Action Spectrum for Cryptochrome-Dependent Hypocotylthe production of an action spectrum, a dose-response curve at multiple wavelengths, for a certain plant activity or physiological

Action Spectrum for Cryptochrome-Dependent HypocotylGrowth Inhibition in Arabidopsis1

Margaret Ahmad*, Nicholas Grancher, Mary Heil, Robert C. Black, Baldissera Giovani, Paul Galland, andDanielle Lardemer

Universite Paris VI, Tour 53 E 5, Casier 156, 4, Place Jussieu, 75252 Paris cedex 05, France (M.A., N.G.,B.G., D.L.); Pennsylvania State University, 25 Yearsley Mill Road, Media, Pennsylvania 19063 (M.A., M.H.,R.C.B.); and FB Biologie/Botanik, Philipps-Universitaet Marburg, Karl-von-Frisch-Strasse, 35032 Marburg,Germany (P.G.)

Cryptochrome blue-light photoreceptors are found in both plants and animals and have been implicated in numerousdevelopmental and circadian signaling pathways. Nevertheless, no action spectrum for a physiological response shown tobe entirely under the control of cryptochrome has been reported. In this work, an action spectrum was determined in vivofor a cryptochrome-mediated high-irradiance response, the blue-light-dependent inhibition of hypocotyl elongation inArabidopsis. 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 nmresults from the activity of cryptochrome with no other photoreceptor having a significant primary role at the fluence rangetested. In both green- and norflurazon-treated (chlorophyll-deficient) seedlings, cryptochrome activity is fairly uniformthroughout its range of maximal response (390–480 nm), with no sharply defined peak at 450 nm; however, activity at longerwavelengths was disproportionately enhanced in CRY1-overexpressing seedlings as compared with wild type. The actionspectrum does not correlate well with the absorption spectra either of purified recombinant cryptochrome photoreceptor orto that of a second class of blue-light photoreceptor, phototropin (PHOT1 and PHOT2). Photoreceptor concentration asdetermined by western-blot analysis showed a greater stability of CRY2 protein under the monochromatic light conditionsused 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.

Blue-light responses are found in organismsthroughout the biological kingdom; in many in-stances, these responses are mediated by photore-ceptors that specifically absorb blue light and are rel-atively ineffective at other wavelengths. In Arabidop-sis, flavoprotein blue-light photoreceptors known ascryptochromes have been implicated in light re-sponses including inhibition of hypocotyl elongation,anthocyanin accumulation, internode and petioleelongation, seed germination, blue-light-regulatedgene expression, initiation of flowering time, photot-ropism, and the entrainment of circadian rhythms(Ahmad and Cashmore, 1996; Ahmad, 1999).Cryptochrome-type photoreceptors have recentlybeen found in animal systems as well wherein theyseem to play essential roles in the entrainment andmaintenance of circadian rhythms (Sancar, 2000).Cryptochromes are characterized by their strikinghomology to certain classes of DNA photolyases, orblue-light-dependent DNA repair enzymes, whichcatalyze a blue-light-dependent electron transfer re-

action (Deisenhofer, 2000). In Arabidopsis thereare two similar genes encoding cryptochromephotoreceptors, CRY1 (or HY4) and CRY2, whoseencoded proteins differ primarily in their respectiveC-terminal domains. In addition, the CRY1 photore-ceptor is stable in plants grown in high intensities ofblue light, whereas CRY2 photoreceptor fails to ac-cumulate and appears to be rapidly degraded underconditions wherein the photoreceptor is active(Ahmad et al., 1998a; Lin et al., 1998). Cryptochromescontain the chromophore-binding domain of photol-yases, and have been shown to bind both a pterin andflavin chromophore; however, they lack the apparentpyrimidine dimer binding or repair activity of pho-tolyases (Malhotra et al., 1995; Lin et al., 1995). Inaddition, some cryptochromes contain C-terminal ex-tensions not found in photolyases, which are shownto be necessary for photoreceptor functions (Ahmadet al., 1995; Yang et al., 2000). Given the strikinghomology of cryptochromes to photolyases, a pri-mary mechanism of action via blue-light-dependentelectron transfer is likely (see Ahmad and Cashmore,1996; Ahmad, 1999; Lin, 2000, and refs. therein). Re-cent studies with fruitfly (Drosophila melanogaster)and animal cryptochromes also suggest electrontransfer mechanisms (Lin et al., 2001).

Although the cryptochrome photoreceptors arewell characterized and have been studied in a variety

1 This work was supported by a Contrat Atipe Blanche from theCentre National de la Recherche Scientifique (to M.A.).

* Corresponding author; e-mail [email protected]; fax133144272916.

Article, publication date, and citation information can be foundat www.plantphysiol.org/cgi/doi/10.1104/pp.010969.

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of plant and animal systems, very little is knownabout their mechanism of action and photochemistry.Photolyases, to which the cryptochrome photorecep-tors are most closely related, are unusual amongflavoproteins in that they are activated with flavinpresent in the reduced form. Reduced flavin, in con-trast to oxidized flavin, has a peak of absorption ataround 360 nm and absorbs very little visible light.As a result, photolyases rely on an antennae pigment,a folate, or deazaflavin derivative for the bulk of theiractivity in blue light (400–500 nm). The peak of ab-sorption of the antennae pigment can be between 380to 440 nm depending on the precise composition ofthe chromophore (Sancar et al., 1987; Ahmad andCashmore, 1996) and seems to occur at near 420 nmfor purified recombinant cryptochrome (Malhotra etal., 1995). By contrast, most flavoenzymes occur inthe oxidized form, showing a peak of absorption at450 nm, as is found in the phototropin-like photore-ceptors (Briggs et al., 2001). Therefore, an importantclue regarding the mechanism of action of the cryp-tochrome photoreceptors would be to ascertain theabsorption characteristics of the photoreceptor in itsactive state within the plant. Such data is provided bythe production of an action spectrum, a dose-response curve at multiple wavelengths, for a certainplant activity or physiological response that is underthe control of this photoreceptor.

Although many action spectra for plant blue-lightresponses exist in the literature, their interpretation iscomplicated by the fact that multiple plant photore-ceptors in addition to cryptochromes are active inblue light. Unrelated blue-light photoreceptors, dis-tinct from the cryptochromes, have recently beenidentified and shown to be flavoproteins, absorbingmaximally at 450 nm (Briggs and Huala, 1999; Briggset al., 2001). In addition, plant phytochrome photo-receptors, which respond principally to red/far-redlight, show some absorption of blue light, renderingthe interpretation of classical blue-light action spec-tra more difficult (Shinomura et al., 2000; Shino-mura et al., 1996). In the case of higher plants, thesituation is even further complicated by the obser-vation that a signal from phytochrome is necessaryfor full activity of the cryptochrome blue-light pho-toreceptors in hypocotyl growth inhibition and an-thocyanin accumulation (Ahmad and Cashmore,1997). Therefore, it is not possible to infer that anygiven published blue-light action spectra representsthe activity of cryptochrome.

In this study, an action spectrum is generated for aresponse demonstrated to be under the control ofcryptochrome, namely blue-light-dependent inhibi-tion of hypocotyl elongation in Arabidopsis. In pre-vious studies in Arabidopsis (Young et al., 1992; Gotoet al., 1993), the extent of growth inhibition resultingfrom cryptochrome activity (both CRY1 and CRY2)had not been determined, nor had there been correc-tion for potential artifacts resulting from shading of

the blue-light receptors by chlorophylls and carote-noids. In the present study, we show that cry1cry2double-mutant seedlings show no measurablegrowth inhibition at wavelengths from 390 to 530 nmunder the given light intensities, indicating crypto-chrome as the primary photoreceptor species in-volved. Growth inhibition biosynthesis studies in thepresence of norflurazon, a carotenoid inhibitor re-sulting in photobleaching of Arabidopsis seedlings,were performed to correct for possible shifts in theaction spectra due to shading by chlorophyll. Furtherclues into possible photoreceptor mechanism of ac-tion and photochemistry is provided by examinationof action spectra of a cryptochrome-1-overexpressingtransgenic line and an analysis of photoreceptor con-centration and stability, in particular that of light-labile CRY2. These action spectra are compared withthe absorption spectra of purified Arabidopsis blue-light photoreceptors, and the results are discussed inrelation to the known photoreceptors that might con-tribute to this response in Arabidopsis and otherplants.

RESULTS

Inhibition of Hypocotyl Elongation in ArabidopsisActivity Spectrum for Cryptochrome in aHigh-Irradiance Response

In Arabidopsis, inhibition of hypocotyl elongationis a sensitive and quantitative light response in whichmultiple photoreceptors participate, including phy-tochromes, CRY1, and CRY2. There are currently twopublished action spectra for blue-light-dependent in-hibition of hypocotyl elongation in Arabidopsis(Young et al., 1992; Goto et al., 1993), but in neithercase did the authors identify the component of theresponse due solely to cryptochrome (CRY1 andCRY2). In the present study, the growth response ofwild-type Arabidopsis seedlings was compared withthat of double cryptochrome cry1cry2 mutants, allow-ing unequivocal determination of an activity spec-trum for cryptochrome. In addition, the responsivityof seedlings overexpressing CRY1 protein was eval-uated for possible wavelength-specific effects, be-cause such seedlings show a hypersensitive responseto blue light under broad band conditions (Lin et al.,1996; Ahmad et al., 1998a). In darkness, the lengths ofthe hypocotyls of wild type, mutant seedlings andCRY1 overexpressors were essentially the same (Fig.1A). Hypocotyl growth inhibition in light-treatedseedlings was measured at multiple light intensitiesat wavelengths of 10- to 20-nm intervals spanning therange of 380- to 450-nm bandwidth, with additionalpoints in the red/far-red and near-UV region (notshown). The degree of inhibition of hypocotylgrowth was plotted as a percentage of the growth ofseedlings retained in complete darkness (no growthinhibition) at wavelengths in which a robust crypto-chrome response was obtained (Fig. 1B). These ex-

Cryptochrome Action Spectrum

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Figure 1. Inhibition of hypocotyl elongation in Arabidopsis seedlings. A, Hypocotyl lengths of seedlings germinated asdescribed in “Materials and Methods” and maintained for the indicated lengths of time in darkness. Error bars represent theSE. Wt, Wild type; Oe, CRY1 overexpressor; C1C2, cry1cry2 double-mutant seedlings. B, Seedlings were plated and placedunder monochromatic light sources as indicated in “Materials and Methods.” Measurements of seedling hypocotyl growthare presented as percentages of dark-grown control seedlings (where there is no growth inhibition). Error bars represent theSE. OE, Transgenic seedlings overexpressing CRY1 protein; Wt, wild-type seedlings; C1C2, cry1cry2 double-mutantseedlings. C, Plot of cryptochrome action spectrum for 20% and 30% hypocotyl growth inhibition, respectively, ascalculated from the data used in B. Oe, Overexpressor of CRY1; Wt, wild type. Peaks represent peak activity (maximalsensitivity) of the photoreceptor.

Ahmad et al.

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periments were repeated in a total of five separatetrials; in all cases, the results were qualitatively sim-ilar and internally consistent. The differential growthinhibition of seedlings lacking all cryptochrome(cry1cry2), and containing a superabundance of cryp-tochrome photoreceptor (Oe) allows the determina-tion of a high-irradiance action spectrum for a cryp-tochrome response.

Significantly, cry1cry2 double-mutant seedlingsdid not show growth inhibition at any wavelengthof monochromatic light tested between 311 and 550nm at the photon fluence rates used in this study.Both wild-type and CRY1-overexpressing seedlingsshowed maximal growth inhibition between 380and 500 nm (Fig. 1B). Very little response was seenbelow 365 nm, using UV sources of light fluence 0.5to 1.0 �mol m�2 s�1. From 500 to 550 nm, littlegrowth inhibition of wild type was observed at theoutput (30–50 �mol m�2 s�1) our lamps could gen-erate, although increased growth inhibition ofCRY1-overexpressing seedlings as compared withwild type or cry1cry2 double mutant was observeduntil 550 nm (not shown). At wavelengths longerthan 570 nm, there were no detectable differences inlight-dependent hypocotyl growth inhibition be-tween cryptochrome-deficient and cryptochrome-overexpressing seedlings, even at quite high lightintensities (80–100 �mol m�2 s�1; not shown). Thisresult is consistent with cryptochromes being activeprincipally in the range from 365 to 550 nm.

To more precisely ascertain the wavelength atwhich cryptochrome is maximally effective in theseseedlings, an action spectrum was generated inwhich the photon fluence rate resulting in 20% or30% growth inhibition was plotted as a function ofwavelength (Fig. 1C). The action spectrum is plottedsuch that peaks occurring in the plot correspond topeak sensitivity of the photoreceptor. In the case ofwild type, the action spectrum is almost flat with nomore than a 2-fold variation in effectiveness of lightbetween 380 and 480 nm. Similar to wild type, theCRY1-overexpressing seedlings showed a fairly flataction spectrum, although they were considerablymore light-sensitive than wild type at all wave-lengths tested. However, in contrast to wild type,maximal activity in the CRY1 overexpressor occurrednear 480 nm, with a greater relative responsivity at500 nm (only 4-fold less than that of wild type, whichshowed a 10-fold drop in activity at 500 nm com-pared with 450 nm; Fig. 1C). Thus, increasing thedosage of cryptochrome photoreceptor appears toresult in shift of photoreceptor sensitivity towardhigher bandwidths.

A concern with the interpretation of action spectrain de-etiolated plant material is shading by non-photoreceptor pigments, in particular chlorophyllsand carotenoids, both of which absorb in blue light. Ithad been previously shown for both Sinapis alba(Beggs et al., 1980) and Chenopodium rubrum (Holmes

and Wagner, 1982) that chlorophyll significantly al-tered the action spectrum of hypocotyl growth inhi-bition in these plant species. To correct for possibleshading artifacts, action spectra for seedling growthinhibition were repeated on petri plates containingthe herbicide norflurazon, an inhibitor of carotenoidbiosynthesis that also prevents chlorophyll accumu-lation in light-grown plants (Beggs et al., 1980;Holmes and Wagner, 1982).

Wild type, cry1cry2 mutant seedlings, and CRY1overexpressors grown on norflurazon showed iden-tical rates of growth in darkness, red light (660 nm),and far-red light (713 nm; Fig. 2A). This indicates thatthere was no activity of cryptochrome under theseconditions, and also that the various mutant lineswere comparable in growth capacity. At wavelengthsfrom 392 to 530 nm, there was no observable growthinhibition in cry1cry2 double-mutant seedlings (Fig.2B), indicating that no additional blue-light photore-ceptor was contributing measurably to the primaryresponse at these wavelengths. As an additional con-trol, the degree of growth inhibition obtained forseedlings at 450 nm was measured after 30 h ofcontinuous growth instead of 60 h. These data werecompared with the data at 450 nm obtained forgrowth inhibition at 60 h in Figure 2B. No differencein fluence response characteristics was observed, in-dicating that no major distortion in the action spectrais likely to occur at least during the latter one-half ofthe growth period (not shown). The action spectrumfor the norflurazon-grown seedlings was calculatedfrom the data used for Figure 2B and plotted for 20%,30%, and 50% growth inhibition for wild-type andCRY1-overexpressing seedlings (Fig. 2C).

The action spectra obtained on norflurazon forwild-type seedlings is also fairly flat in shape with nomore than 2.5-fold variation in peak photon effective-ness in the wavelength range from 380 to 480 nm(Fig. 2C). In contrast to untreated seedlings, somegrowth inhibition was observed for cry1cry2 double-mutant seedlings at 380 nm (Fig. 2B, see 380-nmcurve), indicating the activity of additional photore-ceptors absorbing in the UV that were shaded inuntreated seedlings and that were, thereby, not de-tected. In addition, wild-type seedlings were moresensitive to blue light on norflurazon and respondedto lower light intensities than was the case for un-treated seedlings, again suggesting that chlorophyllsignificantly shades the relevant photoreceptors ingreen tissues. Action spectra obtained on norflurazonfor the cryptochrome-overexpressing lines did notshow a peak near 480 nm as for untreated seedlingsbut, instead, showed a more pronounced shoulderthan seen for the wild type.

Differential Stability of CRY2 Protein in BroadBand and Monochromatic Light

It has been previously shown that both CRY1 andCRY2 contribute to the growth inhibition response in




Arabidopsis (Ahmad et al., 1998a; Lin et al., 1998).However, unlike CRY1, the CRY2 photoreceptor ofArabidopsis appears to be light labile, and levels ofphotoreceptor protein in seedlings are significantlylower in broad band blue, UV-A, and green light (inwhich the photoreceptor is active) than in red light or

dark (in which the photoreceptor shows no activity).To assess the possible contribution of CRY2 proteinto the cryptochrome action spectrum, the stability ofCRY2 protein was evaluated at several wavelengthsof monochromatic light throughout the range of max-imum cryptochrome effectiveness (Fig. 3). Western-

Figure 2. Inhibition of hypocotyl elongation in Arabidopsis seedlings grown on norflurazon. A, Hypocotyl lengths ofseedlings 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 �mol m�2 s�1) or 713 nm far red light (29 �mol 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.” Measure-ments of seedling hypocotyl growth are presented as percentages of dark-grown control seedlings (where there is no growthinhibition). 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% hypocotylgrowth inhibition, respectively, as calculated from the data presented in B. Oe, Overexpressor of CRY1; Wt, wild type. Peaksrepresent peak activity (maximal sensitivity) of the photoreceptor. (Figure continues on facing page.)

Ahmad et al.



blot analysis of seedlings of Arabidopsis were per-formed with anti-CRY2 antibody, and levels of CRY2protein were compared with levels of accumulationin dark. Surprisingly, at 400, 450, and 500 nm, theamount of CRY2 protein did not decrease dramati-cally from that found in dark-grown control seed-lings. The light photon fluences used were relativelyhigh; at 450 nm, the intensity of 8 �mol m�2 s�1 wassufficient to induce more than 50% hypocotyl growthinhibition in wild-type seedlings, for example, indi-cating considerable cryptochrome photoreceptor ac-tivity. These results are in contrast to the markedinstability of CRY2 protein in broad band blue light(Fig. 3).

Cryptochrome Activity Is Modified by ReceptorDosage in a Wavelength-Specific Manner

An unexpected feature of the cryptochrome actionspectrum is that CRY1-overexpressing seedlingsshow relatively greater activity than wild type atwavelengths longer than about 430 nm (Figs. 1C and2C). There is an approximately 4-fold greater sensi-tivity to 400 nm in CRY1-overexpressing seedlings ascompared with wild type, whereas the difference insensitivity is almost 20-fold at 480 nm light. Theobserved shift in peak activity to 480 nm ofcryptochrome-overexpressing seedlings (Fig. 1C) issignificantly less pronounced in norflurazon-treatedseedlings (Fig. 2C), suggesting it may have been par-tially resulting from the higher chlorophyll content ofsuch seedlings in continuous light conditions. Nev-ertheless, a greater sensitivity to higher wavelengthlight in CRY1-overexpressing seedlings holds even inthe presence of norflurazon. For instance, CRY1-overexpressing seedlings on norflurazon are about

4-fold more sensitive than wild-type seedlings be-tween 392 and 413 nm monochromatic light (Fig. 2, Band C). By contrast, the difference in sensitivity isclose to 10-fold at several longer wavelengths, par-ticularly between 472 and 491 nm.

This effect is illustrated in Figure 4A, showing atypical experiment where seedlings of wild-type andoverexpressing lines were subjected to decreasingphoton fluences of monochromatic light at 491 and400 nm light, respectively. Wild-type seedlings showsimilar sensitivity to the initial light intensity chosen.However, CRY1-overexpressing seedlings are con-siderably more sensitive to decreasing intensities of491 nm than of 400 nm light.

A simple explanation for the increased sensitivityto blue light in CRY1-overexpressing Arabidopsislines would be a proportionate increase in the con-centration of the cryptochrome photoreceptors. Totest this possibility, the levels of CRY1 and CRY2protein were compared in wild-type and overex-pressing Arabidopsis lines by western-blot analysisunder the different growth conditions used in Fig-ure 4A (Fig. 4B). At none of the light intensitiesexamined was there any variation relative to darklevels of cryptochrome photoreceptor, either inoverexpressing (Oe) or wild-type lines. Finally, adilution series of protein extracted from crypto-chrome-overexpressing lines indicated the increasein cryptochrome photoreceptor concentration in theCRY1-overexpressing Arabidopsis lines was notgreater than a 5-fold increase as compared with wildtype (Fig. 4C).

Figure 3. Stability of CRY2 protein under monochromatic light con-ditions. Equal amounts of protein from 3-d-old seedlings grownunder the indicated light conditions were loaded onto each lane ofan SDS-polyacrylamide gel, transferred to nitrocellulose, and probedwith anti-CRY2 antibody. Light intensities used are 10 �mol m�2 s�1

for red and blue light under broad band conditions and 10, 8, and 25�mol m�2 s�1 light fluence for 400-, 450-, and 500-nm monochro-matic light, respectively.

Figure 2. (Figure continued from previous page.)




DISCUSSION

In this study we have measured hypocotyl growthinhibition in a number of cryptochrome mutants overthe wavelength range 311 to 730 nm. To correct forpossible shading artifacts by chlorophyll or carote-noids (Beggs et al., 1980; Holmes and Wagner, 1982),the action spectra were also performed with seed-lings grown on the herbicide norflurazon, whicheliminates chlorophyll in light-grown seedlings.Based on comparisons of wild-type, cry1cry2 double-mutant, and CRY1-overexpressing Arabidopsis seed-lings, it was determined that maximal cryptochromeactivity occurs between 380- to 500-nm bandwidth,and that, moreover, there is no significant indepen-dent contribution by other blue-light receptors tostem growth inhibition between 392 to 530 nm underthe conditions used in this study. There was someresidual cryptochrome activity at shorter wave-

lengths (up to 365 nm) and also at longer wave-lengths up to 550 nm, but no cryptochrome-specificactivity in either red (660 nm) or far-red (713 nm)light, consistent with several prior studies (Goto etal., 1993; Lin et al., 1996) and somewhat in contrast torecent suggestions for a role for cryptochrome in redlight (Devlin and Kay, 2000).

In overall shape and fluence threshold, the data wepresent for wild-type seedlings are in agreementwith the results from prior studies investigating hy-pocotyl growth in Arabidopsis as a function of wave-length, involving a comparison of wild-type withphytochrome-deficient hy2 mutant seedlings (Goto etal., 1993) and to blu1, an allele of hy4 (deficient inCRY1; Young et al., 1992). However, the action spec-tra obtained from norflurazon-treated seedlings,though similar in shape to those of untreated seed-lings for wild type, showed significantly greater sen-

Figure 4. CRY1 dosage dependence of hypo-cotyl growth inhibition. A, Relative growth in-hibition of norflurazon-treated wild-type (wt)and cryptochrome-overexpressing seedlings atdecreasing photon fluence rates of 491- and400-nm monochromatic light, respectively. Ini-tial photon fluence rates (designated 100%)were chosen that resulted in identical growthinhibition of wild-type seedlings (7 �mol m�2

s�1 at 491 nm; 2.4 �mol m�2 s�1 at 400 nm).Growth of seedlings was compared at decreas-ing intensities of light (60%, 33%, and 13%),and growth inhibition of CRY1-overexpressingseedlings was compared under the two wave-lengths. Overexpressing seedlings (Oe) showedgreater relative sensitivity to 491-nm than to400-nm light. B, CRY1 cryptochrome photore-ceptor concentration in the seedlings did notchange at any light treatment. Western blotswere prepared from Wt and Oe (overexpress-ing) seedlings from A and compared with seed-lings grown in continuous darkness. C, Com-parison of photoreceptor concentrationbetween cryptochrome-overproducing andwild-type seedlings. Lane 1, Wt and Oe indi-cates equivalent concentrations of total proteinsof wild-type and overexpressing seedlings, re-spectively. Lanes 0.5 and 0.2 represent a dilu-tion of 2- and 5-fold, respectively, of plant ex-tract from the cryptochrome-overexpressingseedlings.

Ahmad et al.



sitivity at all light intensities, in agreement with priorobservations for other plant species (Beggs et al.,1980; Holmes and Wagner, 1982).

The absence of response in cry1cry2 double mu-tants in blue light is important because it suggeststhat no other photoreceptor has a significant directrole in blue-light-dependent hypocotyl growth inhi-bition. Therefore, other photoreceptors that havebeen proposed to play a direct role, in particularphytochrome A (phyA; Casal and Mazella, 1998; Neffand Chory, 1998; Poppe et al., 1998) most likely actindirectly through modification/interaction with thecryptochrome-dependent signaling pathway. An en-hancement of CRY2 function by phytochrome isdemonstrated by pulse experiments, in which red-light treatment increases CRY2-dependent growthinhibition in Arabidopsis seedlings (Fig. 5). In addi-tion, possible interaction between CRY2 and phyto-chrome has been documented both in vitro (Ahmadet al., 1998b) and in vivo (Mas et al., 2000). Recentstudies on the role of phyA in early events of blue-light-mediated stem growth inhibition support anindirect role for phyA in some cryptochrome re-sponses (Folta and Spalding, 2001b).

Action spectra have traditionally been constructedas a tool for proposing the molecular identity of therelevant photoreceptor. The absorption spectra of pu-tative photoreceptors is compared with the actionspectrum of the response, and the best “match” ispresumed to be the photoreceptor. There exist a num-ber of published absorption spectra for recombinantcryptochrome expressed in heterologous systems.Plant cryptochrome expressed heterologously inEscherichia coli shows a pronounced peak of absorp-tion near 420 nm as a result of the pterin chro-mophore (Malhotra et al., 1995), with the absorptionspectrum trailing off rapidly to near zero by 500 nm.CRY1 photoreceptor has, in addition, been expressedin insect cell systems, from which it has been isolatedas an oxidized flavoprotein lacking a secondarypterin chromophore (Lin et al., 1995). However, adifference spectrum between insect cell extracts ex-pressing high levels of CRY1 photoreceptor protein

and control extracts (not expressing CRY1) shows anabsorption spectrum identical to the E. coli expres-sion product, with a major peak near 420 nm (Fig.6A). Therefore, the lack of a secondary chromophorein these earlier insect cell experiments may haveoccurred as a result of the purification process, inwhich the pterin chromophore was lost.

Surprisingly, comparison of the action spectra(Figs. 1C and 2C) with the cryptochrome absorptionspectrum (Fig. 6A) shows little correspondence.There is no pronounced peak at 420 nm in the actionspectrum, and activity at 500 nm is relatively high,particularly in overexpressing seedlings, in compar-ison with the absorption spectrum. This lack of cor-respondence between action spectrum and absorp-tion spectrum is all the more surprising because, inthe case of type I photolyases, to which plant cryp-tochromes show the greatest degree of homology,action spectra for DNA photorepair are essentiallysuperimposable upon the absorption spectra of thepurified proteins (Jorns et al., 1986; Sancar et al.,1987). Cryptochrome may in fact be active in a dif-ferent form (perhaps using oxidized flavin, therebyexplaining the peak at 450 nm) than is photolyase,thus, raising the intriguing possibility of a differingprimary mechanism of action of cryptochrome fromphotolyase.

An alternative explanation for the lack of correspon-dence between absorption spectra and action spectramay be complex events downstream of the point ofphotoreception, which distort the action spectra.This is particularly a possibility with action spec-tra involving long periods of irradiation (HIR spectra).To determine whether distortion might occur overthe period of total irradiation, we measured degree ofgrowth inhibition after a shorter (30 h) growth pe-riod for seedlings in 450 nm light, but observed nosignificant distortion at least after the 1st d ofgrowth (not shown). Nevertheless, only a much morerapid assay for cryptochrome function can defini-tively exclude the possibility of artifact in theseaction spectra. It would also be preferable to devisean assay for cryptochrome function in dark-grown

Figure 5. Enhancement of CRY1 and CRY2 ac-tion by phytochrome. Seedlings of the indicatedgenotypes (wt, wild type; cry1cry2, double mu-tant of cryptochrome; OECRY1, overexpressor ofCRY1; OECRY2, overexpressor of CRY2; Ahmadet al., 1998a) were germinated as described in“Materials and Methods” and then placed for72 h under the following light conditions: BL,0.05 �mol m�2 s�1 blue-light intensity; BL � RL,seedlings were kept at 0.05 �mol m�2 s�1 blue-light intensity and subjected to 10-min red lightpulses once every 3 h at a fluence of 3 �mol m�2

s�1; RL, seedlings were kept in continuous dark-ness and subjected to 10-min red light pulsesonce every 3 h at a fluence of 3 �mol m�2 s�1.Hypocotyl lengths of 20 seedlings per light treat-ment were averaged; error bars represent the SE.




seedlings, because in this way, indirect light effectson concentrations of other photoreceptors and/orsignaling intermediates may be avoided.

A second class of plant blue-light photoreceptor forwhich the absorption spectra have been determinedare the plant phototropins, PHOT1 and PHOT2. Thisclass of photoreceptor bind oxidized flavin and havebeen implicated in a variety of blue-light responsesincluding phototropism and blue-light-dependentchloroplast movements (Briggs et al., 2001). The ab-sorption spectrum of purified recombinant PHOT1has been identified from both insect cell (Christie etal., 1998) and E. coli (Salomon et al., 2000) expressionsystems and shows a marked peak at 450 nm result-ing from oxidized flavin. For the purposes of thisdiscussion, we have expressed a fragment of PHOT2comprising the flavin-binding domains of this pho-toreceptor in E. coli and present the absorption spec-trum below (Fig. 6B). Interestingly, the cryptochromeaction spectrum is somewhat more similar to thephototropin absorption spectra than it is to that ofpurified cryptochrome. The position of the peak (450nm) and shoulders (Figs. 1C and 2C) in wild-typeseedlings corresponds more closely with those of thephototropin absorption spectrum, although the cryp-tochrome curve is much flatter. We have determinedthat there is a small effect of PHOT1 on hypocotylelongation at high-intensity broad band blue light(M. Ahmad, unpublished data). A role for pho-totropin in inhibition of hypocotyl elongation is also

suggested in recent publications investigating blue-light-dependent activation of ion channels (Folta andSpalding, 2001a). However, given that cry1cry2 dou-ble mutants show no growth inhibition at the condi-tions used in the present work, any contribution bysuch PAS-domain-containing photoreceptors to theaction spectrum is likely to be indirect and/or minor.

Evidence of wavelength-specific effects on thecryptochrome photoreceptors is found in the differ-ential stability of CRY2 protein. Under broad bandblue-light conditions, CRY2 protein is rapidly de-graded and accumulates to only very low concentra-tions in seedlings. This may be due to targeting bydegradative enzymes upon activation of the photo-receptor, by analogy to the situation with phyA.However, under the narrow band light conditionsused in these action spectra, CRY2 protein appears tobe more stable and accumulates even at relativelyhigh blue-light intensities. It is possible that crypto-chromes may cycle between multiple conformationsupon activation by light and that only one of theseforms is recognized by protein degradative enzymes.It will be intriguing to examine whether combina-tions of monochromatic light of different wave-lengths reduce the stability of CRY2 protein andwhether the conformation of CRY2 photoreceptorunder monochromatic light conditions differs fromthat under broad band blue light.

The action spectra of CRY1-overexpressing seed-lings show an unexpected feature in that the respon-sivity varies between a 4-fold to a more than 10-foldincrease compared with wild type, even though theincrease in photoreceptor concentration is not morethan 5-fold at any wavelength. In particular, there isgreater responsivity of cryptochrome-overexpressingseedlings above 430 nm in comparison with wild-type seedlings of both untreated and norflurazon-treated plant material. A similar shift to longer wave-lengths in the action spectrum has been observed fortransgenic Arabidopsis seedlings overexpressingphytochrome (McCormac et al., 1993; Shinomura etal., 1998) and was explained in terms of wavelengthdependent differences in the absolute amounts of Pfrgenerated in overexpressing lines (Shinomura et al.,1998). It is intriguing to speculate that dosage depen-dence in cryptochrome photoreceptor mutants mayalso reflect differential accumulation and/or lifetimeof multiple interchangeable receptor conformations.

In summary, we present here an action spectrumfor a response demonstrated to be under the controlof cryptochrome under the fluence range investi-gated. The action spectrum shows several unex-pected features, in particular a lack of correspon-dence with the absorption spectrum of the purifiedcryptochrome photoreceptor from several heterolo-gous systems. This may indicate some difference inthe biochemical mode of action between crypto-chromes and photolyases, where absorption spectraand action spectra coincide closely. Furthermore, ex-

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 asin published plant cryptochrome spectra (Malhotra et al., 1995). B,Absorption spectrum of amino terminal NPL1 or PHOT-2 proteinfragment expressed in E. coli. Characteristics of absorption spectraare as in published NPH1 spectra (Christie et al., 1998; Salomon etal., 2000).

Ahmad et al.



amination of cryptochrome responses in CRY1-overexpressing seedlings shows a complex dosagedependence at different wavelengths, suggesting acomplex mechanism of activation. Finally, the stabil-ity of CRY2 photoreceptor is affected differentially bybroad band and monochromatic light, further sug-gesting complex events within the photoreceptor atthe point of absorption of the light signal. It will be ofgreat interest to sort out the molecular basis of thisintriguing physiology, both by biophysical investiga-tion of the purified photoreceptor and by detailedexamination of cryptochrome interactions with po-tential substrates as a function of wavelength.

MATERIALS AND METHODS

Arabidopsis Seedling Germination and Growth

For all light irradiation experiments Arabidopsis seedswere surface sterilized by a brief wash in 100% (v/v)ethanol, followed by air drying under a laminar flow hood.Seeds were sown on one-half-strength Murashige andSkoog salts medium (Sigma, St. Louis) on petri plates con-taining 2% (w/v) Suc and 0.8% (w/v) agar. Norflurazonwas added at a final concentration of 5 � 10�6 M and wasa kind gift of Dr. Klaus Kreuz (Novartis, Basel). Plates werestored for 2 d at 4°C to break dormancy, and were subse-quently transferred to white light (30 �mol m�2 s�1) for24 h to induce germination. Hy4-2.23N and fha alleles wereused for CRY1 and CRY2 mutations, respectively; CRY1-and CRY2-overexpressing seedlings were as described(Ahmad et al., 1998a).

Interference Filters and Light Sources

Monochromatic light was produced by slide projectors(Prado Universal 31047, Ernst Leitz GmBH, Wetzler, Ger-many) in combination with 5-mm KGI heat-absorbing fil-ters (Schott Glaswerke, Mainz, Germany) placed beforeinterference filters of 8- to 10-nm half-bandwidth (SchottGlaswerke, Mainz, Germany). Broad band light was pro-duced by using cool-white fluorescent bulbs (Philips, Eind-hoven, The Netherlands) in association with broad bandplexiglass filters of 100-nm bandwidth as used in priorstudies (Ahmad et al., 1998a).

Inhibition of Hypocotyl Elongation Assays

Approximately 50 seeds of wild-type, cryptochrome-overexpressing, and cryptochrome-deficient Arabidopsislines were plated within a single petri dish for each lighttreatment. In this way, hypocotyl growth of seedlings isdirectly comparable and did not suffer from possible in-consistencies in light treatments or experimental manipu-lation between plates. Seedlings were plated an average of1 to 2 mm apart to minimize self-shading through thecourse of the light irradiations. Plates were stacked incolumns under light sources, and irradiated from above,such that the light intensity at subsequent positions in thestack were reduced by a defined amount. Beams of light

from slide projectors were shone through interference fil-ters by objectives that penetrated the wall at each position.Irradiations were performed for a continuous 60 h after the24-h white-light treatment to induce germination of seed-lings (see above). For each experiment, a number of plateswere wrapped in foil and maintained for 60 h as darkcontrols. Light intensities at the surface of the petri plateswere measured for each plate in a stack after the comple-tion of the experiment. In all instances, dark controlsshowed no evidence of differential growth between thedifferent genotypes, indicating that this growth regimedoes not activate the cryptochrome photoreceptors in theabsence of light. Subsequent to termination of the lighttreatments, samples for determination of protein concen-trations were taken immediately. Additional plates of seed-lings were stored at 4°C for a period not greater than 8 h,during which time from 10 to 20 seedlings were measuredper plate per Arabidopsis line. Error bars represent the se;in general, growth was very uniform, and variation withina population was not more than 10% of total seedlinglength. Light treatments under broad band filters werecarried out under light conditions (filters and fluorescentbulbs) as described previously (Ahmad et al., 1998a).

Western-Blot Analysis

Western-blot analysis of seedlings was performed usinganti-CRY1 and anti-CRY2 antibodies prepared to the Cterminus of the respective proteins expressed in recombi-nant form, essentially as described previously (Ahmad etal., 1998a). All seedlings used for western-blot analysiswere quick-frozen in liquid nitrogen subsequent to lighttreatments. Samples were then ground in SDS samplebuffer (Laemmli) and equivalent amounts of protein run oneach lane of an SDS polyacrylamide gel, before westerntransfer and detection by antisera.

Absorption Spectra of Recombinant Photoreceptors

Cryptochrome-1 protein was expressed in insect cell(Sf9) system as described previously (Lin et al., 1995).Insect cell cultures expressing recombinant cryptochromewere precipitated and lysed as described (Lin et al., 1995)in parallel with an equal amount of uninfected Sf9 cells.The cell suspensions were clarified by ultracentrifugationat 40,000 rpm for 1 h, and the supernatants were adjustedto an equivalent total protein concentration (1 mg mL�1).The absorption spectrum of the extract from bothcryptochrome-expressing and control cells was taken in aDU7400 spectrophotometer (Beckman Coulter, Inc., Fuller-ton, CA). Insect cell control cultures not expressing recom-binant cryptochrome showed no measurable absorption atthe concentration used in visible (380–800 nm) light. Theabsorption spectrum of cryptochrome was assessed as thedifference spectra between extracts from cryptochrome-expressing and uninduced cell cultures, similarly to thedetermination of recombinant PHOT1 absorption spectrafrom insect cell culture (Christie et al., 1998).




An N-terminal fragment of PHOT2 photoreceptor com-prising a flavin-binding or LOV domain (amino acids1–900) was expressed in Escherichia coli using the PET21vector expression system (Novagen, Madison, WI). Recom-binant truncated NPL1 protein was purified via an HISaffinity tag on a nickel affinity column by methods recom-mended by the manufacturer. The purified recombinantprotein was eluted, and the absorption spectrum taken in aBeckman Coulter DU7400 spectrophotometer. The spec-trum is identical to published spectra (Salomon et al., 2000).

ACKNOWLEDGMENTS

We thank Alfred Batschauer for use of action spectros-copy facilities and critical reading of the manuscript; Jean-Pierre Bouly for critical reading of the manuscript; andEmile Miginiac, Jean-Claude Kader, and the members ofthe plant science laboratory at the University of Paris fortheir assistance and support.

Received October 23, 2001; returned for revision December26, 2001; accepted March 12, 2002.

LITERATURE CITED

Ahmad M (1999) Seeing the world in red and blue: insightinto plant vision and photoreceptors. Curr Opin PlantBiol 2: 230–235

Ahmad M, Cashmore AR (1996) Seeing blue: the discoveryof cryptochrome. Plant Mol Biol 30: 851–861

Ahmad M, Cashmore AR (1997) The blue-light receptorcryptochrome 1 shows functional dependence on phyto-chrome A or phytochrome B in Arabidopsis thaliana. PlantJ 11: 421–427

Ahmad M, Jarillo JA, Cashmore AR (1998a) Chimericproteins between cry1 and cry2 Arabidopsis blue lightphotoreceptors indicate overlapping functions and vary-ing protein stability. Plant Cell 10: 197–208

Ahmad M, Jarillo J, Smirnova O, Cashmore AR (1998b)The CRY1 blue light photoreceptor of Arabidopsis inter-acts with phytochrome A in vitro. Mol Cells 1: 939–948

Ahmad M, Lin C, Cashmore AR (1995) Mutations through-out an Arabidopsis blue-light photoreceptor impair blue-light-responsive anthocyanin accumulation and inhibi-tion of hypocotyl elongation. Plant J 8: 653–658

Beggs CJ, Holmes MG, Jabben M, Schafer E (1980) Actionspectra for the inhibition of hypocotyl growth by contin-uous irradiation in light and dark-grown Sinapis alba L.seedlings. Plant Physiol 66: 615–618

Briggs W, Huala E (1999) Blue-light photoreceptors inhigher plants. Annu Rev Cell Dev Biol 15: 33–62

Briggs WR, Beck CF, Cashmore AR, Christie JM, HughesJ, Jarillo JA, Kagawa T, Kanegae H, Liscum E, NagataniA et al. (2001) The phototropin family of photoreceptors.Plant Cell 13: 993–997

Casal JJ, Mazella MA (1998) Conditional synergism be-tween cryptochrome 1 and phytochrome B is shown bythe analysis of phyA, phyB, and hy4 simple, double, andtriple mutants in Arabidopsis. Plant Physiol 118: 19–25

Christie JM, Reymond P, Powell GK, Bernasconi P,Raibekas AA, Liscum E, Briggs W (1998) ArabidopsisNPH1: a flavoprotein with the properties of a photore-ceptor for phototropism. Science 282: 1698–1701

Deisenhofer J (2000) DNA photolyases and crypto-chromes. Mutat Res 460: 143–149

Devlin PF, Kay SA (2000) Cryptochromes are required forphytochrome signaling to the circadian clock but not forrhythmicity. Plant Cell 12: 2499–2510

Folta KM, Spalding EP (2001a) Unexpected roles for cryp-tochrome 2 and phototropin revealed by high-resolutionanalysis of blue light-mediated hypocotyl growth inhibi-tion. Plant J 26: 471–478

Folta KM, Spalding EP (2001b) Opposing roles of phyto-chrome A and phytochrome B in early cryptochrome-mediated growth inhibition. Plant J 28: 333–340

Goto N, Yamamoto K, Watanabe M (1993) Action spectrafor inhibition of hypocotyl growth of wild-type plantsand of the hy2 long-hypocotyl mutant of Arabidopsis thali-ana. Photochem Photobiol 57: 867–871

Holmes MG, Wagner E (1982) The influence of chlorophyllon the spectral control of elongation growth in Chenopo-dium rubrum L. hypocotyls. Plant Cell Physiol 23: 745–750

Jorns MS, Baldwin ET, Sancar GB, Sancar A (1986) Actionmechanism of Escherischia coli DNA photolyase. J BiolChem 262: 486–491

Lin C (2000) Plant blue-light receptors. Trends Plant Sci 5:337–342

Lin C, Ahmad M, Cashmore AR (1996) Arabidopsis cryp-tochrome 1 is a soluble protein mediating blue light-dependent regulation of plant growth and development.Plant J 10: 893–902

Lin C, Robertson DE, Ahmad M, Raibekas AA, Jorns MS,Dutton PL, Cashmore AR (1995) Association of flavinadenine dinucleotide with the Arabidopsis blue lightreceptor CRY1. Science 269: 968–970

Lin C, Yang H, Guo H, Mockler T, Chen J, Cashmore AR(1998) Enhancement of blue-light sensitivity of Arabi-dopsis seedlings by a blue-light receptor cryptochrome 2.Proc Natl Acad Sci USA 95: 7686–7690

Lin FJ, Song W, Meyer-Bernstein E, Naidoo N, Sehgal A(2001) Photic signaling by cryptochrome in the Drosoph-ila circadian system. Mol Cell Biol 21: 7287–7294

Malhotra K, Sang-Tae K, Batschauer A, Dawut L, SancarA (1995) Putative blue-light photoreceptors from Arabi-dopsis thaliana and Sinapis alba with a high degree ofsequence homology to DNA photolyase contain the twophotolyase cofactors but lack DNA repair activity. Bio-chemistry 34: 6892–6899

Mas P, Devlin PF, Panda S, Kay SA (2000) Functionalinteraction of phytochrome B and cryptochrome 2. Na-ture 408: 207–211

McCormac AC, Wagner D, Boylan MT, Quail PH, SmithH, Whitelam GC (1993) Photoresponses of transgenicArabidopsis seedlings expressing introduced phyto-chrome B-encoding cDNA: evidence that phytochrome Aand phytochrome B have distinct photoregulatory func-tions. Plant J 4: 19–27

Ahmad et al.



Neff MM, Chory J (1998) Genetic interactions betweenphytochrome A, phytochrome B, and cryptochrome 1during Arabidopsis development. Plant Physiol 118: 27–36

Poppe C, Sweere U, Drumm-Herrel H, Schafer E (1998)The blue-light receptor cryptochrome 1 can act indepen-dently of phytochrome A and B in Arabidopsis thaliana.Plant J 16: 465–471

Salomon M, Christie JM, Knieb E, Lempert U, Briggs WR(2000) Photochemical and mutational analysis of theFMN-binding domains of the plant blue light receptor,phototropin. Biochemistry 39: 9401–9410

Sancar A (2000) CRYPTOCHROME: the second photoac-tive pigment in the eye and its role in circadian photo-reception. Annu Rev Biochem 69: 31–67

Sancar GB, Jorns MS, Payne G, Fluke DJ, Rupert CS,Sancar A (1987) Action mechanism of Escherischia coliDNA photolyase. J Biol Chem 262: 492–498

Shinomura T, Hanzawa H, Schafer E, Furuya M (1998)Mode of phytochrome B action in the photoregulation of

seed germination in Arabidopsis thaliana. Plant J 13:583–590

Shinomura T, Nagatani A, Hanzawa H, Kubota M, Wa-tanabe M, Furuya M (1996) Action spectrum forphytochrome-A and B-specific photoinduction of seedgermination in Arabidopsis thaliana. Proc Natl Acad SciUSA 93: 8129–8133

Shinomura T, Uchida K, Furuya M (2000) Elementaryprocesses of photoperception by phytochrome A forhigh-irradiance response of hypocotyl elongation in Ara-bidopsis. Plant Physiol 122: 147–156

Yang HQ, Wu YJ, Tang RH, Liu D, Liu Y, CashmoreAR (2000) The C termini of Arabidopsis crypto-chromes mediate a constitutive light response. Cell 103:815–827

Young J, Liscum E, Hangarter RJ (1992) Spectral-dependence of light-inhibited hypocotyl elongation inphotomorphogenic mutants of Arabidopsis: evidence for aUV-A photosensor. Planta 188: 106–114




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Action Spectrum for Cryptochrome-Dependent Hypocotylthe production of an action spectrum, a dose-response curve at multiple wavelengths, for a certain plant activity or physiological