Overexpression of rice phytochrome A in tobacco seedlings modifies responsiveness but not competence to phytochrome

To analyse the control of rice phytochrome A (phyA) overexpression (wild type or variously mutated) on gene regulation, transgenic tobacco lines overexpressing various rice phyA constructs were crossed with transgenic tobacco lines containing mustard Lhcb1 or Chs1 promoters fused to the uidA reporter gene (-glucuronidase). It was demonstrated that the temporal pattern of competence to respond to phytochrome was not altered by rice phyA overexpression. Also, overexpression of rice phyA did not change the spatial pattern of gene expression. The responsiveness to red and far-red light, on the other hand, depended on the type of overexpressed rice phyA in a structure-function relation: the serine-to-alanine mutant mediated an enhanced response both under continuous red and far-red light, whereas the N-terminal deletion mutant showed a dominant negative effect under continuous far-red light and even after red light pulses. However, the effectiveness of rice phyA overexpression depended on the promoter construct and the developmental stage of the seedlings. The Lhcb1 promoter also conferred -glucuronidase activity in etiolated seedlings. This dark expression could be decreased by a long-wavelength farred light pulse given early in development (24 h after sowing), indicating that this phenomenon is under the control of stable types of phytochrome.Abbreviations Chs1 chalcone synthase - GUS -glucuronidase - Lhcb1 type 1 light-harvesting chlorophyll a/b-binding protein - NTD N-terminal deletion mutant of rice phyA - phyA phytochrome A - phyB phytochrome B - Pfr far-red absorbing form of phytochrome - Pr red-absorbing form of phytochrome - RW rice wild-type phyA - S/A serine-to-alanine mutant of rice phyA - XAN wild-type tobacco cv. Xanthi We thank N.-H. Chua (Rockefeller Univ., New York, USA) and J. Stockhaus (Heinrich-Heine-Universität, Düsseldorf, Germany) for providing seeds from tobacco lines overexpressing the diverse rice phyA proteins. The work was supported by a grant from the Human Frontier Science Program and a grant from Deutsche Forschungsgemeinschaft (SFB 388). K.E. is a recipient of a Landesgraduierten-förderung fellowship     

The light-induced reduction of the gravitropic growth-orientation of seedlings of Arabidopsis thaliana (L.) Heynh. is a photomorphogenic response mediated synergistically by the far-red-absorbing forms of phytochromes A and B

Hypocotyls of dark-grown seedlings of Ara bidosis thaliana exhibit a strong negative gravitropism, which is reduced by red and also by long-wavelength, far-red light treatments. Light treatments using phytochrome A (phyA)- and phytochrome B (phyB)-deficient mutants showed that this response is controlled by phyB in a red/far-red reversible way, and by phyA in a non-reversible, very-low-fluence response. Crosses of the previously analyzed phyB-1 allele (in the ecotype Landsberg erecta background) to the ecotype Nossen wild-type (WT) background resulted in a WT-like negative gravitropism in darkness, indicating that the previously described gravitropic randomization observed with phyB-1 in the dark is likely due to a second mutation independent of that in the PHYB gene.Abbreviations FR long-wavelength far-red light - phyA phytochrome A (holoprotein) - phyB phytochrome B (holoprotein) - Pr red-absorbing form of phytochrome - WT wild type We thank Dr. A. Nagatani (RIKEN Institute, Wako-City, Japan) and Dr. M. Furuya (Hitachi, Hatoyama, Japan) for the phyA-201/phyB-5 double mutant. The work was supported by Deutsche Forschungsgemeinschaft and Human Frontier Science Program grants to E.S.     

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.).     

Light exaggerates apical hook curvature through phytochrome actions in tomato seedlings

Contrary to the established notion that the apical hook of dark-grown dicotyledonous seedlings opens in response to light, we found in tomato (Solanum lycopersicum L.) that the apical hook curvature is exaggerated by light. Experiments with several tomato cultivars and phytochrome mutants, irradiated with red and far-red light either as a brief pulse (Rp, FRp) or continuously (Rc, FRc), revealed: the hook-exaggeration response is maximal at the emergence of the hypocotyl from the seed; the effect of Rp is FRp-reversible; fluence–response curves to a single Rp or FRp show an involvement of low and very low fluence responses (LFR, VLFR); the effect of Rc is fluence-rate dependent, but that of FRc is not; the phyA mutant (phyA hp-1) failed to respond to an Rp of less than 10⁻² μmol m⁻² and to an FRp of all fluences tested as well as to FRc, thus indicating that the hook-exaggeration response involves phyA-mediated VLFR. The Rp fluence–response curve with the same mutant also confirmed the presence of an LFR mediated by phytochrome(s) other than phyA, although the phyB1 mutant (phyB1 hp-1) still showed full response probably due to other redundant phytochrome species (e.g., phyB2). Simulation experiments led to the possible significance of hook exaggeration in the field that the photoresponse may facilitate the release of seed coat when seeds germinate at some range of depth in soil. It was also observed that seed coat and/or endosperm are essential to the hook exaggeration.     

Photocontrol of anthocyanin biosynthesis in tomato

Juvenile anthocyanin biosynthesis has been studied in dark-grown seedlings of tomato (Lycopersicon esculentum Mill.) wild types (WTs) and photomorphogenic mutants. During a subsequent 24-hr period of monochromatic irradiation at different fluence rates of red light (R) the fluence-rate response relationships for induction of anthocyanin in all the WTs are similar, yet complex, showing a response at low fluence rates (LFRR) followed by a fluence rate-dependent high irradiance response (HIR). In the hypocotyl this response is restricted to the sub-epidermal layer of cells. The high-pigment-1 (hp-1) mutant exhibits a strong amplification of both response components. Theatroviolacea (atv) mutant shows strongest amplification of the HIR component. In contrast, a transgenic line overexpressing an oat phytochrome A gene (PHYA3 ⁺) shows a most dramatic amplification of the LFRR component. The far-red light (FR)-insensitive (fri) mutant, deficient in phytochrome A (phyA), lacks the LFRR component whilst retaining a normal HIR. The temporarily R-insensitive (tri) mutant, deficient in phytochrome B1 (phyB1) retains the LFRR, but lacks the HIR. Thehp-1,fri andhp-1,tri double mutant, exhibit amplified, yet qualitatively similar responses to the monogenicfri andtri mutants. Thefri,tri double mutant lacks both response components in R, but a residual response to blue light (B) remains. Similarly, theaurea (au) mutant deficient in phytochrome chromophore biosynthesis and presumably all phytochromes, lacks both response components in the R and FR regions of the spectrum. Experiments at other wavelengths demonstrate that while there is only a small response in the FR spectral region (729 nm) in tomato, there is an appreciable HIR response in the near FR at 704 nm, which is retained in thetri mutant. This suggests that the labile phyA pool participates in the HIR at this wavelength. The intense pigmentation (Ip) mutant appears to be specifically deficient in the B1 induced anthocyanin biosynthesis. Adult plants, grown under fluorescent light/dark cycles, show a reduction of anthocyanin content of young developing leaves upon application of supplemtary or end-of-day FR. The involvement of different phytochrome species in anthocyanin biosynthesis based on micro-injection studies into theau mutant and studies using type specific phytochrome mutants is discussed.     

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.     

Multiple phytochromes are involved in red-light-induced enhancement of first-positive phototropism in Arabidopsis thaliana.

The amplitude of phototropic curvature to blue light is enhanced by a prior exposure of seedlings to red light. This enhancement is mediated by phytochrome. Fluence-response relationships have been constructed for red-light-induced enhancement in the phytochrome A (phyA) null mutant, the phytochrome B- (phyB) deficient mutant, and in two transgenic lines of Rabidopsis thaliana that overexpress either phyA or phyB. These fluence-response relationships demonstrate the existence of two response in enhancement, a response in the very-low-to-low-fluence range, and a response in the high-fluence range. Only the response in the high-fluence range is present in the phyA null mutant. In contrast, the phyB-deficient mutant is indistinguishable from the wild-type parent in red-light responsiveness. These data indiacate that phyA is necessary for the very-low-to-low but not the high-influence response, and that phyB is not necessary for either response range. Based on these results, the high-fluence response, if controlled by a single phytochrome, must be controlled by aphytochorme other than phyA of phyB. Overexpression of phyA has a negative effect and overexpression of phyB has an enhancing effect in the high-fluence range. These results suggest that overexpression of either phytochrome perturbs the function of the endogenous photoreceptor system in an unpredictable fashion.     

Phytochrome A enhances the promotion of hypocotyl growth caused by reductions in levels of phytochrome B in its far-red-light-absorbing form in light-grown Arabidopsis thaliana.

We sought to determine if phytochrome B (phyB)-mediated responses to the red light (R)/far-red light (FR) ratio are affected by phytochrome A (phyA) activity in light-grown seedlings of Arabidopsis thaliana. Pulses of FR delayed into the dark period were less effective than end-of-day (EOD) FR in promoting hypocotyl growth over a given period in darkness. White light minus blue light interposed instead of darkness between the end of the white-light photoperiod and the FR pulse was sufficient to maintain responsivity to the decrease in phyB in FR-light-absorbing form in wild-type (WT) seedlings, but not in the phyA mutant. Compared with EOD R, hourly R+FR pulses provided throughout the night caused a stronger promotion of stem growth than a single EOD R+FR pulse in WT Arabidopsis, cucumber, mustard, sunflower, tobacco, and tomato, but not in phyA Arabidopsis or in the aurea mutant of tomato. WT seedlings of Arabidopsis responded to a range of high EOD R/FR ratios, whereas the phyA mutant required stronger reductions in the EOD R/FR ratio. In sunlight, phyA seedlings of Arabidopsis showed no response to the "early warning" signals of neighboring vegetation, and hypocotyl-growth promotion occurred at higher plant densities than in the WT. Thus, under a series of light conditions, the sensitivity or responsivity to reductions in the R/FR ratio were larger in WT than in phyA seedlings. A product of phyA is therefore proposed to enhance the hypocotyl-growth response to decreases in phyB in FR-light-absorbing form in light grown seedlings.     

Physiological interactions of phytochromes A, B1 and B2 in the control of development in tomato

The role of phytochrome B2 (phyB2) in the control of photomorphogenesis in tomato (Solanum lycopersicum L.) has been investigated using recently isolated mutants carrying lesions in the PHYB2 gene. The physiological interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phyB2 have also been explored, using an isogenic series of all possible mutant combinations and several different phenotypic characteristics. The loss of phyB2 had a negligible effect on the development of white-light-grown wild-type or phyA-deficient plants, but substantially enhanced the elongated pale phenotype of the phyB1 mutant. This redundancy was also seen in the control of de-etiolation under continuous red light (R), where the loss of phyB2 had no detectable effect in the presence of phyB1. Under continuous R, phyA action was largely independent of phyB1 and phyB2 in terms of the control of hypocotyl elongation, but antagonized the effects of phyB1 in the control of anthocyanin synthesis, indicating that photoreceptors may interact differently to control different traits. Irradiance response curves for anthocyanin synthesis revealed that phyB1 and phyB2 together mediate all the detectable response to high-irradiance R, and, surprisingly, that the phyA-dependent low-irradiance component is also strongly reduced in the phyB1 phyB2 double mutant. This is not associated with a reduction in phyA protein content or responsiveness to continuous far-red light (FR), suggesting that phyB1 and phyB2 specifically influence phyA activity under low-irradiance R. Finally, the phyA phyB1 phyB2 triple mutant showed strong residual responsiveness to supplementary daytime FR, indicating that at least one of the two remaining phytochromes plays a significant role in tomato photomorphogenesis.     

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.     

Hierarchical coupling of phytochromes and cryptochromes reconciles stability and light modulation of Arabidopsis development

In plants, development is a continuing process that takes place under strong fluctuations of the light environment. Here we show that in Arabidopsis thaliana plants grown under intense white light, coupling of the photoreceptor cryptochrome 2 to developmental processes is broader than previously appreciated. Compared to the wild type, the cry2 mutant showed reduced activity of a Lhcb1*2 promoter fused to a reporter, and delayed flowering. The cry2 mutation also reduced the inhibition of hypocotyl growth, the unfolding of the cotyledons, the rate of leaf production during the vegetative phase, and the pace of development after transition to the reproductive stage; but these effects were obvious only in the absence of cryptochrome 1 and in some cases phytochrome A and/or phytochrome B. Complementary, the cry2 mutation uncovered novel roles for cryptochrome 1 and phytochrome A. The activity of the Lhcb1*2 promoter was higher in the cry1 cry2 mutant than in the cry2 mutant, suggesting that cry1 could be involved in blue-light repression of photosynthetic genes. Surprisingly, the phyA cry1 cry2 triple mutant flowered earlier and showed better response to photoperiod than the cry1 cry2 double mutant, indicating that phyA is involved in light repression of flowering. Growth and development were severely impaired in the quadruple phyA phyB cry1 cry2 mutant. We propose that stability and light modulation of development are achieved by simultaneous coupling of phytochrome A, phytochrome B, cryptochrome 1 and cryptochrome 2 to developmental processes, in combination with context-dependent hierarchy of their relative activities.     

Dual effect of phytochrome A on hypocotyl growth under continuous red light

The role of phytochrome A in the control of hypocotyl growth under continuous red light (Rc) was investigated using phyA and phyB mutants of Arabidopsis thaliana, which lack phytochrome A (phyA) or phytochrome B (phyB), respectively, and transgenic seedlings of Nicotiana tabacum overexpressing Avena phyA, compared to the corresponding wild type (WT). In WT seedlings of A. thaliana, hypocotyl growth inhibition showed a biphasic response to the fluence rate of Rc, with a brake at 10^?2μmol m^?2 s^?1. At equal total fluence rate, hourly pulses of red light caused slightly more inhibition than Rc. The response to very low fluences of continuous or pulsed red light was absent in the phyA and phyA phyB mutants and present in the phyB mutant. The second part of the response was steeper in the phyA mutant than in the WT but was absent in the phyB mutant. In WT tobacco the response to Rc was biphasic. Overexpression of Avena phyA enhanced the response only at very low fluence rates of Rc (< 10^?2μmol m^?2 s^?1). In both species, the effect of hourly pulses of far-red light was similar to the maximum inhibition observed in the first phase of the response to Rc. Using reciprocity failure (i.e. higher inhibition under continuous than pulsed light) as the operational criterion, a ‘true’ high-irradiance reaction occurred under continuous far-red light but not under Rc or red plus far-red light mixtures. Native and overexpressed phyA are proposed to mediate very low fluence responses under Rc. In WT A. thaliana, this effect is counteracted by a negative action of phyA on phyB-mediated low-fluence responses.     

Interaction of phytochromes A and B in the control of de-etiolation and flowering in pea

The interactions of phytochrome A (phyA) and phytochrome B (phyB) in the photocontrol of vegetative and reproductive development in pea have been investigated using null mutants for each phytochrome. White-light-grown phyA phyB double mutant plants show severely impaired de-etiolation both at the seedling stage and later in development, with a reduced rate of leaf production and swollen, twisted internodes, and enlarged cells in all stem tissues. PhyA and phyB act in a highly redundant manner to control de-etiolation under continuous, high-irradiance red light. The phyA phyB double mutant shows no significant residual phytochrome responses for either de-etiolation or shade-avoidance, but undergoes partial de-etiolation in blue light. PhyB is shown to inhibit flowering under both long and short photoperiods and this inhibition is required for expression of the promotive effect of phyA. PhyA is solely responsible for the promotion of flowering by night-breaks with white light, whereas phyB appears to play a major role in detection of light quality in end-of-day light treatments, night breaks and day extensions. Finally, the inhibitory effect of phyB is not graft-transmissible, suggesting that phyB acts in a different manner and after phyA in the control of flower induction.     

Interactions of phytochromes A,B1 and B2 in light-induced competence for adventitious shoot formation in hypocotyl of tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> L.)

The interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phytochrome B2 (phyB2) in light-dependent shoot regeneration from the hypocotyl of tomato was analysed using all eight possible homozygous allelic combinations of the null mutants. The donor plants were pre-grown either in the dark or under red or far-red light for 8 days after sowing; thereafter hypocotyl segments (apical, middle and basal portions) were transferred onto hormone-free medium for culture under different light qualities. Etiolated apical segments cultured in vitro under white light showed a very high frequency of regeneration for all of the genotypes tested besides phyB1phyB2, phyAphyB1 and phyAphyB1phyB2 mutants. Evidence is provided of a specific interference of phyB2 with phyA-mediated HIR to far-red and blue light in etiolated explants. Pre-treatment of donor plants by growth under red light enhanced the competence of phyB1phyB2, phyAphyB1 and phyAphyB1phyB2 mutants for shoot regeneration, whereas pre-irradiation with far-red light enhanced the frequency of regeneration only in the phyAphyB1 mutant. Multiple phytochromes are involved in red light- and far-red light-dependent acquisition of competence for shoot regeneration. The position of the segments along the hypocotyl influenced the role of the various phytochromes and the interactions between them. The culture of competent hypocotyl segments under red, far-red or blue light reduced the frequency of explants forming shoots compared to those cultured under white light, with different genotypes having different response patterns.Abbreviations HIR: High irradiance response - LFR: Low fluence response - Pfr: Far-red absorbing form of phytochrome - phyA: Phytochrome A - phyB1: Phytochrome B1 - phyB2: Phytochrome B2 - phyA(B1, B2): Phytochrome mutant deficient in phyA (B1, B2) - phyAphyB1(B1B2,AB2): Double phytochrome mutant deficient in phyA and phyB1(B1, B2) - phyAphyB1phyB2: Triple mutant deficient in phyA, phyB1 and phyB2 - VLFR: Very low fluence response - WT: Wild-type tomato Communicated by R. Reski     

Analysis of the phytochrome gene family in <Emphasis Type="Italic">Ceratodon purpureus</Emphasis> by gene targeting reveals the primary phytochrome responsible for photo- and polarotropism

Using gene targeting by homologous recombination in Ceratodon purpureus, we were able to knock out four phytochrome photoreceptor genes independently and to analyze their function with respect to red light dependent phototropism, polarotropism, and chlorophyll content. The strongest phenotype was found in knock-out lines of a newly described phytochrome gene termed CpPHY4 lacking photo- and polarotropic responses at moderate fluence rates. Eliminating the atypical phytochrome gene CpPHY1, which is the only known phytochrome-like gene containing a putative C-terminal tyrosine kinase-like domain, affects red light-induced chlorophyll accumulation. This result was surprising, since no light dependent function was ever allocated to this unusual gene. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Accession number for CpPHY4: EU122393.     

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.     

Fluence and wavelength requirements for Arabidopsis CAB gene induction by different phytochromes.

The roles of different phytochromes have been investigated in the photoinduction of several chlorophyll a/b-binding protein genes (CAB) of Arabidopsis thaliana. Etiolated seedlings of the wild type, a phytochrome A (PhyA) null mutant (phyA), a phytochrome B (PhyB) null mutant (phyB), and phyA/phyB double mutant were exposed to monochromatic light to address the questions of the fluence and wavelength requirements for CAB induction by different phytochromes. In the wild type and the phyB mutant, PhyA photoirreversibly induced CAB expression upon irradiation with very-low-fluence light of 350 to 750 nm. In contrast, using the phyA mutant, PhyB photoreversibly induced CAB expression with low-fluence red light. The threshold fluences of red light for PhyA- and PhyB-specific induction were about 10 nmol m-2 and 10 mumol m-2, respectively. In addition, CAB expression was photoreversibly induced with low-fluence red light in the phyA/phyB double mutant, revealing that another phytochrome(s) (PhyX) regulated CAB expression in a manner similar to PhyB. These data suggest that plants utilize different phytochromes to perceive light of varying wave-lengths and fluence, and begin to explain how plants respond so exquisitely to changing light in their environment.     

The serine-rich N-terminal region of <Emphasis Type="Italic">Arabidopsis</Emphasis> phytochrome A is required for protein stability

Deletion or substitution of the serine-rich N-terminal stretch of grass phytochrome A (phyA) has repeatedly been shown to yield a hyperactive photoreceptor when expressed under the control of a constitutive promoter in transgenic tobacco or Arabidopsis seedlings retaining their native phyA. These observations have lead to the proposal that the serine-rich region is involved in negative regulation of phyA signaling. To re-evaluate this conclusion in a more physiological context we produced transgenic Arabidopsis seedlings of the phyA-null background expressing Arabidopsis PHYA deleted in the sequence corresponding to amino acids 6–12, under the control of the native PHYA promoter. Compared to the transgenic seedlings expressing wild-type phyA, the seedlings bearing the mutated phyA showed normal responses to pulses of far-red (FR) light and impaired responses to continuous FR light. In yeast two-hybrid experiments, deleted phyA interacted normally with FHY1 and FHL, which are required for phyA accumulation in the nucleus. Immunoblot analysis showed reduced stability of deleted phyA under continuous red or FR light. The reduced physiological activity can therefore be accounted for by the enhanced destruction of the mutated phyA. These findings do not support the involvement of the serine-rich region in negative regulation but they are consistent with a recent report suggesting that phyA turnover is regulated by phosphorylation. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.