Phytochrome-mediated branch formation in protonemata of the mossCeratodon purpureus

We have analyzed light induction of side-branch formation and chloroplast re-arrangement in protonemata of the mossCeratodon purpureus. After 12 hr of dark adaptation, the rate of branch formation was as low as 5%. A red light treatment induced formation of side branches up to 75% of the dark-adapted protonema. The frequency of light induced branch formation differed between cells of different ages, the highest frequency being found in the 5th cell, the most distal cell studied from the apex. We examined the effect of polarized light given parallel to the direction of filament growth. The position of branching within the cell depended on the vibration plane of polarized red light. Branch formation was highest when the electric vector of polarized light vibrates parallel to the cell surface and is fluence rate dependent. The positional effect of polarized red light could be nullified to some extent by simultaneous irradiation with polarized far-red light. An aphototropic mutant,ptr116, shows characteristics of deficiency in biosynthesis of the phytochrome chromophore and exhibits no red-light induced branch formation. Biliverdin, a precursor of the phytochrome chromophore, rescued the red-light induced branching when added to the medium, supporting the conclusion that phytochrome acts as photoreceptor for red light induced branch formation. The light effect on chloroplast re-arrangement was also analyzed in this study. We found that polarized blue light induced chloroplast re-arrangement in wild-type cells, whereas polarized red light was inactive. This result suggests that chloroplast re-arrangement is only controlled by a blue light photoreceptor, not by phytochrome inCeratodon.     

Phytochrome and UV signal transduction pathways

The phytochromes are the best studied plant photoreceptors, controlling a wide variety of responses at both whole plant and single cell levels. Three signal transduction pathways, dependent on cGMP and/or calcium, have been found to be utilized by phytochrome to control the expression of genes required for chloroplast development (e.g., CAB and FNR) and anthocyanin biosynthesis (e.g., CHS). In particular, cGMP is a second messenger positively regulating CHS gene expression whilst calcium and calmodulin act as negative regulators. In addition to phytochrome regulation of CHS we have begun to examine the signal transduction pathways utilized by UV photoreceptors. In contrast to phytochrome-mediated responses, results indicate a role for calcium and calmodulin as positive regulators of CHS gene expression in UV light.     

Blue- and red-light action in photoorientation of chloroplasts in Adiantum protonemata

An action spectrum for the low-fluencerate response of chloroplast movement in protonemata of the fern Adiantum capillus-veneris L. was determined using polarized light vibrating perpendicularly to the protonema axis. The spectrum had several peaks in the blue region around 450 nm and one in the red region at 680 nm, the blue peaks being higher than the red one. The red-light action was suppressed by nonpolarized far-red light given simultaneously or alternately, whereas the bluelight action was not. Chloroplast movement was also induced by a local irradiation with a narrow beam of monochromatic light. A beam of blue light at low energy fluence rates (7.3·10^-3-1.0 W m^-2) caused movement of the chloroplasts to the beam area (positive response), while one at high fluence rates (10 W m^-2 and higher) caused movement to outside of the beam area (negative response). A red beam caused a positive response at fluence rates up to 100 W m^-2, but a negative response at very high fluence rates (230 and 470 W m^-2). When a far-red beam was combined with total background irradiation with red light at fluence rates causing a low-fluence-rate response in whole cells, chloroplasts moved out of the beam area. When blue light was used as background irradiation, however, a narrow far-red beam had no effect on chloroplast distribution. These results indicate that the light-oriented movement of Adiantum chloroplasts is caused by red and blue light, mediated by phytochrome and another, unidentified photoreceptor(s), respectively. This movement depends on a local gradient of the far-red-absorbing form of phytochrome or of a photoexcited blue-light photoreceptor, and it includes positive and negative responses for both red and blue light.Abbreviations BL blue light - FR far-red light - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light - UV ultraviolet     

Phytochrome-mediated control of grana and stroma thylakoid formation in plastids of mustard cotyledons

The etioplast»chloroplast transition in the cotyledons of mustard seedlings (Sinapis alba L.) has been studied by electron microscopy. It was found that the active form of phytochrome, established by a red light pulse pretreatment, increases the initial rate and eliminates the lag of grana and stroma thylakoid formation after the onset of white light 60 h after sowing. The effect of a pretreatment with 15 s red light pulses is fully reversible by 756 nm light pulses. This reversibility is lost within 5 min. Evidence is presented which suggests that the time course of grana and stroma thylakoid formation is not correlated with the time course of the dispersal of the prolamellar body. The different functions of phytochrome and chlorophyll in controlling thylakoid formation are discussed.     

Chloroplast movement in Mougeotia induced by blue light pulses

The profile-to-face chloroplast movement in the green alga Mougeotia has been induced by strong blue and near-ultraviolet light pulses (6 J m^-2). Simultaneously, strong red or far-red light (10 W m^-2) was applied perpendicularly to the inducing beam. The response was measured photometrically. Against the far-red background the reciprocity law was found to hold for pulse durations varying two orders of magnitude. The action spectrum exhibited a maximum near 450 nm and a distinct increase in near-ultraviolet. The time-course and the spectral dependence of pulse responses of chloroplasts in Mougeotia were similar to those recorded for other plants which are sensitive only to blue. This points to an alternative sensor system active in the short-wavelength region in addition to the phytochrome system.Abbreviations FR far-red light - Pr red absorbing form of phytochrome - Pfr far-red absorbing form of phytochrome - R red light This paper is dedicated to the memory of Professor Jan Zurzycki     

Light requirement,phytochrome and photoperiodic induction of flowering of Pharbits nil Chois

The low chlorophyll content of cotyledons of Pharbitis nil grown for 24 h in far-red light (FR) or at 18° C in white light from fluorescent lamps (WL) allows spectrophotometric measurement of phytochrome in these tissues. The (A) measurements utilize measuring beams at 730/802 nm and an actinic irradiation in excess of 90 s. The constancy of the relationship between phytochrome content and sample thickness confirms that, under these conditions of measurement, a true maximum phytochrome signal was obtained. These techniques have been used to follow changes in the form and amount of phytochrome during an inductive dark period for flowering. Following exposure to 24h WL at 18° C with a terminal 10 min red (R), P_fr was lost rapidly in darkness and approached zero in less than 1 h; during this period there was no change in the total phytochrome signal. Following exposure to 24 h FR with a terminal 10 min R, P_fr approached zero in 3 h, and the total phytochrome signal decreased by about half. The relevance of these changes to photoperiodic time measurement is discussed.Abbreviations BCJ irradiation from photographic ruby-red lamps - FR far-red light - P_fr far-red-absorbing form of phytochrome - P_r red-absorbing form of phytochrome - P total phytochrome content - R red light - WL white light from fluorescent lamps     

fhy1 defines a branch point in phytochrome A signal transduction pathways for gene expression

Physiological analysis of the fhy1 mutant of Arabidopsis has led to the proposal that the mutant is deficient in a downstream component of the phytochrome A signal transduction pathway. To define this lesion at the molecular level, we have examined the expression of a range of phytochrome-regulated genes in fhy1. In far-red light, the regulation of genes such as CHS and CHI is blocked in fhy1, whereas the induction of CAB and NR genes is affected minimally. In contrast, the induction of all genes tested is blocked in a phytochrome A-deficient mutant, confirming that gene expression in far-red light is regulated solely by phytochrome A. Thus, fhy1 defines a branch point in phytochrome A signal transduction pathways for gene expression. Contrary to the general opinion that responses to continuous red light are mediated by phytochrome B and other photostable phytochromes, we have shown also that red light-induction of CHS is mediated almost entirely by phytochrome A. Furthermore, phytochrome A-mediated induction of CHS by red light is blocked in fhy1. The induction of CHS by blue light, however, is normal in fhy1, suggesting that although FHY1 is a component of the phytochrome A signaling pathway, it is not a component of the blue-light signaling pathway for CHS expression.     

Analysis of light-controlled accumulation of carotenoids in mustard (Sinapis alba L.) seedlings

Carotenoid accumulation in the cotyledons of the mustard seedling (Sinapis alba L.) is controlled by light. Besides the stimulatory function of phytochrome in carotenogenesis the experiments reveal the significance of chlorophyll accumulation for the accumulation of larger amounts of acrotenoids. A specific blue light effect was not found. The data suggest that light exerts its control over carotenoid biogenesis through two separate mechanisms: A phytochrome regulation of enzyme levels before a postulated pool of free carotenoids, and a regulation by chlorophyll draining the pool by complex-formation.Abbreviations Chl chlorophyll(s) - PChl protochlorophyll(ide) - HIR high irradiance reaction (of phytochrome) - P_fr far-red absorbing, physiologically active form of phytochrome - P_r red absorbing, physiologically inactive form of phytochrome - P_fof total phytochrome, i.e. [P_r]+[P_fr] - [P_fr]/[P_fof], wavelength dependent photoequilibrium of the phytochrome system - red red light - fr far-red light     

Involvement of phytochrome and a blue light photoreceptor in UV-B induced flavonoid synthesis in parsley (Petroselinum hortense Hoffm.) cell suspension cultures

Flavonoid synthesis in cell suspension cultures of parsley (Petroselinum hortense Hoffm.) occurs only after irradiation with ultraviolet light (UV), mainly from the UV-B (280–320 nm) spectral range. However, it is also controlled by phytochrome. A P_fr/P_tot ratio of approximately 20% is sufficient for a maximum phytochrome response as induced by pulse irradiation. Continuous red and far red light, as well as blue light, given after UV, are more effective than pulse irradiations. The response to blue light is considerably greater than that to red and far red light. Continuous red and blue light treatments can be substituted for by multiple pulses and can thus probably be ascribed to a multible induction effect. Continuous irradiations with red, far red and blue light also increase the UV-induced flavonoid synthesis if given before UV. The data indicate that besides phytochrome a separate blue light photoreceptor is involved in the regulation of the UV-induced flavonoid synthesis. This blue light receptor seems to require the presence of P_fr in order to be fully effective.Abbreviations HIR high irradiance response - P_fr far red absorhing form of phytochrome - P_tet total phytochrome - UV ultraviolet light     

Zur Phototaxis von Mougeotia (Mesocarpus)

Summary Mosebach undertook a quantitative study of the light intensity which changes the positive phototaxis of theMougeotia chloroplast into a negative one (this intensity is the so-called, Umschlagspunkt = UP). The UP has no definite value but depends upon the pre-treatment of the plant. The UP is lowered by pre-darkening and is raised by a few hours or even less of pre-illumination.This light effect is a double one: a) the UP depends on the concentration of CO₂ in the medium, which of course is influenced by the ratio of respiration and photosynthesis, and b) in running-water experiments there still exists an influence of the pre-illumination on the UP, which must therefore be a direct light effect and which, it is suggested, is an adaptation.The CO₂-sensitivity of the UP is not an unspecific p_H effect and cannot be duplicated by citric, oxalic or hydrochloric acid. Only acetic acid has a similar effect.The cases in which the chloroplast is found to be oblique to the light beam are discussed from a new point of view.This summary is only a very short review by the editor; for further information compare the German summary by the author, page 41.

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Mit einem Nachwort vonW. Haupt

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Vor seinem letzten Abschied legte mir mein Mann die Manuskripte zweier Arbeiten in die Hand mit der Bitte, wenn nötig, statt seiner für ihre Veröffentlichung zu sorgen. Die erste Arbeit (Über die Polarisierung derEquisetum-Spore durch das Licht, Planta33, 1943) war so gut wie druckfertig. Die zweite, hier vorliegende Arbeit war vonGeorg Mosebach in dieser Fassung noch nicht für die Veröffentlichung bestimmt; trotzdem ist sie nun doch in der ursprünglichen Form erschienen — abgesehen von wenigen oder unbedeutenden Änderungen, die Herr ProfessorBuder und Herr ProfessorRuhland, Leipzig, die Güte hatten vorzunehmen. Herrn Professor Dr.J. Buder und Herrn Professor Dr.W. Ruhland spreche ich auch an dieser Stelle meinen Dank aus für alle selbstlose Hilfe, die mir bei meiner Aufgabe zuteil wurde.Erna Mosebach, 1944.     

Arabidopsis cue mutants with defective plastids are impaired primarily in the photocontrol of expression of photosynthesis-associated nuclear genes

Plant photoreceptors detect light cues and initiate responses ranging from chloroplast differentiation to the control of morphogenesis and flowering. The photocontrol of photosynthesis-related nuclear genes appears closely related to retrograde plastid signals by which the status of the organelle controls the expression of nuclear genes. However, what specific role, if any, plastid-originated signals play in light responses is poorly understood: it has in the past been proposed that plastid signals play a role in all responses to high fluence far-red light perceived by the light-labile phytochrome A, irrespective of whether they involve photosynthesis-related genes. To explore this further, we have re-examined the phenotype of three cue (cab-underexpressed) Arabidopsis mutants, defective in chloroplast development. The mutants have underdeveloped etioplasts, with increasing impairments in cue6, cue8 and cue3. The mutants show only small defects in photocontrol of hypocotyl elongation and cotyledon opening under prolonged far-red or red light, and normal photocontrol under blue. On the other hand, the expression of photosynthesis-associated nuclear genes is much more impaired in the mutants in the dark and following red or far-red light short treatments or continuous light, than that of those phytochrome-dependent genes tested which are not associated with photosynthesis. Furthermore, red/far-red photoreversible responses involving photosynthesis-related genes (induction of Lhcb1–cab promoter activity, and photoreversible extent of greening) mediated by phytochrome B and other photo-stable phytochromes, both show a reduction in the cue mutants, which correlates with the etioplast defect. Our evidence demonstrates that plastid-derived signals need to be operational in order for the phytochrome control of photosynthetic nuclear genes to occur.     

Mapping the interaction sites of <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> phytochrome FphA with the global regulator VeA and the White Collar protein LreB

Aspergillus nidulans senses red and blue-light and employs a phytochrome and a Neurospora crassa White Collar (WC) homologous system for light perception and transmits this information into developmental decisions. Under light conditions it undergoes asexual development and in the dark it develops sexually. The phytochrome FphA consists of a light sensory domain and a signal output domain, consisting of a histidine kinase and a response regulator domain. Previously it was shown that the phytochrome FphA directly interacts with the WC-2 homologue, LreB and another regulator, VeA. In this paper we mapped the interaction of FphA with LreB to the histidine kinase and the response regulator domain at the C-terminus in vivo using the bimolecular fluorescence complementation assay and in vitro by co-immunoprecipitation. In comparison, VeA interacted with FphA only at the histidine kinase domain. We present evidence that VeA occurs as a phosphorylated and a non-phosphorylated form in the cell. The phosphorylation status of the protein was independent of the light receptors FphA, LreB and the WC-1 homologue LreA.     

Visualization of a rapid,red/far-red light-dependent reaction by centrifuge microscopy

Summary Using a centrifuge microscope with stroboscopic illumination, we examined the effects of light irradiation on the passive movement of chloroplasts in dark-adapted mesophyll cells ofVallisneria gigantea. While irradiation with red light accelerates the passive gliding of chloroplasts produced by centrifugal force, irradiation with far-red light negates this effect. Irradiation with blue light does not accelerate the passive gliding, while red light is completely effective even in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosynthesis. An apparently active movement of chloroplasts can be induced by irradiation with red or blue light only in the presence of the far-red light-absorbing form of phytochrome. The significance of the reaction in the light with respect to the regulation of cytoplasmic streaming is discussed.Abbreviations APW artificial pond water - CMS centrifuge microscope of the stroboscopic type - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Pfr phytochrome, far-red light-absorbing form - Pr phytochrome, red light-absorbing form     

Phytochrome- and blue-light-absorbing pigment-mediated directional movement of chloroplasts in dark-adapted prothallial cells of fernAdiantum as analyzed by microbeam irradiation

When prothalli ofAdiantum capillus-veneris L. were kept for 2 d in the dark, chloroplasts gathered along the anticlinal walls (Kagawa and Wada, 1994, J Plant Res 107: 389–398). In these dark-adapted prothallial cells, irradiation with a microbeam (10 gm in diameter) of red (R) or blue light (B) for 60 s moved the chloroplasts towards the irradiated locus during a subsequent dark period. Chloroplasts located less than 20 gm from the center of the R microbeam (18 J·m^–2) moved towards the irradiated locus. The higher the fluence of the light, the greater the distance from which chloroplasts could be attracted. The B microbeam was less effective than the R microbeam. Chloroplasts started to move anytime up to 20 min after the R stimulus, but with the B microbeam the effect of the stimulus was usually apparent within 10 min after irradiation. The velocity of chloroplast migration was independent of light-fluence in both R and B and was about - 0.3 m·min^–1 between 15 min and 30 min after irradiation. Whole-cell irradiation with far-red light immediately after R- and B-microbeam irradiations demonstrated that these responses were mediated by phytochrome and a blue-light-absorbing pigment, respectively. Sequential treatment with R and B microbeams, whose fluence rates were less than the threshold values when applied separately, resulted in an additive effect and induced chloroplast movement, strongly suggesting that signals from phytochrome and the blue-light-absorbing pigment could interact at some point before the induction of chloroplast movement.Abbreviations B blue light - FR far-red light - IR infrared light - R red light     

Signal storage in phytochrome action on nitrate-mediated induction of nitrate and nitrite reductases in mustard seedling cotyledons

Application of nitrate leads to an induction of nitrate reductase (NR; EC 1.6.6.1) and nitrite reductase (NIR; EC 1.7.7.1) in the cotyledons of dark-grown mustard (Sinapis alba L.) seedlings, and this induction can strongly be promoted by a far-red-light pretreatment — operating through phytochrome — prior to nitrate application. This light treatment is almost ineffective — as far as enzyme appearance is concerned — if no nitrate is given. When nitrate is applied, the stored light signal potentiates the appearance of NR and NIR in darkness, even in the absence of active phytochrome, to the same extent as continuous far-red light. This action of previously stored light signal lasts for approx. 12 h.Storage of the light signal was measured for NR and NIR. The process shows enzyme-specific differences. Storage occurs in the absence as well as in the presence of nitrate, i.e. irrespective of whether or not enzyme synthesis takes place. The kinetics of signal transduction and signal storage indicate that the formation and action of the stored signal are a bypass to the process of direct signal transduction. Signal storage is possibly a means of enabling the plant to maintain the appropriate levels of NR and NIR during the dark period of the natural light/dark cycle.Abbreviations cD continuous darkness - cFR continuous far-red light - D darkness - FR far-red light - NIR nitrite reductase (EC 1.7.7.1) - NR nitrate reductase (EC 1.6.6.1) - Pfr phytochrome (far-red absorbing) - Pr phytochrome (red absorbing) - R red light - RG9-light long wavelength far-red light obtained with RG9 glass filter - - Ptot total phytochrome (Pr+Pfr) Professor Wilhelm Nultsch mit guten Wünschen zum 60. Geburtstag     

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)     

Guanosine triphosphate-binding proteins on the plasmalemma of spinach leaf cells

The molecular mechanism of light perception through phytochrome is not well understood. This red-light photosensor has been implicated in various physiological processes, including the photoinduction of flowering. A few recent studies have shown that phytochrome initiates signal transduction chains via guanosine triphosphate (GTP)-binding proteins (G-proteins). We show here by different approaches that G-proteins exist in spinach (Spinacia oleracea L. cv. Nobel). Binding of GTP on the plasmalemma has been partially characterized and its possible regulation by red light examined by in-vitro assays. These experiments indicate a clear regulation of GTP binding by red light and also by Mastoparan. At least three G-proteins or protein subunits were found to be associated with the plasmalemma of leaf cells. The use of an antibody raised against an animal Gβ subunit confirmed the presence of heterotrimeric G-proteins. Separation of a crude membrane extract by free-flow electrophoresis also showed that some G-proteins could exist on the tonoplast.     

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     

The GRAS protein SCL13 is a positive regulator of phytochrome-dependent red light signaling, but can also modulate phytochrome A responses

Phytochrome photoreceptors enable plants to perceive divergent light signals leading to adaptive changes in response to differing environmental conditions. However, the mechanism of light signal transduction is not fully understood. Here we report the identification of a new signaling intermediate from Arabidopsis thaliana, Scarecrow-like (SCL)13, which serves as a positive regulator of continuous red light signals downstream of phytochrome B (phyB). SCL13 antisense lines exhibit reduced sensitivity towards red light, but only a distinct subset of phyB-mediated responses is affected, indicating that SCL13 executes its major role in hypocotyl elongation during de-etiolation. Genetic evidence suggests that SCL13 is also needed to modulate phytochrome A (phyA) signal transduction in a phyB-independent way. The SCL13 protein is localized in the cytoplasm, but can also be detected in the nucleus. Overexpression of both a nuclear and cytoplasmic localized SCL13 protein leads to a hypersensitive phenotype under red light indicating that SCL13 is biologically active in both compartments. SCL13 is a member of the plant-specific GRAS protein family, which is involved in various different developmental and signaling pathways. A previously identified phytochrome A signaling intermediate, PAT1, belongs to the same subbranch of GRAS proteins as SCL13. Although both proteins are involved in phytochrome signaling, each is specific for a different light condition and regulates a different subset of responses.