From The Cover: A role for Arabidopsis cryptochromes and COP1 in the regulation of stomatal opening

Abstract

Cryptochromes (CRY) are blue light photoreceptors that mediate various light-induced responses in plants and animals. Arabidopsis CRY (CRY1 and CRY2) functions through negatively regulating constitutive photomorphogenic (COP) 1, a repressor of photomorphogenesis. Water evaporation and photosynthesis are regulated by the stomatal pores in plants, which are closed in darkness but open in response to blue light. There is evidence only for the phototropin blue light receptors (PHOT1 and PHOT2) in mediating blue light regulation of stomatal opening. Here, we report a previously uncharacterized role for Arabidopsis CRY and COP1 in the regulation of stomatal opening. Stomata of the cry1 cry2 double mutant showed reduced blue light response, whereas those of the CRY1-overexpressing plants showed hypersensitive response to blue light. In addition, stomata of the phot1 phot2 double mutant responded to blue light, but those of the cry1 cry2 phot1 phot2 quadruple mutant hardly responded. Strikingly, stomata of the cop1 mutant were constitutively open in darkness and stomata of the cry1 cry2 cop1 and phot1 phot2 cop1 triple mutants were open as wide as those of the cop1 single mutant under blue light. These results indicate that CRY functions additively with PHOT in mediating blue light-induced stomatal opening and that COP1 is a repressor of stomatal opening and likely acts downstream of CRY and PHOT signaling pathways.

Fig. 1.

Reduced wilting of the cry1 cry2 double mutant plants during drought stress. (A) The spectrum of experimental cool white light shown as the relative spectral irradiance in the wavelength range of 300-900 nm. (B) WT, cry1, cry2, and cry1 cry2 mutant plants were grown under normal watering conditions for ≈21 days and then subjected to drought stress by completely terminating irrigation. Photo shows 2 representative plants of 32 after 14 days of drought stress. (C) Water loss is least and greatest from detached leaves of the cry1 cry2 mutant and CRY1-ovx plants, respectively. Water loss is expressed as the percentage of initial fresh weight. Values indicate a mean of three measurements with standard deviations, each with a sample size of five to eight leaves. One of the triplicate trials is shown. Regression analysis confirmed that the WT curve differs significantly from the CRY1-ovx and cry1 cry2 responses (^**, P ≤ 0.01, Student's t test).

Fig. 2.

Stomatal opening under blue light. (A) Schematic diagrams displaying constructs expressing CRY1 and Myc-tagged CRY2. (B) Six-day-old Arabidopsis seedlings of the WT, cry1, cry1 cry2, CRY1-ovx, and CRY2-ovx plants grown under 5 μmol·m^-2·s^-1 blue light. (Scale bar, 1 mm.) (C) Immunoblot showing expression of CRY1 by using α-CCT1 antibody. Line 4 [CRY1-ovx (4), lane 3] is shown in B and used for all of the phenotypic analysis throughout this study. ^*, a band nonspecifically recognized by the antibody. (D) Western blot showing expression of Myc-CRY2 by using α-Myc antibody. Line 9 [CRY2-ovx (9), lane 3] is shown in B and used to generate the data shown in E and F. (E) Confocal images of stomata of the WT, cry1, cry2, cry1 cry2, CRY1-ovx, and CRY2-ovx plants. Epidermal strips were illuminated with 5 μmol·m^-2·s^-1 blue light under background 50 μmol·m^-2·s^-1 red light for 3 h. (Scale bars in this and other confocal images represent 10 μm.) (F) Stomatal apertures under different light conditions in the WT, cry1, cry2, cry1 cry2, CRY1-ovx, and CRY2-ovx plants. Stomatal opening was induced by 50 μmol·m^-2·s^-1 red light and 20 μmol·m^-2·s^-1 blue light plus 50 μmol·m^-2·s^-1 red light. Stomata of the WT plants open significantly wider than those of the cry1 single and cry1 cry2 double mutant at ^*, P ≤ 0.05 and ^**, P ≤ 0.01 under 20 μmol·m^-2·s^-1 blue light plus 50 μmol·m^-2·s^-1 red light (Student's t test), respectively. (G) Fluence rate dependency of stomatal opening in response to blue light. The measurements represent stomatal apertures obtained at different fluence rates of blue light under background 50 μmol·m^-2·s^-1 red light.

Fig. 3.

Stomata opening of the cop1 mutant, GUS-CCT1, and GUS-CCT2 plants in darkness and under different light conditions. (A) Confocal images showing that stomata of the cop1-4 mutant, GUS-CCT1, and GUS-CCT2 plants are constitutively open in darkness, and open wider than those of the WT under 50 μmol·m^-2·s^-1 red, 50 μmol·m^-2·s^-1 far-red, and 5 μmol·m^-2·s^-1 blue light plus 50 μmol·m^-2·s^-1 red light. (B) Stomatal apertures in the cop1 mutant, GUS-CCT1, and GUS-CCT2 plants under the same conditions in A.

Fig. 4.

Expression of full-length COP1 in the cop1 mutant complements the constitutive stomatal opening phenotype. (A) Confocal images of stomata in the WT, cop1 mutant, transgenic plants expressing COP1 in the cop1 mutant background (COP1-ovx), and plants expressing GUS-COP1 in the WT background in the dark and under 20 μmol·m^-2·s^-1 blue light plus 50 μmol·m^-2·s^-1 red light. (B) Stomatal apertures of the WT, cop1 mutant, COP1-ovx, and GUS-COP1 plants under the same conditions in A. Stomata of the WT plants open significantly wider than those of the GUS-COP1 plants (^*, P ≤ 0.05, Student's t test).

Fig. 5.

Additive roles of cryptochromes and phototropins in the regulation of stomatal opening. (A and B) Confocal images of stomata (A) and stomatal apertures (B) in the cry1 cry2, phot1 phot2, cry1 cry2 phot1 phot2, cry1 cry2 cop1, phot1 phot2 cop1, and phot1 phot2 CRY1-ovx mutant plants under 20 μmol·m^-2·s^-1 blue light plus 50 μmol·m^-2·s^-1 red light. Stomata of the cry1 cry2 phot1 phot2 quadruple mutant opened significantly less wide than those of the phot1 phot2 double mutant (^**, P ≤ 0.01, Student's t test). (C) Blue light fluence rate response analysis of stomata in the cry1 cry2, phot1 phot2, cry1 cry2 phot1 phot2, and phot1 phot2 CRY1-ovx mutants. Epidermal strips were illuminated with different fluence rates of blue light plus 50 μmol·m^-2·s^-1 red light. Stomata of the phot1 phot2 mutant open significantly wider than those of the cry1 cry2 phot1 phot2 mutant under fluence rates >10 μmol·m^-2·s^-1 blue light (^*, P ≤ 0.05 at 10 μmol·m^-2·s^-1; ^**, P ≤ 0.01 at 30 μmol·m^-2·s^-1, Student's t test). (D) Signaling pathways illustrating coactions of CRY and PHOT in the regulation of stomatal opening presumably through negative regulation of COP1. Solid line indicates the defined direct CRY-COP1 interaction (28, 29), and the dashed line denotes the presumptive interactions. X, postulated intermediate signaling partner(s) acting between phototropins and COP1.