Guard-Cell-Specific Expression of Phototropin2 C-Terminal Fragment Enhances Leaf Transpiration

Abstract

Phototropins (phot1 and phot2) are plant-specific blue light receptors that mediate chloroplast movement, stomatal opening, and phototropism. Phototropin is composed of the N-terminus LOV1 and LOV2 domains and the C-terminus Ser/Thr kinase domain. In previous studies, 35-P2CG transgenic plants expressing the phot2 C-terminal fragment-GFP fusion protein (P2CG) under the control of 35S promoter showed constitutive phot2 responses, including chloroplast avoidance response, stomatal opening, and reduced hypocotyl phototropism regardless of blue light, and some detrimental growth phenotypes. In this study, to exclude the detrimental growth phenotypes caused by the ectopic expression of P2C and to improve leaf transpiration, we used the PHOT2 promoter for the endogenous expression of GFP-fused P2C (GP2C) (P2-GP2C) and the BLUS1 promoter for the guard-cell-specific expression of GP2C (B1-GP2C), respectively. In P2-GP2C plants, GP2C expression induced constitutive phototropin responses and a relatively dwarf phenotype as in 35-P2CG plants. In contrast, B1-GP2C plants showed the guard-cell-specific P2C expression that induced constitutive stomatal opening with normal phototropism, chloroplast movement, and growth phenotype. Interestingly, leaf transpiration was significantly improved in B1-GP2C plants compared to that in P2-GP2C plants and WT. Taken together, this transgenic approach could be applied to improve leaf transpiration in indoor plants.

Keywords: Arabidopsis thaliana; BLUS1 (BLUE LIGHT SIGNALING1); phototropin2; stomatal opening; transpiration.

Figure 1

Production of P2-GP2C and B1-GP2C transgenic Arabidopsis plants. (A) Schematic diagrams of the P2-GP2C and B1-GP2C expression vectors used for Agrobacterium-mediated Arabidopsis transformation. The detailed cloning procedures are discussed in the Material and Methods section. (B) Immunoblot analysis of P2-GP2C and B1-GP2C plants. Total proteins were extracted from the rosette leaves of 3-week-old WT, P2-GP2C, and B1-GP2C plants. The blots were probed with anti-Flag (top), anti-phot1 (middle), and anti-phot2 (bottom) antibodies, respectively. (C) Tissue-specific expression patterns of GP2C in P2-GP2C and B1-GP2C plants. GFP fluorescence was observed by confocal microscopy. Note that the fluorescent signals for GFP obtained from palisade mesophyll cells also contained autofluorescent signals from chlorophyll. GFP fluorescence was confirmed in the plasma membrane of mesophyll cells in P2-GP2C plant (white arrow) but not in WT and B1-GP2C plants (yellow arrow). Scale bar = 10 μm.

Figure 2

Chloroplast photorelocation movement in WT, P2-GP2C, and B1-GP2C plants. (A) Chloroplast positioning in different light conditions. The third and fourth rosette leaves were detached from 3-week-old Arabidopsis plants and kept on agar plates containing 0.8% gellan gum in the dark for 14 h or under low intensity of blue light (LB, 2 μmol m⁻² s⁻¹) for 3 h or high intensity of blue light (HB, 50 μmol m⁻² s⁻¹) for 2 h. The rosette leaves were kept in the fixation buffer before observation. Chloroplast positioning was observed using confocal laser scanning microscopy. Scale bar = 10 μm. (B) Chloroplast number in the palisade mesophyll cells of rosette leaves of WT, P2-GP2C, and B1-GP2C plants shown in (A). Data represent the mean ± SE (n = 10 cells). (C) Changes in the red-light (RL) transmittance were traced in the third and fourth rosette leaves of 3-week-old Arabidopsis plants under different light conditions. The rosette leaves were sequentially irradiated with blue light at different intensities of 0 μmol m⁻² s⁻¹ for 20 min, 3 μmol m⁻² s⁻¹ for 100 min, 21 μmol m⁻² s⁻¹ for 40 min, 45 μmol m⁻² s⁻¹ for 40 min, and 0 μmol m⁻² s⁻¹ for 40 min. Data represent the mean ± SE of three independent experiments (n = 3).

Figure 3

Hypocotyl growth and phototropic responses in WT, P2-GP2C, and B1-GP2C plants. (A) Photos showing the phototropic phenotypes of WT, P2-GP2C, and B1-GP2C plants. Three-day-old dark grown Arabidopsis seedlings were irradiated with low intensity of blue light (LB, 2 μmol m⁻² s⁻¹) and high intensity of blue light (HB, 40 μmol m⁻² s⁻¹) for 16 h. Scale bar = 1 cm. (B) Phototropic curvature was measured as the change in hypocotyl angle as determined from an analysis of stacked images of (A). Data represent the mean ± SE (n = 15). (C) WT, P2-GP2C, and B1-GP2C seedlings were grown in the dark or blue light (10 μmol m⁻² s⁻¹) for 5 days. Scale bars = 2 mm. (D) Quantitative analyses of the hypocotyl length of seedlings shown in (C). Data represent the mean ± SE of three independent experiments (n > 25). Asterisks indicate statistical differences detected by Student’s t-test (* not significant p > 0.05; ** significant p < 0.0001).

Figure 4

Vegetative growth phenotypes in P2-GP2C and B1-GP2C plants. (A–E) Comparisons of the rosette leaf growth (A), fresh weight (B), dry weight (C), leaf growth (D), and total leaf area (E) of 6-week-old WT, P2-GP2C, and B1-GP2C plants. Scale bars = 1 cm. Data represent the mean ± SE (n = 15 in B and C, n = 10 in E). Asterisks indicate statistical differences detected by Student’s t-test (* not significant p > 0.05; ** significant p < 0.0001).

Figure 5

Stomatal apertures in P2-GP2C and B1-GP2C plants. (A) Confocal images showing the stomatal opening of the WT, P2-GP2C, and B1-GP2C plants. Epidermal strips were detached and preincubated for 1 h in darkness and then illuminated by red light (R50, 50 μmol m⁻² s⁻¹) with or without blue light (B15, 15 μmol m⁻² s⁻¹) for 3 h. Scale bar = 5 μm. (B) Stomatal apertures in the WT, P2-GP2C, and B1-GP2C plants under the same conditions as A. Data represent the mean ± SE (n = 20). (C) Stomatal density of 4-week-old plants. Data represent the mean ± SE (n = 10 independent leaves). Asterisk indicates statistical difference detected by Student’s t test (* not significant p > 0.05).

Figure 6

Leaf transpirations in P2-GP2C and B1-GP2C plants. (A) Temporal changes in weight loss by leaf transpiration measured at 30 min intervals for 90 min under red light (R50, 50 μmol m⁻² s⁻¹) and for an additional 90 min under red light (15 μmol m⁻² s⁻¹) superimposed on red light (R50 + R15, 50 μmol m⁻² s⁻¹). (B) Temporal changes in weight loss by leaf transpiration measured at 30 min intervals for 90 min under red light (R50, 50 μmol m⁻² s⁻¹) and for an additional 90 min under blue light (15 μmol m⁻² s⁻¹) superimposed on red light (R50 + B15, 50 μmol m⁻² s⁻¹). Weight loss ratios were obtained from five plants at each time point. (C) Actual water reduction in the condition of (A). (D) Actual water reduction in the condition of (B). Data represent the mean ± SE (n = 5).