Drought resistance in a genetic/physiological context refers to the ability of one genotype to yield ‘better’ than another during severe drought stress. On the other hand WUE is defined as the ratio between diffusion of CO2 into the leaf (photosynthesis) and loss of H2O through transpiration, indicated as WUE = A/E, where A is carbon assimilation and E is transpiration. It is positively correlated with carbon isotope discrimination (Δ13C) based on the stable carbon isotope ratio, δ13C (12C/13C relative to a standard, i.e. PeeDee Belemnite), in the plant tissue relative to the atmospheric ratio and is calculated as: Δ13C = δ13C in air - δ13C of the plant/1- δ13C of the plant. Since most gas exchange occurs via the stomata, it is expected that guard cell function would be closely associated with WUE. Indeed, the size and density of stomates correlates well with water use efficiency [8-10]. For drought resistance, yield is not necessarily adversely affected by resistance, whereas for WUE reduced transpiration through stomatal closure is often accompanied by reduced yield potential through reduced carbon assimilation, particularly in herbaceous C3 plants (however, see below). Other parameters, such as root depth, leaf size, and trichome size and density have also been linked to water use efficiency , but they have also been linked to drought resistance as well .
Different methods have been used to measure drought resistance and WUE . These methods measure the energy status of water in plant tissues and the trans-port processes into and out of the soil-plant-atmosphere continuum. In general, these methods isolate specific plant tissues at instantaneous moments in time, whereas Δ 13C represents a time-integrated value of the ratio of Ci (intercellular CO2 concentration) to ambient CO2 which, as previously indicated, reflects the plant’s capacity for gas exchange via stomata  and discrimination of rubisco against 13C. The use of carbon isotope discrimination to select in-dividuals with higher WUE has been applied successfully to cereal breeding programs (13). The extent of δ 13C varies substantially among wheat genotypes, and heritability is high because genotype X environment interactions are relatively low [14, 15]. Rebetzke et al.  reported on the selection of plants with greater biomass, harvest index and kernel weight using results from contrasting high and low Δ13C groups in combination with a backcrossing program. There were significant correlations between Δ13C and yield and between Δ13C and biomass. The resulting high yielding strain, ‘Drysdale’, produces around 10% more grain under drought conditions than other dry-area wheat varieties.
3. Adaptation and the relationship of δ13c to yield
Adaptive changes in populations growing in different environments have been amply demonstrated in a variety of plants . Divergence among populations associated with different environments provides the raw material for speciation and differentiation among closely related species. Higher fitness of genotypes in their native environment compared to genotypes transplanted from contrasting environments provides evidence of local adaptation . For example, when two populations of
WUE in trees adapted to different environments has been documented in several forest species [21-24]. For example, a study of four birch (
Poplar species are differentially adapted to a variety of environments, and because poplar is a rapidly growing tree with heavy water use, there is growing interest in developing lines that are drought resistant. Links between productivity and Δ13C varied in a study comparing different poplar genotypes, suggesting that genotypes displaying simultaneous high productivity and improved water use efficiency could be selected . To obtain more practical information regarding productivity and WUE, a field study was conducted on the same genotypes analyzed in the previous study. Significant clonal diversity was observed for several traits related to productivity and for Δ which showed high heritability (H2 = 0.71) . A lack of correlation between above ground biomass and Δ was reflected in several clones where high productivity was combined with improved WUE. This observation supports previous studies with cereals indicating that WUE and yield can be inherited as separate traits.
Yield of deciduous tree fruit crops is not measured as total biomass yield in commercial production as are agronomic crops such as corn, wheat and rice or forest trees. In commercial orchards it is common practice to reduce yield potential of fruit trees by as much as 50% to insure large fruit size and high fruit quality . Consequently, the paradigm that increased WUE is tied to reduced yield potential is not necessarily valid for tree fruit production. For example, Glenn et al.  have demonstrated the practicality of identifying peach cultivars with high WUE without compromising productivity. This study, taken together with those cited previously, demonstrates the feasibility of selecting for improved WUE without loss in productivity.
4. Adaptation, WUE and δ 18O
Transpiration rate (E) affects water loss to the atmosphere and is negatively correlated with WUE. Despite the fact that atmospheric 18O is low (ca. 0.2% of total oxygen), plants tend to accumulate 18O and 2H in leaf water due to the difference in vapor pressure between heavy water and ‘normal’ water and to differences in diffusivity with air. However, the relationship between E and isotopic enrichment is complex. For example, variation in E can be caused by changes in evaporative demand and/or changes in stomatal conductance, gs . If the source of variation is evaporative demand, then as E increases, 18O enrichment increases. On the other hand, if stomates are the source of variation, then as E decreases (stomates close), leaf water enrichment increases. How does this relate to 18O enrichment of plant organic matter? Plants accumulate 18O in their tissues as a result of the exchange of oxygen between water and carbonyl oxygens in triose phosphates. An enrichment of about 27 parts per thousand (ppt) has been observed in the organic material of several different plants relative to leaf water . This suggests that differences in 18O enrichment can be used to distinguish genotypes with favorable yields and stomatal function. In fact Barbour et al.  recently demonstrated a reliably negative correlation between yield and δ18O in wheat which was used to identify water use efficient varieties for breeding.
5. Specific genes associated with WUE and/or drought responses in apple and other plants
The recent advent of global gene expression methodology has spawned a number of studies of abiotic stress responses, including drought, in several plant species [34-38]. In Arabidopsis, a compilation study of microarray analyses on plants subjected to a variety of stress treatments highlighted overlap among genes up-regulated in the early stages of all the stress responses . Studies on WD stress in cereals and dicots have cataloged a large number of genes up-regulated during treatment [35-37, 40, 43]. Comparisons among these studies reveal that a number of genes are reproducibly up-regulated in response to WD regardless of how the stress was imposed or what plant system was involved, including apple (Table 1) .
leaves and roots
leaves and roots
leaves and roots
6. Genes associated with wue
Recent reports of genes associated with regulation of transpiration demonstrate the complexity of water use and transport, as well as the overlap in gene response to other stresses.
Using a suppression subtractive hybridization (SSH) approach to drought-responsive gene isolation in a commercial apple, ‘Royal Gala’, we identified numerous genes commonly found to be WD responsive in other plants. We also identified several up-regulated genes unique to apple roots (Table 2; manuscript submitted). Some of these genes may reflect the role of roots in nutrient transport during stress, including a copper chaperone and a high affinity nitrate transporter that is a putative Arabidopsis NRT2.4 homolog.
|BYPASS1||Anthocyanin reductase||Auxin/Aluminum-induced protein|
|Serine acetyltransferase||GAST1-like gene||Proteasome maturation factor|
|High affinity nitrate transporter||Asparagine synthetase||Asparagine synthetase|
|NPR1||Chloroplast membrane protein Tic40||Glyoxylate aminotransferase|
7. Genes associated with drought avoidance and escape
An example of the rapid evolution of a drought escape mechanism (early flowering) was demonstrated in a population of
In alfalfa a gene encoding a zinc-finger motif is expressed in roots [50, 51]. The protein encoded by
8. Genes associated with drought tolerance and resistance
Studies of specific genes associated with dehydration responses have been conducted in a number of plants, and roles for many of these genes have been correlated with specific morphological or physiological traits known to be involved in drought resistance. Abscisic acid (ABA) signaling and stomatal function are correlated with WUE and drought resistance, so it is not surprising that several genes involved in ABA perception and stomata opening/closing respond to severe dehydration. Two calcium-dependent protein kinases from Arabidopsis have been implicated in slow-type anion channel activation . In the double mutants, ABA and Ca2+-induced stomatal closing were impaired, but not completely. These genes may contribute to a rapid Ca2+-reactive response resulting in stomatal closure, as opposed to the slower Ca2+-programmed response which maintains long-term stomatal closure. Similar studies have also implicated a G protein-coupled receptor, GCR1, in ABA signaling perception in guard cells and during seed germination . To examine stomatal function in more detail, Klein et al.  used a T-DNA insertion disrupting At
The cuticle is an important barrier to moisture loss in plants, therefore genes associated with cuticle synthesis and turnover are expected to contribute to plant water status. A recent study in alfalfa demonstrated that increased wax production activated by a putative TF (transcription factor) [
Constitutive expression of a barley Group III LEA (late embryogenesis abundant) protein placed in wheat under control of the maize
9. Regulation of pathways/signaling networks associated with dehydration
Most studies of dehydration responsive signaling pathways implicate ABA directly in altering specific gene expression . In fact genes that respond to ABA usually have multiple copies of an ABA response element (ABRE) or a combination of an ABRE with other motifs such as Myb, Myc or coupling elements [for example, 64]. A second pathway involves drought response element binding (DREB) proteins, particularly
Compelling evidence indicates that the ABA pathway likely involves Ca2+ signal transduction as an early step and important relay system for dehydration responses. ABA can also increase reactive oxygen species through higher levels of H2O2 [67, reviewed in 68]. Other studies have suggested stress-responsive pathways that operate through osmotic sensing independently of ABA . An osmotic sensor similar to bacterial two-component receptors has been identified in Arabidopsis . The gene was able to complement several mutations in yeast osmosensors and activated the
Numerous studies support the existence of extensive cross-talk between plant hormone signaling pathways [71-74]. It is therefore expected that both the salicylic acid and Jasmonic acid (JA)/ethylene pathways indirectly influence the expression of genes that respond to drought. Along these lines an Arabidopsis mutant (
10. Studies in
Genetic polymorphisms from twenty populations of
Evaluation of six
A study of the contribution of rootstock source to drought resistance was conducted using ‘Gale Gala’ apple scions grafted onto
Several studies of
|GMAL3975.k||6||250 mm||-27.09 ppt||R||R||dry||0|
|GMAL3685.e||6||250 mm||-29.30 ppt||R||S||dry||10%|
|GMAL3623.f||9||450 mm||-26.33 ppt||S||S||moderate||1%|
|GMAL4455||4||800 mm||-26.30 ppt||R||R||medium||20%|
To begin analyzing
Total RNA was isolated, cDNA prepared and SSH performed using the protocol reported by Bassett et al.  for peach. For gene analysis, we designed primers for several genes shown to be associated with dehydration responsiveness in apple [44; manuscript submitted]. Each primer pair was quality tested and used to prime RT-qPCR reactions in order to quantitate gene expression in different tissues. The qPCR reactions were conducted using a kit containing all reagents (Life Technologies, Applied Biosystems, Grand Island, NY) and the reaction parameters were as follows: 95°C 5 min, followed by 35 cycles of 95°C 1 min, 60-65°C 1 min, 72°C 1 min and a final extension of 72°C for 10 min. Primers for a translation elongation factor (TEF2) was used as an internal control for the qPCR experiments . The relative standard curve method was used to analyze the data.
10.1. Status and results
Most of the genes examined regardless of tissue origin showed around a two-fold difference between watered and water-deficit treaments. A few genes were substanitally up-regulated in response to drought. including the auxin-induced gene from leaves (8-fold increase) and asparagine synthase from bark (4-fold increase). A few genes in roots were also significantly up-regulated in response to drought treatment, one of which was NPR1 (3-fold increase; manuscript submitted). From this information, including the expression of genes not shown here, we have generated a list of potential up-regulated genes responding to a relatively long term drought that can be used to determine if there is any correlation of expression in
10.2. Promoter comparison of NRT2.4 from apple, peach and Arabidopsis
A high affinity nitrate transporter gene from the ‘Royal Gala’ root SSH library (see Table 2) was shown by RT-qPCR to be elevated in roots and bark in response to drought treatment (manuscript submitted). Approximately 700 bp upstream of the ATG start codon was obtained from the genomic sequence of ‘Golden Delicious’ (Genome Database for Rosaceae; http://www.rosaceae.org/). Several
Leaf size and number have been shown to respond negatively to drought, resulting in longer intervals between newly initiated leaves and smaller sizes, all features designed to reduce transpiration to conserve water. Leaf morphological features and stomatal characteristics were examined in the site 6 subpopulation . GMAL3683.o had the smallest leaves by all traits measured, whereas GMAL3687.d and GMAL3989.f had the largest leaves by area. Within the GMAL3683 sibling group, GMAL3683.o leaf area (8.3 cm2) clearly segregated from the other three members (average = 20.3 cm2).
Stomatal density has also been linked to drought tolerance and sensitivity. Therefore, we also examined stomata size and density in the site 6
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