Mutant lines in the AT3G55990 gene were obtained either from the INRA Resource Centre for Arabidopsis thaliana Genomics http://dbsgap.versailles.inra.fr/portail/: esk1-6 in the WS genetic background [68], or from The Salk Institute in the Col-0 genetic background: SALK_078275 (esk1-4) and SALK_089531 (esk1-5) [69]. The WS and Col-0 lines used were from the INRA Versailles Resources Centre: 530AV and 186AV.
Arabidopsis plants were grown following standard procedures established by Loudet et al. [70]. Seeds were stratified for 4 days in a 0.1% (w/v) agar solution at 4°C in the dark. Germination occurred 2 days after sowing on propagation plugs (4 cm height × 4 cm radius – 70% blond peat, 20% perlite and 10% vermiculite, Fertiss®). Plants were grown under long day conditions with a 16 h photoperiod, in a controlled environment chamber (22°C, 70% RH, PPFD approximately 150 μmol m2 s-1) and watered with nutritive solution as described in Bouchabke et al. [71]. The relationship between soil volumetric water content and soil suction was previously assessed [71].
During plant growth prior to starting the stress experiments the propagation plugs were saturated with nutritive solution (100% at t0). During the stress experiments, however, propagation plugs were weighed daily from t0. Once the target saturation was reached, this was then maintained for the duration of the experiment. For controls, soil water content was fixed at 60% of substrate maximal water content (SMWC). The mild water-deficit treatment was fixed at 30% SMWC whereas severe water-deficit corresponded to 20% SMWC. Two approaches were used to control the saturation level, either an averaged or an individually monitored stress, as indicated in Table 2. For the averaged determination method, the weight of ~10% of the propagation plugs was measured and all the plugs adjusted with the average volume calculated to reach the targeted saturation. For the individual determination method each propagation plug, within a set watering regime, was maintained at the same saturation level based on its actual weight. In this way, the substrate saturation of all the genotypes within a watering regime was identical regardless of their water consumption. The individual determination, which is more labour intensive was employed for the experiments where result reproducibility was most likely to be affected by slight differences in the stress imposed, namely the transcriptome and carbon isotope discrimination analyses (Table 2, Figure 11). To measure integrative parameters such as TLA or CRWL, stress was monitored using the averaged determination of the propagation plugs. Experimental start points varied depending on the phenotype examined; for TLA and CRWL t0 was when leaf number 6 emerged (growth stage 1.6 according to Boyes et al. [72]), For transcriptome and carbon isotope discrimination, treatments were applied from floral bud emergence onwards (growth stage 5.10 according to Boyes et al. [72]).
Salt stress experiments were conducted in parallel to those for drought stress. The propagation plugs for treated plants were first reduced to 60% saturation before the salt stress was applied by watering with 0.5× nutritive solution supplemented with 150 mM NaCl. In this way, plants were subjected to each stress for the same time period.
Plants were cultured in peat moss in pots (length 60 mm, width 65 mm, height 60 mm), filled equally with a homogeneous non-enriched compost (Terf® Substrat: 37% blond peat, 60% brown peat, 10% volcanic sand). The pH of this compost was stabilised between 5.5 and 6.1.
Plants were grown in the same environmental conditions as described above. Progressive drought was applied on 9 randomly selected one-month-old plants of each line (stage 5.10 according to Boyes et al. [72]) by stopping watering. As a control, the same number of plants of each line was grown under standard irrigation conditions and watered twice a week. Pictures of the canopy were taken at day 0, 6 and 10 of stress exposure to calculated the Photosynthetic Leaf Area. PLA is equal to TLA minus chlorotic areas.
Seeds were stratified as described earlier (Monitored stress applied in propagation plugs). Then a large-scale screen to test cold tolerance was performed as follows: rows of plants were sown in square pots containing organic substrate and irrigated with mineral nutrient solution once a week and watered every four days. Plants were grown in the greenhouse for 14 days at which time they had reached the 6–8 leaf stage (stage 1.04 according to Boyes et al. [72]). Plants were then transferred to a growth chamber at 5°C under 12 h photoperiod, 70 μM m-1s-1 light intensity and 70% relative humidity for 7 days. Acclimated plants were then exposed to freezing temperatures of -8°C for 48 h. After this cold treatment, plants were put back in the greenhouse. Tolerance to freezing was determined by evaluating the percentage of viability after freezing exposure: viable and dead plants were counted and the percentage viability was calculated. Four rows of the mutant and two rows of the reference strain were put in each square pot to optimise viability comparisons by reducing undesirable environmental variation. At least 400 plants were tested per line. Parallel experiments were carried out without the acclimation period.
Plants were grown in pots as described for the water starvation experiment. For 12 days, 3 × 10 plants of each line were watered every two days with a concentrated saline solution (NaCl 200 mM). Every three watering cycles plants were watered with non-saline water. Results were compared with the same number of plants grown with standard irrigation. Pictures were taken at day 0, 6 and 10 of stress exposure.
For culture on agar plates, seeds were sterilised, stratified four days at 4°C in the dark and then transferred onto 3 × 10 plates of solid medium [73]. Plants were cultivated in growth chambers under long-day conditions (16 h/d) at a photon flux density of 120 μmol m-2 s-1. Temperature (21°C) and relative humidity (70%) were constant in the growth chamber. For in vitro salt stress induction, the solid medium was supplemented with 3 different NaCl concentrations (100; 150; 200 mM). The same number of standard media plates was prepared as a control. For leaf development studies, 9 × 3 seeds from each line were randomized at regular intervals inside the plate. For root development analysis, six seeds were placed (from each genotype) on each plate, close to one of the edges. Plates were laid horizontally for 48 hours and then placed vertically in a rack, with the seeds at the top. All plates were collected at day 12 and scanned with a desktop scanner (Epson scan Photo 4990) using the “transparent object” mode at 300 dpi.
Osmotic stress was induced following the same protocol as for salt stress in solid media, but supplemented with 60 or 75 mM mannitol.
Fresh weight (FW) was obtained by harvesting and weighing freshly cut rosettes (stage 3.70 to 3.90 according to Boyes et al. [72]).
Rosette dry weight was recorded after 48 h at 75°C in a dry oven.
Relative Water Content (RWC) was calculated according to the formula: [(FW-DW)/DW] × 100.
Cut Rosette Water Loss (CRWL) indicating the amount of water lost from freshly cut tissues during the first 60 minutes, was determined by harvesting and weighing freshly cut rosettes. Rosettes were maintained in the growth chamber conditions then weighed every 10 minutes. CRWL was then calculated as the ratio between water loss and plant initial fresh weight, expressed in %.
To assess transpiration in planta the rosettes of 6 plants per genotype and per soil water treatment were isolated from the soil with a plastic film. The entire propagation plug was also covered with plastic film preventing any soil evaporation. Propagation plugs without rosettes were also included in the experiment to assess water evaporation from empty propagation plugs. Two hours after the beginning of the light period, plants were weighed every two hours for 36 hours. Transpiration per unit of dry weight was then calculated as the ratio between transpiration (weight of the propagation plug at tx time, minus weight of the propagation plug at t0, minus evaporation from empty propagation plugs) and the plant dry weight.
The number of stomata per leaf area was determined on the 10th or 11th leaf of five plants grown in control conditions in short days, in the greenhouse (stage 1.13 to 1.14 according to Boyes et al. [72]). Leaves were fixed in ethanol/acetic acid (3/1) for one hour, and then washed three times with pure water. After this step, they were bleached in NaOH 8 M for one hour, then washed three times with pure water. The surplus water was wiped away. The leaves were mounted in a 0.1% calcofluor solution and observed with a F.I.S.H (Fluorescent In Situ Hybridization) microscope at a 350 nm wavelengh (UV light), magnification 12.5×. Six pictures of each leaf were taken on the whole leaf surface excluding the central nervure. The stomata were counted with ImageJ software using the “cell counter” plugin.
Gas exchange was measured from one leaf of three independent plants of each genotype (Col-0 and esk1-5) using a portable gas exchange system (Li-6400; LI-COR) with a standard leaf chamber (6400-40 with red and blue LED light source; LI-COR). Leaf chamber conditions were 400 μmol mol-1CO2, 21% O2, 51.47% relative humidity, 22°C and photosynthetic photon flux density (PPFD) of 500 μmol m-2 s-1 with 10% of blue light. Leaves were kept under this condition for approximately 30 min until parameters were stabilized before recording. Because Col-0 and especially the esk1-5 leaf were too small to fill the entire area of the leaf chamber, the portion enclosed parts of leaves were marked. The leaves were then cut from plants, scanned and the total leaf area (which had been enclosed in the chamber) was evaluated by image analysis using a similar procedure as described in the data processing section below. Net photosynthesis [74] and transpiration (ET) were calculated by using equations derived by Caemmerer and Farquhar [75]. WUE was estimated by the net photosynthesis/evapo-transpiration ratio (NP/ET).
After 7 days of individual determination of treatments, frozen samples were lyophilized and then ground. For each sample, about 1 mg of powder was transferred into tin cups (Courtage analyse service, Mont Saint-Aignan, France) and analysed in an elemental analyser (NA-1500, Carlo Erba, Milan, Italy) coupled to an Isotope Ratio Mass Spectrometer (VG Optima, Fison, Villeurbanne, France). Carbon isotope compositions were calculated as deviations of the carbon isotope ratio (13C/12C, called R) from international standards (Pee Dee Belemnite) according to Farquhar et al.:[56] δ13C = |103 [(Rsample – Rstandard)/Rstandard]|.
For the transcriptome analysis, RNA was extracted with the RNeasy extraction kit from Qiagen® including the DNase treatment. Microarray analysis was carried out at the Unité de Recherche en Génomique Végétale (Evry, France), using the CATMA array [76, 77], containing 24,576 Gene-Specific Tags from Arabidopsis. RNA samples from two independent biological replicates were used. For each biological replicate, RNA samples for a condition were obtained by pooling RNA from 3 plants. For each comparison, one technical replicate with fluorochrome reversal was performed for each biological replicate (i.e. four hybridisations per comparison). RT on RNA in the presence of Cy3-dUTP or Cy5-dUTP (Perkin-Elmer-NEN Life Science Products), hybridisation of labelled samples to the slides, and the scanning of the slides were performed as described in Lurin et al. [78]. Microarray data from this article were deposited at Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/; accession No. GSE10384) and at CATdb (http://urgv.evry.inra.fr/CATdb/; Project RA06-02_StayGreen) according to the “Minimum Information About a Microarray Experiment” standards.
Rosette surface measurements were performed as followed: rosette surfaces were selected using Photoshop® software (selection/colour range) and saved as .tif files. Files were then opened with ImageJ software and transformed to 8-bit. The global scale was set with the help of a ruler in the initial picture, the threshold was adjusted to optimise the selection and the area was measured. Statistical analyses were performed with Statgraphics® software. We used the term Total Leaf Area (TLA) for a green rosette and Photosynthetic Leaf Area for a damaged rosette. PLA corresponds to the area of non-damaged leaves.
For the transcriptome analysis, experiments were designed with the statistics group of the Unité de Recherche en Génomique Végétale. Statistical analysis was based on two dye swaps (i.e. four arrays, each containing 24,576 GSTs and 384 controls) [78]. Controls were used for assessing the quality of the hybridisation, but were not included in the statistical tests or the graphic representation of the results. For each array, the raw data comprised the logarithm of median feature pixel intensity at wavelengths 635 (red) and 532 nm (green). No background was subtracted. In the following description, log ratio refers to the differential expression between two conditions. It is either log2 (red/green) or log2 (green/red) depending on the experimental design. Array-by-array normalisation was performed to remove systematic biases. First, we excluded spots that were considered badly formed features. Then, we performed global intensity-dependent normalisation using the LOESS (locally weighted scatterplot smoothing) procedure to correct the dye bias. Finally, for each block, the log ratio median calculated over the values for the entire block was subtracted from each individual log ratio value to correct print tip effects on each metablock. To identify differentially expressed GSTs, we performed a paired t-test on the log ratios, assuming that the variance of the log ratios was the same for all genes. Spots displaying extreme variance (too small or too large) were excluded. The raw p-values were adjusted by the Bonferroni method, which controls the FWER (Family-Wise Error Rate). We considered genes as being differentially expressed with a FWER of 5%. We used the Bonferroni method (with a type I error equal to 5%) in order to keep strong control of false positives in a multiple-comparison context [79]. A manual clustering step was carried out only considering GSTs with the same expression pattern in the two biological replicates. In this manuscript, any differentially expressed GST that hybridises with two genes (genes with an identification number in The Arabidopsis Information Resource or TAIR) is accounted as two distinct genes. A Perl script was developed to select genes which were highly expressed or highly repressed between wild type and mutant, and expressed at around the background level in wild type or mutant (with a high log2 (ratio) range and with a log2 (red or green intensity values) of around 7.5). Results are presented in the Additional file 1. Analysis of the functional categories of genes according to the Gene Ontology and the whole Arabidopsis thaliana genome annotation was made with the TAIR GO annotation tool http://www.arabidopsis.org/tools/bulk/go/index.jsp September 3rd, 2008.
