Separate functions for nuclear and cytoplasmiccryptochrome 1 during photomorphogenesisof Arabidopsis seedlingsGuosheng Wu and Edgar P. Spalding†

Department of Botany, University of Wisconsin, 430 Lincoln Drive, Madison, WI 53706

Edited by Anthony R. Cashmore, University of Pennsylvania, Philadelphia, PA, and approved October 3, 2007 (received for review May 30, 2007)

Cryptochrome blue-light receptors mediate many aspects of plantphotomorphogenesis, such as suppression of hypocotyl elongationand promotion of cotyledon expansion and root growth. Thecryptochrome 1 (cry1) protein of Arabidopsis is present in thenucleus and cytoplasm of cells, but how the functions of one pooldiffer from the other is not known. Nuclear localization and nuclearexport signals were genetically engineered into GFP-tagged cry1molecules to manipulate cry1 subcellular localization in a cry1-nullmutant background. The effectiveness of the engineering wasconfirmed by confocal microscopy. The ability of nuclear or cyto-plasmic cry1 to rescue a variety of cry1 phenotypes was deter-mined. Hypocotyl growth suppression by blue light was assessedby standard end-point analyses and over time with high resolutionby a custom computer-vision technique. Both assays indicated thatnuclear, rather than cytoplasmic, cry1 was the effective molecule inthese growth inhibitions, as was the case for the mechanisticallylinked membrane depolarization, which occurs within several sec-onds of cry1 activation. Petiole elongation also was inhibited bynuclear, but not cytoplasmic, cry1. Conversely, primary rootgrowth and cotyledon expansion in blue light were promoted bycytoplasmic cry1 and inhibited by nuclear cry1. Anthocyanin pro-duction in response to blue light was strongly stimulated bynuclear cry1 and, to a lesser extent, by cytoplasmic cry1. Animportant step toward elucidation of cry1 signaling pathways isthe recognition that different subcellular pools of the photorecep-tor have different functions.

blue light � hypocotyl � nuclear localization � membrane depolarization �anion channel

P lants employ at least three families of photoreceptor proteinsto monitor light. Through signaling pathways that are being

intensively studied, these photoreceptors convert the quality,quantity, and direction of incident light into developmental andphysiological responses that best adapt the plant to the prevailinglight environment. The cryptochrome photoreceptors of plantsabsorb UV-A/blue wavelengths (1) and mediate the suppressionof seedling stem growth (2), promotion of leaf and cotyledonexpansion (3, 4), f lowering time (5, 6), resetting of the circadianoscillator (7), chlorophyll and anthocyanin synthesis (8), pro-grammed cell death (9), and other processes. Widespread inbiology, cryptochromes are known to regulate circadian rhythmsin many organisms, including mammals (10).

The subject of this article is the cryptochrome 1 (cry1) ofArabidopsis thaliana. It was the founding member of the cryp-tochrome family of photoreceptors, originally identified by agenetic lesion that impaired blue-light suppression of hypocotylelongation. Although some mechanistic details of cry1 actionhave been determined, an integrated view of how its absorptionof blue light results in the various previous responses is lacking.The structural similarity between cry1 and DNA photolyases,light-activated enzymes that repair DNA dimers, indicated anuclear localized function (11). Indeed, cry1 is present in thenucleus (12), but it is not known to bind or repair DNA (1). Atleast some of the cry1 action has been attributed to its physical

interaction with the COP1 E3 ligase, a regulator of seedlingdevelopment (13, 14) that is present in the nucleus until lightstimulates its export to the cytoplasm (15). As a COP1 interactor,cry1 is believed to exert at least some of its effects by influencingthe levels of the HY5 transcription factor, which interacts withthe promoters of some light-regulated genes (16). Comparativetranscript-profiling studies performed on cry1 mutant and wild-type seedlings exposed to blue light identified a large number ofgenes exhibiting cry1-dependent expression at the point in timewhen cry1 begins to influence the rate of hypocotyl elongation,which is �45 min after the onset of irradiation (17). Anothermicroarray expression study identified suites of genes thatchanged expression levels after 6 days of blue light in a mannerthat depended on cry1 and/or cry2 (18). Proteomic analysisidentified 61 specific proteins that were present at differentlevels in a cry1 mutant, compared with the wild type, after ablue-light treatment (19). Thus, there is ample evidence consis-tent with cry1 action being nuclear-localized and manifested bychanges in gene expression. Phosphorylation of cry1, either by anautocatalytic mechanism or a separate kinase, seems to be arequired element of the response mechanism (20–22).

Other findings about cry1 action do not as easily fit a scenarioin which a nuclear-localized photoreceptor fairly directly affectsgene expression to affect photomorphogenesis. The presence ofcry1 in the cytoplasm is one such observation (23). Another isthat blue light activates anion channels at the plasma membrane,causing a depolarization after a lag time of only a few secondsin a cry1/cry2-dependent fashion (24, 25). This channel activa-tion has been causally linked to the onset of cry1-dependentgrowth inhibition, which occurs 30–40 min after the onset ofirradiation and involves changes in auxin and gibberellin levelsand/or signaling (17). Determining the contribution of cytoplas-mic or nuclear cry1 to these processes is the main theme of thepresent work, which is analogous to previous studies of thephytochrome B photoreceptor (26). The general experimentalapproach is to (i) engineer cry1 to contain a short sequence ofamino acids that has a nuclear-localizing effect or a differentshort sequence that results in export from the nucleus, and (ii)determine the extent to which the differently localized photo-receptors restore function to a cry1 mutant in a number ofdifferent photomorphogenesis assays.

ResultsTo visualize the nuclear versus cytoplasmic distribution of thecry1 photoreceptor, the DNA-coding sequence of GFP was

Author contributions: G.W. and E.P.S. designed research, performed research, analyzeddata, and wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

†To whom correspondence should be addressed. E-mail: spalding@wisc.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0705082104/DC1.

© 2007 by The National Academy of Sciences of the USA

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attached to the N terminus of the CRY1 coding sequence (Fig.1A). The N terminus was selected because previous workindicated that modifying the C terminus was likely to affectfunction (23). The decision to place the chimeric gene under thecontrol of the 35S promoter was based on the need for a strongGFP signal to visualize the low-abundance cry1 protein (27) andthe previous finding that cry1 expressed by this strong promoterfunctions well without severe pleiotropic effects in the seedlings(27). To manipulate the cytoplasmic distribution of the cry1photoreceptor, a nuclear localization signal (NLS) or nuclear

export signal (NES) used by Matsushita et al. (26) for a similarpurpose was inserted between the GFP and the cry1-codingsequences (Fig. 1 A). Not shown in Fig. 1 A is the negative controlconstruct in which Arg-611 of cry1 was changed to lysine.Previous studies showed that this mutation (hy4-24) blocksfunction without affecting protein levels (8). The cry1-304 allele,previously demonstrated to lack cry1 protein (20), was trans-formed with these various constructs. Fig. 1B shows that, in theroot apex, GFP-cry1 (hereafter cry1cont for control) was presentin both the cytoplasm and nucleus, as reported before for thenative protein (28). Fig. 1C shows that the GFP-NLS-cry1(hereafter cry1NLS) was concentrated in the nucleus, and levelsin the cytoplasm were below the detection limit. Fig. 1D showsthat GFP-NES-cry1 (hereafter cry1NES) was expressed well inthe cytoplasm, but could not be detected in nuclei, which werevisualized by staining the roots with propidium iodide (Fig. 1E).Essentially the same results were observed in cells of etiolatedhypocotyls (Fig. 1 F–I), light-grown hypocotyls (Fig. 1 J–M), andlight-grown cotyledons (Fig. 1 N–Q). These localization patternswere independent of blue-light treatment. They represent the

Fig. 2. GFP-cry1 quantification in the transgenic seedlings. (A) Nuclear andcytoplasmic GFP-cry1 levels in root apices measured as fluorescence intensityby confocal microscopy. (B) Overall GFP-cry1 levels in the indicated organs inthe three transgenic lines. The roots were imaged with a C-Apochromat �40objective lens so their fluorescence levels in B are not comparable to the otherorgans, which were imaged with a Plan-Aprochromat �20 objective lens. Eachmeasurement in A or B is the mean of measurements obtained from four toseven seedlings. Error bars represent standard errors.

Fig. 1. Schematics of CRY1 transgene constructs and subcellular localizationof the proteins. (A) Schematics of the GFP-tagged chimeric genes with whichcry1 was transformed to test the function of nuclear and cytoplasmicallylocalized cry1. (B–D) Root tips of 4-day-old light-grown seedlings expressingcry1cont (B), cry1NLS (C), or cry1NES (D). (E) Propidium iodide staining highlightsthe nuclei in red. (F–H) Dark-grown hypocotyls expressing cry1cont (F), cry1NLS

(G), or cry1NES (H). (I) Cell in H stained with Vybrant DyeCycle Orange to showthe nucleus in red. (J–L) Light-grown hypocotyls expressing cry1cont (J), cry1NLS

(K), or cry1NES (L). (M) Cell in L stained with Vybrant DyeCycle Orange to showthe nucleus in red. (N–P) Light-grown cotyledons expressing cry1cont (N),cry1NLS (O), or cry1NES (P). (Q) Cell in P stained with Vybrant DyeCycle Orangeto show the nucleus in red. Propidium iodide staining in C, G, K, and O showsthe cell structure in red. (Scale bars: B–Q, 20 �m.)

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steady-state levels of cry1 in the different compartments, but donot provide information about the rates of exchange betweencompartments. An analysis of GFP fluorescence intensity wasperformed to quantify the cytoplasmic and nuclear cry1 levels inthe root tips of plants expressing the different constructs. Theresults, shown in Fig. 2A, are consistent with the confocal images,in that the NLS sequence tag had a profound nuclear-accumulation effect. The NES tag effectively excluded cry1 fromthe nucleus. Importantly, the level of cry1 in the nucleus ofcry1NLS plants was similar to the levels of cry1 in the cytoplasmof cry1NES plants, and the cry1cont situation was similar to thesum of the cry1NES and cry1NLS levels. At the tissue level, ratherthan the subcellular level, cry1 expression levels in the three lineswere similar in each of the organs studied (Fig. 2B). Homozygouslines that faithfully maintained these desired subcellular cry1distributions in the T4 generation were used to compare theextent to which cry1NES and cry1NLS rescued the cry1–304 defectsin blue-light responses.

Among the obvious changes in growth and shape that anetiolated seedling undergoes in response to light are suppressionof hypocotyl elongation, expansion of the cotyledons, elongationof the cotyledon petioles, and promotion of root elongation (1).The cry1 photoreceptor participates in all of these responses, soeach was quantified in the different mutant and transgenic linesafter 7 days of growth in a range of blue photon fluence rates todetermine whether cry1NLS or cry1NES was as able to rescue thecry1–304 defects more or less well as the cry1cont control. Fig. 3Ashows that blue light suppressed hypocotyl elongation in wild-type seedlings and that, at the higher fluence rates of 10, 50, and100 �mol�m�2�s�1, cry1 seedlings were �4-fold taller than wild

type. This iconic cry1 phenotype was completely suppressed bycry1cont, showing that the GFP-cry1 molecule was functional.The results obtained with the cry1NLS and cry1NES lines wereunequivocal. Complete suppression of the phenotype was ob-served in cry1NLS seedlings; however, cry1NES was ineffective.Therefore, nuclear, but not cytoplasmic, cry1 can explain thelong-term result of cry1 action on hypocotyl growth in blue light.Opposite to its effect on hypocotyl elongation, blue light pro-moted cotyledon petiole elongation in wild-type seedlings. Thepromotive effect of blue light was much greater in cry1 than wildtype (Fig. 3B), indicating that cry1 negatively regulates thisphotomorphogenic response, which is presumably initiated by adifferent photoreceptor. Suppression of the cry1 long petiolesbeyond the wild-type levels was achieved by cry1cont and cry1NLS,but cry1NES produced no suppression (Fig. 2B).

Some of the root growth promotion by blue light is because ofcry1 (29). Fig. 3C shows the root of cry1 is shorter than wild type.Shifting the distribution of cry1 to the cytoplasm stimulated rootgrowth (Fig. 3C) at all f luence rates. Conversely, concentratingcry1 in the nucleus inhibited root growth (Fig. 3C). The presenceof cry1 in both compartments (cry1cont) produced promotion atlow fluence rates and inhibition at the higher fluence rates,relative to the wild type. The main conclusion to draw from Fig.3C is that cry1NES promotes root growth in blue light (not indarkness) and that cry1NLS has the opposite effect.

Blue light promotes cotyledon expansion by cry1 signaling (3,4). The effect is evidenced here by the reduced cotyledon areaof cry1 seedlings grown in blue light, especially at higher fluencerates (Fig. 3D). Unlike the other processes presented in Fig. 3,cry1NES was most effective at promoting cotyledon expansion

Fig. 3. Phenotypic analysis of CRY1 transgenic plants. Plants were grown in darkness or under continuous blue light with various intensities for 7 days. Thenhypocotyl lengths (A), cotyledon petiole lengths (B), root lengths (C), and cotyledon areas (D) were measured. (C Inset) Important differences in root lengths at1 �mol�m�2�s�1 blue light. The mean values from �15 plants are shown. Error bars represent standard errors.

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and correcting the cry1 defect. Concentrating cry1 in the nucleusand excluding it from the cytoplasm (cry1NLS) prevented thephotoreceptor from rescuing the mutation. As in roots, cry1NESpromoted cotyledon expansion relative to wild type, cry1NLS, orthe control line (cry1cont), which takes into account the effect ofthe 35S promoter. The stimulatory effect of cytoplasmic cry1 oncotyledon expansion is probably because of a true function of thephotoreceptor; disabling cry1 function through the R611K mu-tation (cry1*) abolished its ability to modify any of the cry1phenotypes tested in any of the subcellular contexts [supportinginformation (SI) Fig. 8]. Thus, all of the effects in Fig. 3 are morelikely related to cry1 signaling, rather than indirect effects suchas titrative reduction of interacting proteins or nonspecificinterferences caused by extraordinary levels or locales ofexpression.

Previous studies using high-resolution tools designed forstudying Arabidopsis seedling growth demonstrated that growthinhibition induced by blue light is initiated not by cry1, but by

phototropin 1 (phot1), an unrelated photoreceptor (25). After30–40 min of blue-light irradiation, a cry1-mediated phase ofgrowth inhibition replaces the phot1 phase (25). The onset of thecry1 phase, but not the initial phot1 phase, has been mechanis-tically linked to an anion channel-mediated depolarization of theplasma membrane that is complete �2 min after the onset ofblue-light irradiation (25). It seemed possible that some portionsof these earliest effects of cry1 on cell physiology and hypocotylgrowth may be more dependent on cytoplasmic cry1 thannuclear cry1. Therefore, electrophysiology and the latest com-puter-vision technique for measuring hypocotyl growth fromtime series of electronic images (30) were used to investigate thefunctions of cry1NLS and cry1NES. Fig. 4 shows that the mem-brane depolarization defect of cry1 was not rescued by cry1NES,but was completely rescued by cry1NLS and cry1cont. If cry1NLSmediates the depolarization and the depolarization is causal tothe onset of cry1-dependent growth inhibition (25), then bothshould be rescued by the same form of cry1. If the ionic eventsare rescued by one form and the growth response by the other,the two will have been uncoupled. Hypocotyl growth rates weredetermined by using the morphometric algorithms developed byMiller et al. (30). The abrupt escape from inhibition after�30–40 min of irradiation typical of cry1 was not corrected bycry1NES (Fig. 5). However, cry1cont and cry1NLS completelyrestored wild-type growth inhibition to cry1. Thus, the electro-physiological results and high-resolution growth measurements(Figs. 4 and 5) are consistent with the model that rapid ionicresponses to blue light and the subsequent cry1-mediated phaseof growth inhibition are causally connected and both mediatedby nuclear cry1. Variations of this conclusion are considered inDiscussion.

Anthocyanin pigments accumulate in Arabidopsis seedlings inresponse to light primarily through a cry1-dependent blue-lightpathway. Genes in the biosynthetic pathway are up-regulated ina cry1-dependent manner. However, Noh and Spalding (31)found that a posttranslational step involving cry1-dependentanion channel activation was required for proper anthocyaninaccumulation. To test the possibility that this complexity was theresult of combinations of nuclear and cytoplasmic cry1 activities,anthocyanin accumulation in the transgenic lines grown in bluelight was measured. As expected, cry1 seedlings were unable toaccumulate anthocyanin (Fig. 6). Wild-type anthocyanin levelswere restored by cry1NES, but cry1NLS and cry1cont were muchmore potent, promoting anthocyanin accumulation at least5-fold higher than the wild-type level.

DiscussionThe majority of available evidence from animal and plant studiesbefore this study pointed to the nucleus as the primary site of

Fig. 4. Blue light-induced membrane depolarizations in wild type, cry1, andtransgenic plants. Membrane depolarization induced by a 20-s pulse of 200�mol�m�2�s�1 blue light in hypocotyls started at 20 s and ended at 40 s. Themean values from more than eight responses were plotted. Error bars at thepeak of each line were shown.

Fig. 5. Morphometric analysis of seedling hypocotyl growth in blue light.The growth kinetics of 2-day-old etiolated wild type, cry1, cry1cont, cry1NLS, andcry1NES respond to 50 �mol�m�2�s�1 blue-light treatment. All growth rateswere normalized to the average dark growth rate during the 2 h precedingblue-light treatment. Data represent the average of more than eight plantsper genotype. Standard error was given every hour.

Fig. 6. Anthocyanin levels in 7-day-old seedlings grown under 100�mol�m�2�s�1 blue light.

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cryptochrome action. However, evidence of a nuclear functionfor these blue-light receptors typically does not exclude a cyto-plasmic function. For example, in animals, cryptochromes het-erodimerize in the cytoplasm with PERIOD (PER) proteins(encoded by the period genes) before the complex translocatesto the nucleus, where it represses key clock genes (32, 33).Although the nuclear gene-regulatory role is clearly important,the cryptochromes also may perform a yet-to-be discoveredfunction while in the cytoplasm. Likewise, the evidence that cry1of Arabidopsis interacts with the COP1 E3 ligase to control levelsof the HY5 transcription factor points to primary cry1 actionbeing nuclear localized, but COP1 moves to the cytoplasm inresponse to light where, in combination with cytoplasmic cry1,it could conceivably perform a photomorphogenic function(13–15). Indeed, a C-terminal portion of cry1 fused to �-glucu-ronidase (GUS) was cytoplasmic in the light and nuclear in thedark (23). The presence of an effective NLS in the amino acidsequence of cry2 in Arabidopsis (28, 34) and the absence of onein cry1 may indicate that cry1 in the cytoplasm is there for afunctional reason. The two cry1-like cryptochromes in rice whenfused to GFP and overexpressed were present in both the nucleusand cytoplasm (35). The five different cryptochromes in the fernAdiantum capillus-veneris when fused to GUS and expressed ingametophytic cells displayed a range of nuclear:cytoplasm dis-tributions, some of which of were light-dependent (36). Thus,cytoplasmic cryptochrome is not an anomaly, and studies aimedat determining its relevance to seedling photomorphogenesiscould produce a better understanding of how this photoreceptoroperates across the biological spectrum. The results presentedhere demonstrate that cytoplasmic cry1 functions during seed-ling photomorphogenesis in ways that can be separated from thefunctions of nuclear cry1.

The long hypocotyl displayed by cry1 mutants grown in bluelight for prolonged periods was the phenotype that led to thediscovery of cryptochromes. The cry1-dependent phase ofgrowth inhibition depends on the activation of anion channels atthe plasma membrane, which depolarizes the membrane (37).Blocking the anion channels pharmacologically in wild-typeseedlings phenocopies the cry1 mutant for a few hours, but notfor the long term (37). This finding raised the possibility of theirbeing at least two cry1-dependent phases–the first one depen-dent on anion channels and the second not. Although cytoplas-mic cry1 would seem to be better positioned to activate anionchannels at the plasma membrane than nuclear cry1, the dataclearly indicated otherwise. Cytoplasmic cry1 suppressed neitherthe membrane depolarization defect nor the defect in hypocotylgrowth control. Complementary to this result is the finding thatcry1NLS restored both the membrane depolarization and theinitial cry1 phase of growth inhibition. It seems somewhatcounterintuitive, but nuclear localized cry1 is responsible for theactivation of plasma membrane anion channels and the atten-dant phase of cry1-dependent growth inhibition.

One direct model that could accommodate these results is thatnuclear cry1 generates a signal that reaches the anion channelsat the membrane within the several seconds lag time. The timingis too fast to be mediated by gene expression, but light-drivenredox changes (38) or ion fluxes could conceivably connectnuclear and plasma membrane activities. A much less directmodel also can be envisioned. Perhaps cry1 mutants grown in thedark underexpress the anion channels or some component of thesignaling chain so that blue light results in a weaker depolariza-tion. This indirect model depends on cry1 influencing theexpression of genes in dark-grown seedlings [i.e., in the absenceof the signal (blue light) believed to be necessary for cry1activation].

The present study found no evidence of a role for cytoplasmiccry1 in the control of hypocotyl or cotyledon petiole elongation,but cotyledon expansion, root growth, and anthocyanin accu-

mulation all displayed a dependence on cry1NES. In some cases,the cry1 phenotypes could be completely explained by a partic-ular form of cry1, but other cases were more complicated. In thecase of cotyledon expansion, cytoplasmic cry1 appeared tooppose the action of nuclear cry1. Cotyledon expansion in cry1was faithfully rescued by cry1cont, not by cry1NLS, and wasstimulated beyond the wild-type level by cry1NES. The anthocy-anin assays also provided evidence of different forms contrib-uting in different ways. Anthocyanin accumulation was rescuedby cry1NES, yet hyperstimulated by cry1NLS, a complexity that isperhaps related to the previous finding that transcriptionalcontrol and posttranslational steps regulated the biosyntheticpathway in response to blue light (31).

The present work (Fig. 3C) indicates that the promotive effectof cry1 on root growth noted by Canamero et al. (29) was due tocytoplasmic cry1. At high fluence rates, blue light inhibits rootelongation, which is caused by nuclear cry1 (Fig. 3C). Redistri-bution of cry1 between the nucleus and cytoplasm was notobserved in response to changes in light conditions (data notshown). A better explanation is that cytoplasmic cry1 dominatesthe signaling pathway to promote root growth in low fluence-rateblue light, whereas the inhibitory effect of nuclear cry1-mediatedsignal transduction predominates at high fluence rates. Theseand the other results presented here can be summarized in adiagrammatic model (Fig. 7) that integrates cry1 subcellularlocalization and the effect the photoreceptor has on seedlingphotomorphogenesis.

MethodsEngineering of the CRY1 Gene and Generation of Transgenic Plants. Togenerate the chimeric fusion construct GFP-CRY1, the completeCRY1 coding sequence was amplified by PCR from an Arabi-dopsis cDNA clone (U12079) by using a forward primer 5�-GGCTCGAGTCTGGTTCTGTATCTGGTTGTGGTTC-3�and a reverse primer 5�-GGCTCGAGTTACCCGGTTTGT-GAAAGCCGTCTCC-3�. The italicized regions denote XhoIsites that were introduced to both primers to facilitate thecloning of amplified DNA. To introduce NLS or NES signalpeptide DNA into the construct, the forward primers5�-GGCTCGAGcctaagaagaagagaaaggttTCTGGTTCTGTAT-CTGGTTGTGGTTC-3� or 5�-GGCTCGAGcttgctcttaagttggct-ggacttgatattTCTGGTTCTGTATCTGGTTGTGGTTC-3�,respectively, were used. The signal sequences are shown inlowercase. After digestion with XhoI, the fragment was fused inframe to the C-terminal end of eGFP in pEGAD vector.

A point mutation in the CRY1-coding region that substitutesArg-611 with a lysine residue (R611K) in the protein sequencewas previously shown to create a protein that accumulatesnormally, but has no detectable function (8). This point mutationwas introduced into the cDNA clone (U12079) by a QuikChangeII site-directed mutagenesis kit (Stratagene) by using the primers

Fig. 7. Model integrating cry1 localization with cry1-mediated signal trans-duction induced by blue light.

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5�-CCAGAATTTAATATCAAAATTGTTGCAGAGAGC-3�and 5�-GCTCTCTGCAACAATTTTGATATTAAATTC-TGG-3�. The resulting plasmid was used as the template to clonethe mutated CRY1 cDNA with NLS or NES tag into pEGAD asdescribed before. The mutated, nonfunctional cry1 produced isdesignated cry1*.

The resulting constructs were introduced into Agrobacteriumtumefaciens GV3101 and used to transform cry1–304 mutantplants by using the floral dip method (39). Plants carrying thetransgene were isolated through basta selection. The homozy-gous lines for the transgene were obtained in the T4 generation.

Plant Materials and Growth Analysis. A. thaliana ecotype Columbiawas used as a wild type in all experiments. Seeds were surface-sterilized with 75% ethanol and plated on half-strength Murash-ige and Skoog agar plates [2.15 g/liter MS salts, 0.8% bacto-agar(pH 5.7)] (Sigma–Aldrich). The plates were maintained indarkness at 4°C for 2–3 days and then placed under variousintensities of constant blue light at 22°C for 7 days. To quantifygrowth rates, seedlings were rearranged on agar plates, andimages were obtained with a flatbed scanner. Hypocotyls, pet-ioles primary roots, and the cotyledon area were determined byusing National Institutes of Health IMAGE software.

Subcellular Localization Studies. Confocal microscopy was per-formed with a Zeiss LSM 510 laser scanning confocal micro-scope equipped with a meta-detector. Propidium iodide wasused to stain the nucleus and the cell wall to delineate the rootstructure. Vybrant DyeCycle Orange stain (Invitrogen) was usedto stain the nucleus in the hypocotyl and cotyledon. Either aC-Apochromat �40 water immersion lens or a Plan-Apochromat �63 oil immersion lens was used. The sample wasexcited with the 488-nm laser line from a 30-mW argon gas laser.The fluorescence was captured in 10-nm bandwidths, and thenlinear unmixing was performed to isolate the GFP signal fromthe stained or endogenous fluorescence background.

Mean fluorescence intensities were calculated within manu-ally selected regions of interest with Zeiss LSM 510 software. Inthe primary root nucleus, a circular area of �50 �m2 covering thewhole nucleus was selected. One representative nucleus per rootwas recorded, and the values reported are the means of at least

four separate roots. For the cytoplasmic values, a field of similarsize adjacent to the nucleus was chosen. To measure overall GFPintensity in the different organs, regions of �0.01–0.02 mm2 forthe root, 0.1–0.15 mm2 for the hypocotyl, 0.05–0.1 mm2 for thepetiole, and 0.15–0.2 mm2 for the cotyledon were used. Seedlingswere 4 days old.

Morphometric Analysis of Hypocotyl Growth. Two-day-old seedlingsgrown in the dark on 1% agar containing 1 mM KCl and 1 mMCaCl2 were used to measure hypocotyl growth with a recentlydeveloped computer-vision method (30). Briefly, electronicimages acquired at 10-min intervals by CCD cameras usinginfrared light were analyzed with custom morphometric algo-rithms designed to extract information such as growth rate (30).Hypocotyl inhibition was induced by 50 �mol�m�2�s�1 blue light.

Surface Potential Measurements. Four-day-old etiolated seedlingsgrown on 1% agar containing 1 mM KCl and 1 mM CaCl2 wereused to measure surface potential induced by a pulse of 200�mol�m�2�s�1 blue light as described previously (24), except thatthe xenon arc light source and delivery mechanism were thoseused by Folta et al. (40).

Anthocyanin Extraction and Quantification. Anthocyanin measure-ment was conducted according to a previously published method(31). Seven-day-old seedlings grown under 100 �mol�m�2�s�1

blue light were harvested from the agar plates, quickly weighed,and placed into microcentrifuge tubes containing 350 �l of 18%1-propanol, 1% HCl, and 81% water. The tubes were placed inboiling water for 3 min and then incubated in darkness overnightat room temperature. After a brief centrifugation to pellet thetissue, 300 �l of the solution was removed and brought to a finalvolume of 600 �l by adding solvent. The amount of anthocyaninsin the resulting extract was quantified spectrophotometrically.The values are reported as (A535–A650) per gram of fresh weight.

We thank Tessa L. Durham for assistance with membrane potentialmeasurements, Daniel R. Lewis for assistance with confocal microscopy,and Nathan D. Miller and Candace R. Moore for assistance with thecomputerized growth measurements. This work was supported by Na-tional Science Foundation Grants IOB-0517350 and DBI-0421266(to E.P.S.).

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