Plant Photoreceptors Research Articles

Immunogold electron microscopy of phytochrome in Avena: identification of intracellular sites responsible for phytochrome sequestering and enhanced pelletability.

Using monoclonal antibodies to the plant photoreceptor, phytochrome, we have investigated by immunogold electron microscopy the rapid, red light-induced, intracellular redistribution (termed "sequestering") of phytochrome in dark-grown Avena coleoptiles. Pre-embedding immunolabeling of 5-micron-thick cryosections reveals that sequestered phytochrome is associated with numerous, discrete structures of similar morphology. Specific labeling of these structures was also achieved by post-embedding ("on-grid") immunostaining of LR-White-embedded tissue, regardless of whether the tissue had been fixed chemically or by freeze substitution. The phytochrome-associated structures are globular to oval in shape, 200-400 nm in size, and are composed of amorphous, granular material. No morphologically identifiable membranes are present either surrounding or within these structures, which are often present as apparent aggregates that approach several micrometers in size. An immunogold labeling procedure has also been developed to identify the particulate, subcellular component with which phytochrome is associated in vitro as a consequence of irradiation of Avena coleoptiles before their homogenization. Structures with appearance similar to those identified in situ are the only components of the pelletable material that are specifically labeled with gold. We conclude that the association of phytochrome with these structures in Avena represents the underlying molecular event that ultimately is expressed both as red light-induced sequestering in vivo and enhanced pelletability of phytochrome detected in vitro.

Photoperiodic time signals during twilight

Abstract. Although daylength has a major effect on flowering and several other aspects of plant development, the actual environmental time signals for the beginning and the end of day are obscure. An intensive spectroradiometric study was carried out in three contrasting environments: namely, unshaded sites, a mature oak woodland and a sugar beet crop. Spectral photon distributions were obtained describing numerous twilight phases and intervening photoperiods throughout the year. From each, absolute photon fluence rates, photon fluence rate ratios and phytochrome photoequilibria were calculated. Although substantial changes in spectral composition occurred during twilight, they were less capable of providing reliable and accurate time signals than the absolute fluence rate; this was especially apparent beneath the canopies. Thus, spectral changes are unlikely to be valuable in photoperiodic perception. The results are discussed in relation to the possible involvement of the known plant photoreceptors in photoperiodism.

Blue-light-absorbing photoreceptors in plants

Evidence is presented that more than one blue-light photoreceptor plays a role in morphogenesis, and that there are at least three, distinguishable on the basis both of action spectra and other criteria, which may be found both in green plants and fungi. One of these has been tentatively identified as a flavoprotein-cytochrome complex, most probably located in the plasma membrane. Studies with oat seedlings suggest that it may be involved in photoreception for phototropism, at least for the first positive curvature response. Both photoreduction of the cytochrome, via excitation of the flavin, and phototropic sensitivity in the first positive curvature range are similarly affected by diphenylether herbicides. The second class of photoreceptors can be distinguished from the first inNeurosporaby both genetic and physiological evidence, as well as by the action spectrum. It could be either flavin or carotenoid, although a different moiety is not excluded. The third class, distinguished only by action spectroscopy, shows a single sharp action peak near 475 mm, and seems unlikely to be either a flavin or a carotenoid, though they are not rigorously excluded. The first positive phototropic curvature response in maize shows a redistribution of growth consonant with the Cholodny-Went hypothesis for tropic responses, with an increase in the growth rate of the shaded side over dark controls, a concomitant decrease on the illuminated side, and no net change in overall growth rate.

Do plant photoreceptors act at the membrane level?

All of the photoreceptors involved in the absorption and transduction of light energy in photosynthesis are integral (carotenoid, chlorophyll) or peripheral (phycobilin) membrane proteins. The informational photoreceptors (phytochrome) and the flavoprotein (carotenoprotein?) cryptochrome, could be integral (carotenoprotein, flavoprotein) or peripheral or soluble (phytochrome, flavoprotein) pigment-protein complexes. The primary activity of the informational photoreceptors is unlikely to involve energization of primary active transport: the solute fluxes produced in this way would not form a quantitatively significant link in the perception-transduction-response sequence. By contrast, regulation of mediated solute fluxes at the plasmalemma could effect a substantial amplification of the absorbed photon signal, i.e. a large change in moles of solute transported could result from the absorption of 1 mol of photons. Modulation of the passive influx (or active efflux) of protons or calcium ions at the plasmalemma are likely targets for regulation by photoreceptors. Calcium flux regulation is particularly attractive in view of the ubiquity of calmodulin activity in eukaryotes, although problems could arise in maintaining the uniqueness of phytochrome messages vis-à-vis cryptochrome messages. Temporal analysis of the relation between photoreceptor changes and electrical effects resulting from changes in ion fluxes cannot, in general, rule out the involvement of intermediates between the redox or conformational change in the photoreceptor and the observed change in ion flux. Although slow in terms of the potential rate of change on solute fluxes resulting from direct interaction of a photoreceptor and a solute porter, the observed rates of signal transduction are well in excess of any obvious ‘need’ on the part of the plant in terms of rates of response to environmental changes.

THE USE OF A COMPUTERIZED SPECTRORADIOMETER TO PREDICT PHYTOCHROME PHOTOEQUILIBRIA UNDER POLYCHROMATIC IRRADIATION

Abstract A method is described for predicting the effect of polychromatic irradiation upon the photo‐stationary equilibrium of the plant photoreceptor phytochrome. This method follows from the rate equations for phototransformation and utilizes the in vivo action spectra for phytochrome phototransformation (Pratt and Briggs, 1966). A scanning spectroradiometer interfaced with a microcomputer is used to determine a spectral photon distribution from 360 to 800 nm. The products of the photon fluence rate and the relative quantum efficiencies at 2‐nm intervals are summed over the entire visible range to yield a predicted percentage of the pigment in the Pfr form. This value was determined under eight different polychromatic light sources and was generally within 7% Pfr of the value measured in etiolated corn coleoptiles under the same light sources.

Binding properties of the plant photoreceptor phytochrome to membranes.

AbstractPhytochrome (P), a chromoprotein of 120,000 MW, occurs at low concentrations in all higher plants. The chromophore is an open tetrapyrrole. The pigment exists in two light‐absorbing forms: Pr, which absorbs at 660 nm, and Pfr, which absorbs at 730 nm. These forms are interconvertible by light. Pr, the physiologically inactive form, exists in dark‐grown plants; Pfr, the active form, appears after irradiation with red light, P‐mediated responses, of which about 80 are known, range from short‐time effects (sec) such as bioelectric potentials, to long‐time effects (hr) such as increases in enzymatic activity. Measurements of phototransformation in vivo with polarized light suggest that P is localized in the plasma membrane. Particulate cell fractions contain about 70% of total extractable P if Pfr is present and only 4% if Pr is present. Evidence indicates that the fraction containing Pfr may be the plasma membrane. One can isolate a partially solubilized membrane system, which can be reversibly reconstituted by adding Mg. The reformed vesicles bind Pfr in vitro. Pfr binding increases with decreasing pH and decreases with increasing monovalent cation concentration. Pfr is released from the membrane by far red light (Pr is formed) and by Triton X‐100. We suggest that Pfr binding to a membrane induces conformational changes; the functional properties of this membrane are altered, which might lead to the observed phytochrome‐mediated responses.

Chlorophyll interactions and light conversion in photosynthesis.

Chlorophyll a is a molecule with an unusual combination of electron donor-acceptor properties. The Ring V keto C=O group can function as donor, the central Mg atom as acceptor. In the absence of extraneous nucleophiles, donor-acceptor interactions form chlorophyll dimers, (Chl.,), and oligomers, (Chl2),. With monofunctional electron donors, monomeric chlorophyll species Chl �9 L 1 and Chl �9 L 2 form. Bifunctional donors may cross-link chlorophylls through Mg atoms to form large polynuclear adduets of colloidal dimensions. Examination of the visible absorption spectra by computer deconvolution techniques indicates considerable similarity between bulk or antenna chlorophyll in the plant and (Chl2)n. Electron spin resonance studies suggest that ESR photo-signal I associated with the photosynthetic reaction center of photosynthetic organisms could arise in a special pair of chlorophyll molecules (Chl H20 Chl)- +. Introduclion There is universal agreement that chlorophyll is the primary photo-receptor in plants, but the details of the mechanisms whereby the energy of light quanta are made available for subsequent chemical purposes remain for the most part to be established. The term "chlorophyll" is generic for a small group of closely related substances invariably present in all organisms that carry out oxidation-reduction reactions with energy supplied by light. The principal chlorophylls are chlorophyll a (Chl a) and b (green plants, algae), bacteriochlorophyll (BChl, purple photosynthetic bacteria), and chlorobium chlorophyll (green photosynthetic bacteria). Chlorophyll q and c~ It, 2] (diatoms, marine brown algae) and chlorophyll d (marine red algae) are minor or auxiliary chlorophylls. Chlorophyll a is without exception present in all organisms that carry out photosynthesis with the evolution of molecular oxygen. As the most widely distributed and most important of the chlorophylls, the discussion here will for the most part concern chlorophyll a. The chlorophylls are cyclic tetrapyrroles (Fig. t) and thus belong to the family of porphyrin compounds, which have a very important role in biology as active participants in respiratory pigments, electron transport agents, and oxidative enzymes. The chlorophylls, however, differ in some important respects from the true porphyrins. Although the methyl, ethyl, vinyl and propionic acid side-chains in the chlorophylls are characteristic of porphyrins, the alicyclic Ring V, which contains a keto C=O function at position C-9, is unique to the chlorophylls. The

The Structure of the Chloroplast

Considerable research effort has recently been directed to elucidating the structure of the chloroplast-the site of photosynthesis in plant cells. In 1952 Weier & Stocking (1) reviewed the structure of the chloroplast together with its biochemical and genetic aspects as it had been studied during the previous ten years. They concluded that not only was there a lack of neces­ sary experimental data, but also considerable confusion in the already exist­ ting data. Since 1952 improved techniques in microscopy, particularly in electron microscopy, chromatography, and other analytical methods have provided additional experimental information. This is evident from the numerous research papers and reviews on chloroplast structure and photo­ synthesis that have since appeared. Attention is therefore directed to the recent reviews by Frey-Wyssling (2), Granick (3), Mlihlethaler (4), and Thomas (5,6) j to Rabinowitch for a historical as well as tabular summary of the data (7, 8, 9); and to a symposium of the Brookhaven National Labora­ tories, The Photochemical Apparatus: Its Structure and Function (10). In addition to these reviews, reference should be made to Leyon (11), von Wettstein (12) and Heitz (13) for discussions of more specific structural aspects of chloroplast development. Also to be noted are the reports of two recent symposia in the U.S.S.R., one on The Origin of Life o n Earth (14), and the other the Second All Union Conference on Photosynthesis (15). One hesitates, in this active field of research, to attempt to assess all of the accumulated experimental data or to review the recent excellent reviews. In gathering the information for this review three questions were paramount. (a) Can the structure of the chloroplast now be discussed at a molecular level? (b) What are the new data on the composition of the chloroplast and on the synthesis of its proteins and pigments? (c) How do these data relate to the growth and structural development of the chloroplast? It was with these considerations in mind that the recent available literature was searched. The structure containing the photosynthetic pigments exists in a variety of shapes and sizes from the photosynthetic bacteria to the higher plants. These have been referred to as chromatophores, plastids, free grana, mega­ plasts and chloroplasts. This nomenclature was intended to give some idea of the plant photoreceptor structure as well as its phylogenetic position. Because of certain inconsistencies additional nomenclature has been intro­ duced and suggested. In this discussion of the research literature it was found desirable to call all of these structures chloroplasts unless otherwise indicated or defined.

Photoreceptor structures. III. Drosophila melanogaster.

The eyes of three eye mutants of Drosophila melanogaster were fixed and thin sections studied for its structural detail in the electron microscope. Each ommatidium was found to have seven retinula cells with an equal number of rhabdomeres (visual units). The rhabdomeres average 1.2 micro in diameter and 60 micro in length. Each rhabdomere consists of osmium-fixed dense bands averaging 120 A in thickness, and with less dense interspaces 200 to 400 A. There is an average of 23 dense bands or 46 interfaces per micron within the rhabdomere. The rhabdomere as we have presented it is a single structure of packed rods or tubes. The "fine structure" within the rhabdomere is similar to that observed by electron microscopy for the retinula of the house fly, and to the retinal rods of the vertebrate eye, and to the chloroplasts of plant cells in a variety of animal and plant photoreceptor structures. In addition, the radial arrangements within the ommatidium of radially unsymmetrical units, the rhabdomeres, is probably related to the analysis of polarized light in the insect eye.