Natural variation in stomatal response to closing stimuli among Arabidopsis thaliana accessions after exposure to low VPD as a tool to recognize the mechanism of disturbed stomatal functioning

Abstract

Stomatal responses to closing stimuli are disturbed after long-term exposure of plants to low vapour pressure deficit (VPD). The mechanism behind this disturbance is not fully understood. Genetic variation between naturally occurring ecotypes can be helpful to elucidate the mechanism controlling stomatal movements in different environments. We characterized the stomatal responses of 41 natural accessions of Arabidopsis thaliana to closing stimuli (ABA and desiccation) after they had been exposed for 4 days to moderate VPD (1.17 kPa) or low VPD (0.23 kPa). A fast screening system was used to test stomatal response to ABA using chlorophyll fluorescence imaging under low O2 concentrations of leaf discs floating on ABA solutions. In all accessions stomatal conductance (gs) was increased after prior exposure to low VPD. After exposure to low VPD, stomata of 39 out of 41 of the accessions showed a diminished ABA closing response; only stomata of low VPD-exposed Map-42 and C24 were as responsive to ABA as moderate VPD-exposed plants. In response to desiccation, most of the accessions showed a normal stomata closing response following low VPD exposure. Only low VPD-exposed Cvi-0 and Rrs-7 showed significantly less stomatal closure compared with moderate VPD-exposed plants. Using principle component analysis (PCA), accessions could be categorized to very sensitive, moderately sensitive, and less sensitive to closing stimuli. In conclusion, we present evidence for different stomatal responses to closing stimuli after long-term exposure to low VPD across Arabidopsis accessions. The variation can be a useful tool for finding the mechanism of stomatal malfunctioning.

Keywords: Arabidopsis thaliana; abscisic acid; desiccation.; natural variation; stomata; vapour pressure deficit (VPD).

Fig. 1.

Schematic representation of the experimental setup and conditions which were used for growing plants and measurements. Boxes describe the conditions used for growing plants and measurements. The arrows shows transferring to new conditions.

Fig. 2.

Stomatal conductance (g_s) of 41 Arabidopsis accession after exposure to different VPDs. Plants had been exposed to moderate VPD (1.17 kPa; filled bars) or to low (0.23 kPa; open bars) VPD. The measurements were carried out at 1.40 kPa VPD and 35 µmol m^–2 s^–1 irradiance. g_s was recorded in fully developed leaves of eight plants (one leave per plant) after a 4-day exposure to each VPD. Bars represent the mean of eight leaves±standard error of the mean.

Fig. 3.

Average PSII efficiency (Φ_PSII) (A) and representative images of Φ_PSII (B) for Col-0 leaf discs in response to ABA after prior exposure to different VPDs. Φ_PSII was measured under non-photorespiratory conditions (20 mmol mol^–1 O₂, 380 µmol mol^–1 CO₂ and remainder N₂) in plants that had been exposed for 4 d to moderate (1.17 kPa; black bars in (A)) or to low (0.23 kPa; L; white bars in (A)) VPD in response to ABA. At the end, an image was made after 5min exposure to 20 mmol mol^–1 O₂ and 50000 µmol mol^–1 CO₂ (grey bars for M, cross-hatched bars for L in Fig. 3A and +CO₂ in Fig. 3B). Leaf discs (0.5cm diameter) were put with the abaxial surface up in petri dishes filled with stomata-opening medium with different concentrations of ABA (0, 50, 100, 200 µM ABA), and Φ_PSII was recorded 3h after application of the ABA. Bars represent the mean of Φ_PSII of eight leaf discs±standard error of the mean.

Fig. 4.

Frequency distribution of different accessions according to the relationships between PSII efficiency (Φ_PSII) under non-photorespiratory conditions in response to 50 (A), 100 (B), and 200 µM ABA (C) relative to no ABA (Φ_{PSII ABA}/ Φ_{PSII C}) after 4 d exposure of plants to moderate VPD (1.17 kPa; black bars) or to low VPD (0.23 kPa; grey bars).

Fig. 5.

Fitted curves of the relationship between transpiration rate (E) and leaf relative water content (RWC) for Col-0 (red and blue lines) and Cvi-0 (purple and green lines) Arabidopsis accessions of leaves of plants that had been exposed for 4 d to moderate (red and purple symbols) or to low (blue and green symbols) VPD. The leaves were first saturated in degassed deionized water and after 1h measurements were conducted during desiccation at VPD of 1.40 kPa. The R square of goodness of fits was 0.9±0.1.

Fig. 6.

Slopes of the curves for relationship between transpiration rate (E) and leaf relative water content during 10 000 s desiccation of the leaves of plants that had been exposed for 4 d to moderate (1.17 kPa; filled bars) or to low (0.23 kPa; open bars) VPD. The leaves were first saturated in degassed deionized water and after 1h measurements were conducted during desiccation at VPD of 1.40 kPa.

Fig. 7.

Principle component analysis (PCA) for 41 Arabidopsis accessions that had been exposed for 4 d to moderate VPD (1.17 kPa) or to low VPD (0.23 kPa). The numbers indicate the accessions according to the numbering in Table 1. The PSII efficiency (Φ_PSII) under non-photorespiratory conditions at 200 µM ABA relative to Φ_PSII of the control (0 µM ABA), and the slope of the fitted sigmoidal relationship between transpiration rate and RWC of the leaves were used for the analysis. Component one and two explain 86.3% of the point variability.

Fig. 8.

Dendrogram classification for 41 Arabidopsis accessions that had been exposed for 4 d to moderate VPD (1.17 kPa) or to low VPD (0.23 kPa). The PSII efficiency (Φ_PSII) under non-photorespiratory conditions at 200 µM ABA relative to Φ_PSII of the control (0 µM ABA), and the slope of the fitted sigmoidal relationship between transpiration rate and RWC of the leaves were used for classification. The red boxes indicate accessions with three different type of responses to closing stimuli. The number at the bottom of the dendrograms correspond to the number of PCA grouping.

Fig. 9.

Concentration of ABA in Col-0 (A), Cvi-0 (B), and Map-42 (C) Arabidopsis accessions before (white bars) and after 45min desiccation (black bars). The plants had been exposed for 4 d to moderate VPD (M) (1.17 kPa) or to low VPD (L) (0.23 kPa) before ABA measurements and desiccation treatment. The desiccation was conducted at VPD of 1.40 kPa.

Fig. 10.

Relationship between desiccation response (slope of the E×RWC relationship) and the ABA-concentration before (open symbols) and after (closed symbols) 45min desiccation of the leaves in Col-0, Cvi-0, and Map-42 accessions. The dashed line is 95% confidence interval. R² of the goodness of the fit is 0.94 for closed symbols and 0.12 for open symbols.

Fig. 11.

Relationship between PSII efficiency (Φ_PSII) under non-photorespiratory conditions in response to 200 µM ABA relative to no ABA (Φ_{PSII 200 ABA}/Φ_{PSII C}) and foliar ABA level for plants that had been exposed for 4 d to moderate (1.17 kPa) (closed symbols) or to low VPD (0.23 kPa) (open symbols).