Proc. Natl. Acad. Sci. USA Vol. 93, pp. 8129-8133, July 1996 Plant Biology

Action spectra for phytochrome A- and B-specific photoinduction of seed germination in Arabidopsis thaliana (phytochrome mutants/spectrograph/light effect/very low fluence response) TOMOKO SHINOMURA*, AKiRA NAGATANIt, HIROKo HANZAWA*, MAMORU KUBOTAt, MASAKATSU WATANABEt, AND MAsAKI FURUYA*§ *Advanced Research Laboratory, Hitachi Ltd., Hatoyama, Saitama 350-03, Japan; tMolecular Genetics Research Laboratory, University of Tokyo, Hongo, Tokyo 113, Japan; and tNational Institute for Basic Biology, Okazaki 444, Japan Communicated by Winslow R. Briggs, Carnegie Institution of Washington, Stanford, CA, April 18, 1996 (received for review January 31, 1996)

ABSTRACT We have examined the seed germination in Pfr form and is present at relatively constant levels both in the Arabidopsis thaliana of wild type (wt), and phytochrome A light and in darkness (6). This heterogeneity of phytochromes (PhyA)- and B (PhyB)-mutants in terms of incubation time was explained by the amino acid sequencing of apoproteins and environmental light effects. Seed germination of the wt with type I and type II phytochromes in pea (7) and the cloning and PhyA-null mutant (phyA) was photoreversibly regulated of five phytochrome genes (PHYA to PHYE) in Arabidopsis by red and far-red lights of 10-1,000 ,umol m-2 when incu- thaliana (8, 9). Phytochrome A (PhyA) and phytochrome B bated in darkness for 1-14 hr, but no germination occurred in (PhyB) have been indicated, using PhyA-null mutants (phyA) PhyB-null mutant (phyB). When wt seeds and thephyB mutant (10-12) and PhyB-null mutants (phyB) (13), to be the most seeds were incubated in darkness for 48 hr, they synthesized important members of the family for regulation of hypocotyl PhyA during dark incubation and germinated upon exposure elongation. Recent analysis of these mutants have suggested to red light of 1-100 nmol m-2 and far-red light of0.5-10 ,umol very limited significance of PhyA under continuous white light, m-2, whereas the phyA mutant showed no such response. The regardless of the fact that PhyA is the predominant molecular results indicate that the seed germination is regulated by PhyA species in dark-grown tissues (14) and indispensable for the and PhyB but not by other phytochromes, and the effects of response of etiolated seedlings to continuous far-red light (10, PhyA and PhyB are separable in this assay. We determined 12, 15) and for the red light-enhanced phototropism (16). action spectra separately for PhyA- and PhyB-specific induc- Concerning photoinduction of seed germination, in 1935, tion of seed germination at Okazaki large spectrograph. Flint and MacAlister (17) found that continuous irradiation Action spectra for the PhyA response show that monochro- with light of 580-700 nm was effective in inducing germination matic 300-780 nm lights of very low fluence induced the of lettuce seeds, but that of 700-800 nm, as well as 500 nm, was germination, and this induction was not photoreversible in the inhibitory. In 1952, Borthwick et al. (18) examined the effect range examined. Action spectra for the PhyB response show of brief exposures to red and far-red light in lettuce seeds, and that germination was photoreversibly regulated by alternate discovered the red/far-red photoreversible response. They irradiations with light of 0.01-1 mmol m-2 at wavelengths of measured the action spectra for promotion and inhibition of 540-690 nm and 695-780 nm. The present work clearly germination, finding the maximum sensitivity for promotion in demonstrated that PhyA photoirreversibly triggers the ger- the region of 640-670 nm and that for inhibition in 720-750 mination upon irradiations with ultraviolet, visible and far- nm. Very similar action spectra for photoreversible regulation red light of very low fluence, while PhyB controls the pho- of seed germination were determined in Arabidopsis thaliana toreversible effects of low fluence. of the wild-type (wt) (19) and long-hypocotyl mutants (20). However, it has been an open question which phytochrome Diversification within families of sensory receptors allows species regulates the photoinduction of seed germination. discrimination of distinct but related stimuli. Plants have We recently reported (21), using the Arabidopsis phyA and evolved diverse photoreceptor systems for detection of light phyB mutants, that red/far-red reversible induction of seed intensity, quality, and duration to adjust their life in fluctuating germination is principally regulated by PhyB, but not by PhyA, environmental conditions (1). The best characterized photo- and that the phyB mutant seeds became sensitive to red light transducer in plants is phytochrome (2, 3), which exhibits after dark incubation for 48 hr. The purpose of the present photoreversible interconversion between two spectrally and study is to define different physiological roles of PhyA and biochemically distinct forms, a red light-absorbing form, Pr, PhyB, if any, in terms of incubation time in darkness and and a far-red light-absorbing form, Pfr (4). The earliest and characteristics of in and mutants. simplest hypothesis of phytochrome action was that responses light sensitivity phyA phyB are triggered by a red light pulse, converting biologically We report a novel action spectra for PhyA-specific photoin- inactive Pr to active Pfr, which can be reversed by a subsequent duction of seed germination, demonstrating that PhyA is the brief irradiation with far-red light, converting Pfr back to Pr (4). photoreceptor for very low fluence response (VLFR). Spectrophotometrically detectable amounts or states of phy- tochrome in vivo, however, are not consistent with this simple MATERIALS AND METHODS interpretation of phytochrome action (for reviews, see refs. 5 and 6). More recently, physiological and spectrophotometric Plant Materials. The mutant alleles used in the present evidence has accumulated to indicate that two types of phy- study werephyA-201 (frel-1) (15) andphyB-1 (hy3-Bo64) (22) tochrome are present in plants. Type I phytochrome is syn- in A. thaliana (L.) Heynh. The background ecotype of these thesized as Pr in darkness and decays rapidly in the light as a labile Pfr form. In contrast, type II phytochrome is stable in the Abbreviations: PhyA (or B), spectrally active phytochrome A (or B); Pr, phytochrome in the red light-absorbing form; Pfr, phytochrome in the far-red light-absorbing form; PHYA (or B), apoprotein of the wt The publication costs of this article were defrayed in part by page charge PhyA (or B); phyA (or B), mutant gene and allele of PHYA (or B); payment. This article must therefore be hereby marked "advertisement" inI VLFR, very low fluence response; wt, wild type. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 8129 Downloaded by guest on October 4, 2021 8130 Plant Biology: Shinomura et al. Proc. Natl. Acad. Sci. USA 93 (1996)

mutants and the wt was Landsberg erecta. Seeds were har- fectiveness (EA) was calculated as follows: EA = 1/FA X 100/TA, vested, stored and treated before the imbibition as described where FA is the calculated fluence required for induction of previously (21). germination with a normalized germination index of 50 from Germination Assay and Light Treatments. All seeds were the fluence response curves, and TA is the transmittance of the surface-sterilized and plated in lots of 50-100 individuals in seed coat (%) at each wavelength, as measured with mi- each plastic Petri plate containing aqueous agar medium (6 crospectrophotometer (MPM800, Zeiss). mg-ml-'), then exposed to far-red light (3mmolIm-2), inhib- In the case of photoreversible inhibition of germination, iting PhyB-dependent dark germination as described (21). seeds were exposed to saturating red light (700,umol-m-2) as They were kept in total darkness for appropriate period at 25°C described (21) and subsequently irradiated with monochro- and exposed to monochromatic light with threshold boxes of matic light using the spectrograph. Fluence-response curves Okazaki large spectrograph (23) as shown in Fig.LA (see also for photoreversible inhibition of germination were plotted and Fig. 3A). After the exposure to monochromatic light, seeds photon effectiveness for inhibition of germination with a were kept in darkness for 7 days, and germination percentages normalized germination index of 50 were calculated in the were measured in each population on a plate. same formula as for induction, and action spectra for 50% Determination of Action Spectra. Fluence-response curves inhibition were constructed. were determined at 60 different wavelengths from 300-800 nm Production of mAbs and Immunochemical Detection. mAbs at intervals of 5-20 nm. Each curve was fitted by the least- against recombinant Arabidopsis PhyA apoprotein (PHYA) squares method. To normalize experimental differences in and PhyB apoprotein (PHYB) were newly produced as de- germination percentage, germination index (GI>i) was calcu- scribed in Lopez et al. (24). Consequently, four mAbs, namely lated as follows: GIki = GAi/G667 X 100, where GAi is the mAA1 and mAA2 against the PHYA fragment (residues germination percentage at each wavelength at each photon 514-1122), mBA1 against the PHYB fragment (residues fluence, and G667 is the maximum value of the germination 1-598), and mBA2 against the PHYB fragment (residues percentage calculated from mean value of the germination 594-1172), were obtained. For detection of PHYA and PHYB percentages upon irradiation with the saturating fluence of 667 in seeds, extracts were prepared from about 2 x 103 seeds and nm light at 1-10 pumolM-2 and 1 mmolM-2 in PhyA- and analyzed immunochemically as described previously (15). PhyB-dependent germination, respectively. Action spectra for 50% induction of germination at each RESULTS wavelength were constructed from these curves. Photon ef- Incubation Time and Photon Fluence of Red Light. We A 0 1 3 48h determined the photon fluence of monochromatic red light 0 (667 nm) required for the photoinduction of germination in Ft 667 nm light seeds of wt and thephyA andphyB mutants that were incubated 0 s _ on aqueous agar plates for 3 or 48 hr in darkness (Fig. 1A). FR 667 nm light When wt seeds were kept in darkness for 3 hr and irradiated B with monochromatic light of 667 nm, germination was induced 60 by a fluence of 10,umolIm-2 and higher (Fig. 1B). When those were kept in darkness for 48 hr, however, they germinated as 40 80 under such significantly lower fluence 1-100 nmol-m-2 (Fig. iB). The latter response was not observed in the seeds 00 incubated in darkness for 15 hr or shorter, but became 20 0 0 detectable after 24 hr and reached a maximum at 48 hr at 25°C. o The germination percentages lowered after incubation period 0 longer than 72 hr (data not shown). In contrast, seeds of the phyA mutant never showed this high-sensitivity response after : 60 0 00 incubation for 48 hr or longer, although the less sensitive 0. response was observed irrespective of the dark-incubation time C 40 00. (Fig. 1C). Seeds of the phyB mutant showed only the highly sensitive response after prolonged imbibition (Fig. 1D). Taken (D 20 *0 together with the genetic background of the mutants, we conclude that PhyA is responsible for the high-sensitivity 0- -o0 response, while PhyB controls the higher fluence response in D 0 red light-induced germination. 60 0 To determine the levels of PHYA and PHYB in seeds during 0 the dark incubation, crude extracts were prepared and ana- co lyzed immunochemically. As shown in Fig. 2, the amount of 40 0 0 PHYA increased substantially during the incubation period in -~~~~~ 2 darkness (Fig. 2, Upper), whereas PHYB was detectable from 20 0 the beginning and increased slightly during the incubation (Lower). Similar results were obtained with different antibod- 0 -4 -2 -1 0 3 4 ies (data not shown). These results are consistent with the -3 1 jmol2 m-2 physiological observations that the PhyA-dependent high- log10[photon fluence], sensitivity response appears only after the prolonged dark incubation, whereas PhyB controls the higher fluence response FIG. 1. Effects of incubation time and photon fluence of red light from the beginning of the incubation period (Fig. 1). on seed germination. (A) Light regime of the experiment. Black bars, Incubation Time and Photon Fluence of Far-Red Light. We aqueous agar at + 1°C incubation period on plates in darkness 25 examined the effect of photon fluence of monochromatic white bars with arrows, pretreatments with far-red light (FR) and far-red light (726 nm) on photoinduction of germination in wt exposures to 667 nm light; 0 and 0, germination rates of seeds that were kept in darkness for 3 and 48 hr, respectively. (B-D) Fluence- and thephyA andphyB mutants that were kept on aqueous agar response relationships for the wt (B), the phyA mutant (C), and the plates for 3 or 48 hr in darkness (Fig. 3A). None of seeds phyB mutant (D) seeds. germinated when incubated in darkness for 3 hr and then Downloaded by guest on October 4, 2021 Plant Biology: Shinomura et al. Proc. Natl. Acad. Sci. USA 93 (1996) 8131 phyA phyB the red light was not reversed by the subsequently given far-red WT mutant mutant light (data not shown). (h) 3 48 3 48 3 48 Action Spectra for PhyA- and PhyB-Specific Germination. We confirmed that both the PhyA- and PhyB-dependent mAAl - germination obey the Bunsen-Roscoe law of reciprocity within a fluence range from 0.02-2 ,umol m-2 of red light (a time mBA2 -_ i range from 1-1,000 sec) and that from 10-500 ILmolm-2 (5-600 sec), respectively (data not shown). Thus, they are 1 2 3 4 5 6 primarily regulated by a single photochemical reaction and FIG. 2. Immunoblot detection of PHYA- and PHYB-apoproteins worth determining the action spectra. We then examined extracted from seeds of Arabidopsis. Seeds were homogenized after fluence of monochromatic lights in the spectral range of incubation for 3 or 48 hr in the dark. mAA1 and mBA2 are mAbs 300-800 nm in terms of photoinduction of PhyA-specific against Arabidopsis PHYA and PHYB, respectively. Each lane was germination in wt and the phyB mutant after incubation in loaded with 20 ,ug of total protein for detection of PHYA and 100 ,ug darkness for 30-55 hr. Representative fluence-response for PHYB. curves in the phyB mutant are shown in Fig. 4A. Most of the curves appear to be parallel within the accuracy of measure- exposed to far-red light within a fluence range tested (Fig. 3 ments. The results demonstrate that light in wide range of B-D). Wt and the phyB mutant seeds, however, germinated spectrum from near UV to far-red light is able to induce when kept in darkness for 48 hr and exposed to far-red light PhyA-dependent germination. with a fluence of 0.5 ,umol_m-2 and higher (Fig. 3 B and D). Action spectrum for PhyA-specific induction of seed ger- Seeds of the phyA mutant did not germinate even when kept mination in the phyB mutant was constructed (Fig. 5A) from in darkness for 48 hr or longer and exposed to far-red light the fluence-response curves (Fig. 4A). Essentially the same (Fig. 3C). These results indicate that sufficient fluence of spectrum was obtained with the wt seeds (data not shown), far-red light induce the PhyA-dependent germination but does suggesting that PhyB does not interfere with this PhyA action. not at all stimulate the PhyB-dependent germination. In contrast to the PhyA-dependent germination, we previ- We then examined whether PhyA-dependent germination ously reported that PhyB-specific photoinduction of germina- shows red/far-red reversibility. When seeds of the wt and the tion is red/far-red reversible (21). Thus, fluence-response phyB mutant were incubated in darkness for 48 hr and exposed curves for both the induction and inhibition of seed germina- to far-red light (726 nm) of 40 ,umol m-2 after an exposure to red light (667 nm) of 40 nmol'm-2, the germination induced by 100 A 0 1 3 48h A IA Ft 726 nm light A TAL -- L 50 FR 726 nm light 60 B A 0 40 A -3 A 20

0 C 60 0 co c4-0C 40 A 40 0 0 60

40 1 2 3 4 log 10 [photon fluence], ,u mol m2 20 A ~A FIG. 4. Effect of photon fluence of 300-800 nm light on PhyA- and 0 11111111|_J ,,,,I,iLl J . I. ' PhyB-dependent germination. Fluence-response curves were deter- -4 -3 -2 -1 0 1 2 3 4 mined at 60 different wavelengths, and representative curves are j mol m-2 presented. Wavelengths are shown numerically. (A) Fluence-response log10 [photon fluence], relationships of PhyA-dependent induction of germination for the phyB mutant seeds. Seeds were kept on aqueous agar plates for 30-55 FIG. 3. Effects of incubation time and photon fluence of far-red hr, then exposed to monochromatic lights. (B and C) Fluence-response light on seed germination. (A) Light regime of the experiment. A and relationships of PhyB-dependent induction (B) and photoreversible a, germination rates of seeds that were kept in darkness for 3 and 48 inhibition (C) of germination for the phyA mutant seeds. Seeds were hr, respectively. (B-D) Fluence-response relationships for the wt (B), kept on aqueous agar plates for 3-14 hr before the exposures to the phyA mutant (C) and the phyB mutant (D) seeds. monochromatic lights. Downloaded by guest on October 4, 2021 8132 Plant Biology: Shinomura et al. Proc. Natl. Acad. Sci. USA 93 (1996) 3 phototropic response of coleoptiles in oat (25, 26) and corn (27), elongation growth of etiolated seedlings in oat (28, 29) 2 and corn (30), chlorophyll accumulation in pea seedlings (31)

1 and seed germination in lettuce (32) and Arabidopsis (33). Fluence-response curves for these responses are often mul- 0 tiphasic (28-30, 32, 33), and the most sensitive component, -1 VLFR, is induced with 0.1-10.nmolFm-2 of red light (29, 34). cnC,) It also has been known that VLFR is induced with not only U) -2 an extremely low fluence of red light but also a relatively low U1) cD fluence of green and far-red light, and that no red/far-red 0 -3 C.) reversible effect is observed in VLFR (28, 29, 33). It thus has U1) -4 remained obscure whether phytochrome is acting as a photo- 3 receptor for the VLFR, though the action spectrum for the VLFR in the growth inhibition of oat mesocotyl showed 2 similar peaks with the known absorption maxima of phyto- chrome in Pr form (28). The fluences of red light required for 4- 1 the PhyA response, its photoirreversibility and photoinduction 0 C 0 by a relatively low fluence of green and far-red light observed -IJ in the present study are consistent with the VLFR described -1 above, providing evidence that PhyA acts as a photoreceptor -2 for the VLFR. The sensitivity of seeds to red and far-red light was examined -3 after pretreatments with dark incubation at various tempera- ture inArabidopsis (33) and other plant species (2, 32) before -4 the discovery of different phytochrome species. The present 300 400 500 600 700 800 study, however, clearly demonstrates that dark incubation Wavelength, nm increased the level of PHYA, which resulted in a dramatic increase in the sensitivity of the seeds. FIG. 5. (A) Action spectra for induction of PhyA-dependent Distinct Action Spectra for PhyA- and PhyB-Specific re- germination in the phyB mutant and (B) for induction and inhibition sponses. Several action spectra for phytochrome-mediated of PhyB-dependent germination in thephyA mutant. The experimental responses were reported in the literature since 1952 (2), but sensitivity for photon effectiveness is about 1 x 10-4. Points at this been identified as photorecep- value in the figure mean that the response was below the detection phytochrome species has never limit. tor(s) in any reported action spectra. Using thephyA andphyB mutants and changing incubation time in the dark, we sepa- tion were examined. Representative results in thephyA mutant rately determined for the first time the action spectra for are shown in Fig. 4 B and C. The results demonstrate that specific reactions of single phytochrome species. The action 540-690 nm light and 695-780 nm light with the fluence of spectrum for PhyA-dependent germination obtained in the wt 0.01-1 mmol.m-2 were effective on the photoinduction and the and the phyB mutant is quite different from that for PhyB- photoreversible inhibition, respectively. dependent induction in terms of the fluence requirement and We then determined the action spectra for the PhyB- effective wavelengths (Fig. 5 A and B). dependent induction and photoreversible inhibition in the When the vertical axis of PhyA action spectrum in Fig. SA phyA mutant (Fig. SB). Very similar action spectra were is re-plotted on linear scale, the resultant spectrum fits quite obtained in wt (data not shown) in terms of effective wave- well with the absorption spectrum of purified PhyA in the Pr lengths and fluence, suggesting that PhyA does not interfere form (35-37). This evidence indicates that the absorption of with PhyB action under the present experimental condition. In light by PhyA in the Pr form is the primary action of the contrast to the PhyA-specific germination, the PhyB- response. The photon fluence required for 50% induction of the PhyA reaction (Fig. 4A) was estimated to convert only dependent germination clearly showed a photoreversible reg- 0.01% of Pr to Pfr if calculated on the basis of the extinction ulation, and monochromatic light between 300-520 nm coefficient and quantum yield of purified oat PhyA (36, 37). showed neither an inductive nor an inhibitory effect on the Thus, quite a small amount of Pfr appears to be sufficient to PhyB-specific germination within the range of photon fluence trigger the PhyA-dependent germination ofArabidopsis seeds. examined. It is important to note that the action spectrum for PhyB- response (Fig. SB) is essentially the same as that for red/far- DISCUSSION red reversible regulation of seed germination in lettuce (18) andArabidopsis (19). In the past 4 decades, plant physiologists PhyA Acts as a Photoreceptor for VLFR. The previous study had mainly studied red/far-red reversible effects on growth (21) showed that seeds of the phyB mutant did not respond to and morphogenesis as the major effects of phytochrome (2), a brief irradiation with red light for induction of germination while photobiologists and biochemists had investigated molec- when the light was exposed in early period of incubation in the ular properties of phytochrome that was isolated and purified dark, and that the seeds became gradually photoperceptible from etiolated tissues of diverse monocots and dicots (38). We when incubated for a day or two in the dark. However, we were now know that the former physiological effects mostly result unable to find which phytochrome species was involved in this from PhyB or other type II phytochrome and the latter from photoresponse of the phyB mutant though the induction by PhyA (39). This would be the reason why physiological effects PhyA was strongly suggested (21). The present study has of red and far-red light were often too confusing and too showed that PhyA is the photoreceptor for this response (Fig. complicated to understand on the basis of spectrophotochemi- 1). Furthermore, we found that the PhyA response is 104 times cal and biochemical properties of PhyA. more photosensitive than the previously reported PhyB re- The qualitative difference in the two action spectra is most sponse (Fig. 1). simply be explained by difference in the Pfr requirement It has been well known that very small amount of red light between these two responses. Our results indicate that an energy (0.1-10 nmol.m-2) can induce physiological effects on extremely low ratio of Pfr to total phytochrome (estimated to Downloaded by guest on October 4, 2021 Plant Biology: Shinomura et al. Proc. Natl. Acad. Sci. USA 93 (1996) 8133 be 10-4) is sufficient to cause the PhyA-mediated response. 5. Hillman, W. S. (1967) Annu. Rev. Plant Physiol. 18, 301-324. Thus, any wavelength of light would generate enough Pfr to 6. Furuya, M. (1989) Adv. Biophys. 25, 133-167. promote the response. In contrast, the PhyB response appears 7. Abe, H., Takio, K., Titani, K. & Furuya, M. (1989) Plant Cell to require much higher ratio of Pfr. In this case, light at Physiol. 30, 1089-1097. particular wavelengths such as 700 nm would never generate 8. Sharrock, R. A. & Quail, P. H. (1989) Genes Dev. 3, 1745-1757. 9. Clack, T., Mathews, S. & Sharrock, R. A. (1994) Plant Mol. Biol. enough Pfr for the response because the ratio of Pfr reached 25, 413-427. with such wavelengths at the photoequilibrium is relatively low. 10. Dehesh, K., Franci, C., Parks, B. M., Seeley, K. A., Short, T. W., This apparent difference is attributable partly to the difference Tepperman, J. M. & Quail, P. H. (1993) Plant Cell 5, 1081-1088. in the higher level of PHYA in dark-incubated seeds (Fig. 2). 11. Reed, J. W., Nagatani, A., Elich, T. D., Fagan, M. & Chory, J. However, it is unlikely that the level of PHYA is four orders (1994) Plant Physiol. 104, 1139-1149. of magnitude higher than that of PHYB. Thus, there should be 12. Whitelam, G. C., Johnson, E., Peng, J., Carol, P., Anderson, an additional mechanism to reduce the requirement of Pfr Of M. L., Cowl, J. S. & Harberd, N. P. (1993) Plant Cell 5, 757-768. PhyA for the response. 13. Reed, J. W., Nagpal, P., Poole, D. S., Furuya, M. & Chory, J. Physiological Significance of the PhyA Response. The action (1993) Plant Cell 5, 147-157. spectrum for PhyA response in the present work (Fig. SA) 14. Somers, D. E., Sharrock, R. A., Tepperman, J. M. & Quail, P. H. provides a novel evidence that PhyA can capture lights of (1991) Plant Cell 3, 1263-1274. from 300 to 780 nm to induce the seed germina- 15. Nagatani, A., Reed, J. W. & Chory, J. (1993) Plant Physiol. 102, wavelengths 269-277. tion. Hence, it is quite likely that some of the reported effects 16. Parks, B. M., Quail, P. H. & Hangarter, R. P. (1996) Plant of blue and UV light documented in the literature (40) might Physiol. 110, 155-162. have resulted from the action of PhyA rather than putative 17. Flint, L. H. & McAlister, E. D. (1935) Smithson. Misc. Collect. 94, blue/UV photoreceptors, and it will be worthwhile to re- 1-11. examine the previously reported blue/UV effects using phy- 18. Borthwick, H. A., Hendricks, S. B., Parker, M. W., Toole, E. H. tochrome- and blue/UV-A photoreceptor-deficient mutants & Toole, V. K. (1952) Proc. Natl. Acad. Sci. USA 38, 662-666. (41). 19. Shropshire, W., Jr., Klein, W. H. & Elstad, V. B. (1961) Plant Cell It is now evident that plants sense the light environment in Physiol. 2, 63-69. a wide spectral range with exquisite sensitivity using different 20. Cone, J. W. & Kendrick, R. E. (1985) Planta 163, 43-54. phytochromes. Moreover, PhyA and PhyB modulate the tim- 21. Shinomura, T., Nagatani, A., Chory, J. & Furuya, M. (1994) Plant ing of dormancy-break in seeds by entirely different way. PhyA Physiol. 104, 363-371. photoirreversibly triggers the photoinduction of seed germi- 22. Koornneef, M., Rolff, E. & Spruit, C. J. P. (1980) Z. Pflanzen- physiol. 100, 147-160. nation upon irradiation at extremely low fluence with light of 23. Watanabe, M., Furuya, M., Miyoshi, Y., Inoue, Y., Iwahashi, I. the UV-A, visible and far-red range. In contrast, PhyB medi- & Matsumoto, K. (1982) Photochem. Photobiol. 36, 491-498. ates the well-characterized photoreversible reaction, respond- 24. L6pez-Juez, E., Nagatani, A., Tomizawa, K., Deak, M., Kern, R., ing to red and far-red light at 104-fold higher fluences than Kendrick, R. E. & Furuya, M. (1992) Plant Cell 4, 241-251. those to which PhyA responds. Plants survive under ground as 25. Blaauw-Jansen, G. (1959) Acta Bot. Neerl. 8, 1-39. dormant seeds for long periods, and the timing of seed 26. Zimmerman, B. K. & Briggs, W. R. (1963) Plant Physiol. 38, germination is crucial for optimizing growth and reproduction. 248-253. It therefore seems reasonable for plants to possess two quite 27. Chon, H. P. & Briggs, W. R. (1966) Plant Physiol. 41, 1715-1724. different physiological systems of light sensing with a broader 28. Blaauw, 0. H., Blaauw-Jansen, G. & van Leeuwen, W. J. (1968) range of both wavelength and photon fluence. This redun- Planta 82, 87-104. dancy enhances a plant's chance of survival. 29. Mandoli, D. F. & Briggs, W. R. (1981) Plant Physiol. 67, 733-739. 30. Vanderhoef, L. N., Quail, P. H. & Briggs, W. R. (1979) Plant Note. After submission of this manuscript, we have become aware Physiol. 63, 1062-1067. of a publication by Botto et al. (42), in which the authors examined 31. Raven, C. W. & Shropshire, W., Jr. (1975) Photochem. Photobiol. responses of the phyA and phyB mutants of Arabidopsis to red and 21, 423-429. far-red light pulses for induction of seed germination and presented 32. VanDerWoude, W. J. (1985) Photochem. Photobiol. 42,655-661. conclusions similar to ours. Our results in a preliminary form has been 33. Cone, J. W., Jaspers, P. A. P. M. & Kendrick, R. E. (1985) Plant reported in ref. 43. Cell Environ. 8, 605-612. 34. Smith, H., Whitelam, G. C. & McCormac, A. C. (1991) in This work is dedicated to Prof. Horst Senger on the occasion of his Phytochrome Properties and BiologicalAction, eds. Thomas, B. & 65th birthday and to Prof. Pill-Soon Song on the occasion of his 60th Johnson, C. B. (Springer, Berlin), pp. 217-236. birthday. We thank Prof. N. Murata for hosting us at the National 35. Schafer, E., Lassing, T.-U. & Schopfer, P. (1982) Planta 154, Institute for Basic Biology, Prof. J. Chory and Dr. K Sakamoto for 231-240. providing us cDNA clones of PHYA and PHYB, R. Katayanagi for 36. Kelly, J. M. & Lagarias, J. C. -(1985) Biochemistry 24, 6003-6010. assistance with plant cultivation, and F. Tsunekawa for measuring the 37. Lagarias, J. C., Kelly, J. M., Cyr, K. L. & Smith, W. 0. (1987) transmittance of the seed coat. The work was supported in part by Photochem. Photobiol. 46, 5-13. separate grants from International Human Frontier Science Program 38. Furuya, M. & Song, P.-S. (1994) in Photomorphogenesis in Plants, (M.F. and A.N.), and carried out under the National Institute for Basic eds. Kendrick, R. E. & Kronenberg, G. H. M. (Kluwer, Dor- Biology Cooperative Research Programs for the Okazaki Large drecht, The Netherlands), pp. 105-140. Spectrograph 93-530 and 94-519. 39. Furuya, M. (1993) Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 617-645. 1. Kendrick, R. E. & Kronenberg, G. H. M., eds. (1994) Photomor- 40. Senger, H. & Schmidt, W. (1994) in Photomorphogenesis in phogenesis in Plants (Kluwer, Dordrecht, The Netherlands), 2nd Plants, eds. Kendrick, R. 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