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