Intraspecific variation in gene expression under prolonged drought in Piriqueta hybrids and their parental taxa

Plant Science 178 (2010) 429–439

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Intraspecific variation in gene expression under prolonged drought in Piriqueta hybrids and their parental taxa Heather E. Machado ∗ , Mitchell B. Cruzan Portland State University, Department of Biology, PO Box 751, Portland, OR 97207, United States

a r t i c l e

i n f o

Article history: Received 30 September 2009 Received in revised form 10 February 2010 Accepted 15 February 2010 Available online 20 February 2010 Keywords: Drought Heterologous hybridization Hybrid Transgressive Gene expression

a b s t r a c t Closely related lineages can possess phenotypic variation important for adaptation and the evolution of new species—a phenomenon that can be more clearly elucidated by studying hybrid generations. We compare variation in gene expression in response to drought for two taxa and advanced-generation hybrids of the Piriqueta cistoides ssp. caroliniana complex that differ in their levels of tolerance to water limitation. Drought treatments lasted 36 days, through four cycles of drought. Gene expression in drought and control treatments was assessed using heterologous hybridization to a Glycine max microarray. There was a predominance of down-regulated genes in response to sustained drought in all morphotypes. This pattern was more pronounced in the hybrids, which can exhibit greater drought tolerance under field and greenhouse conditions than the parental morphotypes. Expression response profiles were more similar between the hybrid and the drought-tolerant parental morphotype than they were between the hybrid and the parental morphotype that occurs in more mesic habitats. Predominant down-regulation of gene expression contrasts with studies of response to short-term drought and with studies of drought response in annual and non-drought-tolerant model species, and supports the results of a growing number of studies with other drought-tolerant perennial plants under prolonged drought. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Environmental stress limits the survival and reproduction of organisms and shapes the course of their evolution [1–3]. For plants, the most common and profound of such stresses is water limitation [4–6], eliciting systemic cellular responses, phenotypic changes, and eventual changes in the genotypic composition of populations. Cellular response to drought includes the initial perception of stress, signal transduction, change in transcription factors, and change in gene products [7]. Modification of gene expression may initiate a number of phenotypic responses including increased growth of leaf trichomes [8], change in leaf size and shape [9,10] and reduced stomatal movements [11]. Morphological modifications can contribute to major physiological changes such as increased water-use efficiency, a reduction in photosynthesis, and reduced overall growth rate [10,12,13]. The systemic nature of drought response in plants makes broad assays of gene expression with cDNA microarrays particularly appropriate for the study of waterstress. Gene expression studies have identified several genes that are either induced by the onset of drought or repressed in response to drought stress, including those for detoxifying enzymes, antioxi-

∗ Corresponding author. Tel.: +1 503 725 3888. E-mail address: [email protected] (H.E. Machado). 0168-9452/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2010.02.009

dants, heat-shock proteins, and osmoprotectants [14,15]. Changes in gene expression can be assessed by estimating mRNA transcript abundance using a cDNA microarray. While this is not a measure of end gene product and corresponding physiological change, consistency in transcript level has been routinely validated for candidate genes by quantitative PCR, and gene expression profiling has been shown to be a particularly sensitive indicator of stress (but for limitations see [16]). Much of our understanding of gene expression in response to drought stress is based on studies that have utilized model organisms or agricultural species that are not adapted to water-stress. The majority of these studies investigated annual species such as rice [17], wheat [18,19], barley [71], maize [20], and Arabidopsis thaliana [21–23] that do not naturally experience or survive long periods of drought. Also common is the analysis of tissues subjected to water-stress for short periods of time, from an hour to a few days [17,19,22–25]. While informative, such research has limited relevancy to responses of plants that routinely experience prolonged periods of drought. To understand plant response to arid environments, more studies of stress-tolerant plants exposed to sustained drought are needed. The cost of acquiring genomic tools for many species has contributed to the relative paucity of microarray studies on differentially adapted organisms. While microarray construction has taken place for some drought-tolerant species such as jojoba [25], sunflower [26], Phaseolus [27], and horsegram [28], an

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alternative is the cross-species use of existing arrays produced from model organisms for a target species of interest. These experiments, termed heterologous hybridization, have the added challenge of sequence divergence between the platform species and the focal species. Renn et al. tested seven species of varying phylogenetic distance to the platform species, the cichlid fish Astatotilapia burtoni, and found a decrease in ability to detect target cDNA and changes in gene expression with increasing phylogenetic distances [29]. However, even at the highest level of divergence (Danio rerio, ∼200 MY diverged) more than half of the cDNA targets had sufficient hybridization to array, still providing information on the expression of thousands of genes. With heterologous hybridization in general, less than the total number of features on the array will likely be utilizable, and the quantity of genes found to be regulated will be an underestimate. The utility of cross-species microarray experiments has been demonstrated in several other previous studies, they have become a standard use of cDNA microarrays [30–32], and have been conducted for drought-tolerant species such as alfalfa [24]. In the present study we use a soybean (Glycine max) microarray to look at gene expression patterns under sustained drought in the Piriqueta cistoides L. ssp. caroliniana (Walter) Arbo (Piriqueta caroliniana (Walter) Urban) hybrid complex (Turneraceae family). At the time of this study, Glycine max (98 million years diverged from Piriqueta [33]) was the closest relative for which a microarray was commercially available. Piriqueta cistoides caroliniana is an ideal system for the study of drought response because morphotypes within this taxon display contrasting habitat associations, from arid to periodically flooded environments [34,35]. Using closely related taxa that are found in environments with contrasting levels of water availability allows for the characterization of gene expression profiles associated with plants adapted to different degrees of drought.

1.1. The Piriqueta c. caroliniana complex Piriqueta c. caroliniana can be found from Argentina to the southeastern tip of the United States in areas of seasonal aridity [36]. Plants in this complex are herbaceous perennials with distylous, orange-yellow flowers [37]. In the North American range, P. c. caroliniana extends from Florida to southern Georgia where two ecologically and evolutionarily distinct morphotypes, caroliniana (C) and viridis (V), are found (Fig. 1) [34,38]. These morphotypes represent recently diverged lineages that produce high-fitness F1 hybrids and recombinant generations with relatively minor hybrid breakdown [39,40]. Chloroplast DNA haplotype data suggest that caroliniana immigrated to North America from the Bahamas in the early Pleistocene [34]. Contemporary populations of the C morphotype retain an association with the arid longleaf pine habitats (Fig. 1) and well drained quartz sand soils of Central Florida to Southern Georgia, which are dominated by turkey oak scrub (Quercus laevis) and prickly pear cactus (Opuntia sp.) [41]. The viridis morphotype likely arrived in the poorly drained limestone sands of Southern Florida within the last 5000–7000 years [34]. Populations of the V morphotype occur in habitats characterized by slash pine (Pinus elliottii) and palmetto flatwoods (Fig. 1), which are subject to periods of flooding and hypoxic soils [41]. Intraspecific hybridization has occurred between the C and V morphotypes and has produced a broad hybrid zone in central Florida. These advanced-generation diploid hybrids (hereafter, referred to as the hybrid morphotype H) have been in existence for at least 20, but possibly more than 100 generations, are associated with arid longleaf pine habitats, and appear to have expanded primarily to the north displacing C morphotype populations [34,38,42].

Hybridization can result in low- or high-fitness hybrids, which can be a consequence of the break-up of co-adapted gene complexes, combinations of detrimental interactions between genomic elements of the two parental taxa, and the creation of advantageous novel gene complexes and interactions [43–48]. Piriqueta c. caroliniana hybrids from greenhouse crosses have relatively highfitness compared to parental morphotypes [40], as do many other examples of taxa with weak mating barriers [43]. Another potential consequence of natural hybridization is the appearance of non-additive (transgressive) traits, where morphological or physiological characters fall outside the range of expression found in the parental genotypes [49–52]. When gene expression is considered as a phenotype, it has been shown to have the characteristics of a transgressive phenotype when assayed in hybrids, such that transcript abundance is either well above or well below the range seen for either parental taxon [Senecio: 26, Helianthus: 53, Drosophila: 54, maize: 55, Arabidopsis: 56]. Although some traits of P. c. caroliniana hybrids have been shown to be transgressive, including greater trichome density [10,38] and drought tolerance in greenhouse and field transplant experiments [39,40], it is unknown if it also exhibits non-additive gene expression. We use the Piriqueta c. caroliniana complex to examine gene expression after multiple cycles of water limitation in divergent lineages that vary in drought tolerance. We specifically address the difference in gene expression among water-availability treatments in comparison to variation among vegetative cuttings growing in separate blocks exposed to the same treatment conditions. We conducted significance testing to obtain robust and ecologically relevant tests, addressing three major questions (1) How does prolonged and repeated drought affect patterns of gene expression in Piriqueta; (2) What are the differences in expression profiles for morphotypes associated with habitats differing in their moisture availability; and (3) Does the advanced-generation hybrid morphotype differ in gene expression patterns in comparison to the parental morphotypes? In particular, is there any evidence of transgressive expression? Our results indicate that prolonged drought leads to widespread down-regulation in these plants, and we observed even more pronounced down-regulation in the hybrids in response to drought, such that the transcript abundance for several genes fell below the range observed in either parental morphotype.

Fig. 1. Populations of the Piriqueta c. caroliniana morphotypes in Florida and Southern Georgia, United States. C: caroliniana, H: hybrid, V: viridis.

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Table 2 P. c. caroliniana individuals used for each target RNA sample, three for each morphotype/treatment combination.

2. Methods 2.1. Plant material Representative individuals grown from field-collected seeds and individuals produced from crosses between these field-derived plants were propagated in preparation for the drought experiment. The individuals used represented seven caroliniana (C) populations from Northern Florida and Southern Georgia, six viridis (V) populations from Southern Florida, and five hybrid (H) populations from central Florida (Fig. 1, Table 1). All populations were separated by at least 6 km. Plants were grown to maturity and seven to eight cuttings per plant were propagated from each individual. Cuttings were made by taking a stem section that consisted of one node and leaf, and rooting hormone (Shultz TakeRoot® ) was applied to each cutting prior to placing them in peat pellets (Jiffy-7® ). Cuttings were kept moist for 3 weeks to allow the plants to establish roots.

Morphotype/treatment

1st sample

2nd sample

3rd sample

C/D

210-10-2 213-29 218-42-2 GA7-64-2

210-10-2 213-29 218-42-2 GA7-64-2

210-10-2 212-19 213-29 GA7-64-2

C/W

210-10-2 213-29 218-42-2 GA7-64-2

210-10-2 213-29 218-42-2 GA7-64-2

212-19 218-42-2 FL9-108-1 GA7-64-2

H/D

218-34 FL39A-60-1-2 FL41-52 P20-55-1

218-34 FL39A-60-1-2 FL41-52 P20-55-1

218-34 FL39A-60-1 FL41-52-4 P20-55-1

H/W

218-34 FL39A-60-1-2 FL41-52 P20-55-l

218-34 FL39A-60-1-2 FL41-52-4 P20-55-1

218-34 FL38-50-1 FL41-52-4

V/D

212-6 FL25-50-2 FL32-50-1 FL37-X1103-3

212-6 FL25-50-2 FL32-50-1 FL37-X1103-3

FL25-50-2 FL32-50-1 FL33-57-6-2

V/W

212-6 FL25-50-2 FL32-50-1 FL37-X1103-3

212-6 FL25-50-2 FL32-50-1 FL37-X1103-3

212-6 FL25-50-2 FL33-57-6-2

2.2. Greenhouse drought stress experiment One cutting of each individual was planted in each of six (three for the wet [control] and three for the dry treatment) rectangular plastic containers (also referred to as “blocks”, 85 cm × 40 cm × 12 cm). Holes were drilled in the bottom of each container to facilitate drainage, and landscape fabric lining was placed on the bottom to prevent leaking of sand. Containers were filled to a depth of 7 cm with quartz sand. Peat pellets were gently broken away from healthy cuttings, which were washed to remove as much of the soil as possible. Cuttings were then planted in each container in three rows of five, approximately 15 cm apart, for a total of 14–15 plants per container, with individuals randomized by position within each container. Plants were allowed to establish for 2 weeks in the containers prior to the initiation of treatments. During initial establishment, both the control and experimental treatments were watered every second day to saturation of the soil. After initial establishment, height and number of leaves of each plant were measured to assess vegetative growth during the experiment. It has been shown for Piriqueta that under field conditions the product of height and number of leaves at the end of the growing season is a strong indicator of survival and reproduction in subsequent years [40]. The initial cutting leaf was excluded from all measurements of plant growth, and typically abscised soon after establishment. The temperature in the greenhouse was maintained near 35/27 ◦ C (day/night) with a 12-h day length through the entirety of the experiment. Table 1 The original population(s) for each P. c. caroliniana individual used in microarray experiments. The dates of initial collections or of greenhouse crosses (for those showing two populations) are listed under the Collected/Crossed heading. Morphotype

Individual

Population

Collected/Crossed

C

GA7-64-2 210-10-2 213-29 218-42-2 212-19 FL9-108-1

GA7 GA14 × FL45 GA16 × FL8 GA14 × GA14 GA15 × GA15 FL9

5/1/95 4/1/07 4/1/07 4/1/07 4/1/07 5/1/95

H

218-34 FL41-52 FL39A-60-1-2 P20-55 FL38-50

FL41 × FL42 FL41 FL39 P20 FL38

4/1/07 7/1/00 7/1/00 7/1/96 7/1/00

V

FL25-50 212-6 FL32-50-1 FL37-X1103-3 FL33-57-6-2

FL25 FL36 × FL35 FL32 FL37 FL33

7/1/97 4/1/07 7/1/00 7/1/00 7/1/00

Containers were randomly assigned to either the drought or control (wet) treatment. The control treatment containers were placed in shallow trays (2 cm deep) of standing water and were watered every second day to maintain constantly mesic soil conditions. The drought treatment containers were raised 6 cm from the bench to facilitate drainage. These were watered to saturation at the first sign of wilt in any individual in that container, typically occurring 8 days after watering (approximately 3–4 days after complete drying of the soil). After 36 days (four drought cycles) final morphological measurements were made and leaf tissue was harvested and flash frozen in liquid nitrogen for subsequent RNA extraction. 2.3. RNA preparation Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen) according to manufacturer instructions, with the addition of an initial preparation step in which leaf tissue was ground into a fine powder under liquid nitrogen and incubated for ten minutes at 56 ◦ C in buffer RLT. There was a total of six morphotype/treatment conditions (C dry, H dry, V dry, C wet, H wet, and V wet). Three replicate samples of each morphotype/treatment condition were produced by pooling RNA from cuttings of three to four individual plants taken from the same treatment container (Table 2) using the same individuals for each pool when possible (not possible for the third sample). Therefore, the variation among the three samples of a morphotype/treatment includes the inter-container variation (block effect). While this conservative design will not detect individual variation, the pooling provides a mean measure for each condition and allows accurate detection of between-condition variation even if variation among cuttings in replicate blocks is high. Equal amounts of RNA from each individual were combined for a total of 4 ␮g per target sample and was amplified using MessageAmpIITM aRNA Amplification Kit (Ambion Cat. #AM1751), which amplifies mRNA without substantially biasing the sample towards abundant transcripts [53,54]. 3.5 ␮g of aRNA per sample was reverse transcribed, using both random hexamer and oligoDT primers. The single-stranded cDNA product was amino-allyl labelled with Amersham Cy3 and Cy5 mono-reactive

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Array data have been deposited in the Gene Expression Omnibus (GSE19410). 2.5. Data analysis

Fig. 2. Loop design for microarray hybridizations. An arrow represents the competitive hybridization of two samples onto a microarray slide, with the samples prior to the arrow labelled with Cy3 and following the arrow labelled with Cy5. Each sample is designated by a three-character code; the first letter designates the morphotype (C: caroliniana, H: hybrid, V: viridis), the second letter designates the treatment (D: dry, W: wet/control), and the number (1, 2, or 3) indicates the block number of the cuttings that comprise the sample.

fluorescent dyes (GE Healthcare). Each target sample was used in two microarray hybridizations (technical replicates), once labelled with Cy3 and once labelled with Cy5, according to a loop design (Fig. 2) [55]. Paired samples were combined, cleaned using a PCR Purification Kit (Qiagen), and dried down to 12.5 ␮l in a speed vacuum (ThermoSavant SC210A SpeedVac® Plus). To reduce nonspecific hybridization, 1.5 ␮l of tRNA was added to each sample pair, which were then placed in 95 ◦ C water for 3 min. 14 ␮l of 2× hybridization buffer (50% formamide, 10× SSC, 0.2% SDS) was added to each sample pair prior to application to the microarray slide. 2.4. Microarray hybridization The microarray slides used in this experiment were produced by the Soybean Functional Genomics Project to represent 18,000 unigenes (unique sequences) selected from over 280,000 ESTs [56]. These ESTs represent over 80 cDNA libraries made from a variety of soybean tissues and organs during many developmental stages of the plant. The slides were obtained cross-linked from the University of Illinois, were blocked with a series of washes (two with 0.2% SDS, one with 95 ◦ C Nanopure water, and four with room temperature Nanopure water), and then prepared for hybridization with a 1-h incubation in Prehybridization Buffer (5× SSC, 0.1% SDS, 1% BSA) at 42 ◦ C. Each sample was applied to the top half of one of the microarray slides under 22 mm × 25 mm LifterSlips (Erie Scientific) covering 10,000 features, and allowed to hybridize at 42 ◦ C for 16–20 h. Hybridization conditions were the same as in other studies utilizing these arrays [56]. Slides were washed with a series of three solutions (1—1× SSC, 0.2% SDS, 2—0.2× SSC, 0.2% SDS, 3—0.1× SSC) and spun dry. They were then scanned with a GenePix 4000B scanner (Axon Instruments) and fluorescence data was collected (GenePix Pro 5.1). No secondary method of measuring gene transcript abundance, such as quantitative PCR, was used due to the lack of gene sequence for Piriqueta c. caroliniana and the sensitivity of quantitative PCR to non-sequence specific primers.

Plant size was estimated as the product of stem length and leaf number. Growth over the 5-week experiment was assessed by including initial plant size as a covariate in an ANOVA model that tested for differences in final plant size among morphotypes and treatments. In this model, morphotype and treatment were entered as fixed effects while target sample within each morphotype–treatment combination and container within treatment were declared random using SAS Proc GLM [57]. Differences in plant growth among morphotypes and treatments were tested over the variation among target samples and environments within treatments using the Test option for the Random Statement in Proc GLM. Microarray data were filtered and normalized to correct for variation among slides. Features not significantly greater than background (background + 2 standard deviations) were identified in GenePix Pro 5.1 and discarded. GenePix files were converted into MEV files readable by TIGR MIDAS by Express Converter V.1.8 [58]. LOWESS normalization was used to equalize signal intensities within each block of each slide. Genes that did not pass quality controls for 13 or more of the 18 slides were omitted from further analysis. A total of 7439 genes from the initial set of scanned clones were used in the final analysis. Median fluorescence intensity for each array feature was analyzed for each wavelength separately (single channel analysis) to identify genes differentially expressed in the C, H, and V morphotypes for each environment. To normalize across slides, which is necessary for single channel analysis, we subtracted the average fluorescence value of clones in a slide from each spot intensity and divided by the standard deviation. The original mean fluorescence across slides was added back to each measurement to facilitate log2 transformation of the data. Prior to hypothesis testing, data was screened using a Principle Components Analysis (PCA) of the normalized fluorescence of all genes to visually assess consistency in patterns of gene expression across slides. Nested analyses of variance models, with technical replicates nested within block, were applied to the intensity data using Type III Sums of Squares from SAS proc GLM [57], using the model: where Yijk is the observed expression,  is the mean expression across all observations, MTi is the effect of the six different morphotype–treatment combinations (HW, HD, CW, CD, VW, VD), REPij are the replicate blocks nested within morphotype–treatment, Xijk is the expression of the paired is the error. In this target sample with the alternate dye, and model, the effect of replicate blocks in different environments was declared as random, and morphotype and treatment effects were tested using the mean square for the block effect error. Each gene passing quality control was tested for a difference in expression between drought and control treatments (overall) and between drought and control treatments for each of the three morphotypes. Due to the number of statistical tests conducted, a substantial increase in the type I error rate (erroneously rejecting the null hypothesis) is expected. To account for this, the likelihood of a given result being a false positive will be presented as the Q-value [59] and significance was assessed with a fixed rejection criteria for type II error (P < 0.001). This tends to increase power, while still addressing the problem of multiple testing [59]. A correlation analysis was used to examine similarity in expression profiles among morphotypes. For this analysis, all genes were ranked in order of increasing probability of up-regulation

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in response to drought. For each morphotype, the most downregulated gene was ranked 1, and the most up-regulated gene was ranked 7439. Genes with intermediate ranks (20–7438 in C) did not show significant differential expression in response to drought. Using rank-significance controlled for differences in significance levels among morphotypes, focusing on the patterns produced by each morphotype. Not all of the differentially expressed genes were included in the correlation analyses because the disparity among morphotypes in the number of differentially expressed genes might bias the results and swamp existing patterns. Instead, the 80 most drought-responsive genes in each morphotype were included, totalling 138 genes, as several occurred in multiple morphotypes. These 138 genes were used in correlations between morphotypes to allow for adequate comparison of drought response profiles. Genes represented by microarray clones were determined using BLAST searches conducted by the distributor [56,60]. Functional groups of genes were identified using gene ontology classifications that were downloaded from the DFCI Soybean Gene Index maintained by the Computational Biology and Functional Genomics Laboratory at Harvard. Gene ontology classifications were available for approximately half of the gene accessions on the array. 3. Results 3.1. Growth data All morphotypes showed significantly less growth in drought conditions than in control conditions over the 5-week experiment (Fig. 3) (F = 11.04, P = 0.011, df = 1/39). The V morphotype displayed slower growth under drought than the C and H morphotypes, but this difference was not significant (F = 2.67, P = 0.098, df = 2/18). There were no significant differences in biomass change among morphotypes in the constant wet (control) treatment, and the morphotype-by-treatment interaction was not significant. Within a treatment, there was no significant variation among containers for plant growth. 3.2. Heterologous hybridization success Hybridization of Piriqueta RNA to the soybean microarray was successful, with consistent fluorescence greater than background across slides indicating sufficient sequence homology between the species. There was no substantial difference in the number of genes detected (features with sufficient fluorescence above back-

Fig. 3. Growth measured as an estimate of above ground biomass (height × leaf number) change during the 5-week experiment for morphotypes in the P. c. caroliniana complex. Error bars show ± the standard error. Means having the same lower case letter are not significantly different (Tukey multiple range test). C: caroliniana, H: hybrid, V: viridis.

433

ground) between trial hybridizations of Piriqueta and soybean cDNA to the array (data not shown). This could indicate non-specific hybridization; however, our use of a microarray that consists of long cDNA clones (average > 1000 bp) [56] and hybridization conditions of normal stringency should have prevented substantial non-specific hybridization [31]. Alternatively, the observed similarity in hybridization success may be due to the difference in the cDNA preparations between the Piriqueta and trial soybean hybridizations, as the RNA used in the Piriqueta hybridizations was amplified to increase the mRNA transcript abundance, which tends to produce stronger microarray hybridization [61]. When comparing multiple species on an array of a single species, there enters the possibility that a relative increase in fluorescence for a species (in our case, morphotype) is a result of greater transcript sequence similarity of the species to that of the array [62]. Using the Piriqueta system, we have the benefit of three closely related taxa hybridized to an array of a more distantly related taxon, decreasing the chance that there will be greater transcript similarity to soybean of one morphotype than another. A similar example is seen in the heterologous hybridization of two distinct species of Pachycladon, enysii and fastigiata, to an Arabidopsis array [63]. Analysis of sequence divergence and differential microarray hybridization between the Pachycladon species supported the assumption of sufficiently limited divergence for valid transcript abundance comparisons. In our hybrid complex, the morphotype-specific expression patterns found are not likely artifacts of heterologous hybridization and our confidence in inferences from the experimental data is high. 3.3. Microarray analysis Principle components analysis indicated that 1 of the 18 hybridizations was an outlier and was eliminated from further analysis. Although using 17 of the 18 microarray hybridizations in the analysis resulted in an unbalanced design, the ANOVA (Type III) used is robust to this disparity [57]. The error degrees of freedom for each ANOVA varied depending on the number of times that particular gene was omitted from analysis due to insufficient or aberrant fluorescence. The average error degrees of freedom was 11.98, with 12 degrees of freedom being the greatest possible. 3.4. Expression in response to drought We identified several genes that were regulated in response to drought (Table 3). Approximately 1.5% of genes analyzed (110 of 7439) displayed differences in expression between the control and drought treatments (F = 18.97, P < 0.001, Q = 0.063). When the effect of the variation among containers within a treatment was ignored and significance was tested solely over the technical error, 150 genes were identified as significantly regulated. This indicates that our inclusion of the variation among blocks within treatments provides a more conservative test of significant differential expression. The large majority of genes in these two gene lists (drought regulated including or ignoring the block effect error) were present in both analyses, indicating that the use of either error term did not substantially affect the identity of genes found to be regulated, but affected the total number of genes significantly regulated. The majority of differentially expressed genes were down-regulated in response to drought (82%). Major drought-regulated processes include protein metabolism, nucleic acid metabolism, transport, photosynthesis and cell death (Table 3). 3.5. Morphotype-specific expression profiles Differences in gene expression were also found among the morphotypes. A total of 70 genes (0.9% of genes analyzed)

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Table 3 Genes either up- or down-regulated in response to drought (P < 0.001) in the P. c. caroliniana complex across all morphotypes (“All”) or in the individual morphotype (“C”, “H”, and “V”). GenBank

Gene homology

All

C

H

V

AI748648 AI460572 AW102276 AI496483 AI443163 AW102230 AW707039 AI461236 AI440734 BE021231 AW307543 AW307522 AW102264 AI938891 AI494656 AW278507 AW278982 AI495426 AW101364 AW102175 AI438085 AW306863 AI495723 AI461260 AI442605

Acidic Ribosomal Protein P0 At4g35750 Drought Responsive Element Binding Protein Elongation Factor-1 Alpha Histone H3.2 Protein Pentameric Polyubiquitin Phosphoenolpyruvate Carboxylase Isozyme Pepc2 Putative Water Channel Protein Putative Zinc Finger Protein Repressor Protein Ubiquitin Extension Protein Ubiquitin Extension Protein Unknown Unknown Thaumatin-Like Protein PR-5b 60S Ribosomal Protein L35 Putative RING3 Protein Elongation Factor 1A SMV Resistance-Related Protein Unknown Unknown Histone H4 Glycerol 3-Phosphate Permease Unknown Unknown Unknown

Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up – Up Up – Up Up – – – Up

– – – Up Up – – – – – Up – Up – Up* Up* Up* Up* – – – – – – Up+

– – – – – – – – – – – – – – – – – – Up* Up* Up* – – – Up+

– – Up – – – Up – – – – Up Up – – – – – – – – Up* Up* Up* –

AI855465 AI443426 BE021090 AW472328 AW309223 AW101271 AW132325 AW132774 AW133066 AW132698 AW133145 AW133362 AW132248 AW133030 AW132896 AW132852 AW132576 AW568055 AW132334 AW132903 AW133237 AW132447 AW234049 AI855508 AW432980 AW132339 AW132526 AW132871 AW133296 AW568360 AW831452 AI443846 AI901222 AW100631 AI901136 AW432284 AW132752 AW132833 AW132855 AW132314 AW132567 AW132907 AW831830 AW132845 AW704061 AW507689 AW132933 AW508196

2-Oxoglutarate/Malate Translocator Precursor-Like Protein Alpha-3 Tubulin Alpha-Tubulin 4 Aminoimidazolecarboximide Ribonucleotide Transformylase At1g74340 At3g52500/F22o6 120 ATP:Pyruvate Phosphotransferase Carbonic Anhydrase Carbonic Anhydrase Chlorophyll A/B-Binding Protein CP29 Chlorophyll A/B-Binding Protein CP29 Chlorophyll A/B-Binding Protein CP29 Chlorophyll A/B-Binding Protein Type II Chlorophyll A/B-Binding Protein Type II Chlorophyll A/B-Binding Protein Type II Chlorophyll A/B-Binding Protein Type II Elongation Factor G, Chloroplast Precursor Expressed Protein Expressed Protein Fructose-Bisphosphate Aldolase 1, Chloroplast Precursor Fructose-Bisphosphate Aldolase 1, Chloroplast Precursor Geranylgeranyl Pyrophosphate Synthase-Related Protein Kinase Interacting Family Protein Kinesin Motor Family Protein LEC1-Like Protein LHCII Type III Chlorophyll A/B-Binding Protein Low Affinity Sulphate Transporter Magnesium Chelatase Subunit Magnesium Chelatase Subunit Osjnba0027p08.20 P0004d12.28 Peroxidase ATP2a Photolyase/Blue-Light Receptor Photosystem I Light-Harvesting Chlorophyll A/B-Binding Protein Photosystem II Type I Chlorophyll A/B-Binding Protein Photosystem II Type I Chlorophyll A/B-Binding Protein Photosystem II Type I Chlorophyll A/B-Binding Protein Photosystem II Type I Chlorophyll A/B-Binding Protein Photosystem II Type I Chlorophyll A/B-Binding Protein Photosystem II Type I Chlorophyll A/B-Binding Protein Photosystem II Type I Chlorophyll A/B-Binding Protein Plastidic Aldolase Protein Kinase Family Protein Protein Kinase Family Protein Protochlorophyllide Reductase Putative Cell Division Control Protein Putative Chlorophyll A/B-Binding Protein Precursor Putative DAL1 Protein

Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down

– – – – – Down Down Down – Down Down – Down Down Down Down – Down – Down Down – – – – Down Down – Down – – Down – Down Down Down Down Down Down Down Down Down Down Down – Down Down –

– – – – Down Down – Down Down Down Down – Down Down Down Down – Down – Down Down Down – – – Down Down – Down – – Down Down Down Down Down Down Down Down Down Down Down Down Down – Down Down –

– – – – – Down – – Down Down Down – – Down Down Down – Down – Down Down – – – – Down Down – Down – – – – Down Down Down Down Down Down Down Down Down – Down – – Down –

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Table 3 (Continued ) GenBank

Gene homology

All

C

H

V

AW186341 AI966043 AW471677 AW830883 AW132776 AW508778 AW132410 AI965997 AW133315 AI441594 AW132839 AW132791 AW508823 AW119723 AW164395 AW308940 AW318233 AW830833 AW133262 AW396286 AW396354 AW133157 AI900659 AW102129 AW508395 AW568478 AW132367 AW508140 AW507798 AW277642 AW309223 AW830561 AI494872 AI441466 AW133297 AI965956 AW432492 AI901000 AW132618 AI460402 AW734383 AW133176 AW164617 AW278448 AW133184 AW132401 AW133154 AW279378 AW133316 AW307509 AW101421 AW132504 AI855506 AW101119 AW472208 AW308940 BE020096 AI900654 AW132290

Putative Oxalyl-Coa Decarboxylase Putative Plastid Protein Putative Splicing Factor 3B Subunit 2 Putative Poly(A)-Binding Protein Putative Protein Phosphatase PP2A0 B Subunit Putative Splicing Factor Ribulose-1,5-Bisphosphate Carboxylase Small Subunit Rubisco Activase Rubisco Activase Rubisco Activase 2 Rubisco Activase Small Isoform Precursor Salt Tolerance Protein 6 Seed Specific Protein Bn15D1B Similar To PR-10 Translation Initiation Factor 2 Beta Tubulin A-1 Chain No Overlap W/B-Tubulin Tubulin B-Chain No Overlap W/A-Tubulin Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unnamed Protein Product Vdac1.3 Zinc Finger (C3HC4-Type RING Finger) Family Protein Unknown Unknown Acyl-CoA Synthetase-Like Protein At1g74340 Calmodulin (Cam) Catalase CONSTANS-Like B Dehydroascorbate Reductase Drm3 Gcpe Protein N-Glyceraldehyde-2-Phosphotransferase-Like Phosphoribulokinase Plastidic Aldolase Putative Aquaporin Putative Chlorophyll A/B-Binding Protein Putative Enolase Putative Glutaredoxin Putative Membrane Related Protein Ribulose-1,5-Bisphosphate Carboxylase Small Subunit Ribulose-1,5-Bisphosphate Carboxylase Small Subunit Ribulose-1,5-Bisphosphate Carboxylase Small Subunit Serine Methylase (SHMT) Syringolide-Induced Protein 19-1-5 Unknown Unknown Unnamed Protein Product Lipoxygenase Unknown Tubulin A-1 Chain No Overlap W/B-Tubulin Putative CMP-Sialic Acid Transporter Putative Photosystem II Subunit (22 kDa) Precursor Photosynthetic Glyceraldehyde-3-Phosphate Dehydrogenase

Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down – Down – – – – Down Down – Down Down – Down – – Down – – – – – – – – – Down – Down Down Down

Down – – Down Down – – Down Down Down Down Down – – Down – – – – Down Down Down Down – Down Down – Down* Down* – – – – – – – – – – – – – – – – – – – – – – – – – – – Down+ Down+ Down+

Down – – Down – – Down Down Down Down Down Down – Down Down – – – Down Down Down Down Down – Down Down Down – – Down* Down* Down* Down* Down* Down* Down* Down* Down* Down* Down* Down* Down* Down* Down* Down* Down* Down* Down* Down* Down* Down* Down* Down* – – – Down+ Down+ Down+

Down – – Down – – – – Down – – Down – – Down – – – – Down – Down Down – Down Down – – – – – – – – – – – – – – – – – – – – – – – – – – – Down* Down* Down* – – –

*Regulated exclusively in one morphotype (P > 0.01 in all other morphotypes); +Regulated exclusively in C and H morphotypes (P > 0.01 in the V morphotype).

were differentially expressed among morphotypes in one or both treatments (F = 18.74–19.08, P < 0.001, Q = 0.29–0.99). Nearly four times more genes showed morphotype-specific expression in drought than in the control treatment. In Table 3, genes regulated in response to drought in only one morphotype are denoted with an asterisk. These genes show significant response to drought (P < 0.001) in one morphotype and are not significant or marginally significant (P < 0.01) in either of the other morphotypes. This prevents inclusion of genes that are actually differentially regulated in multiple morphotypes but are identified as regulated in only one due to small differences in significance level. Genes showing both a morphotype-specific response to drought in addition to being sig-

nificantly regulated in the combined analysis are likely to be ones that are up- or down-regulated to the extent that they are driving the finding of significant drought regulation overall. The majority of genes on the array (97%) did not have any significant response (all P > 0.001) to treatment, morphotype, or any morphotype/treatment combination. The hybrid morphotype had a greater number of droughtregulated genes than the V or C morphotypes (Fig. 4). It is not possible that this pattern is driven by testing over a lower error term for the H morphotype, as significance was tested over the average error for all morphotype/treatments. The percentage of downregulated genes in the H morphotype (95%) was also greater than

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parison with microarray studies using short-term water limitation treatments [17,18,22,64]. Compared to these studies, all P. c. caroliniana morphotypes exhibited far more down-regulation of genes in response to water limitation. This trend was most pronounced in the advanced-generation hybrid morphotype, which showed transgressive under-expression in several drought-regulated genes. These findings are consistent with other studies of response to prolonged water limitation in drought-tolerant species [65–67], adding to our confidence in the success of the heterologous microarray hybridization. The pattern of gene expression seen in the hybrid and parental morphotypes supports the idea that general down-regulation of genes expressed in leaf tissue is an adaptive response to drought. Fig. 4. The number of genes with significant change in expression response to drought in the C (58 genes), H (85 genes), and V (44 genes) morphotypes (morphotype-specific analysis) and the number which are shared among morphotypes (P < 0.001, Q: 0.06–0.16). C: caroliniana, H: hybrid, V: viridis.

that of the C and V morphotypes (84% in each). A number of these genes tended to show down-regulation in the hybrid morphotype and up-regulation in viridis and/or caroliniana morphotypes; however, only two of these were significant at the P < 0.001 level (Supplementary Table 1). The two morphotypes associated with more xeric habitats (C and H) shared four significantly regulated genes (P < 0.001) that were not significantly or slightly significantly regulated (P > 0.01) in the morphotype associated with a mesic environment (V; Table 3). Drought expression in the hybrid morphotype H was more strongly correlated with the C morphotype in expression profile (r = 0.709) than with the V morphotype (r = 0.560; Fig. 5). Expression profiles of the parental morphotypes C and V were more strongly correlated (r = 0.782) than either were with the H morphotype. A notable number of genes were strongly down-regulated in the hybrid morphotype and not down-regulated in the parental morphotypes, which distinguished its expression profile from that of the parental morphotypes. 4. Discussion This study provides a unique and ecologically relevant treatment of gene expression in a non-model organism. The patterns of gene expression found in Piriqueta were distinct from those found in other microarray studies of drought responses in model systems and crop species [17–19,22,23,64]. This is also true in com-

4.1. Plant growth Plant growth under drought was lower than in the control treatment, and the V morphotype showed lower growth under drought than the C or H morphotypes. Since these growth patterns are seen in common garden experiments in xeric field sites [40], it is reasonable to assume that the drought treatments were effective in applying stress naturally experienced by the plants. Although the differences in plant growth among morphotypes in drought conditions were not significant in this experiment, an increase in sample size would likely find significantly less growth in the V than in the C or H morphotypes under drought. The nominal difference in growth under drought in the C and H morphotypes could be a reflection of their ecological similarity, as both occur primarily in xeric areas [34,38]. 4.2. Gene expression in drought There were substantially more genes that were down-regulated in response to drought (82%) than up-regulated (18% of 110 genes displaying significant change in expression between treatments). In addition, the amplitude of change in transcript abundance was greater for genes that were down-regulated compared to those that were up-regulated (Supplementary Table 1). The majority of studies examining the effects of water limitation have found the contrary, with a preponderance of up-regulated rather than down-regulated genes [17,23,64,68]; however, these studies typically examine short-term responses to water-stress that often last only a few hours to a few days [17,18,22,64]. The ability to survive periods of drought might have more to do with expression observ-

Fig. 5. Similarity of morphotype gene expression in drought. The correlations of drought expression profiles between morphotypes are presented as the log2 rank in order of increasing up-regulation in response to drought in P. c. caroliniana. Internal lines represent significance cut-offs (P < 0.001) for down-regulated genes (found on and below the lower dashed lines/on or to the left of the internal left line) and up-regulated genes (found on and above the upper dashed lines/on or to the right of the internal right line). Blue/green shaded circles are down-regulated, pink/purple shaded circles are up-regulated, and hollow circles are not significantly regulated in response to drought in either morphotype of that particular correlation (i.e. are only significant in the third morphotype, not included in that correlation). C: caroliniana, H: hybrid, V: viridis.

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able after prolonged and/or repeated drought. This is supported by a recent study that used a drought-adapted barley strain [66]. In this case, dehydration shock (6 h out of soil) resulted in a large proportion of up-regulated genes, while drought stress (11 days without water, a more ecologically relevant treatment) resulted in an increased proportion (∼50%) of down-regulated genes. This suggests that the patterns of gene expression during short-term water-stress are not as pertinent to drought-adaptation. Three other experiments using the perennial species Vitis vinifera, Festuca mairei and the drought-tolerant potato strain SA2563 also found greater amounts of down-regulation in shoot tissue in response to prolonged drought [65,67,69]. Over one-third of the down-regulated genes were photosynthesis related. Reduction in photosynthesis is a typical response of plants experiencing drought. These genes included RUBISCOrelated transcripts, chlorophyll binding proteins and chloroplast precursors, all shown to be down-regulated in other gene expression analyses of drought response. Surprisingly, many of these genes are also found to be up-regulated in other studies, such as that by Huang et al. [70] looking at drought response in Arabidopsis, further highlighting the differences seen between this study and those conducted on non-drought-tolerant annuals. An unexpected result was the down-regulation of tubulin genes. Tubulin has been used as an internal control for expression studies, including drought studies [22,71,72]. If tubulin is drought responsive in Piriqueta, it might not be a suitable control for other species as well. This is supported by Nicot et al. [73], where the stability of popular housekeeping genes as internal controls was examined under various stress conditions. They found tubulin to show differential expression under stress, suggesting that it is a poor choice for an internal control in stress experiments. The exceptional amount of down-regulation seen in Piriqueta morphotypes may be a synergistic effect of using a drought-tolerant species and exposure to prolonged water limitation. Studies mentioned previously used perennial species and prolonged drought; however, this pattern of transcriptional down-regulation has also been demonstrated for both factors separately. Kawaguchi et al. [21] demonstrated greater down-regulation in drought in Arabidopsis thaliana (as cited in [68]). This was under a more extended drought treatment than seen in other studies using A. thaliana [68]. Wang and Bughrara [67] looked at short-term water-stress in a drought-tolerant rice strain. Of two rice cultivars, the one found to be water-stress-tolerant showed down-regulation of a greater number of transcripts in response to a 9-h osmotic stress treatment. These findings support the hypothesis that down-regulation of many genes is a general response to drought that is more exaggerated for species that are associated with arid environments. If a strategy for survival in water-limited environments for Piriqueta and other perennial taxa is resource conservation, this can be initiated by key transcription factors/repressors. In Piriqueta, several genes affecting transcriptional regulation were differentially expressed, including elongation factors, histone proteins and zinc finger proteins. Transcripts encoding protein metabolism genes such as ubiquitin, protein kinases, splicing factors, and protein phosphatase were also differentially expressed in drought. Protein kinases have specifically been implicated in osmotic stress signalling [74]. 4.3. Gene expression in hybrid and parental morphotypes Genetic recombination can produce novel phenotypes and traits that can be acted upon by selection [43,75,76]. The recombination effects on mRNA expression are apparent in the intraspecific hybridization of P. c. caroliniana morphotypes, which have experienced a period of geographic isolation in the relative absence of other mating barriers [34,77]. We found the hybrid morphotype

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to have a greater transcriptional response to drought than either the C or V morphotypes, with 47% more differentially expressed genes than caroliniana and 93% more differentially expressed genes than viridis. This transgressive response is manifested as downregulation of more genes, as the hybrids did not have a greater number of up-regulated genes than caroliniana or viridis. The genes differentially expressed exclusively in the hybrid morphotype are four photosynthesis genes and 23 others of various functions. Eight of these are present in the combined analysis (including all morphotypes) of drought-regulated genes in Piriqueta, indicating that the transgressive expression in the H morphotype is driving 7% of the genes found differentially expressed in that analysis. Despite transcriptional down-regulation of photosynthesis, hybrid plants did not seem to exhibit reduced growth compared to the other morphotypes. One growth variable that was not considered was the below ground biomass, so it is possible that the C and/or V morphotypes are storing starch or exhibiting root growth. While we cannot rule out this possibility, it is notable that above ground biomass is a good indicator of fitness in Piriqueta, as its mass at the end of the drought period (measured as in this study) is a good indicator of over-winter survival and subsequent plant growth and reproduction the following year. Because these are advanced-generation hybrids that have been present for hundreds to thousands of years [34,42], it is possible that this transgressive expression is a result of adaptation to arid environments via selection on the broad range of recombinant hybrid genotypes that were produced after initial contact between the parental morphotypes. Contrary to expectations, gene expression in the C morphotype is more strongly correlated with expression in viridis than with the hybrids, which is likely due to the large number of down-regulated genes in the hybrid morphotype that is not seen in either parental morphotype. Ranz et al. [78] also noticed that the expression profiles of two parental taxa, Drosophila melanogaster and D. simulans, were more similar to each other than to their hybrids. However, in the aforementioned case, it was attributed to incompatibilities in gene regulatory mechanisms in these sterile offspring. In contrast, the P. c. caroliniana hybrids of the current study are viable, high-fitness hybrids resulting from several generations of natural selection on the increased genetic variation. Of the two parental morphotypes, gene expression under drought in the H morphotype was more similar to C than to the V morphotype. The hybrid morphotype has been found to have a similar or greater amount of drought tolerance than caroliniana [40] and occurs in very similar habitats [79], a potential explanation for their greater similarity of expression in drought. The genes that are regulated in both the C and H morphotypes, and not in the V morphotype, which are largely photosynthesis related genes, could be in part responsible for this drought tolerance that they share. A putative CMP-sialic acid transporter gene also down-regulated in the C and H morphotypes could be an indicator of more extensive metabolic rate decrease. Although the role of plant CMP-sialic acid transporter homologs is not confirmed, they are thought to be involved in the transport of the cell wall structural component, CMP-kdo [80]. Among the genes with transient expression between the H morphotype and a parental morphotype are two GTP-binding proteins, both down-regulated in the H and up-regulated in the V morphotype. These proteins are involved in a variety of intracellular transport activities such as H-ATPase and sialyltransferase movement and trans-nuclear protein and RNA movement, and have previously been shown to be drought responsive [81,82]. 5. Conclusions This study answers the important aim of measuring gene expression in non-model organisms and contributes to our understanding of the regulation of gene expression in response to

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sustained drought. In particular, these results are consistent with an emerging trend of large transcriptional repression in response to prolonged drought that emphasizes the contrast between current model plant systems, which are predominantly annuals and non-drought-tolerant perennial species. While initial responses to drought may be more similar to general shock responses [15,22,23], gene expression under prolonged water limitation may have more relevance for plants associated with arid environments. The hybrid morphotype of the Piriqueta c. caroliniana complex has been previously shown to display transgressive growth under drought conditions. The current study shows that it also experiences the most pronounced down-regulation under drought, contributing to other research showing transgressive gene expression in hybrid taxa. Since it has been associated with arid conditions for many generations, it is possible that transgressive gene expression in the hybrid morphotype is a consequence of selection and adaptation to periodically water-limited conditions. Continued investigations of this and other stress-adapted species will continue to provide insights into the mechanisms that allow organisms to survive and reproduce in challenging environments. Acknowledgments The authors wish to thank B. Buckley, T. Cheeke, S. Eppley, T. Musial, J. Picotte, A. Ramakrishnan, S. Renn, L. Vodkin, and two anonymous reviewers for comments on earlier drafts of this manuscript, and S. Brar for assistance in the laboratory. The work was funded by NSF grant DEB-0413854 to MBC and Portland State University Forbes-Lee grant to HEM. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.plantsci.2010.02.009. References [1] R. Bijlsma, V. Loeschcke, Environmental stress, adaptation and evolution: an overview, J. Evol. Biol. 18 (2005) 744–749. [2] D. Schluter, Ecology of Adaptive Radiation Oxford Series in Ecology and Evolution, Oxford University Press, New York, 2002. [3] G.L. Stebbins, Variation and Evolution in Plants, Columbia University Press, New York, 1950. [4] J.S. Boyer, Plant productivity and environment, Science 218 (1982) 443–448. [5] H. Lambers, H. Lambers, T. Pons, Plant Physiological Ecology, Springer-Verlag, New York, 1998. [6] P. Nobel, Physiochemical and Environmental Plant Physiology, Academic Press, New York, 2005. [7] E.A. Bray, Molecular responses to water-deficit, J. Plant Physiol. 103 (1993) 1035–1040. [8] D.R. Sandquist, J.R. Ehleringer, Population- and family-level variation of brittlebush (Encelia farinosa, Asteraceae) pubescence: its relation to drought and implications for selection in variable environments, Am. J. Bot. 90 (2003) 1481–1486. [9] A.C. Gibson, Photosynthetic organs of desert plants, BioScience 48 (1998) 911–920. [10] J.J. Picotte, D.M. Rosenthal, J.M. Rhode, M.B. Cruzan, Plastic responses to temporal variation in moisture availability: consequences for water use efficiency and plant performance, Oecologia 153 (2007) 821–832. [11] E.D. Schulze, R.H. Robichaux, J. Grace, P.W. Rundel, J.R. Ehleringer, Plant water balance, BioScience 37 (1987) 30–37. [12] J. Flexas, J. Bota, J. Cifre, J.M. Escalona, J. Galmes, J. Gulias, E.K. Lefi, S.F. MartinezCanellas, M.T. Moreno, M. Ribas-Carbo, D. Riera, B. Sampol, H. Medrano, Understanding down-regulation of photosynthesis under water stress: future prospects and searching for physiological tools for irrigation management, Ann. Appl. Biol. 144 (2004) 273–283. [13] A. Milbau, L. Scheerlinck, D. Reheul, B. De Cauwer, I. Nijs, Ecophysiological and morphological parameters related to survival in grass species exposed to an extreme climatic event, Physiol. Plant. 125 (2005) 500–512. [14] K. Shinozaki, K. Yamaguchi-Shinozaki, Gene expression and signal transduction in water-stress response, Plant Physiol. 115 (1997) 327–334. [15] W.X. Wang, B. Vinocur, A. Altman, Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance, Planta 218 (2003) 1–14.

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