Water and Salinity Stress in Grapevines: Early and Late Changes in Transcript and Metabolite Profiles

Funct Integr Genomics (2007) 7:111134 DOI 10.
1007/s10142-006-0039-y
ORIGINAL PAPER
Water and salinity stress in grapevines: early and late changes in transcript and metabolite profiles
Grant R. Cramer & Ali Ergl & Jerome Grimplet & Richard L. Tillett & Elizabeth A. R. Tattersall & Marlene C. Bohlman & Delphine Vincent & Justin Sonderegger & Jason Evans & Craig Osborne & David Quilici & Karen A. Schlauch & David A. Schooley & John C. Cushman
Received: 21 July 2006 / Revised: 30 September 2006 / Accepted: 30 September 2006 / Published online: 29 November 2006 # Springer-Verlag 2006
Abstract Grapes are grown in semiarid environments, where drought and salinity are common problems. Microarray transcript profiling, quantitative reverse transcriptionPCR, and metabolite profiling were used to define genes and metabolic pathways in Vitis vinifera cv. Cabernet Sauvignon with shared and divergent responses to a gradually applied and long-term (16 days) water-deficit stress and equivalent salinity stress. In this first-of-a-kind study, distinct differences between water deficit and salinity were revealed. Water deficit caused more rapid and greater
Grant R. Cramer and John C. Cushman contributed equally to this paper. Electronic supplementary material Supplementary material is available in the online version of this article at http://dx.doi.org/ 10.1007/s10142-006-0039-y and is accessible for authorized users. G. R. Cramer (*) : J. Grimplet : R. L. Tillett : E. A. R. Tattersall : M. C. Bohlman : D. Vincent : J. Sonderegger : J. Evans : D. Quilici : D. A. Schooley : J. C. Cushman Department of Biochemistry and Molecular Biology, MS200, University of Nevada, Reno, NV 89557-0014, USA e-mail: cramer@unr.edu A. Ergl Biotechnology Institute, Ankara University, 06500 Besevler-Ankara, Turkey C. Osborne Department of Animal Biotechnology, University of Nevada, Reno, NV 89557-0014, USA K. A. Schlauch Boston University School of Medicine, Department of Genetics and Genomics, Boston University, E632, Boston, MA 02118, USA
inhibition of shoot growth than did salinity at equivalent stem water potentials. One of the earliest responses to water deficit was an increase in the transcript abundance of RuBisCo activase (day 4), but this increase occurred much later in salt-stressed plants (day 12). As water deficit progressed, a greater number of affected transcripts were involved in metabolism, transport, and the biogenesis of cellular components than did salinity. Salinity affected a higher percentage of transcripts involved in transcription, protein synthesis, and protein fate than did water deficit. Metabolite profiling revealed that there were higher concentrations of glucose, malate, and proline in water-deficittreated plants as compared to salinized plants. The metabolite differences were linked to differences in transcript abundance of many genes involved in energy metabolism and nitrogen assimilation, particularly photosynthesis, gluconeogenesis, and photorespiration. Waterdeficit-treated plants appear to have a higher demand than salinized plants to adjust osmotically, detoxify free radicals (reactive oxygen species), and cope with photoinhibition. Keywords Vitis vinifera . Affymetrix oligonucleotide array . Gas chromatographymass spectrometry . Abiotic stress
Introduction Grapes from the genus Vitis rank first among fruit crops in the world in terms of both production and economic importance (Vivier and Pretorius 2002). In 2005, grape production was 66,533,393 Mt on 7,326,445 ha (FAOSTAT data, 2005; http:// faostat.fao.org). Of the approximately 60 species within the
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family Vitaceae, cultivars of Vitis vinifera L. are used mainly for the production of wine and distilled liquor production and have the greatest economic value; however, grapes are also used for juice, dried fruit (raisins), table grapes, concentrate, and pharmaceutical and food supplements after being processed. Cultivated V. vinifera and wild species of grape display a wide and complex genetic diversity with 16 genetic groups that can be divided into three major clusters representing the classical eco-geographical groupings of grape cultivars: occidentalis, pontica, and orientalis (Aradhya et al. 2003). Vitis vinifera, which belongs to the occidentalis group, probably originated in the Tigris Euphrates basin in the area near the Caspian Sea, in southwestern Asia. Vitis vinifera L. grapes are relatively tolerant to waterdeficit. Regulated-deficit irrigation has been used advantageously to inhibit vine growth without fruit yield reductions and to make measurable improvements in grape quality (Matthews and Anderson 1988; Matthews et al. 1990; Sipiora and Granda 1998; Esteban et al. 1999, 2001; Kennedy et al. 2000). More specifically, there is an increase in total phenolic and anthocyanin content in fruit, the major components of the sensory characteristics of wine, resulting in improved aroma, color, and flavor (Matthews et al. 1990), as well as enhanced health benefits conferred by polyphenolic compounds with antioxidant activity (German and Walzem 2000; Dixon et al. 2005). Irrigation is used to overcome water-deficit conditions on 15% of cultivated land, particularly in semiarid regions; however, long-term irrigation can lead to soil salinization. About 20% of the 230 Mha under irrigation worldwide is estimated to suffer from some salinization-affected losses in productivity (FAO 2005). Salinity inhibits plant growth in two phases by (1) limiting water uptake through an osmotic or water-deficit effect arising from relatively high solute concentrations in the soil and (2) ion uptake where it injures cells through ionic stress effects (Munns 2002, 2005). Vitis vinifera varieties are ranked moderately sensitive to salinity stress by most researchers (Maas and Hoffman 1977; Hawker and Walker 1978; Shani et al. 1993; Walker et al. 2002). However, one study ranked V. vinifera varieties as sensitive (Prior et al. 1992). Grapevine appears to be more sensitive to Cl toxicity than Na+ toxicity (Walker et al. 2004). The inhibition of grapevine growth and CO2 assimilation by water-deficit or salinity has been related to changes in stomatal conductance, electron transport rate, leaf water potential, chlorophyll fluorescence, osmotic potential, and leaf ion concentrations (Walker et al. 1981; Schultz and Matthews 1988; Patakas and Noitsakis 1999; Medrano et al. 2002). Improvements in wine grape production efficiency and fruit quality will be possible with a better understanding of the molecular genetic basis of berry development and environmental stress responses (Bisson et al. 2002; Vivier and
Pretorius 2002). Recently, functional genomic resources for V. vinifera and related species have proliferated rapidly. Expressed sequence tag sequencing efforts within the last several years have significantly increased the public availability of cDNA sequence information for V. vinifera and several related species (Terrier et al. 2001; da Silva et al. 2005; Moser et al. 2005). Several mRNA expression profiling studies have been completed for Vitis, including differential screening of cDNA libraries from berry (Davies and Robinson 2000) and monitoring of gene expression profiles in flowers and during berry skin development using cDNA or oligonucleotide microarrays (Terrier et al. 2005; Waters et al. 2005). However, transcript profiling of the response of grapevine to water deficit or salinity stress has not been examined using highdensity microarrays, but was recently used to investigate viral disease in grapes (Espinoza et al. 2006). Most large-scale microarray expression profiling studies that have examined salinity and water-deficit or osmotic stress responses in Arabidopsis (Chen et al. 2002; Kreps et al. 2002; Seki et al. 2002; Maathuis et al. 2003) and/or its halotolerant relative Thelungiella halophila (Taji et al. 2004; Gong et al. 2005), barley (Ozturk et al. 2002; Atienza et al. 2004; Ueda et al. 2004), rice (Kawasaki et al. 2001; Rabbani et al. 2003; Walia et al. 2005), or poplar (Gu et al. 2004) applied salinity in a single step and thus as a salt shock. Very few studies have applied salinity stress (Walia et al. 2005) or water-deficit stress gradually over time in order to track the osmotic or dehydration acclimation responses. In this study, we conducted mRNA expression profiling on one variety of V. vinifera (cv Cabernet Sauvignon) using the new Vitis Affymetrix (Santa Clara, CA, USA) GeneChip oligonucleotide microarray version 1.0, which contains 14,470 unigenes. We present the results from the first large-scale analysis of mRNA expression profiles in vegetative tissues of grapevine under both water deficit and equivalent salinity stress in order to identify mRNA abundance changes in response to these stresses and to differentiate osmotic effects from ionic effects. We monitored both the transcript and metabolite changes to gradual application of water-deficit and salinity over 16 days to mimic the gradual onset of such stresses likely to occur under field conditions. This comparative analysis revealed that there are significant differences and overlaps in the transcript and metabolite profiles to waterdeficit and salinity stress in wine grape shoot tissues.
Materials and methods Plant material Two-year-old rooted cuttings of dormant V. vinifera cv. Cabernet Sauvignon clone 8 were obtained from Inland
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Desert Nursery (Benton City, WA, USA), pruned to two winter buds and roots of 8 to 9 cm in length, and planted in 13.3-l pots containing approximately 10 l Scotts Metromix 200 (Sun Gro Horticulture, Vancouver, British Columbia, Canada). Plants were grown in a greenhouse on a 16-h light (26C, 300400 E m2 s1)/8-h dark (18C) cycle supplemented with illumination from sodium vapor lamps. Vines were watered every other day with Gibeauts nutrient solution composed of 1.25 mM KNO3, 1.50 mM Ca(NO3)2, 0.75 mM MgSO4, and 0.50 mM KH2PO4; and the micronutrients, 50 M KCl, 50 M H3BO3, 10 M MnSO4, 2.0 M ZnSO4, 1.5 M CuSO4, 0.075 M (NH4)6Mo7O24, 0.1 mM Na2O3Si, and 72 M Fe-diethylenetriamine pentaacetate (Sequestrene 330) with pH 6.0 (Gibeaut et al. 1997). Plants were progressively pruned to provide uniform plants as follows: upon producing an unbranched solitary shoot of 80 to 100 cm, the shoot was pruned to 60 cm. Lateral shoots, buds, and young flowers were removed. Plants were fertilized with 1 l Gibeauts solution every 2 days, for a total of five times, to promote growth of this new shoot. The experiment began when these new shoots grew and gave rise to a minimum of two new nodes. The experiment consisted of a completely randomized factorial design consisting of 24 (treatment time) with six to eight individually potted plants per time point and treatment. The effects of water deficit and salinity on the growth of Cabernet Sauvignon over the course of 16 days were studied. Two days before the start of the experiment, all plants were given exactly 4 l of tap water, 4 h after daybreak on each day, which was sufficient to totally flush the soil. For water-deficit treatments, watering was completely stopped at the onset of the experiment (day 0). Control plants and salinized plants were watered daily with 4 l of water or salt solution throughout the experiment. Stem water potentials of the plants were measured and a calculated concentration of salt solution was added to the salinized plants to reduce the stem water potential of these plants to match those of the water-deficit-treated plants. Consequently, salt concentration of the irrigating solution was increased over time as follows: On day 1, salt-treated vines were given 10 mM NaCl and 1 mM CaCl2. From day 2 to day 6, they received 20 mM NaCl and 2 mM CaCl2 daily. During days 7 and 8, 55 mM NaCl and 5.5 mM CaCl2 were supplied to the plants. From day 9 to day 12, 75 mM NaCl and 7.5 mM CaCl2 were daily added to the watering solution. During days 13 and 14, the vines received 175 mM NaCl and 17.5 mM CaCl2, and eventually, 250 mM NaCl and 25 mM CaCl2 on days 15 and 16. Stem water potential measurements Fully mature leaves from between the second and sixth oldest nodes of the main shoot were selected for stem water
potential measurement (McCutchan and Shackel 1992). For each measurement, a single leaf per plant was tightly zipped in a plastic bag to eliminate transpiration, with care taken to not clamp or damage the petiole. Aluminum foil was then placed around the bag, deflecting light and heat. After 2 h of equilibration time, the petiole was cleanly cut and carefully threaded through a rubber gasket in the lid of a pressure chamber (3005 Plant Water Status Console, Soilmoisture Equipment, Santa Barbara, CA, USA). The foil was removed before sealing the bagged leaf in the chamber. The balancing pressure required to visibly push stem xylem sap to the cut surface was recorded. Afterwards, the chamber pressure was raised rapidly to allow collection on a paper disc of sufficient xylem sap to measure the solute potential of the sap with a vapor pressure osmometer (Model 5500, Wescor, Logan, UT, USA). Solute potential was calculated as before (Cramer and Bowman 1991) and the water potential of the xylem was corrected for variation in solute accumulation in the salt-treated plants (data not shown). The water potential was recorded every 2 days, using different plants in each measure (six biological replicates for each time point and treatment). Growth measurements Shoot length (mm) was measured every 2 days. Reference starting points along the shoots for growth measurements were marked with tape, resulting in all shoot tips bearing exactly two leaves from the reference point on day 0. There were eight biological replicates for each time point and treatment. RNA extraction, labeling, and array hybridization For all molecular analyses, six shoot tips from different plants were immediately frozen in liquid nitrogen at harvest. Like all other measurements, plants were harvested each time at exactly the same time of the day (midday) to prevent confounding effects of circadian rhythms. Two shoot tips were pooled together to make three biological replicates at each time point except day 0, where the number of biological replicates was doubled. Total RNA was extracted from abiotically stressed V. vinifera cv. Cabernet Sauvignon shoot tissue using a TrisLiCl protocol (Tattersall et al. 2005). The total RNA was further purified using a Qiagen RNeasy plant mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer s instructions. RNA integrity was confirmed by electrophoresis on 1.5% agarose gels containing formaldehyde and quality was confirmed by analysis on an Agilent 2100 Bioanalyzer using RNA LabChip assays according to the manufacturer s instructions.
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Total RNA samples were labeled according to Affymetrix protocols that can be found at http://www.affymetrix.com/ support/technical/index.affx. cDNA was synthesized from total RNA followed by second-strand DNA synthesis using a 3 One-Cycle cDNA Synthesis kit (Affymetrix p/n 900431). Biotin labeled cRNA was synthesized using a MEGAscript IVT Labeling kit (Affymetrix p/n 900449), fragmented by Mg2+ hydrolysis, and hybridized for 16 h at 45C. Arrays were then washed using the GeneChip Fluidics Station 450 and scanned using the GeneChip 3000 Scanner. Spiking controls were added to the total RNA before cDNA synthesis and additional controls were added to the resulting cRNA prior to hybridization. Computational probe selection, masking, and annotation Probe annotation was updated by nucleotide sequence query of the probe consensus sequence against the UniProt/ TrEMBL protein database (version 28) (Bairoch et al. 2005) using Tera-BLASTP (double-banded SmithWaterman), word size 3, gap open penalty=11, extension penalty=1, and a minimum significance of 1e-05 using DeCypher programmable logic hardware (TimeLogic, Carlsbad, CA, USA) at the Nevada Center for Bioinformatics. Microarray data processing and analysis Data from all GeneChip oligonucleotide arrays were processed first by robust multiarray average (RMA) (Irizarry et al. 2003) using the BioConductor R package affy (Gautier et al. 2004). Specifically, expression values were computed from raw CEL files by first applying the RMA model of probe-specific background correction of perfect match (PM) probes. These corrected probe values were then normalized via quantile normalization across all arrays, and a median polish was applied to compute one expression measure from all probe values. Resulting RMA expression values were log2-transformed (please see the Affymetrix manual at http:// www.bioconductor.org/repository/devel/vignette/affy.pdf for details). To determine whether genes were differentially expressed between the two stress conditions and control across the temporal states, ANOVA was performed on the RMA expression values (Kerr et al. 2000). The following model was used for this analysis: yijk = Eik + Tjk +(ET)ijk + ijk, where yijk denotes the log2 signal measured for experimental state i, time j, and biological replicate k, with 1 i 3, 1 j 5, and 1 k 3. The terms Ei and Tj measure the effect of the environmental condition and time point, respectively, and the interaction term (ET)ij accounts for the interaction between environmental condition and time. An ANOVA was performed on each gene using the linear model above, and four contrasts based on differences
between each stress and control with respect to the last four time points (differences between each stress and control at time 0 were not of interest in this study). The R package limma (Smyth 2005) was used for ANOVA methods (http:// www.bioconductor.org/repository/devel/vignette/affy.pdf). A multiple testing correction (Benjamini and Hochberg 1995) was applied to the p-values of the F-statistics to adjust the false discovery rate. Genes with adjusted Fstatistic p-values <0.05 were extracted for further analysis. Clustering was performed using the Eisen Cluster and Treeview software (Eisen et al. 1998), using the Pearson correlation coefficient distance metric and the average agglomerative method. Probe set sequence details of the Vitis GeneChip oligonucleotide array and the raw data from the microarray experiment can be found at PlexDB (http://www.plexdb.org; experiment: VV2). Quantitative real-time reverse transcription-PCR RNA was extracted and its integrity verified as described above. cDNA was synthesized using an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer s instructions with a uniform 1 g RNA/ reaction volume reverse-transcribed. Primers for genes assayed by quantitative real-time reverse transcription (qRT)-PCR were selected using Primer3 software (Rozen and Skaletsky 2000). Quantitative real-time PCR reactions were prepared using an iTaq SYBR Green Supermix with ROX (Bio-Rad) and performed using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Expression was determined for triplicate biological replicates by use of serial dilution cDNA standard curves per gene. Metabolite analyses Tissues frozen in liquid nitrogen were completely ground and lyophilized. Polar metabolites were extracted and derivatized with a water/chloroform protocol as described (Broeckling et al. 2005). For gas chromatographymass spectrometry (GCMS), ribitol was used as an internal standard. Dried polar extracts were methoximated in pyridine with 120 l of 15.0 g l1 methoxyamine-HCl and derivatized with 120 l of Nmethyl-N-trimethylsilyltrifluoroacetamide +1% trimethylchlorosilane. Samples were analyzed at an injection split ratio of 10:1 with a Thermo Finnigan Polaris Q230 GC MS (Thermo Electron, Waltham, MA, USA). The inlet and transfer lines were held at 240 and 320C, respectively. Separation was achieved with a temperature program of 80C for 3 min, then ramped at 5C min1 to 315C and held for 17 min, using a 60-m DB-5MS column (J&W Scientific, 0.25 mm ID, 0.25 m film thickness) and a
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constant flow of 1.0 ml min1. All organic acids, sugars, and amino acids were verified with standards purchased from Sigma (St. Louis, MO, USA). For anion-exchange chromatography, the polar extractions were run on a Dionex high-performance liquid chromatography (Dionex, Sunnyvale, CA, USA). The column was a Dionex AS11-HC with a gradient of 1 mM to 60 mM NaOH over 40 min. The column was at room temperature with a flow rate of 0.27 ml min1 and the injection volume of each sample was 10 l. The detection methods used consisted of UV set at 210 nm and suppressed conductivity. All peaks were identified using known standards for organic acids and anion salts purchased from Sigma.
Fig. 1 The effects of water-deficit and salinity on the plant stem water potential over time. Data are means SE; n =6
Results Physiological responses This experiment was designed to differentiate water-deficit and salinity responses. This was successfully accomplished by allowing the pots to naturally dry out over time. Plant stem water potentials were measured every 2 days and the salinity of the solution added to the salinized plants was increased to mimic the drop in stem water potential of the nonirrigated plants. Plant stem water potentials decreased slightly in the initial phase of the experiment but dropped significantly relative to irrigated controls after day 8 (Fig. 1). There were no significant differences in stem water potential between water-deficit-treated and salinized plants at any time during the course of the experiment (p 0.29 and treatment day interaction was 0.45). Although stem water potentials were equal on day 16, water-deficit-treated plants began to wilt, but no wilting was observed in salinized plants. Shoot length proved to be a very sensitive measure of plant growth and a good indicator of plant strain (Vincent et al. unpublished results). Shoot length was reduced significantly by day 6 (Fig. 2) before significant differences were detected in stem water potential (day 8 in Fig. 1). Salttreated plants had slightly longer shoots than water-deficittreated plants (p 0.01). The interaction term with time (treatment time) was significant (p 0.01), indicating that the treatment effect depended upon time. The relative elongation rate of the shoot (Fig. 2, inset) was a more sensitive indicator of strain than shoot length. Inhibition of shoot relative elongation rates by the stress treatments was detectable within a day after treatment, indicating that shoot elongation was very sensitive to changes in soil water availability. Water-deficit significantly reduced the relative elongation rate of the shoot compared
to salinity and the irrigated controls (p 0.01). Differences between salinized plants and controls were much smaller, but these differences were compounded over time, resulting in differences in length by day 16. Consistent with shoot length, the interaction term with time (treatment time) was significant (p 0.01), indicating that the response to stress varied with time. Microarray analysis The microarray hybridizations were of high quality. The average present call rate across all 44 arrays was 76.3%. As recommended by the GeneChip Operating Software Users Guide, the levels of average background and noise (RawQ) were examined for consistency across all arrays. Average background was 84.4, well within the recommended levels. Average noise level was 2.57, with standard deviation 0.50, indicating that noise measures were consis-
Fig. 2 The effects of water-deficit and salinity on shoot lengths and relative elongation rate (inset) over time. Data are means SE; n =8
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tent across arrays, as advised. The hybridization controls BioB, BioC, BioD, and Cre were present 100% of the time, following the Affymetrix guidelines for quality control. Additionally, it was verified that signal intensities of BioC, BioD, and Cre increased, respectively. Lastly, 3 to 5 ratios of both actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were verified to be within Affymetrix guidelines: all actin ratios were less than 1.0; GAPDH ratios were consistently below 1.1. Images of all arrays were examined, and no obvious scratches or spatial variations were observed. A visual inspection of the distributions of raw PM probe level intensities for all 44 arrays showed one outlying array (day 12, control); preprocessed and normalized PM intensities [using RMA (Irizarry et al. 2003)] were consistent across all arrays, with no apparent outlying arrays (Supplemental Fig. S1). Digestion curves describing trends in RNA degradation between the 5 end and the 3 end in each probe set were examined, and all 44 proved very similar (Supplemental Fig. S2). Correlation between biological replicates was good (Supplemental Table S1; Supplemental Fig. S3). Pearson correlation coefficients and Spearman rank coefficients were computed on the RMA expression values (log2-transformed) for each set of biological replicates. Spearman coefficients ranged from 0.960 to 0.999; Pearson coefficients ranged between 0.970 and 0.999. Validation of microarray data by qRT-PCR The transcript abundance of 22 probe sets was quantified in shoot tissues by qRT-PCR. The quantitative data were normalized as a ratio to eIF4 expression and then calculated as a ratio of expression from stressed plants to control plants. The data were correlated to expression ratios from the GeneChip microarray. Linear regression indicated that there was a very good correlation between qRT-PCR and the microarray data with a goodness of fit (r2) of 0.91 (Fig. 3). Stress effects on transcript abundance ANOVA methods identified approximately 13,000 probe sets with significant F-statistics (p <0.05) after adjusting the false discovery rate, based on differences between water deficit and the nonstressed control state or salinity and control state (Supplemental Tables S2, S3, S4, S5, and S6). The large number of genes identified as significant in this experiment is likely due to several factors, including the severity of the stress, the reduction of noise within the high-quality data allowing for greater detection of significance, and not using an arbitrary twofold ratio screening filter.
Fig. 3 Linear regression of water-deficit or salinity to control treatment (WD/C or S/C) expression ratios measured by qRT-PCR and the Affymetrix GeneChip microarray
There were increased changes in gene expression with time because of increasing stress levels (Fig. 4). Water deficit had a greater impact than salinity on the number of transcripts that were significantly different from control. There was a small set of 14 transcripts with significant differences between water deficit and control plants on day 8 when stress levels were very low (Fig. 4; Supplemental Table S3); no significant differences were found between salinity and control on day 8. As the stress increased with time, so did the number of stress-responsive transcripts. After day 8, when stem water potentials decreased significantly, gene expression also changed significantly. On day 16, there were massive changes in gene expression with over 5,000 transcripts increased or decreased significantly by both stresses relative to control. For each of these two transcript sets on days 12 and 16, the set was reduced to a manageable level by extracting the top 10% of genes with the largest log2-transformed expression ratios (Supplemental Tables S7, S8, S9, and S10). The top 10% was determined based on the absolute value of the gene expression ratio as a way to impartially include genes with either an increase or a decrease in transcript abundance. The transcripts within these sets were also organized by hierarchical clustering (see Supplemental Tables S7, S8, S9, and S10). There was a large number of transcripts that differed between water deficit and salinity stress on both day 12 and day 16 (Fig. 5). On day 12, there were 858 probe sets common to both water deficit and salinity that were significantly different from control, 549 probe sets unique to salinity that were significantly different from control, and 2,302 probe sets unique to water deficit that were significantly different from control. On day 16, there were 9,064 probe sets common to both water-deficit and salinity that were significantly different from control, 1,290 probe sets unique to salinity that were significantly different from
Funct Integr Genomics (2007) 7:111134 Fig. 4 The number of probe sets in stressed plants with significant changes in gene expression (p <0.05; ANOVA) relative to that in control plants across time for water deficit and saltstressed plants. Overexpressed (Ox) transcripts are plotted above the x-axis, whereas underexpressed (Ux) transcripts are plotted below the x-axis
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control, and 1,877 probe sets unique to water deficit that were significantly different from control. In this study, unique refers to those genes that were statistically significant in one stress compared to control, but not in the other stress. It is possible that transcripts in the other stressed set showed increased or decreased expression ratios (compared to control) but the data were noisier and thus considered statistically insignificant. Most of the probe sets (83%) that were significantly different on day 12 were also significantly different from controls on day 16. There were 2,796 probe sets that were
Fig. 5 Venn diagrams representing the number of probe sets in the top 10% transcript sets of stressed plants that were significantly different from control on day 12 and day 16 (left side). All significant transcripts on day 12 were also compared to all significant transcripts on day 16 (right side). The arrows point to the number of probe sets that were common to both day 12 and day 16 for water-deficit or salinity, respectively
significantly affected by water-deficit and 914 probe sets that were significantly affected by salinity in this common set of probe sets between day 12 and day 16. The reduced sets of transcripts (top 10% of water deficit and salinity on day 12 and day 16) were divided into functional categories using the Munich Information Center for Protein Sequences (http://www.mips.gsf.de/) annotation categories (Fig. 6). Automated functional categorization was found to be unreliable and all functional categories were checked manually. Because the transcript sets were of very different sizes, the data are presented as a percentage of the total number of transcripts within the transcript set to reflect the relative changes for each day and treatment. Within the functional categories, the largest sets were for unclassified proteins (those lacking a homolog in the nonredundant database) metabolism, classification not yet clear-cut (homolog match found, but function remains obscure), and protein fate. The greatest differences in percentage of transcripts between water deficit and salinity were on day 12. By day 16, the percentage of transcripts represented in most functional categories between water deficit and salinity was more similar than on day 12. There was an increase in the percentage of transcripts for metabolism, transport and mechanisms, and biogenesis of cellular components from day 12 to day 16; water deficit affected a larger percentage of transcripts than salinity in these functional categories. More transcripts encoding energy-related functions were affected by salinity on day 12, whereas by day 16, more transcripts with energyrelated functions were affected by water deficit. There was a much larger percentage of transcripts involved in cell cycle and DNA processing that was affected by water-
118 Fig. 6 Analysis of Munich Information Center for Protein Sequences functional categories of the top 10% of all genes differentially expressed in stressed plants relative to control on day 12 and day 16. The percentage is based upon the number of probe sets in each set
deficit than by salinity on day 12 and there was little difference between both stresses on day 16. The percentage of transcripts for transcription, protein synthesis, and protein fate was more affected by salinity than by waterdeficit on day 12 and day 16. While there was little difference between days for transcription, both protein synthesis and protein fate were more affected on day 12 than on day 16. Protein activity regulation, communication/ signaling, and development transcripts were also more affected on day 12 than on day 16; here again, salinity affected these transcript sets more than water-deficit particularly for protein activity regulation. In order to identify differential responses of plants to water deficit and equivalent salinity, we performed an additional statistical comparison. Using ANOVA methods, water-deficit responses were compared to those of salinity (water deficit/salinity expression ratio) for all significantly expressed genes (approximately 13,000 probe sets that were significantly affected by the stresses relative to control). No significant differences were found between stresses on days 4, 8, or 12; however, 1,420 probe sets were differentially expressed between water deficit and salinity on day 16 (Supplemental Table S11). Analysis of the functional categories of the differentially expressed transcript set (Fig. 7) indicates that the largest
differences between these stresses are associated with unclassified or not yet clear-cut categories. Of the known functional categories, there were large differences in the number of transcripts involved in metabolism, protein fate, transport and mechanisms, transcription, cellular defense, and communication/signaling. A small sample of some of the significant transcripts is presented in Table 1. Transcripts were included that had the largest changes in expression on different days and with different stresses. For the most part, these highly expressed genes were associated with functional categories related to transcriptional activation, cellular defense, and stress responses. Almost all of these genes showed increased transcript abundance, but all of the genes in the salinity vs water deficit category showed a decrease in transcript abundance for both stresses, with water deficit resulting in a more severe reduction in transcript abundance than salinity stress. Early changes in gene expression The gradual application of increasing stress in this experiment allowed us to detect significant changes in gene expression at very low stress levels at an early phase of the stress. These early changes could provide insights into the early acclimation responses of the plants to the stress. One of the earliest
Funct Integr Genomics (2007) 7:111134 Fig. 7 Analysis of Munich Information Center for Protein Sequences functional categories of all genes differentially expressed between water-deficit and salinity-treated plants on day 16. The percentage is based on the number of transcripts in the set
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responses to water deficit is the increase in expression of RuBisCo activase (Fig. 8). RuBisCo activase transcript abundance increases in salinized plants, but at a later date. These results and those of several other transcripts were verified by qRT-PCR (Supplemental Fig. S4). The increase in RuBisCo activase transcript abundance indicates that photosynthetic regulation may be one of the earliest responses to decreased water potentials. Similarly, other transcripts involved in glycolysis and osmoprotectant metabolism (e.g., fructose bisphosphate aldolase, galactinol synthase) exhibited increased transcript abundance with water deficit at an earlier date than in salinized plants, suggesting that increased sugar or osmoprotectant production is an important early adaptation to water deficit stress (Fig. 8). Cystatin also exhibits an early (day 4) transient peak in expression followed by increasing transcript accumulation between days 8 and 16. Cystatins play various functional roles, including regulation of endogenous cysteine proteinases and as plant defensive proteins to biotic and abiotic stresses (Rassam and Laing 2004). The abundance of a dehydrin-like transcript (dehydrin 1a) encoding a group 2 late embryogenesis abundant (LEA) protein was higher with salinity stress at day 4 than water deficit, but was higher with water deficit stress at days 8 and 12. Although
the exact role of group 2 LEA proteins in stress adaptation remains enigmatic, this transient responsiveness to salinity stress may suggest a functional role for this LEA protein in osmotic stress adaptation. A putative mitochondrial carrier protein (ADP/ATP translocase) gene exhibits an early transient increase in transcript abundance largely in response to water deficit stress. The ADP/ATP translocator mediates adenine nucleotide exchange across the inner mitochondrial membrane (Millar and Heazlewood 2003). A lipid transfer protein gene also shows early, enhanced expression that increases with time. Although the precise roles of nonspecific lipid transfer proteins remain unclear, they have been implicated to function in cuticle formation, in pathogen defense as efficient antifungal agents, and in long-distance systemic resistance signaling (Blein et al. 2002). Early changes in transcription factors The expression patterns of approximately 200 transcription factors on the Vitis GeneChip were significantly affected by both stresses relative to controls. Approximately twothirds of the transcription factor genes showed increased transcript abundance, whereas one-third showed decreased transcript abundance. Almost all of these transcription
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Table 1 Transcripts with the greatest change in transcript abundance in response to water deficit or salinity stress on days 8, 12, and 16 Treatment day Probe set ID UniProt ID Top BLASTX hit in UniProt database Best identity description (organism) E-value MIPS 2.0 FunCat
Water deficit vs controlday 8 1610772_at Q941D7 1621585_at Q9LRC7 1612845_at Q8H6R2 Water deficit vs controlday 12 1620438_at O23547 1616695_s_at Q9SPE0 1610772_at Q941D7 1613946_s_at Q9M276 1615001_s_at Q941D7 Salinity vs controlday 12 1606901_at Q9LHJ3 1616198_at Q3LOQ8 1613698_at Q6RZW8 1622598_at Q9LXM2 1616990_s_at Q43681 Water deficit vs controlday 16 1607412_at Q6TPK3 1622844_at Q6YTL8 1616555_s_at Q54DD6 1615595_at Q9M563 1611233_at Q4W7I3 Salinity vs controlday 16 1620438_at O23547 1611272_at Q6QB11 1615595_at Q9M563 1621876_at Q52QR5 1611233_at Q4W7I3 Water deficit vs salinityday 16 1607412_at Q6TPK3 1616666_at O82049 1608026_at Q9FIC9 1611891_at Q8L7B2 1622203_at P93378 Salinity vs water deficitday 16 1609154_at O22612 1621879_at Q8LEU7 1611222_at Q9FUP3 1619204_at Q58FS3 1609901_at Q9SA68
Hypothetical protein (Arabidopsis thaliana) BZIP transcriptional activator RSG (Nicotiana tabacum) CTV.15 (Poncirus trifoliata) Expansin-related protein 1 precursor (Arabidopsis thaliana) Thaumatin (Vitis riparia) Hypothetical protein (Arabidopsis thaliana) Homeobox-leucine zipper protein ATHB-12 (Arabidopsis thaliana) Hypothetical protein (Arabidopsis thaliana) Hypothetical protein At3g22240 (Arabidopsis thaliana) ERF4 (Gossypium hirsutum) Putative ERF4 (Vitis vinifera) Putative CCR4-associated factor 1 (Arabidopsis thaliana) Probable nonspecific LTP AKCS9 precursor (Vigna unguiculata) Cystatin (Actinidia deliciosa) Hypothetical protein OSJNBa0067K15.36 (Oryza sativa) Hypothetical protein (Dictyostelium discoideum) Beta-1,3-glucanase (Vitis vinifera) Alpha-L-arabinofuranosidase/beta-D-xylosidase (Pyrus pyrifolia) Expansin-related protein 1 precursor (Arabidopsis thaliana) Little protein 1 (Oryza sativa) Beta-1,3-glucanase (Vitis vinifera) NAC1 (Glycine max) Alpha-L-arabinofuranosidase/beta-D-xylosidase (Pyrus pyrifolia) Cystatin (Actinidia deliciosa) Mitochondrial carrier protein (Ribus nigrum) Germin-like protein subfamily 1 member 15 (Arabidopsis thaliana) Serine carboxypeptidase 1-like protein (Arabidopsis thaliana) Tumor-related protein (Nicotiana tabacum) Dormancy-associated protein (Pisum sativum) Hypothetical protein (Arabidopsis thaliana) Kunitz trypsin inhibitor protein (Phaseolus coccineus) RADIALIS (Antirrhinum majus) F10O3.16 protein (Nicotiana tabacum)
5E-23 3.2E-37 2.9E-40 5.9E-64 3.1E-147 5E-23 8.5E-26 5E-23 7E-07 1.3E-36 2.3E-96 2E-112 8.4E-23 1.4E-08 0 5.7E-08 2.4E-199 8.6E-112 5.9E-64 2.2E-26 2.4E-199 5E-23 8.6E-112 1.4E-08 2E-57 2.4E-37 2.9E-43 1.5E-71 0 6.5E-19 5.4E-27 3.2E-26 1.3E-25
Unclassified Transfactor Stress Cell growth Defense Unclassified Transfactor Unclassified Unclassified Transfactor Transfactor Transfactor Lipid transport Defense Unclassified Unclassified Defense Wall metabolism Cell growth Unclassified Defense Transfactor Wall metabolism Defense Transport Defense Proteolysis Defense Unclassified Unclassified Stress Transfactor Stress
Transcripts with decreased relative abundance are indicated by bold emphasis. Relative abundance of all other transcripts was stressed-induced.
factors showed late changes in gene expression on day 16, when stress was severe. Very few showed large changes before day 16. Both a NAC1 and a NAC3 transcription factor showed substantial upregulation at day 8 under water deficit stress with a similar but lagging response of transcript accumulation under salinity stress (Fig. 8). The response of the NAC3 transcription factor was verified by qRT-PCR (Supplemental Fig. S4). The NAC domain family (having a DNA-binding domain and a dimerization domain) of plant-specific transcriptional regulators is involved
in developmental processes, including the formation of the shoot apical meristem, floral organs, and lateral shoots, as well as in plant hormonal control and defense responses (Olsen et al. 2005). A C-repeat binding factor/dehydration responsive element binding (CBF/DREB) protein homolog showed a slight increase in expression on day 8 and continued to increase on days 12 and 16. CBF/DREB family transcriptional activators play important roles in activation (or repression) of cold and water deficit response pathways (Stockinger et al. 1997; Liu et al. 1998). There
Funct Integr Genomics (2007) 7:111134 Fig. 8 Some of the most significant transcripts with early changes in expression in response to water deficit and salinity. Intensities are expressed relative to control values. Names in the legend refer to the probe set ID on the GeneChip, the UniProt description, and the treatment (WD water deficit). Data are means; n =3
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were 41 transcriptional activators that differed significantly between stresses (Supplemental Table S11); 26 of these were more highly expressed by salinity and 15 of these were more highly expressed with water deficit.
Changes in hormone metabolism A number of genes related to hormone metabolism and response showed increased or decreased transcript abun-
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dance relative to the nonstressed state (Table 2). The largest number of transcripts affected by stress was associated with abscisic acid (ABA) (Table 2). Genes associated with ethylene metabolism and responses were the next most represented class showing increased transcript abundance. Most of the genes affected for these two stress hormones exhibited increased transcript abundance. The two genes associated with upregulation of gibberellic acid metabolism (Table 2) were GA2-oxidases associated with GA1 catabolism. Transcript abundance of 26 hormone-related genes differed significantly between water deficit and salinity (Supplemental Table S11). The temporal mRNA expression of genes encoding key enzymes of four hormone pathways was compared in Fig. 9. Transcript abundance of 9-cis-epoxycarotenoid dioxygenase (NCED), a key regulatory enzyme in ABA biosynthesis (Iuchi et al. 2001), is affected earlier and to a greater amount (compared to control) than the transcripts of genes encoding key biosynthetic enzymes of the three other pathways (e.g., ethylene, jasmonate, and gibberellin). The responses of the NCED and GA2-oxidase genes were verified by qRT-PCR (Supplemental Fig. S4). Water-deficit treatment caused increased transcript abundance for GA2 oxidase to a significantly greater amount than did salinity (Fig. 9). Differential stress effects on osmolytes Metabolite profiling analysis conducted in parallel with the mRNA profiling indicated that the relative abundance of a number of metabolites was substantially altered by stress over time. Of the 12 organic acids, 19 amino acids, and 15 sugars analyzed, malate, proline, and glucose concentrations were increased the most by water deficit and showed the greatest differences when compared with salinized plants (Table 3, Fig. 10). The two- to threefold increase in proline accumulation in V. vinifera leaves (Table 3) was consistent with an increase in transcript abundance for delta 1-pyrroline-5-carboxylate synthetase (P5CS), the enzyme that catalyzes the first two steps in the proline biosynthetic pathway (Table 4; Supplemental Tables S5 and S11). Increased transcript abundance of proline dehydrogenase (BQ792635, Q8W415) also accompanied the induction of P5CS transcripts presumably to facilitate the removal of excess proline accumulation,
which can be toxic to the cell if allowed to over accumulate (Deuschle et al. 2004). Glucose and fructose exhibited increased accumulation in response to water deficit stress, as has been observed in Arabidopsis (Rizhsky et al. 2004). However, unlike Arabidopsis, sucrose amounts did not become elevated in V. vinifera in response to water deficit (Table 3). In V. vinifera, tartrate accumulation, the most abundant organic acid in grapevine leaves (Kliewer and Nassar 1966), increased differentially in response to water deficit relative to salinity (128 vs 96% of control, respectively). Consistent with the relatively minor changes in tartrate concentrations, there were small but significant differences between water deficit and salinity in transcript abundance for L-idonate dehydrogenase (TC45858, Q9MBD7; Supplemental Table S11), the committed biosynthetic step in the L-tartaric acid pathway (DeBolt et al. 2006). In addition, malate, the second most abundant organic acid in grape, exhibited large differential increases in response to water deficit vs salinity stress (473 vs 182%). Clues to the specific isogenes likely to be responsible for the observed increase in malate accumulation under water deficit stress could be derived from mRNA expression data. Glyoxosomal (MDGH; TC39850, Q9ZP05, P46488) and chloroplastic malate dehydrogenases (MDH1; TC39041; O48902) exhibited increased transcript abundance, whereas the cytoplasmic (TC48145, Q645N0; TC46416, Q9M6B3; TC45217, Q9FT00; TC45218, Q9FT00; TC45370, Q9M6B3; CF202356, Q9FT00) and mitochondrial (TC49596, O48906; TC40112, O48906) isogenes exhibited decreased transcript abundance (Table 4; see also Supplemental Table S5). These expression patterns are consistent with the observed increase in malate concentrations under stress. Differential stress effects on transport Both stresses substantially increased the abundance of transcripts encoding for a variety of ion, amino acid, nucleotide, and peptide transporters. In particular, transporters for nitrate, nitrite, sulfate, proline, ATP, amino acids, and organic acids exhibited greater transcript accumulation in response to water deficit compared to salinity (Table 4, Fig. 10, Supplemental Table S11). For example, a plasma membrane proline transporter (TC41212, P92961) exhibited increased transcript
Table 2 Number of known hormone-related probe sets on the Vitis GeneChip corresponding to hormone biosynthetic, catabolic, and response pathways with significant changes in mRNA expression affected by either salinity or water-deficit stress treatments Expression Up Down Auxin 8 8 ABA 25 3 Brassinosteroid 1 3 Cytokinin 4 4 Ethylene 10 3 GA 5 3 JA 4 2 SA 1 1
GA gibberellic acid, JA jasmonic acid, SA salicylic acid
Funct Integr Genomics (2007) 7:111134 Fig. 9 Some significant transcripts in hormone metabolism affected by water deficit and salinity over time. ABA abscisic acid, NCED 9-cis-epoxycarotenoid dioxygenase, JA jasmonic acid, OPR 12-oxophytodienoate reductase, GA gibberellic acid. Names in the legend refer to the probe set ID on the GeneChip, the UniProt description, and the treatment (WD water deficit). Data are means; n =3
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abundance particularly in response to water deficit stress (Table 4; Supplemental Table S5). A plasma membrane sulfate transporter (TC47443, Q6ZZ94) is greater than 2.5fold more induced by water deficit than by salinity stress. In contrast, salinity significantly increased the abundance of transcripts encoding a potassium transporter 4 (AtPOT4; TC41344, Q9LD18) and a plasma membrane intrinsic protein (aquaporin, PIP2-1; TC38138, O24049, Q9AVB5), whereas water deficit significantly reduced the transcript abundance of these genes relative to control plants (Supplemental Table S11). This result is consistent with the observation that mRNA expression of all members of the aquaporin gene family in Arabidopsis exhibit reduced transcript abundance following water deficit stress (Alexandersson et al. 2005). Differential stress effects on energy metabolism Analysis of the genes affecting energy metabolism revealed that there were many differences in transcript abundance between water deficit and salinized plants (Table 4, Fig. 10). Water deficit results in a decrease in foliar photosynthesis (Reddy et al. 2004). This decrease is apparently RuBisCo associated with decreased access to atmospheric CO2 uptake due to stomatal closure, decreases in amount and activity (Parry et al. 2002), and downregulation of photosystem activities. Interestingly, transcripts encoding specific photosystem I and II components and Calvin cycle enzymes were observed to increase twoto threefold relative to controls, especially for water-deficittreated plants on day 16 (Table 4). The relative abundance
Table 3 Relative quantities (percentage of control) of some key metabolites involved in energy metabolism and osmotic adjustment on day 16 Metabolite Malate Citrate Isocitrate Succinate Fumarate Tartrate Nitrate Sulfate Chloride Phosphate Proline Glutamate Glutamine Glycine Serine Asparagine Aspartate Cysteine Fructose Glucose Sucrose Water deficit 473* 144 175 20 84 129 nd 125 103* 120* 295* 125 108 183 115 213 40 100 186 543* 73 Salinity 182 101 125 20 77 96 nd 143 186 207 202 118 120 164 100 190 60 93 179 152 79
Significantly reduced metabolites relative to control are in bold. Data are means; n =3 nd not detectable; concentration was below the limits of detection *Water-deficit metabolite concentration significantly different (p <0.05; ANOVA) from that of salinity.
124 Fig. 10 Map of the effects of water deficit and salinity on transcripts and metabolites involved in energy metabolism and osmotic adjustment on day 16. Metabolites (ovals) are indicated in green if metabolite concentration declined or in orange if metabolite concentration increased due to stress relative to control (percentage of control in Table 3), with darker shading representing increasing concentrations. Each oval is separated in half with the left half representing the water-deficit-treated plants and the right half representing the salinized plants. No color means either no induction or that the metabolite was not measured. If no color differences are apparent, then the concentrations for both treatments were approximately the same. Relative transcript abundance of key enzymes (boxes) is indicated in green if transcript abundance declined and in red if transcript abundance increased, with darker shading representing increasing abundance. No color means either no induction or that the transcript was not measured. Various cell compartments in which these processes are taking place are labeled. Membrane localized transporters are indicated as small ovals and color coded as for enzymes, except that salinity and waterdeficit stress are not distinguished by color coding. Abbreviations are the same as those in bold in Table 4
of many transcripts involved in the photorespiratory pathway, gamma-aminobutyrate (GABA) metabolism, and the associated assimilation of NH 4 into amino acids were also increased highly in the chloroplasts, peroxisomes, and mitochondria (Fig. 10 and Table 4). Although water-deficittreated plants typically had higher transcript abundance relative to salinized plants for many of these transcripts, the differences between the two stresses were not significant. Fatty acid metabolism and glyoxylate cycle enzymes also exhibit elevated transcript abundance (Table 4), presumably to facilitate the conversion of acetyl-CoA formed from lipid catabolism into malate for gluconeogenesis.
Differential stress effects on nonphotochemical quenching and reactive oxygen scavenging Under salinity and water deficit stress, the dissipation of excess absorbed light energy or nonphotochemical quenching of chlorophyll fluorescence, is critical to the prevention of photooxidative damage of the photosynthetic apparatus (Niyogi et al. 1998). Therefore, the observed accumulation of transcripts encoding xanthophylls cycle enzymes, zeaxanthin epoxide, and violaxanthin de-epoxidase (Table 4), particularly under water-deficit stress conditions, is likely to be a response for increased synthesis of xanthophylls
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Table 4 A selection of key transcripts involved in energy metabolism and osmotic adjustment organized by their subcellular compartments that showed significantly increased or decreased abundance by stress on day 16 Cellular compartment Top BLASTX hit in nonredundant UniProt protein database MIPS 2.0 Transcript abundance (% of control) Water deficit Salinity
Probe set ID
UniProt ID
Best identity description (EC)
E-value
FunCat
Chloroplast 1618679_s_at Q9BBT1 Photosystem II 44 kDa reaction center protein (P6 protein) (CP43) 1622286_at Q9XQA8 Photosystem II D2 protein 1614462_at Q9MTN4 Photosystem II CP43 protein 1613786_at Q6QY10 Photosystem I P700 chlorophyll A apoprotein A1 1609916_s_at P00823 ATP synthase alpha chain (EC 3.6.3.14) 1620373_at Q8LD49 Thioredoxin X, chloroplast precursor 1620551_s_at O98997 RuBisCo activase, chloroplast precursor 1613102_s_at P27774 Phosphoribulokinase, chloroplast precursor (EC 2.7.1.19) 1609904_at Q9SAU2 Ribulose-5-phosphate-3-epimerase (EC 5.1.3.1) 1622041_at Q84JG8 Sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37) 1618171_s_at Q5SGC9 Zeaxanthin epoxidase (EC 1.14.13.90) 1615888_at Q8S4C2 Violaxanthin de-epoxidase (EC 1.10.99.3) 1619072_at P52032 Phospholipid hydroperoxide glutathione peroxidase (EC 1.11.1.12) 1614207_at Q9LKQ9 Asparagine synthetase (EC 6.3.5.4) (ASN1) 1609754_s_at Q43859 Glutamate synthase (Ferredoxin) (EC 1.4.7.1) (GLU1) 1614158_at Q6L3J2 Putative nitrite transporter 1611167_at Q93ZN6 MXK3_6 1621397_at P46644 Aspartate aminotransferase, chloroplast precursor (EC 2.6.1.1) 1613433_at Q9C8I0 NADP-specific glutatamate dehydrogenase, putative (EC 1.4.1.4) 1611274_at O48902 Malate dehydrogenase [NADP], chloroplast precursor (EC 1.1.1.82) (MDH) Glyoxysome/peroxisome 1609923_at Q9LRR9 Glycolate oxidase (EC 1.1.3.15) (GOX2) 1610871_s_at P17598 Catalase isozyme 1 (EC 1.11.1.6) (CAT1) 1616659_at O49124 Putative serine-glyoxylate aminotransferase (EC 2.6.1.44) (AGT1) 1617382_s_at Q93XV7 Hydroxypyruvate reductase 1 (EC 1.1.1.29) (HPR) 1615976_at Q08375 Acetyl-CoA acyltransferase (EC 2.3.1.16) (PED1) 1613332_at 1612913_at Mitochondrion 1618497_at 1607193_at 1617293_s_at 1616666_at 1613627_at
1.1E-79 6.7E-95 1.2E-29 0 0 4.2E-12 3.6E-27 2.3E-192 1.1E-71 4.6E-110 4.7E-58 3E-75 7.60E-89 0 3.2E-29 1.6E-55 7.3E-183 8.4E-120 0 0
Photosynthesis Photosynthesis Photosynthesis Photosynthesis Photosynthesis Photosynthesis Photosynthesis Calvin cycle Calvin cycle Calvin cycle Xanthophyll cycle Xanthophyll cycle Radical detoxification N metabolism N metabolism N transport ABC transporter Amino acid metabolism Amino acid metabolism Malate metabolism
335* 311* 252* 210* 217* 169* 949 237 249* 238* 318* 233* 188* 1131 422 230 370* 214 175 196
133 127 120 120 128 102 336 141 100 103 121 137 130 1330 205 158 186 214 164 140
7.1E-50 8.6E-287 3.6E-200 3.5E-203 5E-23
Photorespiration Photorespiration Photorespiration Photorespiration Fatty acid metabolism Glyoxylate cycle Glyoxylate cycle Photorespiration Respiration Proline catabolism Nucleotide transporter GABA shunt TCA cycle Glycolysis Gluconeogenesis Gluconeogenesis N transport
266 542 295 241 165 337 177 288* 756 372* 982* 143 46 49* 202 2535 603*
157 246 257 169 178 139 192 153 614 148 126 176 44 78 222 779 409
Q9ZP05 Malate dehydrogenase, glyoxysomal precursor (EC 1.1.1.37) 1.1E-57 (MDHG) P49299 Citrate synthase, glyoxysomal precursor (EC 2.3.3.1) (CS) 4.2E-54 O49954 Q9M432 Q8W415 O82049 Q84P54 Glycine decarboxylase (EC 1.4.4.2) (GDC) Alternative oxidase Proline dehydrogenase Mitochondrial carrier protein 4.5E-23 8.30E-56 5.8E-46 2E-57 2.1E-110 4.9E-175 3.0E-233 1.50E-129 3.2E-120 9.2E-29
GABA transaminase subunit isozyme 1 (EC 2.6.1.19) (GABAT) 1622059_at Q9M6B3 Mitochondrial malate dehydrogenase (EC 1.1.1.37) Cytoplasm and plasma membrane 1620865_at Q7XBE4 Enolase (EC 4.2.1.11) 1616630_at Q94LX9 Phosphoenolpyruvate carboxykinase (EC 4.1.1.49) 1609678_at Q6RUF6 Fructose-bisphosphate aldolase (EC 4.1.2.13) 1618132_at O81393 Nitrate transporter NTL1
126 Table 4 (continued) Cellular compartment Top BLASTX hit in nonredundant UniProt protein database
MIPS 2.0
Transcript abundance (% of control) Water deficit 272 265* 166 317* 178 125 535* 207 152* 52* 49 Salinity
Probe set ID
UniProt ID Q9FUC2 P51118 P54767 Q84MA5
Best identity description (EC)
E-value
FunCat
1615159_s_at 1612789_at 1608251_at 1612194_at
Nitrate reductase (EC 1.7.1.1) (NIA2) Glutamine synthetase cytosolic isozyme 1 (EC 6.3.1.2) (GS1) Glutamate decarboxylase (EC 4.1.1.15) (GAD) Amino acid transport protein AAT1 Proline transporter 2
1.8E-38 7.5E-22 7.5E-45 5.5E-16 1.3E-37 0 1.60E-29 3.2E-39 8.7e-312 1.7E-241 4.9E-175
1611892_s_at P92961
1619565_at Q9XGC4 Pyrroline-5-carboxylate synthetase (EC 2.7.2.11) (P5CS) 1620065_at Q6ZZ94 Plasma membrane sulfate transporter 1616400_s_at Q6F4I8 Gamma-glutamylcysteine synthetase (EC 6.3.2.2) (GSH1) 1617060_s_at O22511 Glutathione reductase (NADPH) (EC 1.6.4.2) (GSHR)
1611103_at O23946 Phosphoenolpyruvate carboxylase 1 (EC 4.1.1.31) 1606799_s_at Q9FT00 Cytosolic malate dehydrogenase (EC 1.1.1.37)
N assimilation N assimilation GABA shunt Amino acid transporter Amino acid transporter Proline biosynthesis Sulfate transporter Glutathione biosynthesis Glutathione metabolism TCA cycle Malate metabolism
174 109 178 159 122 133 185 181 101 67 52
Abbreviated names are in bold and are used in Fig. 10. Data are means; n =3 *Water deficit transcript abundance significantly different (p <0.05; ANOVA) from that of salinity.
pigments. Transcript abundance of genes involved in reactive oxygen species (ROS) detoxification (Mittler et al. 2004), such as phospholipid hydroperoxide glutathione peroxidase (TC45235, O48646), gamma-glutamylcysteine synthetase, and NADPH glutathione reductase, were also increased by both stresses, but preferentially by water-deficit stress (Table 4, Supplemental Tables S5 and S6). A mitochondrial alternative oxidase (TC49428, Q9M432) is also induced by both stress treatments (Table 4, Supplemental Table S5, Fig. 10). Several photorespiratory enzymes of the glyoxysome/peroxisome exhibit elevated transcript abundance in response to both water deficit and salinity stress. These enzymes carry out important functional roles not only in the metabolism of oxygen free-radicals, but may also participate in abiotic stress signaling (Corpas et al. 2001; Moreno et al. 2005). The elevated expression of the GABA transaminase subunit isozyme 1 (GABAT) (Table 4) under both water deficit and salinity stress suggests the need for elevated activity of the GABA shunt, which provides succinate and NADH to the respiratory machinery and plays a key role in limiting the accumulation of reactive oxygen intermediates during times of environmental stress (Bouche et al. 2003).
Discussion There has been considerable discussion on the comparative effects of salinity and water deficits on plants (Bernstein and Hayward 1958; Wyn Jones et al. 1979; Greenway and
Munns 1980; Yeo 1983; Bohnert and Cushman 2002; Munns 2002). Both water deficit and salinity impose osmotic effects on plants. Salinity differs from water deficit in that in addition to lowering the water potential of the plant, it also imposes high salt concentrations in the root zone, which can lead to additional effects, such as ion toxicity. However, high ion concentrations can be an advantage to plants if the ions are compartmentalized, removing them from important metabolic events. As the vacuole can make up approximately 90% of the cell volume of a mature cell, ions can act as cheap osmolytes in the vacuole. Ions are considered cheap because the plant does not have to synthesize them; therefore, less energy is needed to produce solutes for osmotic adjustment as compared with an organic compatible solute such as proline or glycine betaine (Yeo 1983; Raven 1985). One of the unique aspects of this study was the experimental design that allowed us to compare equivalent salinity and water-deficit effects on grapevine. Plants were stressed in a more natural, gradual way. Water deficit was imposed on the potted plants by withholding all irrigation. As the plants transpired, the water potential of the soil and the plant decreased with time. This is not a unique experiment in itself, but the comparison of this treatment to a parallel, gradually increasing salinity treatment that had equivalent water potentials over time is unique. This experimental design allowed us to separate the responses to water deficit effects (decreases in water potential) from those arising from ionic effects within the plant.
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The experiment revealed that grapevine growth was more sensitive to water deficit than to an equivalent salinity level. This suggests that the uptake of salt may have contributed to osmotic adjustment, facilitating water uptake and growth in the young shoots. The water potential gradient in the growing zone of plants is crucial for sustained growth, particularly with osmotic stress (Tang and Boyer 2002). Solute uptake and osmotic adjustment are necessary for the maintenance of this gradient (Fricke 2002; Fricke and Peters 2002). Grapevine is considered to be sensitive to salinity (Maas and Hoffman 1977; Hawker and Walker 1978; Walker et al. 1981, 2002; Shani et al. 1993) withstanding drought to a greater extent than salinity over much longer periods of time. This difference in tolerance at longer periods of time is likely due to the plants inability to cope with high internal ion concentrations and the resultant ion toxicity (Munns 2002). Stress effects on energy metabolism Long-term salinity can reduce photosynthesis and increase photorespiration in grapevines (Walker et al. 1981). Based on the observed changes in metabolite and transcript abundance, it is clear that photosynthesis and photorespiration were significantly affected in the stressed plants of this study (Fig. 10; Table 4). Transcript abundance of RuBisCo activase was increased very early with only slight differences in water status. RuBisCo activase is a key regulator of photosynthesis (Mott and Woodrow 2000; Parry et al. 2003). Under normal conditions, only 85% of the RuBisCo pool is activated by RuBisCo activase in tobacco (Mott and Woodrow 2000). Photosynthesis is modulated up and down by RuBisCo activase with changing environmental conditions. As stomatal conductance decreases with water deficit, internal CO2 concentrations in the leaf are predicted to be reduced, thus causing a slower rate of photosynthesis. Under these conditions, increases in RuBisCo activase could improve photosynthetic efficiency by increasing the amount of RuBisCo that is activated for CO2 fixation, thus compensating for the reduced stomatal conductance. Decreased RuBisCo activity under water deficit stress has been shown to be due to the presence of tight-binding inhibitors, which are removed by the action of RuBisCo activase and ATP hydrolysis (Tezara et al. 1999; Parry et al. 2002). Rates of photosynthesis could also be impaired by the ability of the plant to regenerate RuBP, which could also arise from inadequate ATP supply and/or NADPH to the Calvin cycle. The increased expression of Calvin cycle enzymes observed in this study, particularly under water-deficit conditions, could be interpreted as a compensatory mechanism under stress conditions to restore RuBP regeneration.
Under water deficit stress conditions that reduce photosynthetic rates, plants in normal high light conditions will suffer from photoinhibition (Wingler et al. 2000; Reddy et al. 2004). Photochemical reactions within photosystem II are known to be susceptible to water deficit stress. In this study, as the stress levels increased, there was a pronounced increase in transcript abundance (especially for water deficit) for genes involved in the electron transport chain of photosystem I and II (Table 4). This response may reflect the need to replace labile components of these photosystems that are susceptible to attack by active oxygen species. The importance of photorespiration during osmotic or water deficit stress was highlighted by the increased transcript accumulation of photorespiratory enzymes (Table 4; Fig. 10). The increase in glycolate oxidase and catalase expression in the peroxisome provided strong evidence that photoinhibition was increased in the stressed plants. As CO2 fixation is decreased during water deficits, plants must cope with the excess light energy or photoinhibition, the production of ROS, and the severe leaf damage that results. Photorespiration provides an alternative route for electron flow that helps prevent photoinhibition and scavenge ROS (via catalase and the production of glutathione) (Wingler et al. 2000; Noctor et al. 2002). Also important to the maintenance of photorespiration is the assimilation of NH 4 in the chloroplasts that was produced in the mitochondria. This is clearly supported by our data (Fig. 10). Interestingly, transcript abundance for nitrate transport and assimilation is also stimulated, indicating that the pathway has additional nitrogen inputs, perhaps for additional amino acid synthesis such as for proline and arginine. As nitrate is assimilated, the synthesis of malate is important to provide charge balance. Thus, malate synthesis may serve to not only enhance osmotic adjustment and energy supply, but also to facilitate amino acid biosynthesis. Stress effects on ROS metabolism In addition to photorespiratory enzymes, our study has revealed several other components of ROS metabolism that play important roles in abiotic stress adaptation. Glutathione is a very important scavenger of ROS (Mittler et al. 2004). The expression of the enzyme for the first step in glutathione biosynthesis from glutamate, gamma-glutamylcysteine synthetase (CF206159, Q6F4I8) was significantly greater with both water deficit and salinity stress in this study (Table 4; Supplemental Tables S5 and S6). Cytosolic glutathione reductase (TC48767, Q43621), which uses NADPH to regenerate oxidized glutathione or ascorbate, was preferentially expressed in response to water deficit stress, as has been demonstrated previously in Arabidopsis
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(Mittler et al. 2004). Phospholipid hydroperoxide glutathione peroxidase (TC45235, O48646) was also significantly induced by water-deficit stress over salinity stress. In addition, the GABA shunt pathway enzyme GABA transaminase and mitochondrial alternative oxidase, which are known to help protect plants from ROS, showed increased mRNA expression in response to both salinity and water in this study (Moller 2001; Bouche et al. 2003; Fiorani et al. 2005; Umbach et al. 2005). The observed increased mRNA expression of the xanthophyll cycle enzymes zeaxanthin epoxide and violaxanthin de-epoxidase (Table 4), particularly under water-deficit stress conditions, is likely to be important to prevent photooxidative damage of the photosynthetic apparatus via nonphotochemical quenching (Niyogi et al. 1998). Finally, several photorespiratory enzymes of the glyoxysome/peroxisome, which play important roles in ROS (Corpas et al. 2001; Moreno et al. 2005), exhibited elevated transcript abundance in response to both water deficit and salinity stress. This study has confirmed that the mRNA expression of many enzymes associated with the glyoxylate cycle, which is important for fatty acid oxidation for energy production, and photorespiration, which is important for the recycling of photorespiration products, are elevated in response to salinity and water deficit stress. However, the exact subcellular compartmentation of these enzymes between peroxisomes and glyoxysomes as diagrammed must remain fuzzy for the time being until additional experimentation can be performed (Fig. 10). This is because peroxisomes are known to be converted into glyoxysomes and vice versa during transitional states depending on the developmental age of the plant organ or upon exposure to environmental stress (Olsen and Harada 1995; Corpas et al. 2001; Kamada et al. 2003; Jeong et al. 2004). Both of these transitional conditions were likely to have been present in this study with the imposition of stress over time on young, developing shoot tips. Stress responses and ABA ABA is a sensitive signaling molecule in plants allowing the plant to adjust to a variety of stressful conditions (Wilkinson and Davies 2002). A large number of transcripts involved in ABA metabolism or responsive to ABA were increased with water deficit or salinity over time (Table 2). While changes in NCED expression were not significant until day 8 in water-deficit-treated plants, increases in ABA are not only dependent upon increases in ABA biosynthesis, but also influenced by ABA redistribution and transport (Wilkinson and Davies 2002). Thus, ABA may be affecting the grapevines earlier than day 8 in this study. Likewise, the observed transcript abundance changes in ethylene biosynthetic, catabolic, and response pathways suggest that this plant growth regulator plays a critical role in abiotic
stress responses as previously documented (Kim et al. 2003; Zhao and Schaller 2004). Very small changes in water status can have significant and rapid effects on leaf elongation and stomatal conductance (Passioura and Munns 2000). In drought-stressed plants, decreases in leaf elongation and stomatal conductance were correlated with increases in xylem sap pH, which led to increases in ABA concentrations in the xylem (Hartung et al. 1988; Bacon et al. 1998). Furthermore, diurnal changes in the accumulation of ABA from the xylem were linked to midday closure of stomata in field-grown grapevines (Loveys and During 1984), indicating that sensitive stomatal regulation occurs in grapevines throughout the day. Stress effects on transcription factors Responses to salinity and water deficit stress are controlled by transcriptional regulatory networks (Yamaguchi-Shinozaki and Shinozaki 2006). In osmotic shock experiments, CBF/ DREB transcription factors were shown to respond early to stress within minutes in the signaling pathway and provided improved stress tolerance (Liu et al. 1998). In this study, the relative transcript abundance of most transcription factors did not change significantly until day 16, with the earliest changes detected only at day 8, well after stress effects on growth were apparent. For example, changes in transcript abundance for a DREB-like transcription factor increased steadily with time, but were not significantly different until day 16 (Fig. 8; Supplemental Tables S5 and S6). CBF/DREB family transcriptional activators are important for the activation (or repression) of cold- and water-deficit-response regulons that control the expression of a large number of downstream response genes (Stockinger et al. 1997; Liu et al. 1998). In contrast to other transcription factors, NAC1 and NAC3 had one of the earliest and largest changes in gene expression in response to stress (Fig. 8). A number of other NAC transcription factors were affected as well (Fig. 8; Supplemental Table S4). NAC transcription factors belong to a large and functionally diverse family (Olsen et al. 2005). Although our knowledge of the role of NAC transcription factors in abiotic stress tolerance remains limited, overexpression of one gene family member from Arabidopsis can lead to ABA hypersensitivity (Fujita et al. 2004) and provided improved drought tolerance (Tran et al. 2004). However, a number of other transcription factors known to have important roles in abiotic stress adaptation were revealed by our study. For example, a V. vinifera homeobox-leucine zipper protein gene (CF202637, Q9M276), whose mRNA is induced by both salinity and water deficit (Supplemental Tables S3, S4, S5, and S6), is related to the ABA- and salt-responsive Arabidopsis ATHB-12 gene, which, when expressed in yeast, results in
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improved salinity tolerance presumably by regulating sodium exclusion (Shin et al. 2004). Other V. vinifera salinity and water-deficit-responsive genes included a MYB-related protein (TC39225, P81392; Supplemental Tables S3, S4, S5, and S6) (Abe et al. 2003), a basic leucine zipper transcription factor protein (TC47329, Q9M4H1) (Davies and Robinson 2000), and an ethylene response factor, ERF4 (TC47273, Q9LW49; Supplemental Tables S4 and S6). Of the 41 transcription factors that were expressed differentially between water deficit and salinity treatments (Supplemental Table S11), the ten that showed the greatest reduction in steady-state transcript abundance in response to water deficit stress compared to salinity stress are implicated as having functional roles in cell development, cell growth and cell wall functions. Four of these transcription factors involved in cell development include a Myb (TC51644, Q58FS3), a WOX4 (TC41847, Q6X7J9), and two basic helix-loop-helix (TC43519, Q8LCD1; and TC40436, Q9M0B9) transcription factors. Others include two ERFs (TC45046, P16146; and TC39828, Q6TKQ3), which belong to a large family of plant-specific transcription factors initially referred to as ethylene response binding proteins that function in a diverse range of processes (Guo and Ecker 2004), and transcription factors (TC49028, Q6AWY6; and TC41565, Q6EPP9) involved in regulating vegetative growth and development (Choi et al. 2004). The responses of these transcription factors are consistent with the greater inhibition of growth in water-deficit-treated plants as compared to salt-stressed plants. Of the transcription factors that showed greater steadystate transcript abundance in response to water deficit as compared to salinity stress, some have not been assigned a function (Supplemental Table S11). One functionally annotated factor is a putative Myb transcription factor (TC40303, Q9M9A3) that appears to be involved in hormone and salt stress responses (Yanhui et al. 2006). Our data indicate that it is more responsive to water deficit than salinity. The responses of this transcription factor are consistent with other transcript responses associated with hormones (Fig. 9). Two other transcription factors of this group, a NAC transcription factor (TC47977, Q8LRL3) and a BEL1-related homeotic protein 29 (TC38709, Q8LLD9), are thought to be involved in cell developmental processes (Chen et al. 2003). Two of the factors were components of the Pol II general transcription complex: transcription factor IIA large subunit (TFIIA-L2), which together with TFIID binds to the promoter regions to form preinitiation complexes, and a eukaryotic transcription elongation factor TFIIS-like component of the plastid transcriptionally active chromosome thought to function in plastid transcription elongation by reactivation of arrested RNA polymerases (da Costa e Silva et al. 2004). The
increased steady-state transcript abundance of these two transcription factors may reflect the need for initiation and reinitiation of water-deficit-stress-related transcriptional regulons in the nucleus and plastid, respectively, in response to water-deficit stress. Other members of this group included a Zinc finger protein CONSTANS-LIKE 6 (TC42939, Q8LG76) and a WRKY transcription factor-b (TC41321, Q5DJU0). Stress effects on sugars Supporting the argument that osmotic adjustment is important during stress, malate, proline, and glucose concentrations were substantially higher in the young shoot tips of water-deficit-treated plants compared to salinized plants (Table 3). Increased gluconeogenesis (Table 4) may have contributed to the higher glucose and fructose concentrations. Transcript abundance of fructose bisphosphate aldolase was substantially higher in waterdeficit-treated plants as compared to salt-stressed plants; however, the differences between stresses were not statistically significant with our small sample size. mRNA expression of genes and activities of enzymes important for the breakdown of soluble sugars, such as hexokinase, are known to increase in response to dehydration stress (Whittaker et al. 2001). Galactinol synthase mRNA expression has also been reported to increase in response to water deficit, cold, and salinity stress in Arabidopsis (Taji et al. 2002). Transgenic plants that overexpress this enzyme produce elevated amounts of galactinol and raffinose, which may function as osmoprotectants and contribute to water deficit stress tolerance (Taji et al. 2002). Under normal conditions, total soluble carbohydrates (glucose, fructose, and sucrose) are estimated to represent approximately 70% of the osmotically active solutes in young grapevine leaves (Patakas 2000); glucose and total inorganic ions each contributed approximately 25% to the pool of osmotically active solutes. Total amino acids made up only about 0.5% of the osmotically active solutes. As grapevine leaves mature from 6 to 35 days of age, the relative contribution of soluble carbohydrates declined, whereas total inorganic ions increased. Note that organic acids were not measured in this study (Patakas 2000) or a subsequent one investigating osmotic adjustment of drought-stressed grapevine leaves (Patakas et al. 2002). In mature, drought-stressed grapevine leaves, osmotic adjustment was largely associated with increases in inorganic ion content and not soluble carbohydrates. In fact, starch and soluble carbohydrate concentrations were substantially reduced in these mature leaves, perhaps because they were utilized for sucrose transport to younger sink tissues like the ones used in this study.
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Stress effects on proline Proline is an important osmoprotectant in drought-stressed and salinized plants (Delauney and Verma 1993; Rhodes et al. 2002) and is the most abundant free amino acid in grapevine leaves (Kliewer and Nassar 1966). Increases in proline concentration are one of the most common responses of organisms to dehydration (Yancey et al. 1982; Delauney and Verma 1993). Proline functions as a compatible solute for osmotic adjustment, where it can accumulate to high concentrations without the detrimental ionic strength effects that ions can have on protein stability or catalytic rates (Yancey et al. 1982), and as an osmoprotectant and in the detoxification of ROS (Kocsy et al. 2005). Proline concentrations in growing apices are particularly affected by water deficit where it has been suggested that proline may act as a source of N or energy (Verslues and Sharp 1999). Increases in proline concentration by overexpression of P5CS improve the tolerance of plants to drought and salinity (Kavi Kishor et al. 1995; Zhu et al. 1998; Yamada et al. 2005). Increased proline concentrations in plants have been associated with increased biosynthesis and decreased catabolism (Peng et al. 1996; Yoshiba et al. 1997; Rhodes et al. 2002), although in root apices, increased proline uptake appears to be the cause for the increase (Verslues and Sharp 1999). In this study, we detected increased expression of P5CS, a cytoplasmic enzyme considered to be the key regulatory step in osmotically induced increases in proline (Yoshiba et al. 1997). Proline concentrations in the shoot tip could also be affected by transport from more mature portions of the plant. There was differential expression of a plasma membrane proline transporter (TC41212) in the shoot tips between the stress treatments; the expression of the proline transporter was 178% of control in waterdeficit-treated plants and 122% in salinized plants. The orthologous ProT2 proline transporter from Arabidopsis also displayed increased transcript abundance in response to water deficit and salinity stress and has been found at the plasma membrane of cells in leaves, roots, and stems of Arabidopsis (Rentsch et al. 1996; Grallath et al. 2005). Several reports indicate that the expression of proline dehydrogenase, a mitochondrial enzyme, is reduced with salt stress (Kiyosue et al. 1996; Peng et al. 1996; Miller et al. 2005) and increased by elevated proline concentrations (Kiyosue et al. 1996; Peng et al. 1996). In contrast to these previous studies, the expression of proline dehydrogenase was increased in this study (Table 4). There are a number of possible explanations for this difference. Proline may be being used as an energy source in young tissues (Verslues and Sharp 1999). Alternatively, these differences could be due to differences in the length or severity of the stress treatment, or simply due to species differences.
Stress effects on organic acids Based on our anion exchange data, organic acids, especially tartrate and malate, represent the most abundant anionic osmolytes in grape leaves. This is consistent with previously published results showing that tartrate and malate are the first and second most abundant organic acids in grapevine leaves, respectively (Kliewer and Nassar 1966). Both of these organic acids increase in response to stress, with water deficit triggering a greater accumulation than salinity for both organic acids (Table 3). Consistent with these observations, malate concentrations have been shown to increase by 60% in drought-stressed cotton leaves and were a major factor contributing to diurnal variations in osmotic potential (Cutler et al. 1977). Malate concentrations have also been shown to increase in leaves of salinized bean and barley (Fricke et al. 1996; FernandezBallester et al. 1997). This was particularly evident after the initial osmotic shock; however, malate concentrations decreased with time as Cl was accumulated. Similar to this study, malate concentrations in bean leaves were substantially higher with osmotic shock (PEG) than with salt shock (Fernandez-Ballester et al. 1997). Increased malate accumulation in V. vinifera shoots was accompanied by large increases in multiple malate dehydrogenase isoenzymes predicted to be localized to the glyoxysome and chloroplast. The increased mRNA expression of these two malate dehydrogenase isozymes is likely a consequence of increased reliance on the malate valve during the day. This malate valve permits the generation of extra ATP in the plastid in conjunction with the production of reducing equivalents in the form of NADPH for biosynthetic reactions, allows malate production and transport to the cytosol to serve as a source of NADH in the cytosol, and contributes to ATP production in the mitochondria (Scheibe 2004). Such increased demands for ATP and NADPH would be expected to exist under stress conditions. At night, a second malate valve is coupled to ATP and NADH by glycolytic reactions in the plastid that generate malate with NADPH being produced independently by the oxidative pentosephosphate pathway or Calvin cycle (Scheibe 2004). Satisfying the increased demand for NADPH by the Calvin cycle would explain the increased transcript accumulation observed for several Calvin cycle enzymes including phosphoribulose kinase, ribulose-5-phosphate-3 epimerase, and sedoheptulose-1,7-bisphosphatase (Table 4). Increased expression of genes encoding fatty acid metabolism and glyoxylate cycle enzymes is consistent with the expected increase in lipid utilization to fuel gluconeogenesis. Consistent with this is the low concentrations of succinate observed in stressed plants (Table 3), which is consumed in the glyoxylate cycle to produce malate, which, in turn, is used to fuel gluconeogenesis.
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In summary, we performed a unique stress experiment with V. vinifera cv. Cabernet Sauvignon comparing the gradual effects of a decreasing water deficit to equivalent salinity. Transcript and metabolite profiling provided consistent and corroborative results with growth assays. At equivalent water potentials, water deficit had a more severe effect than salinity on growth, gene expression, and specific metabolites. Stress broadly affected many gene transcripts involved with metabolism, protein fate, transport, transcription, cellular defense, and communication/ signaling. Gene expression of the ABA and ethylene pathways was particularly increased by stress compared to other hormone pathways and was negatively correlated with stem water potentials. Energy metabolism was strongly increased by stress, particularly in water-deficittreated plants. Higher concentrations of glucose, malate, and proline in water-deficit-treated plants, as compared to salinized plants, were linked to differences in gene expression and other metabolites of the photosynthetic, gluconeogenic, and photorespiratory pathways. Not only are these solutes likely to aid water-deficit-treated plants to adjust osmotically, but they also may help plants cope with ROS detoxification and photoinhibition. Simultaneous monitoring of the abundance of both transcripts and metabolites reinforced the observations made with each metric alone, emphasizing the information synergy derived from the use of integrative functional genomic approaches.
Acknowledgments This work was supported by grants from the American Viticulture Foundation, the USDA Viticulture Consortium, the National Science Foundation (NSF) Plant Genome program (DBI0217653) to G.R.C. and J.C.C. and the Bioinformatics program (DBI0136561) to K.A.S., a graduate student fellowship to E.A.R. Tattersall from the NSF EPSCoR Integrated Approaches to Abiotic Stress program (EPS-0132556), and a TUBITAKNATO B1 fellowship to Dr. A. Ergul. The Nevada Genomics Center is supported by grants from the NIH Biomedical Research Infrastructure Network (NIHNCRR, P20 RR16464) and NIH IDeA Network of Biomedical Research Excellence (INBRE, RR-03-008).
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Funct Integr Genomics (2007) 7:111134 DOI 10.

Funct Integr Genomics (2007) 7:111134

Funct Integr Genomics (2007) 7:111134

Funct Integr Genomics (2007) 7:111134

Funct Integr Genomics (2007) 7:111134

Funct Integr Genomics (2007) 7:111134

Funct Integr Genomics (2007) 7:111134

Funct Integr Genomics (2007) 7:111134

Funct Integr Genomics (2007) 7:111134

GA gibberellic acid, JA jasmonic acid, SA salicylic acid

Funct Integr Genomics (2007) 7:111134

Funct Integr Genomics (2007) 7:111134

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7.1E-50 8.6E-287 3.6E-200 3.5E-203 5E-23

Funct Integr Genomics (2007) 7:111134

UniProt ID Q9FUC2 P51118 P54767 Q84MA5

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1615159_s_at 1612789_at 1608251_at 1612194_at

174 109 178 159 122 133 185 181 101 67 52

Funct Integr Genomics (2007) 7:111134

Funct Integr Genomics (2007) 7:111134

Funct Integr Genomics (2007) 7:111134

Funct Integr Genomics (2007) 7:111134

Funct Integr Genomics (2007) 7:111134

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