The involvement of phytochrome in controlling chlorophyll and 5-aminolevulinate formation in a gymnosperm seedling (Pinus sylvestris)

Chlorophyll a (Chl a) accumulation in the cotyledons of Scots pine seedlings (Pinus sylvestris L.) is much higher in the light than in darkness where it ceases 6 days after germination. When these darkgrown seedlings are treated with continuous white light (3,500 lx) a 3 h lag phase appears before Chl a accumulation is resumed. The lag phase can be eliminated by pretreating the seedlings with 7 h of weak red light (0.14 Wm^-2) or with 14 red light pulses separated by relatively short dark periods (<100 min). The effect of 15s red light pulses can be fully reversed by 1 min far-red light pulses. This reversibility is lost within 2 min. In addition, the amount of Chl a formed within 27 h of continuous red light is considerably reduced by the simultaneous application of far-red (RG 9) light. It is concluded that phytochrome (P_fr) is required not only for the elimination of the lagphase but also to maintain a high rate of Chl a accumulation in continuous light. Since accumulation of 5-aminolevulinate (ALA) responds in the same manner as Chl a accumulation to a red light pretreatment it is further concluded that ALA formation is the point where phytochrome regulates Chl biosynthesis in continuous light. No correlation has been found between ALA and Chl a formation in darkness. This indicates that in a darkgrown pine seedling ALA formation is not rate limiting for Chl a accumulation.Abbreviations Chl chlorophyll(ide) - PChl protochlorophyll(ide) - ALA 5-aminolevulinate - P_r the red absorbing form of phytochrome - P_fr the far-red absorbing form of phytochrome - P_tot total phytochrome ([P_r]+[P_fr])     

Osmotic adjustment in Spirulina platensis

Unilateral irradiation of maize (Zea mays L.) seedlings results in a fluence-rate gradient, and hence below saturation, a gradient of the far-red-absorbing form of phytochrome (Pfr). The Pfr-gradients established by blue, red and far-red light were spectrophotometrically measured in the mesocotyl. Based on these Pfr-gradients and the fluence-response curves of phytochrome photoconversion the fluence-rate gradients were calculated. The fluence-rate gradient in the blue (460 nm) was steeper than that in the red (665 nm), which in turn was steeper than that in the far-red light (725 nm). The fluence-rate ratios front to rear were 1:0.06 (460 nm), 1:0.2 (665 nm), and 1:0.33 (725 nm). The assumption that phytochrome-mediated phototropism of maize mesocotyls is caused by local phytochrome-mediated growth inhibition was tested in the following manner. Firstly, the Pfr response curve for growth inhibition was calculated; these calculations were based on measurements of Pfr-gradients and data from red-light-induced phototropism. Secondly, the Pfr response curve for growth inhibition was used as a basis for calculating fluence-response curves for blue-and far-red-light-induced phototropism. Finally, these calculated results were compared with experimental data. It was concluded that the threshold for phytochrome-mediated phototropism of maize mesocotyls reflects the apparent photoconversion cross section of phytochrome whereas the maximal inducable curvature depends on the steepness of the light (Pfr) gradient across the mesocotyl.Abbreviations Pfr far-red-absorbing form of phytochrome - Ptot total phytochrome - Fr far-red light     

Effects of light on phytochrome in cauliflower curd

The effect of light on the phytochrome content of cauliflower (Brassica oleracea (L.) var. botrytis) curd was studied using in vivo spectrophotometry. It was found that light caused a rapid increase in phytochrome level whereas transfer to darkness caused a rapid loss, regardless of the amount of phytochrome initially present in the far red absorbing form. The amount of phytochrome detectable during continuous irradiation appears to be related to the photoequilibrium , and is thus controlled by phytochrome itself.Abbreviation Pr and Pfr red and far red absorbing forms of phytochrome, respectively     

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.     

Monoclonal antibodies directed to phytochrome from green leaves of Avena sativa L. cross-react weakly or not at all with the phytochrome that is most abundant in etiolated shoots of the same species

Seven monoclonal antibodies (MAbs) have been prepared to phytochrome from green oat (Avena sativa L. cv. Garry) leaves. One of these MAbs (GO-1) cross-reacts with apoprotein of the phytochrome that is most abundant in etiolated oat shoots as assessed by immunoblot assay of fusion proteins expressed in Escherichia coli. The epitope for this MAb is located between amino acids 618 and 686 in the primary sequence of type 3 phytochrome (Hershey et al. 1985, Nucleic Acids Res. 13, 8543–8559), which is one of the predominant phytochromes in etiolated oats. Three other MAbs (GO-4, GO-5, GO-6) immunoprecipitate phytochrome isolated from green oat leaves, as evaluated by photoreversibility assay. GO-1, GO-4, GO-5 and GO-6 are therefore directed to phytochrome. While evidence obtained with the other three MAbs (GO-2, GO-7, GO-8) strongly indicates that they are also directed to phytochrome, this evidence is not as rigorous. Recognition of antigen by any of these seven MAbs is not significantly reduced by periodate oxidation, indicating that their epitopes probably do not include carbohydrate. All but GO-1 bind either very poorly or not at all the phytochrome that is abundant in etiolated oat shoots. These data reinforce earlier observations made with antibodies directed to phytochrome from etiolated oats, indicating (1) that the phytochromes that predominate in etiolated and green oats differ immunochemically and (2) that phytochrome preparations from green oat leaves contain very little of the phytochrome that is abundant in etiolated shoots. An hypothesis that these two immunochemically distinct phytochromes form heterodimers in vitroAbbreviations Da Dalton - DEAE diethylaminoethyl - ELISA enzyme-linked immunosorbent assay - HA hydroxyapatite - Ig immunoglobulin - MAb monoclonal antibody - SDS sodium dodecyl sulfate is supported by comparison of immunoblot data obtained with conventionally purified phytochrome from etiolated oats to that expressed as fusion protein in E. coli. This research was supported by the U.S. Department of Energy (contract DE-AC-09-81SR10925 to L.H.P.). We thank Dr. Lyle Crossland and Ms. Sue Kadwell for their assistance in the construction of the cDNA clones, and Dr. Gyorgy Bisztray for providing us with clone pCBP3712. Dr. Phillip Evans and Dr. Russell Malmberg kindly provided MAbs 4F3, 6F12 and 8C10, as well as a corresponding antigen preparation. The excellent technical assistance of Mrs. Donna Tucker and Mrs. Danielle Neal is gratefully acknowledged.     

Hormones are no causal links in phytochrome-mediated adventitious root formation in mustard seedlings (Sinapis alba L.)

The question of whether or not hormones are causal links in the realization of phytochrome control during photomorphogenesis was investigated using the phytochrome-dependent formation of adventitious roots in hypocotyl cuttings excised from mustard seedlings as a test system. Histological examination of regenerating rest seedlings revealed that phytochrome (operationally, continuous far-red light) mediates the de novo formation of root primordia in the pericycle region of the hypocotyl near the cutting surface withing 12–24 h after excision.Auxin (IAA), gibberellin (GA₃), Cytokinin (kinetin), abscisic acid (ABA), and ethylene had no promotive effect on primordium formation in dark-grown or far-red irradiated rest seedlings. Depending on concentration, the application of these hormones was either ineffective or inhibitory in the rooting response. It is concluded that phytochrome does not operate through changes of hormone (auxin, gibberellin, cytokinin, ABA, ethylene) levels.While externally applied ethylene had no specific effect on primordium formation, the number of primordia produced in darkness could be increased to the far-red light level by removing the endogenously formed ethylene. Since the stimulatory effect of light could not be related to a lower ethylene level, it is concluded that ethylene interferes with primordium formation by modulating the susceptibility of this process to phytochrome control. This ethylene effect takes place in a concentration range below the range that can be manipulated by external application of the hormone.Abbreviations ABA abscisic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - P_r P_fr red and far-red absorbing forms of phytochrome     

The phytochrome system in light-grown Zea mays L.

The phytochrome system is analyzed in light-grown maize (Zea mays L.) plants, which were prevented from greening by application of the herbicide SAN 9789. The dark kinetics of phytochrome are not different in the first, second or third leaf. It is concluded that in light-grown maize plants phytochrome levels are regulated by P_r formation and P_fr and P_r destruction, rather than by P_frP_r dark reversion. P_r undergoes destruction after it has been cycled through P_fr. The consequences of this P_r destruction on the phytochrome system are discussed.Abbreviations SAN 9789 4-chloro-5-(methylamino)-2-(,,-trifluoro-m-tolyl)-3(2H) pyridazinone - P_fr far-red absorbing form of phytochrome - P_r red absorbing form of phytochrome - P_tot P_fr+P_r     

Cross-linking Phytochrome to its Receptor in situ using Imidoesters

Phytochrome can be cross-linked to a particulate fraction usingimidoesters, namely dimethy adipimidate (DMA) and dimethyl suberimidate(DMS). DMS was more effective than DMA. Cross-linking of phytochrometo its in situ receptor effected by DMS occurred in red light-irradiatedcoleoptiles. If DMS cross-linking was carried out prior to redlight irradiation there was very little formation of particulatephytochrome. Phytochrome in the particulate fraction obtainedby in situ DMS cross-linking was totally resistant to the solubilizingeffect of washing with solutions of high salt concentrationand high pH and was indistinguishable spectro-scopically fromthe phytochrome in untreated coleoptiles. DMS cross-linkingof phytochrome to its assumed receptor in situ preferentiallyprotected it from destruction following red light irradiationand also prevented it from dissociating from its receptor followingR/FR¹ irradiation when incubated subsequently in the dark. Thesecharacteristics of phytochrome in DMS-treated coleoptiles matchedthose observed using glutaraldehyde as the cross-linking reagent.It is therefore concluded that earlier results obtained usingglutaraldehyde are not peculiar to that reagent and can be duplicatedreadily using more defined bifunctional cross-linkers.     

Partial purification and initial characterization of phytochrome from the mossAtrichum undulatum P. Beauv. grown in the light

The extraction and partial purification of phytochrome from light-grownAtrichum undulatum P. Beauv., a chlorophyllous moss, is described. Polyethyleneimine and salt fractionation followed by hydroxyapatite and Affi-gel-blue chromatography were used to separate phytochrome from chlorophyll, and to purify the pigment. All steps were performed in the presence of Triton X-100 which improved the yield by a factor of about three. The protein has a molecular weight some-what larger than that ofAvena phytochrome (124 kDa), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. It cross-reacts with a monoclonal antibody against phytochrome from etiolated corn (Zea) and a polyclonal antibody against phytochrome from etiolated oat (Avena), and its photoreversibility is similar to that of phytochrome from greenAvena.Abbreviations EDTA ethylenediaminetetraacetic acid - FMN flavinmononucleotide - PMSF phenylmethylsulfonylfluoride - P_r(P_fr) red(far-red)-absorbing form of phytochrome - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Light-induced changes in the photoresponses of plant stems the loss of a high irradiance response to far-red light

De-etiolation results in phytochrome destruction, greening, and the loss of the far-red high irradiance responses (HIR). Evidence is presented against the hypothesis that the loss of the far-red HIR is a direct consequence of phytochrome destruction. Loss of the far-red HIR for the inhibition of elongation in hypocotyls of Raphanus sativus involves two different, but linked, actions of phytochrome. An induction reaction requires the far-red absorbing form of phytochrome for about 20 min after which accumulation of its product depends only on time. A second reaction requires continuous light or frequent short irradiations and involves cycling of the phytochrome system. This acts on the product of the induction reaction. It is proposed that in green plants an important mode of operation of phytochrome in the light depends on pigment cycling, and that during de-etiolation this system is established under phytochrome control.Abbreviations HIR high irradiance response - R red - FR farred light - P_tot phytochrome, P_r its red absorbing form, P_fr its far-red absorbing form A.M. Jose was the holder of Ministry of Agriculture, Fisheries and Food award AE 6819     

LOCALIZATION OF PHYTOCHROME DURING PEANUT (ARACHIS HYPOGAEA) GYNOPHORE AND OVULE DEVELOPMENT

Previous studies indirectly indicated that phytochrome plays a role in peanut (Arachis hypogaea L. cv. Virginia) gynophore elongation and in ovule and embryo development. Recent advances in the use of monoclonal antibody procedures used in this study have allowed precise localization of phytochrome in the developing peanut gynophore and ovular tissues. Peanut phytochrome from etiolated tissues was found to have a molecular weight of 124 kD as determined by immunoblotting procedures using a monoclonal antibody to pea (Pisum sativum L. cv. Alaska) phytochrome. Immunoblotting procedures revealed that no detectable phytochrome was present in the gynophore tissues or immature ovules during the elongation of the peanut gynophores. After the gynophores penetrated the soil for 8–12 d, phytochrome was detected in increasing amounts in the ovular tissues but not the gynophore tissues. Immunohistological analysis revealed that phytochrome was localized in the developing embryo and adjacent integument tissues. These findings contradict earlier reports that suggested phytochrome was initially present in the gynophore tissues after fertilization where it was believed to inhibit ovular development and stimulate gynophore elongation.     

Photocontrol of stem elongation in light-grown plants of Fuchsia hybrida

Stems of the caulescent long-day plant, Fuchsia hybrida cv Lord Byron, showed 2 types of response to light. In one, internode length was increased by far-red irradiation given at the end of an 8 h photoperiod: the response was no greater with prolonged exposure and was less when the start of far-red was delayed. The effect of far-red was reversible by a subsequent exposure to red light. Internode length was inversely proportional to the P_fr/P ratio established before entry to darkness and there was no evidence for loss of P_fr during a 16 h dark period. The inhibitory effect of P_fr acted at a relatively late stage of internode growth. With the development of successive internodes a second response appeared in which stems lengthened following prolonged daily exposures to red or far-red light, or mixtures of the two, or to brief breaks with red or white light. In these later internodes, a short exposure to far-red near the middle of the night was not reversible by red because red alone promoted elongation at this time. Internode length increased with increase in the daily duration of light and, when light was given throughout an otherwise dark period of 16 h, with increase in illuminance to a saturation value of 200 lx from tungsten lamps. Elongation increased as a linear function of decrease in photostationary state of phytochrome down to P_fr/P0.3; however, internodes were shorter in far-red light than in 25% red/red+far-red. It was concluded that stem length is a net response to two modes of phytochrome action. An inductive effect of P_fr inhibits a late stage in internode expansion, and a phytochrome reaction which operates only in light (and may involve pigment cycling) promotes an early stage of internode development. Stem elongation is thus a function both of the daily duration of light and its red/red+far-red content. The outgrowth of axillary buds was controlled by the first type of phytochrome action only.Abbreviations and symbols FR far red light - R red light - P phytochrome - P_fr phytochrome in the far-red light absorbing form - SD 8 h short days - LDP long-day plant - SDP short-day plant     

Coaction of blue/ultraviolet-A light and light absorbed by phytochrome in controlling the appearance of ferredoxin-dependent glutamate synthase in the Scots pine (Pinus sylvestris L.) seedling

The appearance of NADH- and ferredoxin (Fd)-dependent glutamate synthases (GOGATs) was investigated in the major organs (roots, hypocotyl and cotyledonary whorl) of the Scots pine seedling. It was found that cytosolic NADH-GOGAT (EC 1.4.1.14) dropped to a low level during the experimental period (from 4 to 12 d after sowing) and was not significantly affected by light. On the other hand, plastidic Fd-GOGAT (EC 1.4.7.1) increased strongly in response to light. Whereas similar amounts of NADH-GOGAT were found in the different organs, Fd-GOGAT was mainly found in the cotyledons even in the presence of nitrate. Protein chromatography revealed only a single Fd-GOGAT peak. No isoforms were detected. Experiments to investigate regulation of the appearance of Fd-GOGAT in the cotyledonary whorl yielded the following results: (i) In darkness, neither nitrate (15 mM KNO₃) nor ammonium (15 mM NH₄Cl) had an effect on the appearance of Fd-GOGAT. In the light, nitrate stimulated Fd-GOGAT activity by 30% whereas ammonium had no effect. The major controlling factor is light. (ii) The action of long-term white light (100 W · m^–2) could be replaced quantitatively by blue light (B, 10 W · m^–2). Since the action of long-term far-red light was very weak, operation of the High Irradiance Reaction of phytochrome is excluded. On the other hand, light-pulse experiments with dark-grown seedlings showed the involvement of phytochrome. (iii) Red light, operating via phytochrome, could fully replace B, but only up to 10 d after sowing. Thereafter, there was an absolute requirement for B for a further increase in the enzyme level. It appears that the operation of phytochrome was replaced by the operation of cryptochrome (B/UV-A photoreceptor). (iv) However, dichromatic experiments (simultaneous treatment of the seedlings with two light beams to vary the level of the far-red-absorbing form of phytochrome (Pfr) in blue light) showed that B does not affect enzyme appearance if the Pfr level is low. It is concluded that B is required to maintain responsiveness of Fd-GOGAT synthesis to phytochrome (Pfr) beyond 10 d after sowing.Abbreviations and Symbols B blue light - c continuous - D darkness - Fd-GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1) - FR far-red light - HIR high-irradiance reaction of phytochrome - NADH-GOGAT nicotinamide-dinucleotide-dependent glutamate synthase (EC 1.4.1.14) - R red light - RG9 long-wavelength far-red light defined by the properties of the Schott glass filter (_RG9<0.01) - Pfr/Ptot far-red-absorbing form of phytochrome/total phytochrome, wavelength-dependent photoequilibrium of the phytochrome system Research supported by Deutsche Forschungsgemeinschaft (SFB 46 und Schwerpunkt Physiologie der Bäume). We thank E. Fernbach for his help with the dichromatic experiments.     

Induction versus modulation in phytochrome-regulated biochemical processes

The time course of appearance of competence towards phytochrome (P_fr) was studied in cotyledons of mustard (Sinapis alba L.) with regard to the light-mediated formation of anthocyanin (aglycone cyanidin) and NADP-dependent plastidal glyceraldehyde-3-phosphate dehydrogenase (GPD, EC 1.2.1.13). The experiments were performed to answer the following question: Does phytochrome act to turn responses on (induction), or — as an alternative — does phytochrome cause an amplification of processes already occurring in absolute darkness albeit at low rates once competence is reached (modulation)? The data show that in the case of GPD, phytochrome causes an amplification of the rate of synthesis once the competence point is reached at approximately 36 h after sowing at 25° C. In the case of anthocyanin, it was found that two distinct points of competence exist (26 h and 39 h after sowing, 25° C). In the case of ‘early anthocyanin’ (competence point at 26 h), synthesis does not occur in darkness without P_fr, while in the case of ‘late anthocyanin’ (competence point at 39 h), phytochrome causes an amplification of a process occurring in complete darkness albeit at a very low rate. It is concluded that in phytochrome-mediated photomorphogenesis, modulation as well as induction of biosynthetic processes plays a role.     

Time-dependent changes in the responsiveness to light of phytochrome-mediated anthocyanin synthesis

Abstract. It is well established that seedlings of mustard ( Sinapis alba L.) synthesize juvenile anthocyanin only if treated with light pulses or continuous light. The light effects are considered to be due to the operation of phytochrome. Here we show that the responsiveness of anthocyanin synthesis to a saturating red light pulse or to continuous far-red light varies as a function of time and is strongly influenced by a light pretreatment prior to competence. Competence appears approximately 25 h after sowing. The starting point of anthocyanin synthesis, which is 27 h after sowing, and the lag- phase of this response, which is 2 h, are not affected by light pretreatments prior to competence. It is concluded that quantitative interpretations of phytochrome responses based entirely on properties of phytochrome can no longer be considered adequate.     

Light quantity and quality interactions in the control of elongation growth in light-grown Chenopodium rubrum L. seedlings

The spectral control of hypocotyl elongation in light-grown Chenopodium rubrum L. seedlings has been studied. The results showed that although the seedlings responded to changes in the quantity of combined red and far-red radiation, they were also very sensitive to changes in the quantity of blue radiation reaching the plant. Altering the proportion of red: far-red radiation in broad waveband white light caused marked differences in hypocotyl extension. Comparison of the responses of green and chlorophyll-free seedlings indicated no qualitative difference in the response to any of the light sources used, although photosynthetically incompetent plants were more sensitive to all wavelengths. Blue light was found to act primarily of a photoreceptor which is different from phytochrome. It is concluded that hypocotyl extension rate in vegetation shade is photoregulated by the quantity of blue light and the proportion of red: far-red radiation. In neutral shade, such as that caused by stones or overlying soil, hypocotyl extension appears to be regulated primarily by the quantity of light in the blue waveband and secondarily by the quantity of light in the red and far-red wavebands.Abbreviations B blue - FR far-red - k ₁, k ₂ rate constants for photoconverison of Pr to Pfr and Pfr to Pr, respective - k ₁/k ₁ +k ₂= phytochrome photoequilibrium - k ₁ +k ₂= phytochrome cycling rate - Pr=R absorbing form of phytochrome - Pfr=FR absorbing form of phytochrome - P_tot Pr+Pfr - PAR photosynthetically active radiation = 400–700 nm - R red - WL white light     

Light-induced linear dichroism in photoreversibly photochromic sensor pigments. – V. Reinterpretation of the experiments on in vivo action dichroism of phytochrome

Experiments by several authors on the effects of polarized light on phytochromemediated responses in fern gametophytes and in the green alga Mougeotia have earlier been interpreted as showing that the transition moment of phytochrome in the P_r form is parallel to the plasmalemma, but perpendicular to the plasmalemma for the P_fr form of phytochrome. It is now shown that the experimental results can be interpreted differently, and that they are also consistent with a chromophore rotation of about 30° (instead of 90°), as found for immobilized phytochrome molecules in vitro. Thus there is no evidence for a rotation of the whole phytochrome protein. For the gametophyte of Adiantum it is calculated that the P_r transition moment is inclined 17° to the plasmalemma, and the P_fr transition moment ca 50°, corresponding to an in vivo chromophore rotation of ca 33°; however, these values are very approximate.     

Phytochrome — all regions marked by a set of monoclonal antibodies reflect conformational changes

Monoclonal antibodies to defined locations on six regions of the phytochrome molecule (from Avena sativa L. or Zea mays L.) were each found to have a different affinity toward the farred-absorbing form of phytochrome (Pfr) and the red-absorbing form (Pr). The differences were small, but were consistently shown by antibodies which bind to the vicinity of the aminoterminus, the carboxylterminus and to sequences in between. It seems that the conformational differences between Pr and Pfr extend over the whole molecule in as far as it is represented by these regions and the antibodies binding to them.Abbreviations Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome