Action spectraforphytochromeA-and B-specific ... · 514-1122), mBA1 against the PHYB fragment...

5
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 Arabidopsis thaliana of wild type (wt), and phytochrome A (PhyA)- and B (PhyB)-mutants in terms of incubation time and environmental light effects. Seed germination of the wt and PhyA-null mutant (phyA) was photoreversibly regulated by red and far-red lights of 10-1,000 ,umol m-2 when incu- bated in darkness for 1-14 hr, but no germination occurred in PhyB-null mutant (phyB). When wt seeds and the phyB mutant seeds were incubated in darkness for 48 hr, they synthesized PhyA during dark incubation and germinated upon exposure to red light of 1-100 nmol m-2 and far-red light of 0.5-10 ,umol m-2, whereas the phyA mutant showed no such response. The results indicate that the seed germination is regulated by PhyA and PhyB but not by other phytochromes, and the effects of PhyA and PhyB are separable in this assay. We determined action spectra separately for PhyA- and PhyB-specific induc- tion of seed germination at Okazaki large spectrograph. Action spectra for the PhyA response show that monochro- matic 300-780 nm lights of very low fluence induced the germination, and this induction was not photoreversible in the range examined. Action spectra for the PhyB response show that germination was photoreversibly regulated by alternate irradiations with light of 0.01-1 mmol m-2 at wavelengths of 540-690 nm and 695-780 nm. The present work clearly demonstrated that PhyA photoirreversibly triggers the ger- mination upon irradiations with ultraviolet, visible and far- red light of very low fluence, while PhyB controls the pho- toreversible effects of low fluence. Diversification within families of sensory receptors allows discrimination of distinct but related stimuli. Plants have evolved diverse photoreceptor systems for detection of light intensity, quality, and duration to adjust their life in fluctuating environmental conditions (1). The best characterized photo- transducer in plants is phytochrome (2, 3), which exhibits photoreversible interconversion between two spectrally and biochemically distinct forms, a red light-absorbing form, Pr, and a far-red light-absorbing form, Pfr (4). The earliest and simplest hypothesis of phytochrome action was that responses are triggered by a red light pulse, converting biologically inactive Pr to active Pfr, which can be reversed by a subsequent brief irradiation with far-red light, converting Pfr back to Pr (4). Spectrophotometrically detectable amounts or states of phy- tochrome in vivo, however, are not consistent with this simple interpretation of phytochrome action (for reviews, see refs. 5 and 6). More recently, physiological and spectrophotometric evidence has accumulated to indicate that two types of phy- tochrome are present in plants. Type I phytochrome is syn- 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 Pfr form and is present at relatively constant levels both in the light and in darkness (6). This heterogeneity of phytochromes was explained by the amino acid sequencing of apoproteins with type I and type II phytochromes in pea (7) and the cloning of five phytochrome genes (PHYA to PHYE) in Arabidopsis thaliana (8, 9). Phytochrome A (PhyA) and phytochrome B (PhyB) have been indicated, using PhyA-null mutants (phyA) (10-12) and PhyB-null mutants (phyB) (13), to be the most important members of the family for regulation of hypocotyl elongation. Recent analysis of these mutants have suggested very limited significance of PhyA under continuous white light, regardless of the fact that PhyA is the predominant molecular species in dark-grown tissues (14) and indispensable for the response of etiolated seedlings to continuous far-red light (10, 12, 15) and for the red light-enhanced phototropism (16). Concerning photoinduction of seed germination, in 1935, Flint and MacAlister (17) found that continuous irradiation with light of 580-700 nm was effective in inducing germination of lettuce seeds, but that of 700-800 nm, as well as 500 nm, was inhibitory. In 1952, Borthwick et al. (18) examined the effect of brief exposures to red and far-red light in lettuce seeds, and discovered the red/far-red photoreversible response. They measured the action spectra for promotion and inhibition of germination, finding the maximum sensitivity for promotion in the region of 640-670 nm and that for inhibition in 720-750 nm. Very similar action spectra for photoreversible regulation of seed germination were determined in Arabidopsis thaliana of the wild-type (wt) (19) and long-hypocotyl mutants (20). However, it has been an open question which phytochrome species regulates the photoinduction of seed germination. We recently reported (21), using the Arabidopsis phyA and phyB mutants, that red/far-red reversible induction of seed germination is principally regulated by PhyB, but not by PhyA, and that the phyB mutant seeds became sensitive to red light after dark incubation for 48 hr. The purpose of the present study is to define different physiological roles of PhyA and PhyB, if any, in terms of incubation time in darkness and characteristics of light sensitivity in phyA and phyB mutants. We report a novel action spectra for PhyA-specific photoin- duction of seed germination, demonstrating that PhyA is the photoreceptor for very low fluence response (VLFR). MATERIALS AND METHODS Plant Materials. The mutant alleles used in the present study were phyA-201 (frel-1) (15) and phyB-1 (hy3-Bo64) (22) in A. thaliana (L.) Heynh. The background ecotype of these 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 PhyA (or B); phyA (or B), mutant gene and allele of PHYA (or B); I VLFR, very low fluence response; wt, wild type. §To whom reprint requests should be addressed. 8129 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Downloaded by guest on July 28, 2020

Transcript of Action spectraforphytochromeA-and B-specific ... · 514-1122), mBA1 against the PHYB fragment...

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

Action spectra for phytochrome A- and B-specific photoinductionof 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, Tokyo113, 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 inArabidopsis thaliana of wild type (wt), and phytochrome A(PhyA)- and B (PhyB)-mutants in terms of incubation timeand environmental light effects. Seed germination of the wtand PhyA-null mutant (phyA) was photoreversibly regulatedby red and far-red lights of 10-1,000 ,umol m-2 when incu-bated in darkness for 1-14 hr, but no germination occurred inPhyB-null mutant (phyB). When wt seeds and thephyB mutantseeds were incubated in darkness for 48 hr, they synthesizedPhyA during dark incubation and germinated upon exposureto red light of 1-100 nmol m-2 and far-red light of 0.5-10 ,umolm-2, whereas the phyA mutant showed no such response. Theresults indicate that the seed germination is regulated by PhyAand PhyB but not by other phytochromes, and the effects ofPhyA and PhyB are separable in this assay. We determinedaction spectra separately for PhyA- and PhyB-specific induc-tion of seed germination at Okazaki large spectrograph.Action spectra for the PhyA response show that monochro-matic 300-780 nm lights of very low fluence induced thegermination, and this induction was not photoreversible in therange examined. Action spectra for the PhyB response showthat germination was photoreversibly regulated by alternateirradiations with light of 0.01-1 mmol m-2 at wavelengths of540-690 nm and 695-780 nm. The present work clearlydemonstrated that PhyA photoirreversibly triggers the ger-mination upon irradiations with ultraviolet, visible and far-red light of very low fluence, while PhyB controls the pho-toreversible effects of low fluence.

Diversification within families of sensory receptors allowsdiscrimination of distinct but related stimuli. Plants haveevolved diverse photoreceptor systems for detection of lightintensity, quality, and duration to adjust their life in fluctuatingenvironmental conditions (1). The best characterized photo-transducer in plants is phytochrome (2, 3), which exhibitsphotoreversible interconversion between two spectrally andbiochemically distinct forms, a red light-absorbing form, Pr,and a far-red light-absorbing form, Pfr (4). The earliest andsimplest hypothesis of phytochrome action was that responsesare triggered by a red light pulse, converting biologicallyinactive Pr to active Pfr, which can be reversed by a subsequentbrief irradiation with far-red light, converting Pfr back to Pr (4).Spectrophotometrically detectable amounts or states of phy-tochrome in vivo, however, are not consistent with this simpleinterpretation of phytochrome action (for reviews, see refs. 5and 6). More recently, physiological and spectrophotometricevidence has accumulated to indicate that two types of phy-tochrome are present in plants. Type I phytochrome is syn-thesized as Pr in darkness and decays rapidly in the light as alabile Pfr form. In contrast, type II phytochrome is stable in the

Pfr form and is present at relatively constant levels both in thelight and in darkness (6). This heterogeneity of phytochromeswas explained by the amino acid sequencing of apoproteinswith type I and type II phytochromes in pea (7) and the cloningof five phytochrome genes (PHYA to PHYE) in Arabidopsisthaliana (8, 9). Phytochrome A (PhyA) and phytochrome B(PhyB) have been indicated, using PhyA-null mutants (phyA)(10-12) and PhyB-null mutants (phyB) (13), to be the mostimportant members of the family for regulation of hypocotylelongation. Recent analysis of these mutants have suggestedvery limited significance of PhyA under continuous white light,regardless of the fact that PhyA is the predominant molecularspecies in dark-grown tissues (14) and indispensable for theresponse of etiolated seedlings to continuous far-red light (10,12, 15) and for the red light-enhanced phototropism (16).Concerning photoinduction of seed germination, in 1935,

Flint and MacAlister (17) found that continuous irradiationwith light of 580-700 nm was effective in inducing germinationof lettuce seeds, but that of 700-800 nm, as well as 500 nm, wasinhibitory. In 1952, Borthwick et al. (18) examined the effectof brief exposures to red and far-red light in lettuce seeds, anddiscovered the red/far-red photoreversible response. Theymeasured the action spectra for promotion and inhibition ofgermination, finding the maximum sensitivity for promotion inthe region of 640-670 nm and that for inhibition in 720-750nm. Very similar action spectra for photoreversible regulationof seed germination were determined in Arabidopsis thalianaof the wild-type (wt) (19) and long-hypocotyl mutants (20).However, it has been an open question which phytochromespecies regulates the photoinduction of seed germination.We recently reported (21), using the Arabidopsis phyA and

phyB mutants, that red/far-red reversible induction of seedgermination is principally regulated by PhyB, but not by PhyA,and that the phyB mutant seeds became sensitive to red lightafter dark incubation for 48 hr. The purpose of the presentstudy is to define different physiological roles of PhyA andPhyB, if any, in terms of incubation time in darkness andcharacteristics of light sensitivity in phyA and phyB mutants.We report a novel action spectra for PhyA-specific photoin-duction of seed germination, demonstrating that PhyA is thephotoreceptor for very low fluence response (VLFR).

MATERIALS AND METHODSPlant Materials. The mutant alleles used in the present

study werephyA-201 (frel-1) (15) andphyB-1 (hy3-Bo64) (22)in A. thaliana (L.) Heynh. The background ecotype of these

Abbreviations: PhyA (or B), spectrally active phytochrome A (or B);Pr, phytochrome in the red light-absorbing form; Pfr, phytochrome inthe far-red light-absorbing form; PHYA (or B), apoprotein of the wtPhyA (or B); phyA (or B), mutant gene and allele of PHYA (or B);

I VLFR, very low fluence response; wt, wild type.§To whom reprint requests should be addressed.

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mutants and the wt was Landsberg erecta. Seeds were har-vested, stored and treated before the imbibition as describedpreviously (21).Germination Assay and Light Treatments. All seeds were

surface-sterilized and plated in lots of 50-100 individuals ineach plastic Petri plate containing aqueous agar medium (6mg-ml-'), then exposed to far-red light (3mmolIm-2), inhib-iting PhyB-dependent dark germination as described (21).They were kept in total darkness for appropriate period at 25°Cand exposed to monochromatic light with threshold boxes ofOkazaki large spectrograph (23) as shown in Fig.LA (see alsoFig. 3A). After the exposure to monochromatic light, seedswere kept in darkness for 7 days, and germination percentageswere measured in each population on a plate.

Determination of Action Spectra. Fluence-response curves

were determined at 60 different wavelengths from 300-800 nmat intervals of 5-20 nm. Each curve was fitted by the least-squares method. To normalize experimental differences ingermination percentage, germination index (GI>i) was calcu-lated as follows: GIki = GAi/G667 X 100, where GAi is thegermination percentage at each wavelength at each photonfluence, and G667 is the maximum value of the germinationpercentage calculated from mean value of the germinationpercentages upon irradiation with the saturating fluence of 667nm light at 1-10 pumolM-2 and 1 mmolM-2 in PhyA- andPhyB-dependent germination, respectively.

Action spectra for 50% induction of germination at eachwavelength were constructed from these curves. Photon ef-

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FIG. 1. Effects of incubation time and photon fluence of red lighton seed germination. (A) Light regime of the experiment. Black bars,incubation period on aqueous agar plates in darkness at 25 + 1°Cwhite bars with arrows, pretreatments with far-red light (FR) andexposures to 667 nm light; 0 and 0, germination rates of seeds thatwere kept in darkness for 3 and 48 hr, respectively. (B-D) Fluence-response relationships for the wt (B), the phyA mutant (C), and thephyB mutant (D) seeds.

fectiveness (EA) was calculated as follows: EA = 1/FA X 100/TA,where FA is the calculated fluence required for induction ofgermination with a normalized germination index of 50 fromthe fluence response curves, and TA is the transmittance of theseed coat (%) at each wavelength, as measured with mi-crospectrophotometer (MPM800, Zeiss).

In the case of photoreversible inhibition of germination,seeds were exposed to saturating red light (700,umol-m-2) as

described (21) and subsequently irradiated with monochro-matic light using the spectrograph. Fluence-response curves

for photoreversible inhibition of germination were plotted andphoton effectiveness for inhibition of germination with a

normalized germination index of 50 were calculated in thesame formula as for induction, and action spectra for 50%inhibition were constructed.

Production of mAbs and Immunochemical Detection. mAbsagainst recombinant Arabidopsis PhyA apoprotein (PHYA)and PhyB apoprotein (PHYB) were newly produced as de-scribed in Lopez et al. (24). Consequently, four mAbs, namelymAA1 and mAA2 against the PHYA fragment (residues514-1122), mBA1 against the PHYB fragment (residues1-598), and mBA2 against the PHYB fragment (residues594-1172), were obtained. For detection of PHYA and PHYBin seeds, extracts were prepared from about 2 x 103 seeds andanalyzed immunochemically as described previously (15).

RESULTS

Incubation Time and Photon Fluence of Red Light. Wedetermined the photon fluence of monochromatic red light(667 nm) required for the photoinduction of germination inseeds of wt and thephyA andphyB mutants that were incubatedon aqueous agar plates for 3 or 48 hr in darkness (Fig. 1A).When wt seeds were kept in darkness for 3 hr and irradiatedwith monochromatic light of 667 nm, germination was inducedby a fluence of 10,umolIm-2 and higher (Fig. 1B). When thosewere kept in darkness for 48 hr, however, they germinatedunder such significantly lower fluence as 1-100 nmol-m-2 (Fig.iB). The latter response was not observed in the seedsincubated in darkness for 15 hr or shorter, but becamedetectable after 24 hr and reached a maximum at 48 hr at 25°C.The germination percentages lowered after incubation periodlonger than 72 hr (data not shown). In contrast, seeds of thephyA mutant never showed this high-sensitivity response afterincubation for 48 hr or longer, although the less sensitiveresponse was observed irrespective of the dark-incubation time(Fig. 1C). Seeds of the phyB mutant showed only the highlysensitive response after prolonged imbibition (Fig. 1D). Takentogether with the genetic background of the mutants, we

conclude that PhyA is responsible for the high-sensitivityresponse, while PhyB controls the higher fluence response inred light-induced germination.To determine the levels of PHYA and PHYB in seeds during

the dark incubation, crude extracts were prepared and ana-

lyzed immunochemically. As shown in Fig. 2, the amount ofPHYA increased substantially during the incubation period indarkness (Fig. 2, Upper), whereas PHYB was detectable fromthe beginning and increased slightly during the incubation(Lower). Similar results were obtained with different antibod-ies (data not shown). These results are consistent with thephysiological observations that the PhyA-dependent high-sensitivity response appears only after the prolonged darkincubation, whereas PhyB controls the higher fluence response

from the beginning of the incubation period (Fig. 1).Incubation Time and Photon Fluence of Far-Red Light. We

examined the effect of photon fluence of monochromaticfar-red light (726 nm) on photoinduction of germination in wtand thephyA andphyB mutants that were kept on aqueous agar

plates for 3 or 48 hr in darkness (Fig. 3A). None of seedsgerminated when incubated in darkness for 3 hr and then

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FIG. 2. Immunoblot detection of PHYA- and PHYB-apoproteinsextracted from seeds of Arabidopsis. Seeds were homogenized afterincubation for 3 or 48 hr in the dark. mAA1 and mBA2 are mAbsagainst Arabidopsis PHYA and PHYB, respectively. Each lane wasloaded with 20 ,ug of total protein for detection of PHYA and 100 ,ugfor PHYB.

exposed to far-red light within a fluence range tested (Fig. 3B-D). Wt and the phyB mutant seeds, however, germinatedwhen kept in darkness for 48 hr and exposed to far-red lightwith a fluence of 0.5 ,umol_m-2 and higher (Fig. 3 B and D).Seeds of the phyA mutant did not germinate even when keptin darkness for 48 hr or longer and exposed to far-red light(Fig. 3C). These results indicate that sufficient fluence offar-red light induce the PhyA-dependent germination but doesnot at all stimulate the PhyB-dependent germination.We then examined whether PhyA-dependent germination

shows red/far-red reversibility. When seeds of the wt and thephyB mutant were incubated in darkness for 48 hr and exposedto far-red light (726 nm) of 40 ,umol m-2 after an exposure tored light (667 nm) of 40 nmol'm-2, the germination induced by

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FIG. 3. Effects of incubation time and photon fluence of far-redlight on seed germination. (A) Light regime of the experiment. A anda, germination rates of seeds that were kept in darkness for 3 and 48hr, respectively. (B-D) Fluence-response relationships for the wt (B),the phyA mutant (C) and the phyB mutant (D) seeds.

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the red light was not reversed by the subsequently given far-redlight (data not shown).

Action Spectra for PhyA- and PhyB-Specific Germination.We confirmed that both the PhyA- and PhyB-dependentgermination obey the Bunsen-Roscoe law of reciprocity withina fluence range from 0.02-2 ,umol m-2 of red light (a timerange from 1-1,000 sec) and that from 10-500 ILmolm-2(5-600 sec), respectively (data not shown). Thus, they areprimarily regulated by a single photochemical reaction andworth determining the action spectra. We then examinedfluence of monochromatic lights in the spectral range of300-800 nm in terms of photoinduction of PhyA-specificgermination in wt and the phyB mutant after incubation indarkness for 30-55 hr. Representative fluence-responsecurves in the phyB mutant are shown in Fig. 4A. Most of thecurves appear to be parallel within the accuracy of measure-ments. The results demonstrate that light in wide range ofspectrum from near UV to far-red light is able to inducePhyA-dependent germination.

Action spectrum for PhyA-specific induction of seed ger-mination in the phyB mutant was constructed (Fig. 5A) fromthe fluence-response curves (Fig. 4A). Essentially the samespectrum was obtained with the wt seeds (data not shown),suggesting that PhyB does not interfere with this PhyA action.

In contrast to the PhyA-dependent germination, we previ-ously reported that PhyB-specific photoinduction of germina-tion is red/far-red reversible (21). Thus, fluence-responsecurves for both the induction and inhibition of seed germina-

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FIG. 4. Effect of photon fluence of 300-800 nm light on PhyA- andPhyB-dependent germination. Fluence-response curves were deter-mined at 60 different wavelengths, and representative curves arepresented. Wavelengths are shown numerically. (A) Fluence-responserelationships of PhyA-dependent induction of germination for thephyB mutant seeds. Seeds were kept on aqueous agar plates for 30-55hr, then exposed to monochromatic lights. (B and C) Fluence-responserelationships of PhyB-dependent induction (B) and photoreversibleinhibition (C) of germination for the phyA mutant seeds. Seeds werekept on aqueous agar plates for 3-14 hr before the exposures tomonochromatic lights.

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FIG. 5. (A) Action spectra for induction of PhyA-dependentgermination in the phyB mutant and (B) for induction and inhibitionof PhyB-dependent germination in thephyA mutant. The experimentalsensitivity for photon effectiveness is about 1 x 10-4. Points at thisvalue in the figure mean that the response was below the detectionlimit.

tion were examined. Representative results in thephyA mutantare shown in Fig. 4 B and C. The results demonstrate that540-690 nm light and 695-780 nm light with the fluence of0.01-1 mmol.m-2 were effective on the photoinduction and thephotoreversible inhibition, respectively.We then determined the action spectra for the PhyB-

dependent induction and photoreversible inhibition in thephyA mutant (Fig. SB). Very similar action spectra were

obtained in wt (data not shown) in terms of effective wave-

lengths and fluence, suggesting that PhyA does not interferewith PhyB action under the present experimental condition. Incontrast to the PhyA-specific germination, the PhyB-dependent germination clearly showed a photoreversible reg-ulation, and monochromatic light between 300-520 nm

showed neither an inductive nor an inhibitory effect on thePhyB-specific germination within the range of photon fluenceexamined.

DISCUSSIONPhyA Acts as a Photoreceptor for VLFR. The previous study

(21) showed that seeds of the phyB mutant did not respond toa brief irradiation with red light for induction of germinationwhen the light was exposed in early period of incubation in thedark, and that the seeds became gradually photoperceptiblewhen incubated for a day or two in the dark. However, we wereunable to find which phytochrome species was involved in thisphotoresponse of the phyB mutant though the induction byPhyA was strongly suggested (21). The present study hasshowed that PhyA is the photoreceptor for this response (Fig.1). Furthermore, we found that the PhyA response is 104 timesmore photosensitive than the previously reported PhyB re-

sponse (Fig. 1).It has been well known that very small amount of red light

energy (0.1-10 nmol.m-2) can induce physiological effects on

phototropic response of coleoptiles in oat (25, 26) and corn(27), elongation growth of etiolated seedlings in oat (28, 29)and corn (30), chlorophyll accumulation in pea seedlings (31)and seed germination in lettuce (32) and Arabidopsis (33).Fluence-response curves for these responses are often mul-tiphasic (28-30, 32, 33), and the most sensitive component,VLFR, is induced with 0.1-10.nmolFm-2 of red light (29, 34).

It also has been known that VLFR is induced with not onlyan extremely low fluence of red light but also a relatively lowfluence of green and far-red light, and that no red/far-redreversible effect is observed in VLFR (28, 29, 33). It thus hasremained obscure whether phytochrome is acting as a photo-receptor for the VLFR, though the action spectrum for theVLFR in the growth inhibition of oat mesocotyl showedsimilar peaks with the known absorption maxima of phyto-chrome in Pr form (28). The fluences of red light required forthe PhyA response, its photoirreversibility and photoinductionby a relatively low fluence of green and far-red light observedin the present study are consistent with the VLFR describedabove, providing evidence that PhyA acts as a photoreceptorfor the VLFR.The sensitivity of seeds to red and far-red light was examined

after pretreatments with dark incubation at various tempera-ture in Arabidopsis (33) and other plant species (2, 32) beforethe discovery of different phytochrome species. The presentstudy, however, clearly demonstrates that dark incubationincreased the level of PHYA, which resulted in a dramaticincrease in the sensitivity of the seeds.

Distinct Action Spectra for PhyA- and PhyB-Specific re-sponses. Several action spectra for phytochrome-mediatedresponses were reported in the literature since 1952 (2), butphytochrome species has never been identified as photorecep-tor(s) in any reported action spectra. Using thephyA andphyBmutants and changing incubation time in the dark, we sepa-rately determined for the first time the action spectra forspecific reactions of single phytochrome species. The actionspectrum for PhyA-dependent germination obtained in the wtand the phyB mutant is quite different from that for PhyB-dependent induction in terms of the fluence requirement andeffective wavelengths (Fig. 5 A and B).When the vertical axis of PhyA action spectrum in Fig. SA

is re-plotted on linear scale, the resultant spectrum fits quitewell with the absorption spectrum of purified PhyA in the Prform (35-37). This evidence indicates that the absorption oflight by PhyA in the Pr form is the primary action of theresponse. The photon fluence required for 50% induction ofthe PhyA reaction (Fig. 4A) was estimated to convert only0.01% of Pr to Pfr if calculated on the basis of the extinctioncoefficient and quantum yield of purified oat PhyA (36, 37).Thus, quite a small amount of Pfr appears to be sufficient totrigger the PhyA-dependent germination ofArabidopsis seeds.

It is important to note that the action spectrum for PhyB-response (Fig. SB) is essentially the same as that for red/far-red reversible regulation of seed germination in lettuce (18)andArabidopsis (19). In the past 4 decades, plant physiologistshad mainly studied red/far-red reversible effects on growthand morphogenesis as the major effects of phytochrome (2),while photobiologists and biochemists had investigated molec-ular properties of phytochrome that was isolated and purifiedfrom etiolated tissues of diverse monocots and dicots (38). Wenow know that the former physiological effects mostly resultfrom PhyB or other type II phytochrome and the latter fromPhyA (39). This would be the reason why physiological effectsof red and far-red light were often too confusing and toocomplicated to understand on the basis of spectrophotochemi-cal and biochemical properties of PhyA.The qualitative difference in the two action spectra is most

simply be explained by difference in the Pfr requirementbetween these two responses. Our results indicate that anextremely low ratio of Pfr to total phytochrome (estimated to

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be 10-4) is sufficient to cause the PhyA-mediated response.Thus, any wavelength of light would generate enough Pfr topromote the response. In contrast, the PhyB response appearsto require much higher ratio of Pfr. In this case, light atparticular wavelengths such as 700 nm would never generateenough Pfr for the response because the ratio of Pfr reachedwith such wavelengths at the photoequilibrium is relatively low.This apparent difference is attributable partly to the differencein the higher level of PHYA in dark-incubated seeds (Fig. 2).However, it is unlikely that the level of PHYA is four ordersof magnitude higher than that of PHYB. Thus, there should bean additional mechanism to reduce the requirement of Pfr OfPhyA for the response.

Physiological Significance of the PhyA Response. The actionspectrum for PhyA response in the present work (Fig. SA)provides a novel evidence that PhyA can capture lights ofwavelengths from 300 to 780 nm to induce the seed germina-tion. Hence, it is quite likely that some of the reported effectsof blue and UV light documented in the literature (40) mighthave resulted from the action of PhyA rather than putativeblue/UV photoreceptors, and it will be worthwhile to re-examine the previously reported blue/UV effects using phy-tochrome- and blue/UV-A photoreceptor-deficient mutants(41).

It is now evident that plants sense the light environment ina wide spectral range with exquisite sensitivity using differentphytochromes. Moreover, PhyA and PhyB modulate the tim-ing of dormancy-break in seeds by entirely different way. PhyAphotoirreversibly triggers the photoinduction of seed germi-nation upon irradiation at extremely low fluence with light ofthe UV-A, visible and far-red range. In contrast, PhyB medi-ates the well-characterized photoreversible reaction, respond-ing to red and far-red light at 104-fold higher fluences thanthose to which PhyA responds. Plants survive under ground asdormant seeds for long periods, and the timing of seedgermination is crucial for optimizing growth and reproduction.It therefore seems reasonable for plants to possess two quitedifferent physiological systems of light sensing with a broaderrange of both wavelength and photon fluence. This redun-dancy enhances a plant's chance of survival.

Note. After submission of this manuscript, we have become awareof a publication by Botto et al. (42), in which the authors examinedresponses of the phyA and phyB mutants of Arabidopsis to red andfar-red light pulses for induction of seed germination and presentedconclusions similar to ours. Our results in a preliminary form has beenreported in ref. 43.

This work is dedicated to Prof. Horst Senger on the occasion of his65th birthday and to Prof. Pill-Soon Song on the occasion of his 60thbirthday. We thank Prof. N. Murata for hosting us at the NationalInstitute for Basic Biology, Prof. J. Chory and Dr. K Sakamoto forproviding us cDNA clones of PHYA and PHYB, R. Katayanagi forassistance with plant cultivation, and F. Tsunekawa for measuring thetransmittance of the seed coat. The work was supported in part byseparate grants from International Human Frontier Science Program(M.F. and A.N.), and carried out under the National Institute for BasicBiology Cooperative Research Programs for the Okazaki LargeSpectrograph 93-530 and 94-519.

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