Elementary processes of photoperception by phytochrome A for high-irradiance response of hypocotyl elongation in Arabidopsis

Elementary processes of photoperception by phytochrome A (PhyA) for the high-irradiance response (HIR) of hypocotyl elongation in Arabidopsis were examined using a newly designed irradiator with LED. The effect of continuous irradiation with far-red (FR) light could be replaced by intermittent irradiation with FR light pulses if given at intervals of 3 min or less for 24 h. In this response, the Bunsen-Roscoe law of reciprocity held in each FR light pulse. Therefore, we determined the action spectrum for the response by intermittent irradiation using phyB and phyAphyB double mutants. The resultant action spectrum correlated well with the absorption spectrum of PhyA in far-red-absorbing phytochrome (Pfr). Intermittent irradiation with 550 to 667 nm of light alone had no significant effect on the response. In contrast, intermittent irradiation with red light immediately after each FR light pulse completely reversed the effect of FR light in each cycle. The results indicate that neither red-absorbing phytochrome synthesized in darkness nor photoconverted Pfr are physiologically active, and that a short-lived signal is induced during photoconversion from Pfr to red-absorbing phytochrome. The mode of photoperception by PhyA for HIR is essentially different from that by PhyA for very-low-fluence responses and phytochrome B for low-fluence responses.     

Seed germination of Arabidopsis thaliana phyA/phyB double mutants is under phytochrome control.

We examined the photocontrol of seed germination in the phyA/phyB double mutants of Arabidopsis thaliana seeds. Dormant phyA/phyB seeds showed a red/far-red light (R/FR)-reversible induction of seed germination. This suggests the involvement of at least one other phytochrome, phyC, D, and/or E, in controlling seed germination. We designated this spectrally active phytochrome in phyA/phyB as phyX. The full reversibility of the R-induced germination by subsequent FR pulses, and the observation that the response is reversible by FR, even after a 3-h R treatment, indicates that this phyX response belongs to the low-fluence-response type. Thus, this phyX response is functionally related to phyB-mediated responses. However, in contrast to phyB-controlled seed germination, this phyX-mediated response needs a prolonged imbibition period and exhibits reversibility kinetics different from that needed for phyB. Furthermore, this phyX response requires a prolonged irradiation time and shows a fluence rate response dependency, showing a similarity to the high irradiance response of photomorphogenesis. Thus, phyX, with regard to its control of seed germination, is a functionally new phytochrome that shares some characteristics of both phyA- and phyB-mediated responses.     

Coupling of phytochrome B to the control of hypocotyl growth in Arabidopsis

Etiolated seedlings of the wild-type (WT) and of the phyB-1 mutant of Arabidopsis thaliana (L.) Heynh. were exposed to red-light (R) and far-red light (FR) treatments to characterize the action of phytochrome B on hypocotyl extension growth. A single R or FR pulse had no detectable effects on hypocotyl growth. After 24-h pre-treatment with continuous FR (FRc) a single R, compared to FR pulse inhibited (more than 70%) subsequent hypocotyl growth in the WT but not in the phyB-1 mutant. This effect of FRc was fluence-rate dependent and more efficient than continuous R (Rc) or hourly FR pulses of equal total fluence. Hypocotyl growth inhibition by Rc was larger in WT than phyB-1 seedlings when chlorophyll screening was reduced either by using broadband Rc (maximum emission 610 nm) or by using narrow-band Rc (658 nm) over short periods (24 h) or with seedlings bleached with Norflurazon. Hourly R or R + FR pulses had similar effects in WT and phyB-1 mutant etiolated seedlings. It is concluded that phytochrome B is not the only photoreceptor of Rc and that the action of phytochrome B is enhanced by a FRc high-irradiance reaction. Complementary experiments with the phyA-201 mutant indicate that this promotion of a phytochrome B-mediated response occurs via co-action with phytochrome A.Abbreviations D darkness - FR far-red light - FRc continuous FR - Pfr FR-absorbing form of phytochrome - HIR high-irradiance reaction - Pfr/P proportion of phytochrome as Pfr - phyA phytochrome A - phyB phytochrome B - R red light - Rc continuous R - WT wild-type I thank Professors R.E. Kendrick and M. Koornneef (Wageningen Agricultural University, The Netherlands) and Professor J. Chory (Salk Institute, Calif., USA) for their kind provision of the original WT and phyB-1 and phyA-201 seed, respectively. This work was financially supported by grants PID and PID-BID from CONICET, AG 040 from Universidad de Buenos Aires and A 12830/1-000019 from Fundación Antorchas.     

Co-action between phytochrome B and HY4 in Arabidopsis thaliana

A combination of physiological and genetic approaches was used to investigate whether phytochromes and blue light (BL) photoreceptors act in a fully independent manner during photomorphogenesis of Arabidopsis thaliana (L.) Heynh. Wild-type seedlings and phyA, phyBand hy4 mutants were daily exposed to 3 h BL terminated with either a red light (R) or a far-red light (FR) pulse. In wild-type and phyA-mutant seedlings, BL followed by an R pulse inhibited hypocotyl growth and promoted cotyledon unfolding. The effects of BL were reduced if exposure to BL was followed by an FR pulse driving phytochrome to the R-absorbing form (Pr). In the wild type, the effects of R versus FR pulses were small in seedlings not exposed to BL. Thus, maximal responses depended on the presence of both BL and the FR-absorbing form of phytochrome (Pfr) in the subsequent dark period. Impaired responses to BL and to R versus FR pulses were observed in phyB and hy4 mutants. Simultaneous irradiation with orange light indicated that BL, perceived by specific BL photoreceptors (i.e. not by phytochromes), required phytochrome B to display a full effect. These results indicate interdependent co-action between phytochrome B and BL photoreceptors, particularly the HY4 gene product. No synergism between phytochrome A (activated by continuous or pulsed FR) and BL photoreceptors was observed.Abbreviations BL blue light - D darkness - FR far-redlight - FRc continuous FR - Pfr FR-absorbing form of phytochrome - Pfr/P proportion of phytochrome as Pfr - phyA phytochrome A - phyB phytochrome B - R red light - WT wild type We thank Professors R.E. Kendrick and M. Koornneef (Wageningen Agricultural University, The Netherlands), Professor J. Chory (Salk Institute, Calif., USA) and the Arabidopsis Biological Resource Center (Ohio State University, Ohio, USA) for their kind provision of the original seed batches. This work was financially supported by CONICET, Universidad de Buenos Aires (AG 040) and Fundación Antorchas (A-12830/1 0000/9)     

Photoresponses of transgenic Arabidopsis seedlings expressing introduced phytochrome B-encoding cDNAs: evidence that phytochrome A and phytochrome B have distinct photoregulatory functions

The photocontrol of hypocotyl elongation has been studied in two transgenic lines of Arabidopsis thaliana which contain elevated levels of phytochrome B encoded by either an introduced rice- or Arabidopsis -derived cDNA driven by the 35S CaMV promoter. Inhibition of hypocotyl growth in etiolated seedlings of the phyB -transformed lines was saturated at photon fluence rates of continuous red light (R) which were markedly lower than those required for inhibition of growth in seedlings of the isogenic wild-type (WT). Inhibition of hypocotyl growth in etiolated seedlings of the phyB -transgenic lines under continuous far-red irradiation (FR), however, showed the same relationship with fluence rate as WT. Light-grown seedlings of the phyB -transgenic lines responded to end-of-day FR by an acceleration of growth, in a manner comparable with WT. This response was unaltered when the end-of-day FR was extended from a 15 min pulse to 14 h of continuous irradiation. The response of light-grown, phyB -transformed seedlings to decreasing R:FR ratio was also qualitatively similar to WT, i.e. increased elongation growth of the hypocotyl and petioles occurred under low R:FR quantum ratio. However, absolute elongation growth was markedly less in the transgenic seedlings at all R:FR ratios tested than in WT. Together, these data indicate that seedlings over-expressing phytochrome B are more responsive to R than are WT, but are unaltered in their responsiveness to FR. By contrast, seedlings overexpressing phytochrome A are more responsive than WT to both R and FR; whereas the phytochrome B-deficient mutant hy3 is unresponsive to R while retaining WT-like responsiveness to FR. These data indicate that in WT etiolated seedlings phytochrome A mediates the effects of continuous FR, and phytochrome B the effects of continuous R. The evidence thus supports the conclusion that these two molecular species of the photoreceptor have differential regulatory roles in the plant.     

Phytochrome A Mediates the Promotion of Seed Germination by Very Low Fluences of Light and Canopy Shade Light in Arabidopsis

Seeds of the wild type (WT) and of the phyA and phyB mutants of Arabidopsis thaliana were exposed to single red light (R)/far-red light (FR) pulses predicted to establish a series of calculated phytochrome photoequilibria (Pfr/P). WT and phyB seeds showed biphasic responses to Pfr/P. The first phase, i.e. the very-low-fluence response (VLFR), occurred below Pfr/P = 10-1%. The second phase, i.e. the low-fluence response, occurred above Pfr/P = 3%. The VLFR was similarly induced by either a FR pulse saturating photoconversion or a subsaturating R pulse predicted to establish the same Pfr/P. The VLFR was absent in phyA seeds, which showed a strong low-fluence response. In the field, even brief exposures to the very low fluences of canopy shade light (R/FR ratio < 0.05) promoted germination above dark controls in WT and phyB seeds but not in the phyA mutant. Seeds of the phyA mutant germinated normally under canopies providing higher R/FR ratios or under deep canopy shade light supplemented with R from light-emitting diodes. We propose that phytochrome A mediates VLFR of A. thaliana seeds.     

Negative interference of endogenous phytochrome B with phytochrome A function in Arabidopsis

To study negative interactions between phytochromes, phytochrome B (phyB) overexpressor lines, the mutants phyA-201, phyB-4, phyB-5, phyD-1, phyA-201 phyB-5, phyA-201 phyD-1, and phyB-5 phyD-1 of Arabidopsis were used. Endogenous phyB, but not phytochrome D (phyD), partly suppressed phytochrome A (phyA)-dependent inhibition of hypocotyl elongation in far-red light (FR). Dichromatic irradiation demonstrated that the negative effect of phyB was largely independent of the photoequilibrium, i.e. far-red light absorbing form of phytochrome formation. Moreover, phyB-4, a mutant impaired in signal transduction, did not show a loss of inhibition of phyA by phyB. Overexpression of phyB, conversely, resulted in an enhanced inhibition of phyA function, even in the absence of supplementary carbohydrates. However, overexpression of a mutated phyB, which cannot incorporate the chromophore, had no detectable effect on phyA action. In addition to seedling growth, accumulation of anthocyanins in FR, another manifestation of the high irradiance response, was strongly influenced by phyB holoprotein. Induction of seed germination by FR, a very low fluence response, was suppressed by both endogenous phyB and phyD. In conclusion, we show that both classical response modes of phyA, high irradiance response, and very low fluence response are subject to an inhibitory action of phyB-like phytochromes. Possible mechanisms of the negative interference are discussed.     

Photoresponses of transgenic tobacco plants expressing an oat phytochrome gene

The physiological responses of transgenic tobacco (Nicotiana tabacum L.) plants that express high levels of an introduced oat (Avena sativa L.) phytochrome (phyA) gene to various light treatments are compared with those of wild-type (WT) plants. Seeds, etiolated seedlings, and light-grown plants from a homozygous transgenic tobacco line (9A4) constructed by Keller et al. (EMBO J, 8, 1005–1012, 1989) were treated with red (R), far-red (FR), or white light (WL) with or without supplemental FR light, revealing major perturbations of the normal photobiological responses. White light stimulated germination of both WT and transgenic seed, but addition of FR to the WL treatment suppressed germination. In the WT, all fluence rates tested inhibited germination, but in the transgenics, reduction effluence rate partially relieved germination from the FR-mediated inhibition. It is suggested that the higher absolute levels of the FR-absorbing form of phytochrome (Pfr) in the irradiated transgenics, compared to the WT, may be responsible for the reduced FR-mediated inhibition of germination in the former. Hypocotyl extension of dark-grown seedlings of both WT and transgenic lines was inhibited by continuous R or FR irradiation, typical of the high-irradiance response (HIR). After 2 d of de-etiolation in WL, the WT seedlings had lost the FR-mediated inhibition of hypocotyl extension, whereas it was retained in the transgenics. The FR-mediated inhibition of hypocotyl extension in the transgenic seedlings after de-etiolation may reflect the persistence of an, FR-HIR response mediated by the overexpressed oat PhyA phytochrome. Light-grown WT seedlings exhibited typical shade-avoidance responses when treated with WL supplemented with high levels of FR radiation. Internode and petiole extension rates were markedly increased, and the chlorophyll ab ratio decreased, in the low-R: FR treatment. The transgenics, however, showed no increases in extension growth under low-R: FR treatments, and at low fluence rates both internode and petiole extension rates were significantly decreased by low R FR. Interpretation of these data is difficult. The depression of the chlorophyll ab ratio by low R FR was identical in WT and transgenic plants, indicating that not all shade-avoidance responses of light-grown plants were disrupted by the over-expression of the introduced oat phyA gene. The results are discussed in relation to the proposal that different members of the phytochrome family may have different physiological roles.Abbreviations FR far-red light - PAR photosynthetically active radiation - Pr, Pfr red- and FR-absorbing forms of phytochrome - Ptot total phytochrome - PhyA (PhyA) gene (encoded protein) for phytochrome - R red light - WL white light - WT wild type This work was supported by an Agricultural and Food Research Council research grant to H.S. and A.C.M.; the production of the transgenic seed was funded by the U.S. Department of Energy (DE-F602-88ER13968) to R.D.V., and by E.I. du Pont de Nemours; Dr. G.C. Whitelam is thanked for the provision of monoclonal antibodies for the immunoblot analyses.     

Phytochrome A-mediated inhibition of seed germination in tomato

Far-red light (FR) inhibition of seed germination of tomato (Solanum lycopersicum L.) was studied with the phytochrome (phy)-hypersensitive mutants, hp-1w, hp-1w,fri1, a phyA-deficient double mutant, and hp-1w,tri1, a phyB1-deficient double mutant. Seeds of all mutants germinated readily in the dark at 25 degrees C, and the germination was retarded by a single 100-s FR pulse given 1-3 h after sowing. The effect of an FR pulse was red-light reversible in all mutants used. After 24 h where a single FR pulse was no longer effective, prolonged FR exposure or hourly FR pulses suppressed germination in hp-1w and hp-1w,tri1, whereas in hp-1w,fri1 the suppressive effect of FR was almost absent. The effect of the prolonged FR was greater than that of the hourly 3-min FR pulses having equal photon fluence, and was fluencerate dependent. Thus we conclude that the germination inhibition by FR in tomato seed consists of a low-fluence response and a high irradiance response (HIR); the latter is controlled by phyA, but not phyB1. This is the first indication of phyA being involved in the HIR of seed germination inhibition.     

Intramolecular uncoupling of chromophore photoconversion from structural signaling determinants drive mutant phytochrome B photoreceptor to far-red light perception

The phytochrome (phy) photoreceptor family regulates almost all aspects of plant development in a broad range of light environments including seed germination, onset of the photomorphogenic program in seedling stage, the shade avoidance syndrome in competing plant communities, flowering induction and senescence of adult plants. During evolution two clearly distinct classes of phy-s emerged covering these very different physiological tasks.¹ PhyA is rapidly degraded in its activated state. PhyA functions in controlling seed germination at very low light intensities (very low fluence response, VLFR) and seedling establishment under photosynthetic shade conditions (high irradiance response, HIR) where the far-red portion of the transmitted light to understorey habitats is substantially enhanced. Arabidopsis phyB together with phyC, D and E belongs to the relatively stable sensor class in comparison to the light labile phyA. PhyB functions at all stages of development including seed germination and seedling establishment, mediates classical red/far-red reversible low fluence responses (LFR) as well as red light high irradiance responses, and it is considered to be the dominating phytochrome sensor of its class.     

Photoresponses of Light-Grown phyA Mutants of Arabidopsis (Phytochrome A Is Required for the Perception of Daylength Extensions)

Several aspects of the photophysiology of wild-type Arabidopsis thaliana seedlings were compared with those of a phytochrome A null mutant, phyA-1, and a mutant, fhy1, that is putatively involved in the transduction of light signals from phytochrome A. Although phyA seedlings display a near wild-type phenotype when grown in white light (W), they nevertheless display several photomorphogenic abnormalities. Thus, whereas the germination of wild-type and fhy1 seeds is almost fully promoted by a pulse of red light (R) or by continuous far-red light (FR), phyA seed germination is responsive only to R. Following growth under day/night cycles, but not under continuous W, the hypocotyls of light-grown phyA and fhy1 seedlings are more elongated than those of wild-type seedlings. For seedlings grown under low red/far-red (R/FR) ratio light conditions, phyA and fhy1 seedlings display a more marked promotion of hypocotyl elongation than wild-type seedlings. Similarly, seedlings that are doubly null for phytochrome A and phytochrome B(phyA phyB) also have more elongated hypocotyls under low R/FR ratio conditions than phyB seedlings. This indicates that phytochrome A action in light-grown seedlings is antagonistic to the action of phytochrome B. Although wild-type, fhy1, and phyA seedlings flower at essentially the same time under both short-day and long-day conditions, an obvious consequence of phytochrome A deficiency is a pronounced late flowering under conditions where a short day of 8 h of fluorescent W is extended by 8 h of low-fluence-rate incandescent light. The evidence thus indicates that phytochrome A plays a role in seed germination, in the control of elongation growth of light-grown seedlings, and in the perception of daylength.     

Regulation of hormone metabolism in Arabidopsis seeds: phytochrome regulation of abscisic acid metabolism and abscisic acid regulation of gibberellin metabolism

In a wide range of plant species, seed germination is regulated antagonistically by two plant hormones, abscisic acid (ABA) and gibberellin (GA). In the present study, we have revealed that ABA metabolism (both biosynthesis and inactivation) was phytochrome-regulated in an opposite fashion to GA metabolism during photoreversible seed germination in Arabidopsis. Endogenous ABA levels were decreased by irradiation with a red (R) light pulse in dark-imbibed seeds pre-treated with a far-red (FR) light pulse, and the reduction in ABA levels in response to R light was inhibited in a phytochrome B (PHYB)-deficient mutant. Expression of an ABA biosynthesis gene, AtNCED6, and the inactivation gene, CYP707A2, was regulated in a photoreversible manner, suggesting a key role for the genes in PHYB-mediated regulation of ABA metabolism. Abscisic acid-deficient mutants such as nced6-1, aba2-2 and aao3-4 exhibited an enhanced ability to germinate relative to wild type when imbibed in the dark after irradiation with an FR light pulse. In addition, the ability to synthesize GA was improved in the aba2-2 mutant compared with wild type during dark-imbibition after an FR light pulse. Activation of GA biosynthesis in the aba2-2 mutant was also observed during seed development. These data indicate that ABA is involved in the suppression of GA biosynthesis in both imbibed and developing seeds. Spatial expression patterns of the AtABA2 and AAO3 genes, responsible for last two steps of ABA biosynthesis, were distinct from that of the GA biosynthesis gene, AtGA3ox2, in both imbibed and developing seeds, suggesting that biosynthesis of ABA and GA in seeds occurs in different cell types.     

Light-grown plants of transgenic tobacco expressing an introduced oat phytochrome A gene under the control of a constitutive viral promoter exhibit persistent growth inhibition by far-red light

A comparison of the photoregulation of development has been made for etiolated and light-grown plants of wild-type (WT) tobacco (Nicotiana tabacun L.) and an isogenic transgenic line which expresses an introduced oat phytochrome gene (phyA) under the control of a constitutive viral promoter. Etiolated seedlings of both the WT and transgenic line showed irradiance-dependent inhibition of hypocotyl growth under continuous far-red (FR) light; transgenic seedlings showed a greater level of inhibition under a given fluence rate and this is considered to be the result of the heterologous phytochrome protein (PhyA) functioning in a compatible manner with the native etiolated phytochrome. Deetiolation of WT seedlings resulted in a loss of responsiveness to prolonged FR. Light-grown transgenic seedlings, however, continued to respond in an irradiance-dependent manner to prolonged FR and it is proposed that this is a specific function of the constitutive PhyA. Mature green plants of the WT and transgenic lines showed a qualitatively similar growth promotion to a brief end-of-day FR-treatment but this response was abolished in the transgenic plants under prolonged irradiation by this same FR source. Growth inhibition (McCormac et al. 1991, Planta 185, 162–170) and enhanced levels of nitrate-reductase activity under irradiance of low red:far-red ratio, as achieved by the FR-supplementation of white light, emphasised that the introduced PhyA was eliciting an aberrant mode of photoresponse compared with the normal phytochrome population of light-grown plants. Total levels of the oat-encoded phytochrome in the etiolated transgenic tobacco were shown to be influenced by the wavelength of continuous irradiation in a manner which was qualitatively similar to that seen for the native, etiolated tobacco phytochrome, and distinct from that seen in etiolated oat tissues. These results are discussed in terms of the proposal that the constitutive oat-PhyA pool in the transgenic plants leads to a persistence of a mode of response normally restricted to the situation in etiolated plants.Abbreviations FR far-red light - R red light - WL white light - WL + FR white light supplemented with FR - HIR high-irradiance response - PAR photosynthetically active radiation - Pr, Pfr R- and FR-absorbing forms of phytochrome - Ptot total phytochrome - phyA (PhyA) gene (encoded protein) for phytochrome - WT wild type This work was supported by an Agricultural and Food Research Council research grant to H.S. and A.M.; J.R. Cherry and R.D. Vierstra, (Department of Horticulture, University of Wisconsin-Madison, USA) are thanked for the provision of the transgenic tobacco line.     

Overexpression of Arabidopsis phytochrome B inhibits phytochrome A function in the presence of sucrose

Overexpression of phytochrome B (phyB) in Arabidopsis has previously been demonstrated to result in dominant negative interference of phytochrome A (phyA)-mediated hypocotyl growth inhibition in far-red (FR) light. This phenomenon has been examined further in this study and has been found to be dependent on the FR fluence rate and on the availability of metabolizable sugars in the growth medium. Poorly metabolized sugars capable of activating the putative hexokinase sensory function were not effective in eliciting the phytochrome interference response. Overexpressed phyB lacking the chromophore-binding site was also effective at inhibiting the phyA response, especially at higher fluence rates of FR. Overexpressed phyB produces the dominant negative phenotype without any apparent effect on phyA abundance or degradation. It is possible that phyA and phyB interact with a common reaction partner but that either the energy state of the cell or a separate sugar-signaling mechanism modulates the phytochrome-signaling interactions.     

Is the far-red-absorbing form of Avena phytochrome A that is present at the end of the day able to sustain stem-growth inhibition during the night in transgenic tobacco and tomato seedlings?

Avena phytochrome A (phyA) overexpressed in tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon sculentum Mill) was functionally characterised by comparing wild-type (WT) and transgenic seedlings. Different proportions of phytochrome in its far-red-absorbing form (Pfr/P) were provided by end-of-day (EOD) light pulses. Stem-length responses occurred largely in the range of low Pfr/P (3–61%) for WT seedlings and in the range of high Pfr/P (61–87%) for transgenic seedlings. A similar shift was observed when the photoperiod was interrupted by short light pulses providing different Pfr/P ratios and followed by 1 h dark incubation. In other experiments, Avena phyA was allowed to re-accumulate in darkness and subsequently phototransformed to Pfr but no extra inhibition of stem extension growth was observed. In transgenic tomato seedlings the response to EOD far-red light was faster and the response to a far-red light pulse delayed into darkness was larger than in the WT. Avena phyA Pfr remaining at the end of the photoperiod appears intrinsically unable to sustain growth inhibition in subsequent darkness. Avena phyA modifies the sensitivity and the kinetics of EOD responses mediated by native phytochrome.Abbreviations EOD end-of-day - FR far-red light - Pfr/P pro-portion of phytochrome in its FR-absorbing form - phyA phyto-chrome A - phyB phytochrome B - R red light - RFR R to FR ratio - WT wild type We thank Dr Brian Thomas for providing the antibodies used in this work, and Federico Guerendiain for his excellent technical assistance. This work was financially supported by grants UBA AG 040 and Fundacion Antorchas A-12830/1-19 (both to J.J.C.), PID-CONICET (to R.A.S. and J.J.C.), United States Department of Energy DE-FG02-88ER13968 (to R.D.V.).     

Photoregulation of germination in seed of transgenic lines of tobacco and Arabidopsis which express an introduced cDNA encoding phytochrome A or phytochrome B

Photoinduction and photoinhibition of germination in seed from a homozygous tobacco (Nicotiana tabacum L.) line containing an introduced oat phyA cDNA (encoding phytochrome A) is compared with that of isogenic wild-type (WT) tobacco. Under continuous irradiation by a light source with a low redfar-red (RFR) ratio the transgenic tobacco seed appeared to be less susceptible to photoinhibition of germination compared with WT seed. However, induction of germination following a short pulse by R (666 nm) was not enhanced in the genotype transformed by oat phyA cDNA compared with the WT; neither did germination of the transgenic tobacco seed show an increased sensitivity to saturating pulses of light of longer wavelengths (666–730 nm). In seeds of transgenic Arabidopsis thaliana (L.) Heynh. which contained an introduced phytochrome-B-encoding cDNA, levels of dark germination were enhanced, consistent with mediation of response by phytochrome B-P_fr. The germination behaviour of Arabidopsis genotypes wich contained an introduced cDNA encoding phytochrome A, however, did not significantly differ from that of the WT.Abbreviations ABO seed transformed with Arabidopsis phyB - cDNA; CaMV cauliflower mosaic virus - FR far-red light - P_fr far-red-absorbing form of phytochrome - P_tot total phytochrome - P_fr/P_tot phytochrome photoequilibrium - R red light - RBO seed transformed with rice phyB cDNA - RFR quantum ratio of red and far-red light - WL white light - WL + FR whitelight supplemented with far-red light - WT wild type The authors wish to thank R.D. Vierstra (Department of Horticulture, University of Wisconsin-Madison, USA) for providing the transgenic tobacco line, and M.T. Boylan, D. Wagner and P.H. Quail (U.C. Berkeley/USDA Plant Gene Expression Center, Albany, Calif. USA) for providing the transgenic Arabidopsis lines. The work presented in this paper was funded by grants from the Agricultural and Food Research Council (H.S., A.C.M., G.C.W.).     

Far-red light-insensitive, phytochrome A-deficient mutants of tomato

We have selected two recessive mutants of tomato with slightly longer hypocotyls than the wild type, one under low fluence rate (3 mol/m²/s) red light (R) and the other under low fluence rate blue light. These two mutants were shown to be allelic and further analysis revealed that hypocotyl growth was totally insensitive to far-red light (FR). We propose the gene symbol fri (far-red light insensitive) for this locus and have mapped it on chromosome 10. Immunochemically detectable phytochrome A polypeptide is essentially absent in the fri mutants as is the bulk spectrophotometrically detectable labile phytochrome pool in etiolated seedlings. A phytochrome B-like polypeptide is present in normal amounts and a small stable phytochrome pool can be readily detected by spectrophotometry in the fri mutants. Inhibition of hypocotyl growth by a R pulse given every 4 h is quantitatively similar in the fri mutants and wild type and the effect is to a large extent reversible if R pulses are followed immediately by a FR pulse. After 7 days in darkness, both fri mutants and the wild type become green on transfer to white light, but after 7 days in FR, the wild-type seedlings that have expanded their cotyledons lose their capacity to green in white light, while the fri mutants de-etiolate. Adult plants of the fri mutants show retarded growth and are prone to wilting, but exhibit a normal elongation response to FR given at the end of the daily photoperiod. The inhibition of seed germination by continuous FR exhibited by the wild type is normal in the fri mutants. It is proposed that these fri mutants are putative phytochrome A mutants which have normal pools of other phytochromes.     

Phytochrome regulation of phytochrome A mRNA levels in the model short-day-plant Pharbitis nil

The exposure of dark-grown Pharbitis nil seedlings to continuous R induces a rapid decrease in PHYA mRNA abundance with a half-life of about 2 h. A 5 min R pulse also induces this decline, and the effect is partially reversible by subsequent FR irradiation, confirming that the regulation of expression is mediated via the Pfr form of a phytochrome. When de-etiolated seedlings are returned to darkness after a W photoperiod, PHYA mRNA slowly reaccumulates from 20% to 50% of the dark level within 24 h. The rate of reaccumulation is greatly accelerated by the removal of Pfr with a FR pulse, resulting in reaccumulation to 100% within approximately 11 h. Without FR irradiation PHYA mRNA expression remains fully repressed for at least 11 h after the end of the photoperiod, suggesting that the controlling Pfr is highly stable.     

Ultraviolet B radiation enhances a phytochrome-B-mediated photomorphogenic response in Arabidopsis

Ultraviolet B radiation (UV-B, 290-315 nm) can cause damage and induce photomorphogenic responses in plants. The mechanisms that mediate the photomorphogenic effects of UV-B are unclear. In etiolated Arabidopsis seedlings, a daily exposure to 2.5 h of UV-B enhanced the cotyledon opening response induced by a subsequent red light (R) pulse. An R pulse alone, 2.5 h of UV-B terminated with a far-red pulse, or 2.5 h of continuous R caused very little cotyledon opening. The enhancing effect of UV-B increased with fluence rate up to approximately 7.58 micromol m(-2) s(-1); at higher fluence rates the response to UV-B was greatly reduced. The phyA, phyA cry1, and cry1 cry2 mutants behaved like the wild type when exposed to UV-B followed by an R pulse. In contrast, phyB, phyB cry1, and phyB phyA mutants failed to open the cotyledons. Thus, phytochrome B was required for the cotyledon opening response to UV-B --> R treatments, whereas phytochrome A and cryptochromes 1 and 2 were not necessary under the conditions of our experiments. The enhancing effect of low doses of UV-B on cotyledon opening in uvr1 uvr2 and uvr1 uvr3 mutants, deficient in DNA repair, was similar to that found in the wild type, suggesting that this effect of UV-B was not elicited by signals derived from UV-B-induced DNA lesions (cyclobutane pyrimidine dimers and 6-4 photoproducts). We conclude that low doses of UV-B, perceived by a receptor system different from phytochromes, cryptochromes, or DNA, enhance a de-etiolation response that is induced by active phytochrome B.     

Phytochrome Action during Prechilling Induced Germination of Betula papyrifera Marsh

Seeds of paper birch (Betula papyrifera Marsh.) were induced to germinate by prechilling at 3 C or by red light. The light requirement was mediated by phytochrome and the action of phytochrome during prechilling was investigated. Red irradiation (R) prior to prechilling markedly enhanced the effectiveness of the prechilling treatment in inducing subsequent germination at 18 C. Reversal of this enhancement by far-red irradiation (FR) was more effective when FR was supplied after a 1-week prechill treatment than after a 2-week treatment. The R enhancement effect exhibited a sharp drop as prechilling temperature was increased from 5 to 7 C. This decline is consistent with a membrane phase transition at about 7 C where Pfr action is diminished by a loss in sensitivity of its receptor sites. Although phytochrome action was observed during prechilling treatments, the seeds failed to germinate at prechilling temperatures. Therefore, it was concluded that while potentiation of germination by Pfr occurred during prechilling, some other reaction(s) leading to radicle protrusion requires higher temperatures. In one seed source loss of germination potential was observed with protracted storage at 3 C. This was prevented by R supplied during the prechilling treatment. Taken collectively the data suggest that action of phytochrome during prechilling is accentuated in these seeds by two factors: (a) an increase in the sensitivity (or number) of Pfr receptor sites; and (b) preservation of Pfr by deferment of thermal reversion.