Volume 16, Issue 9, Pages 882-887 (May 2006) The Identification of Genes Involved in the Stomatal Response to Reduced Atmospheric Relative Humidity  Xiaodong Xie, Yibing Wang, Lisa Williamson, Geoff H. Holroyd, Cecilia Tagliavia, Erik Murchie, Julian Theobald, Marc R. Knight, William J. Davies, H.M. Ottoline Leyser, Alistair M. Hetherington  Current Biology  Volume 16, Issue 9, Pages 882-887 (May 2006) DOI: 10.1016/j.cub.2006.03.028 Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 1 Infrared Images of 5-Week-Old Wild-Type Col-2 and the 30A and 7C Mutants 40 Minutes after They Experienced a Drop in Relative Humidity from 65% to 25% Methods are as follows. 20,000 seeds from an Arabidopsis thaliana (ecotype Col-2) ethyl methanesulphonate (EMS) M2 population representing 40 independent pools (each pool corresponding to approximately 1000 M1 plants) [24] were germinated and grown in a 3:1 mix of peat-based compost (SHL, Multi purpose, William Sinclaire Horticulture, UK) and washed horticultural silver sand (Gem Horticulture, UK) in acrylic horticultural propagators (William Sankey Products, UK). The propagators were placed in a growth room (10.5 hr photoperiod, PPFD 150 μmol m−2 s−1; air temperature 23°C ± 2°C; RH 25% ± 5%), and the plants were grown until they were 3 weeks old. The relative humidity inside the propagators was 65%. 40 min prior to infrared thermography, the acrylic lid was removed from the propagator. This caused the plants to experience a drop in relative humidity from 65% to 25%. Plants displaying aberrant leaf temperatures were detected by infrared thermography conducted with an Inframetrics ThermaCam SC1000 focal plane array (256 × 256 pixel platinum silicide) imaging radiometer (3.4–5 μm) fitted with a 16° lens (Flir Systems Inc), and the data were analyzed with ThermaGRAM 95 Pro image analysis software (Thermoteknix Systems Ltd., UK). Mutants exhibiting altered leaf surface temperature compared to wild-type were selected, and self-pollinated and seed (M3) was collected for further investigation. Backcross seed (F1s) were obtained by using the mutant lines as female and Columbia as male, and the F2 was used for segregation analysis. Mutants segregating in F2 were backcrossed to wt Columbia for another two generations before being used for fine mapping and phenotypic analysis. Current Biology 2006 16, 882-887DOI: (10.1016/j.cub.2006.03.028) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 2 The Morphology of 6-Week-Old Wild-Type, 7C, and 30A Mutants The backcrossed 7C and 30A lines were outcrossed to Landsberg-erecta (Ler) plants, and the resulting progeny were self-fertilized. Mutant individuals were selected from the F2 and DNA was prepared from leaf samples with Dneasy 96 plant kits according to the manufacturer's instructions (Qiagen, Germany). The DNA samples were used to genotype the F2s with respect to known CAPS and SSLP markers (http://www.Arabidopsis.org/), allowing cosegregation and relative position to be assessed for each marker. Current Biology 2006 16, 882-887DOI: (10.1016/j.cub.2006.03.028) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 3 Typical Responses of Stomatal Conductance after a Drop in Atmospheric Humidity for Wild-Type Col-2 and the 7C and 30A Mutants To measure stomatal conductance, plants were grown in propagators as described above to 6 weeks old. Gas exchange measurements were made with a Licor 6400 (Licor, NE) photosynthesis system with a 6400-15 Arabidopsis leaf chamber attachment. Illumination was provided to the adaxial side by a fiberoptic attached to a Schott lamp and was 90 μmol m−2s−1 at the leaf surface. Air temperature within the leaf chamber was maintained at 22°C throughout. Plants were taken directly from the growth chamber and placed in the leaf chamber, where the humidity was set at 75% (RH) ± 10%. Stomatal conductance was monitored graphically on a computer, and data were collected automatically every 5 s. Stabilization of stomatal conductance typically took 25–35 min. After stabilization, humidity was reduced to 5% RH over a period of 7 min, and measurements resumed immediately after this period, continuing until g was stable once more at a lower value. Analysis of the stomatal conductance response curves took place with Sigmaplot 9.0 (Point Richmond, CA) and showed that the data fitted an equation for exponential decay. Converting these data to a logarithmic scale gave straight lines (example shown here). Experiments were repeated four times for each line: the slope of the linearized response was statistically significantly greater for wild-type than for both 7C and 30A (0.0043 ± 0.0007, 0.0017 ± 0.0002, 0.003 ± 0.0007, respectively). Before the decline in humidity, the initial steady-state values for stomatal conductance in 7C and 30A were 0.56 ± 0.02 mol m−2s−1and 0.58 ± 0.12 mol m−2s−1, respectively, more than twice that of wild-type (0.23 ± 0.05 mol m−2s−1). After the decline in humidity, however, the final values as a percentage of the initial were 53%, 55%, and 32% for wild-type, ost1-4, and aba2-13, respectively. Current Biology 2006 16, 882-887DOI: (10.1016/j.cub.2006.03.028) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 4 ABA Signaling in Mutant and Wild-Type Plants (A) Effect of withdrawing water on endogenous ABA levels in wt, ost1-4, and aba2-13. ABA levels were measured in 6-week-old plants (five from each genotype) from which water had been withheld (gray bars) for the final 6 days of the experiment and in plants (five from each genotype) that had been well watered throughout the experiment (black bars). Data are the mean ± SE of two separate experiments. Methods are as follows: wild-type and mutant plants were grown until 6 weeks old. Water was then withheld from five plants of each genotype for 6 days, while a further five plants from each genotype received water during this period. On day 6, the aerial parts of all plants were harvested individually. The concentration of ABA in leaf tissue was determined by radioimmunoassay as described in Quarrie et al. [25]. In brief, freeze-dried leaves were ground to a fine powder and extracted with distilled water 1:20 (leaf DW:solvent) overnight at 5°C–10°C. Each sample (50 μl) was incubated for 1 hr with 100 μl of DL-cis,trans-[G-3H]ABA (TRK644; Amersham Biosciences, UK) and 100 μl of MAC 252 monoclonal antibody against (S)-cis,trans-ABA (obtained from Dr. Geoff Butcher, Babraham Institute, Cambridge, UK). Excess label was removed by washing the bound complex twice with 100% and then 50% saturated ammonium sulfate. The pellet was resuspended in 100 μl of water with 1.5 ml of Ecoscint-H scintillation cocktail (National Diagnostics, Hull, UK) for counting (Tri-Carb 1600TR, Packard Instrument Company, Meriden, CT). The concentration of ABA in samples was calculated by interpolation of radioactive counts from a curve of standards that had been linearized by plotting the logit-transformation of the data against the ln of unlabeled ABA. The experiment was repeated twice. (B) The effect of exogenous ABA on stomatal aperture. The ability of ABA to inhibit stomatal opening in wt (white bars), aba2-13 (gray bars), and ost1-4 (black bars). Plants were grown and the inhibition of stomatal opening on isolated epidermal strips from 5- to 6-week-old plants was investigated by the procedures described by Webb and Hetherington [23]. The experiment was repeated three times and the results are the means ± SE of 120 stomata. Stomatal density determinations were carried out with the dental impression procedure described in Gray et al. [26]. Current Biology 2006 16, 882-887DOI: (10.1016/j.cub.2006.03.028) Copyright © 2006 Elsevier Ltd Terms and Conditions