Planta (1992)188:106-114

P l a n t a ((~ Springer-Verlag 1992

Spectral-dependence of light-inhibited hypocotyl elongation in photomorphogenic mutants of Arabidopsis: evidence for a UV-A photosensor Jeff C. Young, Emmanuel Liscum, and Roger P. Hangarter*

Department of Plant Biology, Ohio State University, 1735 Neff Ave., Columbus, OH 43210, USA Received 26 February; accepted 14 April 1992 Abstract. Photon fluence rate-response curves at different wavelengths were generated for the light-induced inhibition of hypocotyl elongation in seedlings of wildtype and photomorphogenic mutants of Arabidopsis thaliana. (L.) Heynh. Treatment of wild-type seedlings with continuous low-fluence-rate light (< 1.0 gmol photons, m - 2 - s -1) induced some inhibition of hypocotyl elongation at all wavelengths tested, with maximum inhibition in blue light. At higher fluence rates, inhibition reached a maximum of 70-80% in UV-A, blue, and far-red light. Fluence rate-response curves for seedlings of blul, a blue light-response mutant, showed a specific reduction in their response to blue light, but their response to UV-A, red, and far-red light was similar to that in wild-type seedlings. In contrast, the phytochromedeficient mutant hy6 showed a loss of response to lowfluence-rate light at all wavelengths, as well as to highfluence-rate far-red light. However, hy6 seedlings retained sensitivity to high-fluence-rate blue and UV-A light. The data support the conclusion that blue-lightand phytochrome-dependent photosensory systems regulate hypocotyl elongation independently and in an additive manner. Furthermore, hypocotyl inhibition in wild-type, blul, hy6 and blul-hy6 double mutants was indistinguishable in U V - A light, whereas marked differences were observed at other wavelengths, indicating the involvement of a third photosensory system with an absorption maximum in the UV-A. Key words: Arabidops& - Photomorphogenesis - Blue/ U V - A light - Hypocotyl elongation - Phytochrome

Introduction

Light regulates numerous aspects of plant growth and development through the action of several photorecep* To whom correspondence should be addressed; FAX (614) 292-7162

tors such as phytochrome and the blue/UV-A and UV-B photoreceptors. Of these, only the phytochrome system has been extensively studied at the physiological, genetic, and biochemical level (Sharrock and Quail 1989; for a review see Quail 1991). Recent work has identified multiple species of phytochrome with different transcriptional and post transcriptional regulation (Furuya 1989; Sharrock and Quail 1989; Dehesh et al. 1991; Wang et al. 1991). Moreover, it appears that different phytochrome chromoproteins control different responses (Adamse et al. 1988a; Smith and Whitelam 1990; Ballare et al. 1991 ; Nagatani et al. 1991; Whitelam and Smith 1991). Characterization of the photosensory system(s) that mediate blue/UV-A light responses in higher plants is complicated, in part, because blue and UV light can be absorbed by phytochrome and may result in phytochrome-induced responses. For example, high-irradiance responses, such as the inhibition of hypocotyl elongation, frequently have activity maxima in the blue/UV-A region as well as the red/far-red region of the spectrum. Thus, it is difficult to determine conclusively to what extent a phytochrome or a blue-light photoreceptor mediates the response to blue/UV-A light. However, research with phytochrome-deficient and blue-lightresponse mutants has demonstrated that inhibition of hypocotyl elongation by blue and UV-A light is not phytochrome-dependent (Koornneef et al. 1980; Adamse et al. 1988a, b; Lercari et al. 1990; Liscum and Hangarter 1991). Although the photoreceptors involved in blue/UV-A light responses in plants have not been biochemically identified, it has been suggested that flavin-type chromophores with absorption maxima near 360 nm and 450 nm are involved (e.g. Galland and Senger 1988a). However, investigations of the chromoproteins involved in the blue/UV-A light mediated phototaxis in Euglena (Galland et al. 1990; Brodhun and Hfider 1990; H/ider and Brodhun 1991) and the photosensitization of D N A photolyase in Escherichia coli (Wang et al. 1988; Jorns et al. 1990) indicate that these responses do not occur through the exclusive action of flavin chromophores. In

J.C. Young et al. : Light-inhibited hypocotyl elongation in Arabidopsis mutants particular, these studies indicate that pterins with absorption maxima in the U V - A may act as a photoreceptor in the near-UV, or perhaps as an element in ravinmediated electron-transfer reactions in both U V - A and blue light (Galland and Senger 1988b). Thus, the diversity of blue and U V - A light responses might involve multiple chromoproteins, as with phytochrome-dependent responses, or multiple chromophores on a single protein. Photomorphogenic m u t a n t plants are powerful tools for dissecting the complex network of events controlling light-dependent development and can provide a more accurate analysis of the functions of the various photosensory systems than can be obtained from wildtype plants alone (e.g. K o o r n n e e f et al 1980; Chory et al. 1989; Liscum and Hangarter 1991). For example, several of the hy mutants in Arabidopsis lack spectrophotometrically detectable phytochrome (Chory et al. 1989; Parks et al. 1989; Parks and Quail 1991), and are useful for distinguishing between phytochrome-dependent and phytochrome-independent processes (Koornneef et al. 1980; Liscum and Hangarter 1991). Similarly, the blu mutants have altered sensitivity to blue light, and are useful for investigating the complexity of blue-light responses (Liscum and Hangarter 1991). In this paper, comparative studies on the lightinduced inhibition of hypocotyl elongation in the bluelight-response mutant blul and the phytochrome-deficient mutant hy6 are presented. These studies show that blue light- and phytochrome-dependent photosensory systems operate independently in inhibiting hypocotyl elongation and indicate that there m a y be a distinct U V - A photosensory system that functions in this highirradiance response.

Materials and methods

Plant materials and growth conditions. Wild-type Arabidopsis thaliana (L.) Heynh. ecotype "Columbia", and mutant strains homozygous for blul (Liscurn and Hangarter 1991) and hy6 (Chory et al. 1989) were used. In addition, a carotenoid-deficient mutant strain

am45 was used (Orbovi6 and Poff 1991). Because the lesion in strain am45 is a recessive nuclear mutation that is seedling-lethal when homozygous, the strain was maintained as a heterozygote. Seeds from self-fertilization of these heterozygotes were used in the experiments reported here. For all lines, seeds were surface sterilized in 1.5% sodium hypochlorite for 20 rain, rinsed with sterile H20, and planted in polystyrene Petri dishes on medium containing 0.5 x Murashige and Skoog salts (Murashige and Skoog 1962) and 1.0% (w/v) agar. For experiments with UV-A light, glass Petri dishes were used to eliminate trace amounts of blue fluorescence produced by the polystyrene dishes. The seeds were incubated at 44- 1~ C for 2-3 d, then exposed to red light for 30 min to induce uniform germination as described by Liscum and Hangarter (1991). The red-light-treated seeds were incubated in darkness for an additional 23.5 h and then placed in the indicated light conditions for an additional 48 h at 23~ C. To inhibit carotenoid biosynthesis, Seeds were handled as above except that the herbicide 4-chloro-5(methylamino)-2-(7,y,y-trifluorom-tolyl)-3(2H)-pyridazinone (SAN 9789; Sandoz, Richmond, Cal., USA), was added to the growth medium. SAN 9789 prevents carotenoid synthesis, but does not affect ravin synthesis (Vierstra

107

and Poff 1981) or the effectiveness of phytochrome in dark-grown seedlings (Jabben and Deitzer 1979).

Light sources. For wavelengths greater than 400 nm, light from halogen flood lamps (150 W Quartzline; General Electric Co., Cleveland, O., USA) was filtered through 6.5 cm of water-cooled 1.5% CuSO4, a layer of clear Plexiglas (6.4 mm thick) and interference filters of the indicated wavelength maxima (8-10 nm halfbandwidth). For far-red treatments, the CuSO4 was replaced with water. For UV-A treatments, light from black-light fluorescent bulbs (F15T8BLB; Sylvania, Danvers, Mass., USA) was filtered through a layer of yellow Roscolux No. 11 producing light with a peak intensity at 360 nm and a 30-nm half-bandwidth. Fluence rates were controlled by changing the voltage and-or the distance between the seedlings and the light source such that the spectral quality remained constant. Fluence rates were measured with a LI-189 quantum photometer or a LI-1800 portable spectroradiometer (LICOR, Inc., Lincoln, Neb., USA).

Growth analysis. After 2 d of growth in the continuous light treatments, seedlings were carefully pulled from the agar and placed onto transparent tape. Hypocotyl lengths were measured at a resolution of 0.05 mm and cotyledon areas at a resolution of 0.01 mm/ using SigmaScan (Jandel, Sausalito, Cal., USA) to digitize images of the seedlings projected from a photographic enlarger (10 • magnification) onto a digitizing tablet. For each treatment, 25-150 seedlings were measured. Light-induced inhibition of hypocotyl elongation was considered to be saturated by a light treatment when inhibition exceeded 70% because maximum inhibition of hypocotyl elongation in wild-type Arabidopsis is reached in continuous white light when hypocotyls are about 70-80% inhibited compared with dark-grown controls (Liscum and Hangarter 1991). Thus, in some experiments fluence rates were not tested above those that caused greater than 70 % inhibition. Chlorophyll measurements were made as described previously (Liscum and Hangarter 1991).

Results

Fluence rate-responses for inhibition of hypocotyl elongation. Wild-type and blul seedlings responded similarly to red and far-red light (Fig. 1). In far-red light (738 nm), inhibition o f hypocotyl elongation exhibited a log-linear relationship with p h o t o n fluence rate. The threshold for the response occurred around 0.1 lamol" m -2" s-a and the response became saturated at about 10 gmol 9m - 2 . s-1. Some sensitivity to far-red light was observed in the hy6 seedlings, but the threshold for this response was near 1 gmol 9m - 2 . s-1 and only reached 20% inhibition at the highest fluence rate measured (10 ~tmol 9m -2 " S-a). Wild-type and blul seedlings also showed similar fluence rate-responses to wavelengths between 600 and 660 nm with a maximum response of 47% inhibition (Fig. 1). In contrast, hy6 seedlings showed no inhibition by red light. The response of wild-type and blul seedlings to green and red light was similar (Fig. 1, 2), although the maximum response in green light was a little lower. At 530 nm, hypocotyl inhibition in wild-type seedlings reached saturation at a low fluence rate (0.3 I~mol 9m -2 9 s-1). Green light did not inhibit hypocotyl elongation in hy6 seedlings. The response o f the different genotypes to blue light (410-500 nm) was complex (Fig. 2). For example, wildtype seedlings had multi-phasic fluence rate-response

J.C. Young et al. : Light-inhibited hypocotyl elongation in Arabidopsis mutants

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Fig. 1. Photon fluence rate-response curves for the inhibition of hypocotyl elongation in wild-type and mutant Arabidopsis seedlings by 48 h of monochromatic light at wavelengths between 738 and 550 nm. The vertical error bars represent the product of the SEs for the dark control and light-treated seedlings. Curves were drawn by inspection, o, wild type; o, blul; v, hy6

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curves that saturated near 10 gmol . m - 2 . s - t . This multi-phasic behavior was m o s t apparent at 450 and 486-nm, where there is a m a j o r inflection near 1 gmol 9m - 2. s - 1. However, neither of the mutants showed such a complex response: rather, by6 was responsive to high-fluence-rate light while bluI responded to lowfluence rate light only. Although hypocotyl elongation was partially inhibited in blul seedlings by 450- and 486-nm light, inhibition reached a m a x i m u m of only 37 % at 1 gmol 9 m - 2. s - 1. When c o m p a r e d with the response of wild-type and by6 seedlings, this indicates a partial loss of sensitivity to low-fluence-rate blue light and a complete loss o f sensitivity to high-irradiance blue light. Addition of the blul and by6 fluence rate-response curves results in a curve with essentially the same multi-phasic shape as the wild-type curve (not shown).

The fluence rate-response relationships obtained with 360- and 410-nm light in wild-type, blul and hy6 seedlings were virtually identical within the resolution of our experiments (Fig. 2). To determine if photosensory systems other than phytochrome and the blue-light system play a role in hypocotyl inhibition caused by 360-nm light, the response o f blul-hy6 double mutants was examined. As previously shown, blul-hy6 double mutants lack sensitivity to white, blue, red, and far-red light as would be expected for plants that are missing phytochromes and the blue-light-dependent system (Liscum and H a n garter 1991). However, 360-nm light inhibited hypocotyl elongation in the double m u t a n t to nearly the same extent as in wild-type, blul, and hy6 seedlings (Figs. 2, 6, Table 1).

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Table 1. Growth features of 3-d-old wild-type, blul, hy6, and blulhy6 double mutant Arabidopsis seedlings in 360-nm light. After 1 d of growth in the dark, 360-nm light was provided from above for 48 h at 15.0• ~tmol" m -2 9s -1. Chlorophyll measurements (~tmol Chl 9(~tg protein) -1) were obtained from pooled samples of 20 cotyledon pairs. Chlorophyll levels in dark controls were less

than 0.001 I~mol' (~tg protein) -1. Cotyledon areas represent the m e a n s i SE for 20 cotyledons pairs. Dark controls had a mean cotyledon area of 0.19• mm 2 for all genotypes. Hypocotyl lengths represent the means • SE of at least 50 seedlings, except for the double mutants where only 36 seedlings were used. Hypocotyl lengths of the dark controls are shown in parentheses

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J.C. Young et al. : Light-inhibited hypocotyl elongation in Arabidopsis mutants

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Activity spectra for inhibition of hypocotyl elongation. Relative responsiveness can be compared by measuring the response at equivalent fluence rates at each wavelength as shown in Fig. 3. The wild-type response to 1 gmol 9m - 2 . s-1 of light showed inhibition at all wavelengths tested, with peak activities in the blue, red and far-red regions. However, at 10 g m o l - m - z . s -1 inhibition increased in the UV-A, blue, and far-red regions only. The hy6 mutant showed reduced sensitivity to 1 gmol 9m - 2. s- 1 irradiation at all wavelengths tested except 360 nm. At 10 gmol. m -2- s -1, hy6 retained near-normal levels of inhibition in the U V - A and blue regions, but showed reduced sensitivity to far-red light. The blul mutant was about as sensitive as wild-type seedlings to low-fluence-rate irradiation at all wavelengths and had a normal response to high-fluencerate U V - A and far-red light. However, the blue component (i.e. 450 to 500 nm) of the wild-type high-fluencerate response was absent in blul (Fig. 3).

Unfortunately, true action spectra are difficult to obtain for high-irradiance responses such as the inhibition of hypocotyl elongation. Specifically, inhibition of hypocotyl elongation does not follow the law of reciprocity, which is a requirement for constructing true action spectra (Shropshire 1972), and involves multiple photoreceptors with overlapping absorbtion spectra. This results in different spectra for each level of response because the fluence rate-response curves are not all log-linear and parallel (Figs. 1, 2). In addition, because action spectra are normalized to the most effective wavelength for each genotype and level of inhibition, it is difficult to compare the relative photon-rate effectiveness directly within and between genotypes. In spite of these difficulties, activity spectra (Fig. 4) were calculated from the fluence-response curves (Figs. 1, 2) to facilitate comparison of the results presented here for Arabidopsis with results obtained with other plants. The activity spectra show that 450-nm light was most effective for achieving 30% inhibition in wild-type plants, although wild-type plants were sensitive to light throughout most of the spectral range tested. The 60% level of inhibition was obtained most easily in wild-type plants by wavelengths below 500 nm and above 700 nm, but there was little sensitivity between 500 and 700 nm. Of the numerous activity spectra that have been reported for light-induced inhibition of hypocotyl elongation in wild-type plants (e.g. Evans et al. 1965; Hartman 1967; Jose and Vince-Prue 1977; Beggs et al. 1980; Holmes and Schafer 1981), those for lettuce and Petunia (Evans et al. 1965) are most similar to the activity spectra reported here for hypocotyl inhibition in wild-type Arabidopsis (Fig. 4) in that maximal activity was observed near 410 nm and in red/far-red light. Analysis of the mutant plants showed that it was more difficult to achieve a 30% response in the phytochromedeficient hy6 seedlings than in blul or wild-type seedlings. However, hy6 plants were relatively sensitive to wavelengths below 500 nm at both 30% and 60% levels of inhibition. In addition, hy6 was much less sensitive to far-red light than were wild-type or blul plants. In contrast to hy6, blul could be inhibited to 30% across much of the wavelength range tested. However, 60 % inhibition was most easily reached in blul with far-red light and at wavelengths below 436 nm. All of the genotypes tested were sensitive to 360-nm light and highly sensitive to 410-nm light for all levels of inhibition.

Effect of carotenoids on inhibition of elongation. Carotenoids are generally not considered as candidates for the chromophore of blue-light photoreceptors because carotenoid-deficient mutants and plants treated with inhibitors of carotenoid biosynthesis retain blue-light responses (Jabben and Deitzer 1979; Vierstra and Poff 1981 ; Galland and Senger 1988a). Carotenoids have also been dismissed as photoreceptors for blue/UV-A responses because they do not generally absorb U V - A light. The observation that the blul-hy6 double mutants respond to U V A light but not blue light prompted us to re-examine the possibility that carotenoids might play a role in the blue-light-sensitive hypocotyl-inhibition re-

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J.C. Young et al. : Light-inhibited hypocotyl elongation in

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Fig. 6. Photograph of representative seedlings of Arabidopsis wildtype (WT), blul, hy6, and blul-hy6 double (dbl) mutants grown for 72 h in 360-nm light and in darkness

112

J.C. Young et al. : Light-inhibited hypocotyl elongation in Arabidopsis mutants

sponse. Wild-type seedlings grown in the presence of 10 ~tM SAN-9789, an inhibitor of carotenoid synthesis, and a carotenoid-deficient mutant (strain am45) (Orbovi6 and Poff 1991) were found to exhibit more inhibition of hypocotyl elongation in blue light than the corresponding controls (Fig. 5). Although blul seedlings grown on medium containing SAN-9789 were slightly shorter than seedlings grown without SAN-9789, they were for the most part not inhibited by blue light. Thus, other than acting as a blue-light screen, carotenoids do not appear to be involved in blue-light inhibition of hypocotyl elongation unless extremely low concentrations of carotenoids are sufficient.

De-etiolation responses induced by UV-A. In addition to inhibition of hypocotyl elongation, de-etiolation processes such as apical-hook opening, cotyledon expansion, and chlorophyll biosynthesis were induced by 360-nm light in wild-type seedlings (Table 1, Fig. 6). Of these responses, chlorophyll biosynthesis was the only response altered in the hy6 seedlings and cotyledon expansion was the only response altered in blul seedlings in 360 nm light. In the blul-hy6 double mutants, hypocotyl elongation was inhibited as in wild-type seedlings but the apical hooks remained closed, cotyledons did not expand, and chlorophyll biosynthesis was only slightly induced (Table 1, Fig. 6). The induction of inhibition of hypocotyl elongation and other de-etiolation processes by UV-A light are most probably physiological responses, rather than a result of cell damage. Damage to plants by UV light is primarily limited to wavelengths below 320 nm (Giese 1968; Coohill 1989), and many non-damaging effects have been observed in UV-B as well as U V - A light (Baskin and Iino 1987; Drumm-Herrel and Mohr 1981; Lercari et al. 1990).

Discussion

Contributions of phytochrome and blue-light photosensory systems to light-induced inhibition of hypocotyl elongation. Compared with wild-type seedlings, the phytochromedeficient hy6 seedlings were less sensitive to wavelengths greater than 486 nm. However, hy6 seedlings were sensitive to U V - A and high-fluence-rate blue light. These results indicate that the inhibition of hypocotyl elongation in U V - A light is not exclusively phytochrome dependent and sensitivity to blue light is only partially a result of phytochrome activity. Because hy6 presumably represents a lesion in a gene affecting chromophore biosynthesis or assembly of phytochrome holoprotein (Chory et al. 1989), all phytochromes are affected. However, by6 seedlings retain about 5 % of wild-type levels of spectrophotometrically detectable phytochrome (Chory et al. 1989) and this residual phytochrome may be responsible for the low level of inhibition observed with high fluence rates of far-red light (Fig. 1). These observations are consistent with and extend previous reports that showed hypocotyl elongation in the phytochrome-deficient hyl, hy2, and hy6 seedlings was not inhibited by red

or far-red light at fluence rates that saturated the wildtype response (Koomneef 1980; Chory et al. 1989). Although not tested in these studies, the hy3 mutant was previously shown to lack inhibition of hypocotyl elongation in red light while retaining wild-type sensitivity to far-red light (Koornneef 1980). Recently, by3 seedlings were shown to be deficient in phyB, a species of phytochrome that is stable in light-grown tissues, while phyA, a phytochrome species that is labile in light-grown tissues, is present at normal levels (Nagatani et al. 1991 ; Somerset al. 1991). Thus, phyB may be the phytochrome species responsible for inhibition of hypocotyl elongation caused by low-fluence-rate light between 450 and 660 nm and phyA probably functions in the inhibition observed with high-irradiance far-red light. In contrast to hy6, the fluence rate-response relationships for blul show a specific reduction in the inhibition of hypocotyl elongation in blue light between 410 nm and 500 nm, but sensitivity to red and far-red light is the same as in wild-type seedlings. Comparison of the fluence rate-response curves at 450 and 486 nm for wild-type seedlings with the curves for the mutant seedlings (Fig. 2), show that the blue-light-dependent photosensory system functions independently of the phytochrome system. At these wavelengths, the complex curve obtained in wild-type seedlings is separated into its component parts in the two mutants.

Contribution of a UV-A photosensory system to light~ induced inhibition of hypocotyl elongation. The fluence rate-response relationships for inhibition of hypocotyl elongation in the mutants and wild-type seedlings were essentially the same in the UV-A region. However, the relationships were strikingly different with wavelengths above 436 nm (Figs. 1, 2). The observation that the phytochrome-deficient hy6 seedlings retain wild-type sensitivity to UV-A light (Fig. 2) but only partial sensitivity to far-red light (Fig. 1) indicates that the UV A response is not dependent on phytochrome even though the response in UV-A light was previously considered to be a high-irradiance phytochrome response (e.g. Hartmann 1967). Evidence for a UV photoreceptor separate from phytochrome was also observed in tomato (Lercari et al. 1990). Similarly, the observation that blul seedlings are altered in their sensitivity to blue but not U V - A light indicates the involvement of more than a single blue/ U V - A photoreceptor for inhibition of hypocotyl elongation. Furthermore, other de-etiolation responses induced by U V - A light in wild-type, hy6, and blul seedlings (Fig. 6, and Table 1) were similar to those induced by white and blue light (Liscum and Hangarter 1991). For example, cotyledon expansion, apical-hook opening, and chlorophyll biosynthesis occur along with inhibition of hypocotyl elongation (Fig. 6, Table 1), indicating that U V - A light elicits a variety of developmental responses like those mediated by the phytochrome and blue-light systems. However, of the de-etiolation processes observed in wild-type, blul, and hy6 seedlings, only the inhibition of hypocotyl elongation was observed in blulhy6 double mutants (Fig. 6, Table 1). This is complementary to the earlier observation that blue light induces

J.C. Young et al.: Light-inhibited hypocotyl elongation in Arabidopsis mutants several de-etiolation processes in the blul mutant, but does not inhibit hypocotyl elongation (Liscum and Hangarter 1991). Thus, U V - A light appears to affect hypocotyl elongation through a chromophore that is different from the blue-light- and phytochrome-dependent chromophores that appear to control other de-etiolation responses. Carotenoids, which absorb blue but not UV light, do not appear to act as a chromophore in the blue-light photoreceptor for inhibition of hypocotyl elongation because a carotenoid-deficient Arabidopsis mutant and wild-type seedlings treated with the carotenoid biosynthesis inhibitor, SAN-9789, retain blue-light-dependent inhibition of hypocotyl elongation (Fig. 5). Similarly, carotene-lacking mutants of Phycomyces (Presti et al. 1977) and corn seedlings treated with SAN-9789 (Vierstra and Poff 1981) retain blue-light sensitivity. Although carotenoids do not appear to act as a photoreceptor for blue-light responses, they can modify such responses by screening blue light (Vierstra and Poff 1981). For example, an increased amount of carotenoid could lead to a loss of the blue response without affecting the U V - A response. However, elimination of carotenoids in the blul seedlings without a loss of the blul phenotype argues against a carotenoid screen (Fig. 5). Although the nature of the blue/UV-A photoreceptor pigments has not been determined, they are generally considered to be fiavoproteins because many blue-light responses show maximal activities in blue and U V - A light that are consistent with light absorption by a flavoprotein. However, the separation of UV-A-dependent activity from blue-light-dependent activity shown here in the Arabidopsis mutants indicates that there are two different photoreceptors, one with peak activity in the U V - A region and one with peak activity in the blue region. These findings do not rule out flavoproteins as photoreceptors for these responses because flavins and flavoproteins are known that lack U V - A absorption peaks and U V - A activity maxima are not universal (Galland and Senger 1988a, b). In addition, some flavoproteins can have higher U V - A absorbance relative to blue when they are semireduced (Massey and Palmer 1966). Because of their chemical and photophysical properties, pterins have also been considered as photoreceptor pigments for some responses that are induced by blue and U V - A light (Galland and Senger 1988b). Indeed, it has been demonstrated that both flavins and pterins act as photoreceptors for phototaxis in Euglena (Brodhun and H/ider 1990; Galland et al. 1990; Hfider and Brodhun 1991) and various photoresponses in Phycomyces (Hohl et al. 1992ab). In addition, E. coli D N A photolyase contains both pterin and flavin and either chromophore can function as a photosensitizer in catalysis (Wang et al. 1988; Jorns et al. 1990). The complexity in the phytochrome-independent blue and U V - A responses described here are consistent with a model in which blueand UV-A-light-induced responses are mediated by a diversity of photoreceptors containing flavin, pterin, and-or other chromophores. In summary, comparison of the fluence rate- and wavelength-dependence for light-induced inhibition of

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hypocotyl elongation in wild-type Arabidopsis and the blul and hy6 photomorphogenic mutants shows that the phytochrome and blue photosensory systems affect hypocotyl elongation independently and in an additive manner. Specifically, inhibition of hypocotyl elongation caused by phytochrome activity is primarily a low-fluence-rate response between 436 and 680 nm light and a high-irradiance response in far-red light, whereas inhibition by high-irradiance light between 436 and 500 nm is primarily due to blue-photosensor-dependent activity. Moreover, there appears to be a U V - A photosensory system that can cause inhibition of hypocotyl elongation independently of the phytochrome and blue-light photosensory systems. Taken together, the observations reported here indicate that a multitude of photoreceptor pigments, including several different phytochromes and a number of different blue- and UV-A-sensitive pigments, function together to coordinate development of the de-etiolation program in seedlings. We thank N.K. Peters and W.D. Bauer for critical reading of the manuscript and K.L. Poff for providing SAN 9789, seeds of am45, and several interference filters. This work was supported by National Science Foundation Grant No. DCB9106697 to R.P.H.

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