Action spectrum for cryptochrome-dependent hypocotyl growth inhibition in Arabidopsis

Abstract

Cryptochrome blue-light photoreceptors are found in both plants and animals and have been implicated in numerous developmental and circadian signaling pathways. Nevertheless, no action spectrum for a physiological response shown to be entirely under the control of cryptochrome has been reported. In this work, an action spectrum was determined in vivo for a cryptochrome-mediated high-irradiance response, the blue-light-dependent inhibition of hypocotyl elongation in Arabidopsis. Comparison of growth of wild-type, cry1cry2 cryptochrome-deficient double mutants, and cryptochrome-overexpressing seedlings demonstrated that responsivity to monochromatic light sources within the range of 390 to 530 nm results from the activity of cryptochrome with no other photoreceptor having a significant primary role at the fluence range tested. In both green- and norflurazon-treated (chlorophyll-deficient) seedlings, cryptochrome activity is fairly uniform throughout its range of maximal response (390-480 nm), with no sharply defined peak at 450 nm; however, activity at longer wavelengths was disproportionately enhanced in CRY1-overexpressing seedlings as compared with wild type. The action spectrum does not correlate well with the absorption spectra either of purified recombinant cryptochrome photoreceptor or to that of a second class of blue-light photoreceptor, phototropin (PHOT1 and PHOT2). Photoreceptor concentration as determined by western-blot analysis showed a greater stability of CRY2 protein under the monochromatic light conditions used in this study as compared with broad band blue light, suggesting a complex mechanism of photoreceptor activation. The possible role of additional photoreceptors (in particular phytochrome A) in cryptochrome responses is discussed.

Figure 1

Inhibition of hypocotyl elongation in Arabidopsis seedlings. A, Hypocotyl lengths of seedlings germinated as described in “Materials and Methods” and maintained for the indicated lengths of time in darkness. Error bars represent the se. Wt, Wild type; Oe, CRY1 overexpressor; C1C2, cry1cry2 double-mutant seedlings. B, Seedlings were plated and placed under monochromatic light sources as indicated in “Materials and Methods.” Measurements of seedling hypocotyl growth are presented as percentages of dark-grown control seedlings (where there is no growth inhibition). Error bars represent the se. OE, Transgenic seedlings overexpressing CRY1 protein; Wt, wild-type seedlings; C1C2, cry1cry2 double-mutant seedlings. C, Plot of cryptochrome action spectrum for 20% and 30% hypocotyl growth inhibition, respectively, as calculated from the data used in B. Oe, Overexpressor of CRY1; Wt, wild type. Peaks represent peak activity (maximal sensitivity) of the photoreceptor.

Figure 2

Inhibition of hypocotyl elongation in Arabidopsis seedlings grown on norflurazon. A, Hypocotyl lengths of seedlings germinated as described in “Materials and Methods” and maintained for the indicated lengths of time in darkness (upper panel) or for 60 h continuous growth in 660-nm red light (41 μmol m⁻² s⁻¹) or 713 nm far red light (29 μmol m⁻² s⁻¹). Error bars represent the se. Wt, Wild type; Oe, CRY1 overexpressor; C1C2, cry1cry2 double-mutant seedlings. B, Seedlings were plated and placed under monochromatic light sources as indicated in “Materials and Methods.” Measurements of seedling hypocotyl growth are presented as percentages of dark-grown control seedlings (where there is no growth inhibition). Error bars represent the se. Oe, Transgenic seedlings overexpressing CRY1 protein; Wt, wild-type seedlings; C1C2, cry1cry2 double-mutant seedlings. C, Plot of cryptochrome action spectrum for 20%, 30%, and 50% hypocotyl growth inhibition, respectively, as calculated from the data presented in B. Oe, Overexpressor of CRY1; Wt, wild type. Peaks represent peak activity (maximal sensitivity) of the photoreceptor.

Figure 2

Inhibition of hypocotyl elongation in Arabidopsis seedlings grown on norflurazon. A, Hypocotyl lengths of seedlings germinated as described in “Materials and Methods” and maintained for the indicated lengths of time in darkness (upper panel) or for 60 h continuous growth in 660-nm red light (41 μmol m⁻² s⁻¹) or 713 nm far red light (29 μmol m⁻² s⁻¹). Error bars represent the se. Wt, Wild type; Oe, CRY1 overexpressor; C1C2, cry1cry2 double-mutant seedlings. B, Seedlings were plated and placed under monochromatic light sources as indicated in “Materials and Methods.” Measurements of seedling hypocotyl growth are presented as percentages of dark-grown control seedlings (where there is no growth inhibition). Error bars represent the se. Oe, Transgenic seedlings overexpressing CRY1 protein; Wt, wild-type seedlings; C1C2, cry1cry2 double-mutant seedlings. C, Plot of cryptochrome action spectrum for 20%, 30%, and 50% hypocotyl growth inhibition, respectively, as calculated from the data presented in B. Oe, Overexpressor of CRY1; Wt, wild type. Peaks represent peak activity (maximal sensitivity) of the photoreceptor.

Figure 3

Stability of CRY2 protein under monochromatic light conditions. Equal amounts of protein from 3-d-old seedlings grown under the indicated light conditions were loaded onto each lane of an SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with anti-CRY2 antibody. Light intensities used are 10 μmol m⁻² s⁻¹ for red and blue light under broad band conditions and 10, 8, and 25 μmol m⁻² s⁻¹ light fluence for 400-, 450-, and 500-nm monochromatic light, respectively.

Figure 4

CRY1 dosage dependence of hypocotyl growth inhibition. A, Relative growth inhibition of norflurazon-treated wild-type (wt) and cryptochrome-overexpressing seedlings at decreasing photon fluence rates of 491- and 400-nm monochromatic light, respectively. Initial photon fluence rates (designated 100%) were chosen that resulted in identical growth inhibition of wild-type seedlings (7 μmol m⁻² s⁻¹ at 491 nm; 2.4 μmol m⁻² s⁻¹ at 400 nm). Growth of seedlings was compared at decreasing intensities of light (60%, 33%, and 13%), and growth inhibition of CRY1-overexpressing seedlings was compared under the two wavelengths. Overexpressing seedlings (Oe) showed greater relative sensitivity to 491-nm than to 400-nm light. B, CRY1 cryptochrome photoreceptor concentration in the seedlings did not change at any light treatment. Western blots were prepared from Wt and Oe (overexpressing) seedlings from A and compared with seedlings grown in continuous darkness. C, Comparison of photoreceptor concentration between cryptochrome-overproducing and wild-type seedlings. Lane 1, Wt and Oe indicates equivalent concentrations of total proteins of wild-type and overexpressing seedlings, respectively. Lanes 0.5 and 0.2 represent a dilution of 2- and 5-fold, respectively, of plant extract from the cryptochrome-overexpressing seedlings.

Figure 5

Enhancement of CRY1 and CRY2 action by phytochrome. Seedlings of the indicated genotypes (wt, wild type; cry1cry2, double mutant of cryptochrome; OECRY1, overexpressor of CRY1; OECRY2, overexpressor of CRY2; Ahmad et al., 1998a) were germinated as described in “Materials and Methods” and then placed for 72 h under the following light conditions: BL, 0.05 μmol m⁻² s⁻¹ blue-light intensity; BL + RL, seedlings were kept at 0.05 μmol m⁻² s⁻¹ blue-light intensity and subjected to 10-min red light pulses once every 3 h at a fluence of 3 μmol m⁻² s⁻¹; RL, seedlings were kept in continuous darkness and subjected to 10-min red light pulses once every 3 h at a fluence of 3 μmol m⁻² s⁻¹. Hypocotyl lengths of 20 seedlings per light treatment were averaged; error bars represent the se.

Figure 6

Absorption spectra of purified blue-light photoreceptors. A, Absorption spectrum of CRY1 protein expressed in baculovirus-infected Sf9 insect cells. Characteristics of absorption spectra are as in published plant cryptochrome spectra (Malhotra et al., 1995). B, Absorption spectrum of amino terminal NPL1 or PHOT-2 protein fragment expressed in E. coli. Characteristics of absorption spectra are as in published NPH1 spectra (Christie et al., 1998; Salomon et al., 2000).