Figure 1.
Structure and subcellular localization of NFXL2 proteins.
A. Exon-intron structure and domain organisation of NFXL2 proteins. Exons are represented by boxes, untranslated regions by thick lines, and introns by thin lines. T-DNA insertions in the nfxl2-1 (SALK_140301) and nfxl2-2 (GABI-Kat line 668B09) mutants are indicated. B. Subcellular localisation of GFP:NFXL2-100, GFP:NFXL2-97, and GFP:NFXL2-78 proteins. GFP-fluorescent images and bright field images of roots of stably transformed Arabidopsis plants. The scale bar represents 25 µm.
Figure 2.
A. Real-time RT-PCR analysis of NFXL2 transcript levels in shoots of wild-type (Col-0) plants. For drought treatment, four-week-old soil-grown plants were withheld from water for 3 d. Higher bars indicate higher transcript levels. A difference of one indicates a fold change of two. Error bars: SE of three technical replicates. B. Real-time RT-PCR analysis of NFXL2 transcript levels in shoots of four-week-old nfxl2-1/35S::NFXL2-78, nfxl2-1/35S::NFXL2-97, and nfxl2-1/35S::NFXL2-100 plants in comparison to the wild type and nfxl2-1 mutant. Wild-type transcript levels were arbitrarily adjusted to a relative value (i.e. 4). The nfxl2-1 mutant is not able to produce the NFXL2-78/97/100 mRNAs. Transcript levels of the transgenic lines were given relative to the wild type. Higher bars indicate higher transcript levels. A difference of one indicates a fold change of two. Error bars: SE of three technical replicates.
Figure 3.
A. 4-week-old soil-grown plants were withheld from water for 7 d. Survival rates were determined 7 d after water resupply. The number of surviving plants in five independent experiments is represented as the percentage (mean±SE) of the number of total plants (approximately 35 plants per genotype and experiment). Mutant values denoted with an asterisk are significantly different from those of their wild type (t test, P<0.01). B. For drought treatment, 4-week-old soil-grown plants were withheld from water for 9 d. Photos were taken 7 d after water resupply. Plants were arranged according to the visual phenotype. Wild-type plants were not able to survive the prolonged drought period, but approximately 60% of nfxl2-1 plants were still turgescent and viable.
Figure 4.
Stomatal conductance and leaf temperature under drought stress.
A. Stomatal conductance was determined with a Decagon SC-1 porometer at day 3 of a drought stress experiment. Data are given as mean±SE (n = 10 to 15 leaves per genotype). Mutant values denoted with an asterisk are significantly different from those of their wild type (t test, P<0.001).B. Leaf temperature calculated from the quantification of infrared images (means±SE; n = 10 to 15 leaves per genotype). Values denoted with an asterisk are significantly different from the wild type (t test, P<0.001).
Figure 5.
Stomatal aperture and stomatal conductance of well-watered plants grown under long day growth conditions.
A. Stomatal aperture was determined from images of silicone rubber imprints of the abaxial surfaces of rosette leaves of five-week-old plants. Plants were grown in a greenhouse under standard conditions (see Methods for details) or in a controlled growth chamber (16 h light, 21°C, 120 µmol m−2 s−1, 60% humidity; 8 h dark, 19°C, 75% humidity). Data are given as mean±SE of at least 60 stomata per genotype and condition. Values denoted with an asterisk are significantly different from those of their wild type (t test, P<0.001). B. Stomatal conductance of rosette leaves of five-week-old plants was determined with a Decagon SC-1 porometer. Plants were grown in a greenhouse under standard greenhouse conditions. Data are given as mean±SE (n = 10 to 15 leaves per genotype). Mutant values denoted with a positive sign are significantly different from their wild type (t test, P<0.05).
Figure 6.
Stomatal aperture of wild-type and nfxl2-1 plants under different environmental conditions.
Stomatal aperture was determined from images of silicone rubber imprints of the abaxial surfaces of rosette leaves (A to C) or determined directly from epidermal peels using a microscope (D). Data are given as mean±SE of at least 60 stomata per genotype and condition. Mutant values denoted with an asterisk are significantly different from their wild type (t test, P<0.001). A. Stomatal aperture of 37-d-old plants grown in soil in a greenhouse under standard conditions. B. Stomatal aperture of 26-d-old wild-type and nfxl2-1 plants grown in a controlled growth chamber at 85% relative humidity (further climate parameters as given in Figure 5A). C. Stomatal aperture of 14-and 21-d-old plants grown in jars (0.5xMS medium supplemented with 1% sucrose). D. Stomatal response to fusicoccin. Epidermal peels were dissected from rosette leaves of soil-grown plants and incubated for 4 h in darkness either in stomata opening buffer (SOB) or SOB plus 10 µM fusicoccin (SOB+10 µM FC).
Figure 7.
Stomatal conductance in response to light and CO2.
4-week-old soil-grown plants were investigated by gas exchange measurements. Graphs represent H2O conductance rates obtained during a stepwise change of PFD (A) or CO2 concentration (B). Each data point represents the average of 5-10 individual plants±SE.
Figure 8.
Stomatal aperture in rosette leaves, cotelydon greening, and root growth in the presence of exogenous ABA.
A. Stomatal aperture after ABA application. Stomatal aperture was determined from images of silicone rubber imprints of the abaxial surfaces of rosette leaves. Three droplets of an ABA solution (100 µM in SOB, solidified with 0.1% agarose) were supplied to leave petioles. Inset: Imprint of representative wild-type and nfxl2-1 stomata after 6 h ABA treatment (each photo represents a width of 50 µm).B. Stomatal aperture after ABA application. Three droplets of an ABA solution (100 µM in SOB, solidified with 0.1% agarose) were supplied to leave petioles. Details as in A.C. Stomatal aperture after ABA application. Three droplets of an ABA solution (10 µM in SOB, solidified with 0.1% agarose) were supplied to leave petioles. Further details as in A.D. Cotyledon greening in the presence of exogenous ABA. Seeds were allowed to germinate on half-concentrated MS medium supplemented with 1% sucrose at 4°C. 4-d-old seedlings were transferred to the same medium supplemented with 0.2 and 0.3 µM ABA and cultured at 22°C, with 14 h light (140 µmol m−2 s−1)/10 h dark photoperiod. After 5 d, the percentage of seedlings with green cotyledons was scored (mean±SE). Values denoted with an asterisk are significantly different from that of their wild type (t test, P<0.001).E. 4-d-old seedlings were transferred to half-concentrated MS medium supplemented with 1% sucrose and 0, 5, and 10 µM ABA. Root length of 14-d-old plants was determined and is given as percentage (mean±SE) of control plants. Values denoted with an * or+are significantly different from that of their wild type (t test, P<0.001 or P<0.03).
Figure 9.
Endogenous ABA levels and imaging of hydrogen peroxide production.
A. For drought treatment, soil-grown plants were withheld from water for 3 d. Two independent experiments were analyzed. Results are mean ng ABA per gram fresh weight±SE (n = 3 pools of 10 to 15 plants per genotype and experiment). Values denoted with an * are significantly different from that of their wild type (t test, P<0.001).B. Rosette leaves were infiltrated with 3,3-diamino-benzidine (DAB). Formation of brown polymerisation product indicates H2O2 formation [75]. Plants were grown in parallel to the plants used in A. Supplemental data are given in Figure S1.