Plant Physiol. (1999) 120: 605-614

Arabidopsis Contains at Least Four Independent Blue-Light-Activated Signal Transduction Pathways¹

Gérard Lascève, Juliette Leymarie, Margaret A. Olney, Emmanuel Liscum, John M. Christie, Alain Vavasseur, and Winslow R. Briggs^*

Cadarache, Commissariat á l'Energie Atomique, Département d'Ecophysiologie Végétale et Microbiologie, Laboratoire de Bioénergetique Cellulaire, F-13108, St. Paul lez Durance cedex, France (G.L., J.L., A.V.); Department of Plant Biology, Carnegie Institution of Washington, 260 Panama Street, Stanford, California 94305 (M.A.O., J.M.C., W.R.B.); and Department of Biological Sciences, University of Missouri, Columbia, Missouri 65211 (E.L.)

	ABSTRACT

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We have investigated the stomatal and phototropic responses to blue light of a number of single and double mutants at various loci that encode proteins involved in blue-light responses in Arabidopsis. The stomatal responses of light-grown mutant plants (cry1, cry2, nph1, nph3, nph4, cry1cry2, and nph1cry1) did not differ significantly from those of their wild-type counterparts. Second positive phototropic responses of etiolated mutant seedlings, cry1, cry2, cry1cry2, and npq1-2, were also similar to those of their wild-type counterparts. Although npq1 and single and double cry1cry2 mutants showed somewhat reduced amplitude for first positive phototropism, threshold, peak, and saturation fluence values for first positive phototropic responses of etiolated seedlings did not differ from those of wild-type seedlings. Similar to the cry1cry2 double mutants and to npq1-2, a phyAphyB mutant showed reduced curvature but no change in the position or shape of the fluence-response curve. By contrast, the phototropism mutant nph1-5 failed to show phototropic curvature under any of the irradiation conditions used in the present study. We conclude that the chromoproteins cry1, cry2, nph1, and the blue-light photoreceptor for the stomatal response are genetically separable. Moreover, these photoreceptors appear to activate separate signal transduction pathways.

	INTRODUCTION

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In the past decade, studies of mutants have been of great value in delineating the functions of various photoreceptors in their independent, interdependent, and antagonistic roles in mediating the developmental and functional events of photomorphogenesis in plants (Jenkins et al., 1995; Chamovitz and Deng, 1996; Fankhouser and Chory, 1997; Quail, 1998; Whitelam et al., 1998). Although there are numerous studies of mutants at the various phytochrome loci and loci that encode components downstream from the phytochrome photoreceptors, there are fewer with mutants involving processes regulated by blue light. Because the number of known or postulated blue-light photoreceptors has climbed from zero to four (cry1, Ahmad and Cashmore, 1993; nph1, Christie et al., 1998; cry2, Hoffmann et al., 1996; Lin and Cashmore, 1996; Lin et al., 1996b; zeaxanthin, Zeiger and Zhu, 1998), it is now possible to use mutants to investigate which pathways are activated by these photoreceptors (Liscum and Hangarter, 1994; Parks et al., 1998; Briggs and Huala, 1999), as has been done with the phytochromes and their signal transduction pathways. In the present study we have used mutants in an attempt to determine which photoreceptors are involved in blue-light-induced stomatal opening and which are involved in phototropism. We have included single mutants at the NPH1, CRY1, CRY2, and NPQ1 loci, the first three encoding photoreceptors and the fourth encoding an enzyme involved in zeaxanthin biosynthesis. We have also included the double mutants nph1cry1, cry1cry2, and phyAphyB in several experiments.

Extensive biochemical studies have been carried out on nph1, a protein associated with the plasma membrane of phototropically sensitive tissues of etiolated seedlings (Short and Briggs, 1994; Briggs and Liscum, 1996, 1997). This protein becomes rapidly and extensively phosphorylated both in vivo and in vitro upon irradiation with blue light. Biochemical and genetic evidence show that this phosphoprotein, which is a Ser/Thr kinase (Reymond et al., 1992a; Huala et al., 1997), participates early in phototropism (for review, see Short and Briggs, 1994). Recently, Liscum and Briggs (1995, 1996) described a series of mutants at four loci in Arabidopsis, designated nph1 through nph4 (non-phototropic hypocotyl), all impaired in their phototropic responses. In nph1 mutants carrying alleles generated by fast-neutron bombardment (nph1-1, nph1-3, nph1-4, and nph1-5), no detectable light-induced phosphorylation was observed, and these mutants appeared to lack the target protein for phosphorylation (Liscum and Briggs, 1995; Huala et al., 1997). The nph1-5 allele contains a deletion that removes the region coding for the C-terminal half of the gene (Huala et al., 1997). No NPH1 mRNA is detectable in nph1-5 (J.M. Christie and W.R. Briggs, unpublished results).

With the exception of the weak allele nph1-2 (mutant JK224; Khurana and Poff, 1989), which shows impaired first positive curvature but normal second positive curvature (Khurana and Poff, 1989), all known mutants at the NPH1 locus lack first and second positive curvature of etiolated hypocotyls in response to blue light. They also lack second positive curvature of roots and hypocotyls of light-grown seedlings. Liscum and Briggs (1995) hypothesized that the NPH1 gene encodes the photoreceptor apoprotein, a hypothesis borne out by the cloning and characterization of the gene and complementation of the nph1-5 mutant with the wild-type gene (Huala et al., 1997). A recent study showed that NPH1 expressed in insect cells binds FMN as a chromophore and retains photosensitivity for light-induced phosphorylation similar to that of the native Arabidopsis protein and supports the hypothesis that nph1 functions as the photoreceptor for phototropism (Christie et al., 1998). The NPH2, NPH3, and NPH4 loci encode proteins that function downstream from the nph1 protein (Liscum and Briggs, 1995, 1996).

In the past few years, the genes HY4 and CRY2 that encode, respectively, the apoproteins for the blue-light photoreceptors cry1 (Ahmad and Cashmore, 1993) and cry2 (Hoffmann et al., 1996; Lin et al., 1996b, 1998) in Arabidopsis have been cloned. (Note: hy4 is the original designation for mutants at the CRY1 locus. Subsequent isolates have been given the designation cry1 instead of hy4. Following the conventions for phytochrome, the wild-type holoproteins for these photoreceptors are designated here cry1, cry2, and nph1, and the apoproteins CRY1, CRY2, and NPH1.) The cry1 protein has been partially characterized (Lin et al., 1995a, 1995b, 1996a; Malhotra et al., 1995; Lin and Cashmore, 1996); it encodes a protein with significant homology to prokaryote DNA photolyases. cry1 has two chromophores: one FAD (Lin et al., 1995a, Malhotra et al., 1995) and the other probably a pterin (Malhotra et al., 1995). The CRY2 apoprotein also has homology with prokaryotic DNA photolyases and is reported to bind at least a flavin as a chromophore (Lin et al., 1996b). Both cry1 and cry2 serve as photoreceptors mediating blue-light-induced inhibition of hypocotyl growth in Arabidopsis (Ahmad and Cashmore, 1993; Lin et al., 1995a, 1995b, 1996a, 1998), with cry2 also playing a role in flowering (Guo et al., 1998).

Recently, Ahmad et al. (1998) published evidence that a cry1cry2 double mutant was deficient in first positive phototropic curvature and postulated a role for cry1 and cry2 in phototropism. However, both biochemical and genetic evidence indicate that at least the cry1 and nph1 proteins participate in separable signal transduction pathways (Liscum and Briggs, 1995). The hy4-105 mutant fails to show blue-light-inducible inhibition of hypocotyl elongation but does show normal phototropism. By contrast, nph1 null mutants show normal blue-light inhibition of hypocotyl elongation but lack any phototropic response to unilateral blue light. In addition, the nph1hy4-105 F₁ hybrid shows both responses, and the double mutant shows neither response (Liscum and Briggs, 1995). Finally, the null mutant hy4-2.23N (Koornneef et al., 1980; Ahmad and Cashmore, 1993) has wild-type amounts of the plasma membrane phosphoprotein and shows wild-type levels of phosphorylation upon blue-light irradiation (Liscum and Briggs, 1995).

Another well-known response of higher plants to light is the blue-light activation of stomatal opening (Zeiger, 1983). Stomata respond to blue light at very low photon-flux densities with insufficient energy to drive stomatal opening directly (Sharkey and Ogawa, 1987). With whole plants, a blue-light pulse that was added to a strong red light background saturating for photosynthesis triggered a transient increase in stomatal opening (Commelina communis, Zeiger et al., 1985; wheat, Karlsson, 1986). Similar results were obtained with C. communis seedlings placed in darkness and CO₂-free air (Lascève et al., 1993). Thus, under saturating red light or CO₂-free air and darkness, blue light acts as a signal independent of phytochrome to promote stomatal opening; it must do so by activating a specific blue-light photoreceptor. The Arabidopsis mutant npq1 has been shown to be defective in its violaxanthin de-epoxidase (Niyogi et al., 1998), the enzyme mediating the first step in the formation of zeaxanthin. This mutant fails to show blue-light-induced stomatal opening (Zeiger and Zhu, 1998). Thus, zeaxanthin may serve as the chromophore for a fourth blue-light-activated photoreceptor system.

Because the nph1 and cry1 pathways are known to be genetically separable, as are the cry1 and cry2 pathways, our first objective was to investigate, by studying the responses of mutants, whether any of these photoreceptors plays a role in the stomatal response to blue light. Mutants carrying lesions in genes encoding elements downstream from nph1 were also included in the study. Chory (1992) and Liscum and Hangarter (1994) mention that light-induced stomatal opening in hy4 null mutants of Arabidopsis appears to be unimpaired, but they provide no details. To our knowledge, there is no information yet available concerning whether nph1 and/or cry2 is required for light-activated stomatal opening or, alternatively, plays a role in regulating the magnitude of the response. Our aim, therefore, was to investigate light-induced stomatal opening in available mutants at all of the CRY and NPH loci.

Our second objective was to carry out a detailed study of the phototropic responses and blue-light-induced phosphorylation of a number of different mutant alleles at the CRY1 and CRY2 loci. We used both single and double mutants in an effort to investigate a possible role for the cry1 and cry2 photoreceptors in these responses. Because phyAphyB double mutants have also shown reduced phototropism (Hangarter, 1997), a phyAphyB double mutant was included in this study. Phytochromes in Arabidopsis have been shown to affect the magnitude of phototropic curvature (Hangarter, 1997; Janoudi et al., 1997), without having a role in mediating detection of light direction; this could provide a model for the action of cryptochromes in phototropism. Because zeaxanthin was also hypothesized to be a photoreceptor (Quiñones and Zeiger, 1994; Quiñones et al., 1996), we included the mutant npq1-2 in the phototropism study.

	MATERIALS AND METHODS

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Plant Material and Growth Conditions

The nph1-5 mutant is a true null (Huala et al., 1997), and neither cry1-304 (hy4-304) nor cry2-1 produced detectable CRY1 or CRY2 apoprotein, respectively, based on western analysis (Guo et al., 1998). The hy4-B104 mutant produced a trace of CRY1 mRNA, detectable by northern analysis, and a trace of CRY1 apoprotein, detectable by western analysis (M.A. Olney and J.M. Christie, unpublished data). The cry2-1 and the hy4-B104cry2-1 mutants showed neither CRY2 mRNA nor CRY2 apoprotein by northern or western analysis, respectively (M.A. Olney and J.M. Christie, unpublished data). The hy4-2.23N mutant produced a truncated transcript (Ahmad and Cashmore, 1993) and is presumably a protein null. The nature of the lesions in the mutants hy4-105, nph3-1, and nph4-1 is presently unknown. Seeds for the hy4-B104, cry1-304, and cry2-1 single mutants and the hy4-B104cry2-1 and cry1-304cry2-1 double mutants were kindly provided by Dr. Chentao Lin (University of California, Los Angeles). The phyAphyB double mutant was kindly provided by Dr. Peter H. Quail (University of California, Berkeley). The npq1-2 mutant was the kind gift of Dr. Krishna Niyogi (University of California, Berkeley).

For investigations of light regulation of stomatal aperture, seeds were germinated and grown in sand and watered with one-half-strength Hoagland solution. The pots were placed in a growth chamber (16-h light period, 23°C, and RH 75%; 8-h dark period, 20°C, and RH 85%). For membrane preparations from etiolated seedlings to be used in the phosphorylation experiments, sterilized seeds (30% commercial bleach for 15 min followed by three rinses with sterile distilled water) were sown on moist filter lying on one-half-strength Murashige and Skoog medium in 1% agar (Sigma). After cold treatment (5°C) in darkness for 2 to 4 d and a 2-h red-light treatment to induce germination, the plates were kept horizontal, wrapped in foil, and in complete darkness for 3 d at 22°C before harvest under a dim-red light. For the phototropism studies, the sterilized seeds were placed in rows directly on one-half-strength Murashige and Skoog medium in 1% agar in square Petri plates. After the same cold and red-light treatments, the plates were placed on edge, wrapped in aluminum foil, and kept in complete darkness at 22°C for 2 or 3 d before phototropic induction. Seedling age was determined from the start of the red-light treatment.

Light Sources

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View larger version (23K): [in this window] [in a new window]   Figure 1. Spectral distribution of the blue- and red-light sources for experiments of light effects on stomatal aperture. For the blue (solid line) and red (dashed line) spectra, the fluence rate at the leaf surface was near 60 µmol m2 s1.

Light for induction of second positive phototropic curvature was provided by a cool-white fluorescent lamp (F20T12/CW, Sylvania) filtered through one layer of blue Plexiglas (no. 2424, Rohm and Haas, Philadelphia, PA), fluence rate approximately 2 µmol m² s¹. Light for induction of first positive curvature was provided by a 500-W tungsten light source (33-89-39, Bausch & Lomb, Rochester, NY) filtered through a broad wavelength band, blue-glass filter (5-60, Corning Inc., Corning, NY). For fluence-response studies of first positive curvature, the fluence rate was adjusted with neutral density filters, and exposure times varied from 1 to 100 s to cover 4 orders of magnitude of fluences. Red light for both induction of germination and testing its effects on phototropism was obtained by filtering the light from two red fluorescent lamps (F20T12/R, 20 W, Phillips, Eindhoven, The Netherlands) through Shinkolite 102 red plastic (Argo Plastics, Los Angeles, CA). The fluence rate was approximately 2 µmol m² s¹.

Whole-Plant Gas-Exchange Measurements

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Plants were left in the chamber for a few days before the beginning of each experiment. A single plant was used for several days, and its leaf surface area was measured daily from enlarged photographs. Because of the small size of the Arabidopsis leaves, the leaf temperature was not measured. However, under the low-light fluence rates used, we assumed, for our calculations of leaf conductances, that it remained close to air temperature. Any error introduced by this assumption would be almost constant between experiments and should not affect the conclusions.

The light responses were measured with the following procedure: After a standard dark period of 14 h at 21°C, leaf conductances were recorded during a 45-min exposure to blue light, followed by 45 min of exposure to red light of the same fluence. A second blue-light exposure was given before returning the plants to white light. Experiments were duplicated, and the data are presented as mean leaf conductance, g (millimoles per square meter per second) and CO₂ flux (micromoles per square meter per second), both calculated for each plant.

Phototropism Studies

For induction of first positive curvature, the lids of the Petri dishes were removed just before irradiation and the plates were fixed vertically and aligned so that the plane of the Petri dish was about 15° from the plane parallel to the light beam and there could be no shading of one seedling by another. The agar surface was kept vertical with the seedlings in the same orientation in which they had been growing. Thus, the Petri dish wall did not attenuate the light striking the seedlings save for a few closest to the wall. The technique permitted calculation of the inductive fluences with an error of less than 5%. The error introduced because the curvature was 15° out of the imaged plane was small and the same for all samples. The Petri dish lid was replaced immediately after irradiation. For induction, we first followed the multiple-pulse technique of Steinitz and Poff (1986), a procedure that amplified first positive curvature in Arabidopsis without altering threshold and peak values for the curvature response. A given total fluence was administered at 20-min intervals in five successive pulses, each one-fifth the total fluence.

Janoudi and Poff (1992) found that a pulse of red light given 2 h before a single inductive pulse of blue light strongly enhanced subsequent first positive phototropic curvature of dark-grown Arabidopsis hypocotyls. Therefore, we also used this technique with the two cry1cry2 double mutants to determine whether they showed amplified first positive curvature under those irradiation conditions. Curvatures were imaged as they were for the second positive curvature, 120 min after the start of the first pulse. All seedlings were measured except those few that touched one another; the curvature over the apical 1 cm of the image of each hypocotyl was recorded (1 cm on the image = 0.386 cm of hypocotyl). Apart from the blue-light treatment, all manipulations were carried out in total darkness until imaging. Where required, hypocotyl-length measurements were made directly from the seedling images and then corrected for magnification by the imaging system.

Phosphorylation Studies

	RESULTS

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Stomatal Responses in Wild-Type and Mutant Seedlings

View larger version (33K): [in this window] [in a new window]   Figure 2. Changes in mean leaf stomatal conductance (solid lines) and CO2 uptake (dashed lines). After a dark period (14 h), plants were successively irradiated with blue (60 µmol m2 s1), red (60 µmol m2 s1), blue (60 µmol m2 s1), and then white light (230 µmol m2 s1) for 45-min exposures. Each experiment was done at least twice.

For each of the genotypes, changes in stomatal conductance and absolute values of mean leaf stomatal conductance at the end of each irradiation treatment are given in Table I. The time courses for changes in stomatal conductance (Fig. 2) and the mean leaf stomatal conductances at the end of the irradiation periods indicate that for all the genotypes studied, blue light (60 ± 5 µmol m² s¹) was far more effective than red light in promoting stomatal opening. Red light, by contrast, was unable to sustain the level of stomatal opening reached under blue light. With all genotypes, the mean leaf stomatal conductances after 45 min of red light treatment were similar to those measured in darkness.

The amplitude of the stomatal opening induced by white light (250 µmol m² s¹) depended on the genotype (Table I). The source of this variability is unknown, but may reflect variations in leaf temperature among plants, inaccuracy in estimation of exposed leaf surface (difficult to obtain accurately without destroying the plant), or some consequence of the mutations. However, when the responses to blue or red light are plotted relative to the response to white light (Fig. 3), the blue-light responses are similar for all genotypes. Under the same fluence rates (60 µmol m² s¹), the rate of CO₂ uptake was significantly lower under blue than under red light (Table II). Despite this difference, the low-fluence-rate blue light (60 µmol m² s¹) induced stomatal opening similar to that induced by white light at a fluence rate (230 µmol m² s¹), which had sustained approximately a 7-fold greater rate of photosynthesis. These results, consistent with those obtained with Commelina communis (Travis and Mansfield, 1981; Schwartz and Zeiger, 1984; Gautier et al., 1992) and fava bean (Shimazaki et al., 1986), suggest that photosynthesis does not play a major role in blue-light-induced stomatal opening in these species and that a specific blue-light-activated system must be involved.

View larger version (48K): [in this window] [in a new window]   Figure 3. Relative mean conductances measured in darkness before experiments and at the end of the first blue- and red-light irradiations. Leaf conductances under the different conditions were normalized in each case to conductances measured after 45 min of white light.

Phototropic Responses of Wild-Type and Mutant Seedlings

Second Positive Curvature

View larger version (37K): [in this window] [in a new window]   Figure 4. Second positive phototropic curvatures of wild-type and various mutant seedlings of Arabidopsis after 6 (gray bars) and 24 (black bars) h of continuous irradiation with unilateral blue light of 2 µmol m2 s1 (n = 18-39, except that for npq1-2, n = 70-97). Seedlings were 3 d old at the onset of irradiation.

Both phyA and phyB have been shown to play a role in determining the magnitude of the Arabidopsis second positive phototropic response (Hangarter, 1997; Janoudi et al., 1997). Development of curvature in phyA or phyB single mutants is slower than in the wild type, and phyAphyB double mutants show dramatically impaired curvature development during the first few hours of continuous unilateral irradiation (Hangarter, 1997). However, by 6 h the phyAphyB double mutant seedlings showed almost as much curvature as their wild-type counterparts (Fig. 4) and by 24 h they differed little from the wild type.

First Positive Curvature

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View larger version (18K): [in this window] [in a new window]   Figure 5. Fluence-response curves for first positive curvature for etiolated 3-d-old wild-type and mutant seedlings of Arabidopsis (n = 57-102; except that for phyAphyB, n = 32-56; for nph1-5, n = 29-37; and for npq1-2, n = 34-47). Error bars represent ±SE.

All of the 3-d-old cry mutants, both single and double, showed reduced curvatures in comparison to the wild type. This reduction may reflect either a real genetic difference between the mutants and the wild type, implicating the cry1 and cry2 photoreceptors in modulating the magnitude of the phototropic response, which is known to be the case with phyA and phyB (Hangarter, 1997; Janoudi et al., 1997), or inherent differences in growth rates from different seed lots related to different conditions during seed maturation and harvest. Therefore, we measured hypocotyl lengths of 2- and 3-d-old cry1cry2 double mutants, npq1-2, and the wild type (Table III). These three mutants were chosen because cry1cry2 double mutants failed to show first positive curvature in the Ahmad et al. (1998) study and because zeaxanthin was implicated in phototropism by the correlative studies of Quiñones and Zeiger (1994) and Quiñones et al. (1996). The results indicate that all three mutants grew more rapidly than their wild-type counterparts. Given the difference in early growth rates, we measured fluence-response curves for the 2-d-old seedlings for the cry1cry2 double mutants and npq1. All three of these younger mutants showed strong curvatures that were indistinguishable in magnitude from those of 3-d-old wild-type seedlings (Fig. 6). Threshold, peak, shoulder, and saturation fluence levels for the double mutants and for npq1-2 were again the same as those for the wild type. No curvature response was detected in the shorter 2-d-old wild-type seedlings (data not shown).

View larger version (29K): [in this window] [in a new window]   Figure 6. Fluence-response curves for first positive curvature for the etiolated 2-d-old npql-2, hy4-B104cry2-1, and cry1-304cry2-1 seedlings (n = 36-96) and for 3-d-old etiolated wild-type seedlings (n = 57-80).

Ahmad et al. (1998) failed to detect first positive curvature in response to a single pulse of blue light in the first-positive-fluence range from a cry1cry2 double mutant. We therefore attempted to induce first positive curvature in the wild type and in the two cry1cry2 double mutants with a single pulse instead of five multiple pulses. The fluence chosen was 3 µmol m² (log 0.5), where first positive curvature induced by multiple pulses was near its maximum (Fig. 5). Initial efforts with 3-d-old etiolated seedlings of Columbia wild type, hy4-B104cry2-1, and cry1-304cry2-1 gave curvatures of only 3.9° ± 0.7°, 1.3° ± 0.7°, and 3.6° ± 0.7° (n = 91, 98, and 66), respectively. We used a 10-min pulse of red light starting 2 h before phototropic induction, a technique demonstrated by Janoudi and Poff (1992) to amplify first positive curvature strongly. In this case, the curvatures were 16.2° ± 2.6°, 11.3° ± 1.3°, and 12.1° ± 1.7° (n = 74, 72, and 69), respectively. Thus, in our experiments cry1cry2 double mutants show first positive phototropic responses to both single and multiple blue-light pulses.

Blue-Light-Induced Autophosphorylation of nph1 in Wild-Type and Mutant Seedlings

View larger version (61K): [in this window] [in a new window]   Figure 7. Light-induced phosphorylation of nph1 in etiolated seedlings from the wild-type (WT) and various mutant lines. Protein load was 20 µg per lane. A, Autoradiograms of dark-control (lanes D) membranes from the various cry mutants and nph1-5 and those given saturating blue light (lanes L, 103.3 µmol m2) immediately before phosphorylation. B, Same as A, except for npq1-2. C, Fluence-response curve for light-induced phosphorylation of nph1 from etiolated wild-type and hy4-B104cry2-1 and cry1-304cry2-1 mutant seedlings (n = 3).

	DISCUSSION

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All of the mutants tested, both single and double, showed strong stomatal regulation by blue light (Fig. 2). The nph1-1hy4-105 double mutant showed a somewhat reduced response, but its conductance in darkness was lower than that of all of the other wild-type and mutant plants; and when all of the responses were normalized to the white-light response (Fig. 3), its responses fell within the range of the other mutants and the wild type. Hence, we conclude that the action of blue light in inducing stomatal opening must be mediated by a photoreceptor and a signal transduction pathway genetically separable from the pathways mediated through cry1, cry2, or nph1a photoreceptor probably using zeaxanthin as its chromophore. We further conclude that cry1, cry2, and nph1 do not play a major role in modulating the magnitude of the stomatal response to blue light.

With the exception of the phototropism null mutant nph1-5, all of the mutants tested, both single and double, showed strong second positive curvature (Fig. 4). These results support the conclusion that cry1 and cry2, either alone or together, cannot serve as the photoreceptor detecting light direction for second positive phototropism. They could modulate curvature magnitude; one of the double mutants, cry1-304cry2-1, showed slightly reduced curvature after 24 h of unilateral light. The result with npq1-2 likewise indicates that zeaxanthin cannot serve to detect light direction for second positive phototropism. The only mutant failing to respond was nph1-5, supporting the argument that nph1 alone detects the light direction.

As was the case with second positive curvature, all of the mutants tested, except for nph1-5, displayed first positive curvature over the same fluence range (Fig. 5). The threshold, peak, shoulder, and saturation fluence values did not differ among the mutants and wild type. The reduced response magnitude obtained from the mutants in comparison to that of wild-type seedlings could reflect action by cry1, cry2, or zeaxanthin downstream from nph1, which appears to be the case with phyA and phyB (Fig. 5; Hangarter, 1997; Janoudi et al., 1997). On the other hand, the reduced magnitude of curvature reported here for npq1-2 and the single and double cry mutants may reflect physiological differences between the seedlings arising from conditions during seed maturation and/or storage or from differences in growth rates (Table III). Seedlings of npq1-2 and both cry1cry2 double mutants only 2-d-old showed first positive curvature that is as strong as that of 3-d-old wild-type seedlings (Fig. 6). By contrast, 2-d-old wild-type seedlings were much shorter than 2-d-old seedlings of the double mutants or npq1-2 (Table III) and failed to show measurable phototropic curvature (data not shown). Hence, in first positive phototropism, as with the stomatal response and second positive phototropism, there must be a separate photoreceptor and signal transduction pathway independent of zeaxanthin, cry1, or cry2 and their downstream signaling components. Again, the only mutant failing to respond was nph1-5, supporting the argument that only nph1 detects the light direction.

The reason that Ahmad et al. (1998) did not obtain measurable first positive curvature in their cry1cry2 double mutant may be that their growth conditions somewhat desensitized phototropism in their seedlings. Evidence for this is suggested by the very narrow fluence range, little more than 1 order of magnitude, of their curvatures. The full fluence-response curve for first positive phototropism normally extends over at least 3 orders of magnitude (Fig. 5; Iino, 1990).

Quiñones and Zeiger (1994) reported a correlation between the level of zeaxanthin in maize coleoptiles and phototropic sensitivity and proposed that zeaxanthin was the photoreceptor for this response. Subsequently Palmer et al. (1996) showed that maize coloeptiles devoid of all carotenoids, as the result of either a genetic lesion or herbicide treatment, nevertheless showed strong second positive curvature and normal blue-light-induced phosphorylation. Horwitz and Berrocal (1997) have hypothesized that these two papers address separate phenomena. Under the appropriate fluence conditions, the curve for first positive curvature shows two peaks (Konjevi et al., 1989, 1992). Horwitz and Berrocal (1997) hypothesized that they might involve two different photoreceptors, zeaxanthin for the higher-fluence peak and some other chromophore for the lower-fluence peak. However, in the present study the mutant nph1-5 failed to show either peak of first positive curvature, although the other mutants and the wild type showed both. Therefore, both peaks must require a functional nph1-photoreceptor holoprotein.

In vitro blue-light-activated phosphorylation of nph1 in npq1-2 and the cry single and double mutants appears to be similar to that of the protein from wild-type seedlings (Fig. 7). Microsomal membranes from these mutants plus cry1 and cry2 single mutants showed significant increases in phosphorylation on irradiation with saturating blue light. A fluence-response curve for phosphorylation in both of the cry1cry2 double mutants is not distinguishable from that of the wild type (Fig. 7). Ahmad et al. (1998) reported phosphorylation in wild-type membranes at fluences below those at which we have detected light-induced phosphorylation (Short and Briggs, 1990; Reymond et al., 1992b; Palmer et al., 1993; Christie et al., 1998). This discrepancy may be the consequence of differences in growth conditions. From our results, however, we conclude that neither cry1 nor cry2 serve as photoreceptors for the blue-light-induced phosphorylation of nph1.

As mentioned above, Zeiger and Zhu (1998) have shown that the Arabidopsis npq1 mutant fails to show blue-light-induced stomatal opening. They also reported correlations between zeaxanthin content and sensitivity of the stomatal response to blue light. Their conclusion, based on these results, is that zeaxanthin functions as the photoreceptor chromophore for blue-light-induced stomatal opening. Their findings indicate that the stomatal photoreceptor contains a carotenoid chromophore rather than one involving FMN (nph1; Christie et al., 1998), or FAD and a pterin (for cry1 at least, but not known for cry2; Lin et al., 1995a, 1996b; Malhotra et al., 1995). Thus, unlike the phytochromes, which carry a bilitriene chromophore, different blue-light photoreceptors use different chromophores.

In summary, our results support the conclusion that the blue-light photoreceptors cry1, cry2, nph1, and zeaxanthin (the likely photoreceptor chromophore for blue-light-induced stomatal opening) all activate genetically separable pathways and that Arabidopsis must contain at least four different photoreceptors and signal transduction pathways. Our results also support the conclusion that only nph1 functions to detect light direction in phototropism, both first positive and second positive, although other photoreceptors may modulate subsequent differential growth response.

	FOOTNOTES

   Received November 18, 1998; accepted March 9, 1999.

	LITERATURE  CITED

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Ahmad M, Cashmore AR (1993) HY4 gene of A. thaliana encodes a protein with characteristics of a blue-light photoreceptor. Nature 366: 162-166 [CrossRef][Medline]

Ahmad M, Jarillo JA, Smirnova O, Cashmore AR (1998) Cryptochrome blue-light photoreceptors implicated in phototropism. Nature 392: 720-723 [CrossRef][Medline]

Briggs WR, Huala E (1999) Blue-light photoreceptors in higher plants. Annu Rev Cell Dev Biol (in press)

Briggs WR, Liscum E (1996) Blue light-activated signal transduction in higher plants. In P Aducci, eds, Signal Transduction in Plants. Birkhauser Verlag, Basel, Switzerland, pp 107-135

Briggs WR, Liscum E (1997) The role of mutants in the search for the photoreceptor for phototropism in higher plants. Plant Cell Environ 20: 768-771 [Medline]

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Chory J (1992) A genetic model for light-regulated seedling development in Arabidopsis. Development 115: 167-172

Christie JM, Reymond P, Powell GK, Bernasconi P, Raibekas A, Liscum E, Briggs WR (1998) Arabidopsis NPH1: a flavoprotein with the properties of a photoreceptor for phototropism. Science 282: 1698-1701 [Abstract/Free Full Text]

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Guo H, Yang H, Mockler CT, Lin C (1998) Regulation of flowering time by Arabidopsis photoreceptors. Science 279: 1360-1363 [Abstract/Free Full Text]

Hangarter RP (1997) Gravity, light and plant form. Plant Cell Environ 20: 796-800 [CrossRef][Medline]

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Huala E, Oeller PW, Liscum E, Han I-S, Larsen E, Briggs WR (1997) Arabidopsis NPH1: a protein kinase with a putative redox-sensing domain. Science 278: 2120-2123 [Abstract/Free Full Text]

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Janoudi A-K, Gordon WR, Wagner D, Quail P, Poff KL (1997) Multiple phytochromes are involved in red-light-induced enhancement of first-positive phototropism in Arabidopsis thaliana. Plant Physiol 113: 975-979 [Abstract]

Janoudi A-K, Poff KL (1992) Action spectrum for enhancement of phototropism by Arabidopsis thaliana seedlings. Photochem Photobiol 56: 655-659

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Karlsson PE (1986) Blue light regulation of stomata in wheat seedlings. I. Influence of red background illumination and initial conductance level. Plant Physiol 66: 202-206

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Konjevi R, Khurana JP, Poff KL (1992) Analysis of multiple photoreceptor pigments for phototropism in a mutant of Arabidopsis thaliana. Photochem Photobiol 55: 789-792 [Medline]

Konjevi R, Steinitz B, Poff KL (1989) Dependence of the phototropic response of Arabidopsis thaliana on fluence rate and wavelength. Proc Natl Acad Sci USA 86: 9876-9880 [Abstract/Free Full Text]

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Lascève G, Gautier H, Jappé J, Vavasseur A (1993) Modulation of the blue light response of stomata of Commelina communis by CO₂. Physiol Plant 88: 453-459 [CrossRef]

Lin C, Ahmad M, Cashmore AR (1996a) Arabidopsis cryptochrome is a soluble protein mediating blue light-dependent regulation of plant growth and development. Plant J 10: 893-902 [CrossRef][ISI][Medline]

Lin C, Ahmad M, Chan J, Cashmore AR (1996b) CRY2. A second member of the Arabidopsis cryptochrome gene family (accession no. U43397) (PGR 96-001). Plant Physiol 110: 1047 [CrossRef][Medline]

Lin C, Ahmad M, Gordon D, Cashmore AR (1995a) Expression of an Arabidopsis cryptochrome gene in transgenic tobacco results in hypersensitivity to blue, UV-A, and green light. Proc Natl Acad Sci USA 92: 8423-8427 [Abstract/Free Full Text]

Lin C, Cashmore AR (1996) Cryptochrome and plant photomorphogenesis. In WR Briggs, RL Heath, EM Tobin, eds, Regulation of Plant Growth and Development by Light. American Society of Plant Physiologists, Rockville, MD, pp 30-41

Lin C, Robertson DE, Ahmad M, Raibekas AA, Jorns MS, Dutton PL, Cashmore AR (1995b) Association of flavin adenine dinucleotide with the Arabidopsis blue light receptor CRY1. Science 269: 968-970 [Abstract/Free Full Text]

Lin C, Yang H, Guo H, Meckler T, Chen J, Cashmore AR (1998) Enhancement of blue-light sensitivity of Arabidopsis seedlings by a blue light receptor cryptochrome 2. Proc Natl Acad Sci USA 95: 2686-2690 [Abstract/Free Full Text]

Liscum E, Briggs WR (1995) Mutations in the NPH1 locus of Arabidopsis disrupt the perception of phototropic stimuli. Plant Cell 7: 473-485 [Abstract]

Liscum E, Briggs WR (1996) Mutants of Arabidopsis in potential transduction and response components of the phototropic signaling pathway. Plant Physiol 112: 291-296 [Abstract]

Liscum E, Hangarter R (1991) Arabidopsis mutants lacking blue light-dependent inhibition of hypocotyl elongation. Plant Cell 3: 685-694 [Abstract/Free Full Text]

Liscum E, Hangarter R (1994) Mutational analysis of blue-light sensing in Arabidopsis. Plant Cell Environ 17: 639-648

Malhotra K, Kim S-T, Batschauer A, Dawut L, Sancar A (1995) Putative blue-light photoreceptors from Arabidopsis thaliana and Sinapis alba with a high degree of sequence homology to DNA photolyase contain the two photolyase cofactors but lack DNA photolyase activity. Biochemistry 34: 6892-6899 [CrossRef][Medline]

Mockler TC, Guo H, Yang H, Duong H, Lin C (1999) Antagonistic actions of the Arabidopsis cryptochromes and phytochrome B in the regulation of floral induction. Development (in press)

Niyogi KK, Grossman AR, Björkman O (1998) Arabidopsis mutants define a central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion. Plant Cell 10: 1121-1134 [Abstract/Free Full Text]

Palmer JM, Short TW, Briggs WR (1993) Correlation of blue light-induced phosphorylation to phototropism in Zea mays L. Plant Physiol 102: 1219-1225 [Abstract]

Palmer JM, Warpeha KMF, Briggs WR (1996) Evidence that zeaxanthin is not the photoreceptor for phototropism in maize. Plant Physiol 110: 1323-1328 [Abstract]

Parks BM, Cho MH, Spalding E (1998) Two genetically separable phases of growth inhibition induced by blue light in Arabidopsis seedlings. Plant Physiol 118: 609-615 [Abstract/Free Full Text]

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Reymond P, Short TW, Briggs WR (1992a) Blue light activates a specific protein kinase in higher plants. Plant Physiol 100: 655-661 [Abstract/Free Full Text]

Reymond P, Short TW, Briggs WR, Poff KL (1992b) Light-induced phosphorylation of a membrane protein plays an early role in signal transduction for phototropism in Arabidopsis thaliana. Proc Natl Acad Sci USA 89: 4718-4721 [Abstract/Free Full Text]

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Shimazaki K, Iino M, Zeiger E (1986) Nature 319: 324-326 [CrossRef]

Short TW, Briggs WR (1990) Characterization of a rapid, blue light-mediated change in detectable phosphorylation of a plasma membrane protein from etiolated pea (Pisum sativum L.) seedlings. Plant Physiol 92: 179-185 [Abstract/Free Full Text]

Short TW, Briggs WR (1994) The transduction of blue light signals in higher plants. Annu Rev Plant Physiol Plant Mol Biol 45: 143-171 [CrossRef][ISI]

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Vavasseur A, Lascève G, Couchat P (1988) Oxygen-dependent stomatal opening in Zea mays leaves: effect of light and carbon dioxide. Physiol Plant 73: 547-552 [CrossRef]

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Zeiger E, Iino M, Ogawa T (1985) The blue light response of stomata: pulse kinetics and some mechanistic implications. Photochem Photobiol 42: 759-763

Zeiger E, Zhu J (1998) Role of zeaxanthin in blue-light photoreception and the modulation of light-CO₂ interactions in guard cells. J Exp Bot 49: 433-442 [Abstract]

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