Association Of Phytochrome Research Articles

Red light-induced appearance of phosphotyrosine-like epitopes on nuclear proteins from pea (Pisum sativum L.) plumules.

As assayed by western blot analysis, red light induces the appearance of epitopes recognized by anti-phosphotyrosine antibodies in several pea nuclear proteins. The immunostaining is blocked by preadsorbing the antibodies with phosphotyrosine but not by preadsorbing them with phosphoserine or phosphothreonine. This light response is observed whether the red light irradiation is given to pea plumules or nuclei isolated from the plumules. The red-light-induced response seen in plumules is reversible by a subsequent far-red-light irradiation, indicating that the likely photoreceptor for this response may be phytochrome. By immunoblot analysis pea phytochrome A, but not phytochrome B, can be detected in proteins extracted from pea nuclear chromatin-matrix preparations. Phytochrome A and the protein bands immunostained by anti-phosphotyrosine antibodies can be solubilized from unirradiated pea chromatin by 0.3 M NaCl, but irradiating this preparation with red light does not induce the appearance of phosphotyrosine-like epitopes in any nuclear proteins. These results suggest that the association of phytochrome with purified pea nuclei is such that its conversion to the far-red light-absorbing form can induce a post-translational epitope change in nuclear proteins in vivo.

Photoreversible association of phytochrome with membranes. II. Reciprocity tests and a model for the binding reaction

Photoreversible association of phytochrome with membranes. II. Reciprocity tests and a model for the binding reaction

Photoreversible association of phytochrome with membranes. I. Distinguishing between two light-induced binding responses

Photoreversible association of phytochrome with membranes. I. Distinguishing between two light-induced binding responses

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.

Integral association of phytochrome with a membranous fraction from etiolatedAvena shoots: red/far-red photoreversibility and in vitro characterization

The results reported in this paper provide strong evidence to support the belief that the small percentage of phytochrome recovered in low-speed centrifugation pellets, when prepared in the absence of divalent cations after various in vivo irradiations, is not simply a manifestation of non-specific co-precipitation of soluble phytochrome.The far-red reversibility of the observed near-doubling of phytochrome pelletability after in vivo red irradiation indicates that phytochrome pelletability in the absence of divalent cations is a phytochrome-controlled response. The characteristics of the pelleted phytochrome indicate a strong, hydrophobic interaction with membranes. A tentative proposal to explain the observed characteristics of the association of phytochrome with membranous material in the absence of divalent cations after different in vivo irradiations has been put forward.

Integral association of phytochrome with a membranous fraction fromAvena shoots: in vivo characterization and physiological significance

Phytochrome in the far-red light absorbing form (Pfr) was observed to disappear in vivo more rapidly from the non-cation-requiring pelletable phytochrome population than from the supernantant phytochrome population of oat seedlings given an increasing dark incubation after red irradiation. The amount of pelletable phytochrome in the red light absorbing form (Pr) remained relatively stable while supernatant Pr was lost. These observations indicated that supernant Pfr was subject to loss during the incubation, while pelletable Pfr was subject to both dark reversion and loss.During the incubation, the ability of far-red irradiation to reverse the red-induced increase in phytochrome pelletability was lost, with kinetics similar to those of the loss of pelletable Pfr.Far-red reversibility of the red-induced increase in coleoptile elongation correlated with the change intotal Pfr in both supernatant and pelletable phytochrome populations, but with the change in the ratio of Pfr to total phytochrome only in the pelletable phytochrome population.The possible significance of these results is discussed with reference to the action of phytochrome in the photocontrol of physiological growth responses.

Evidence for a natural inhibitor of phytochrome pelletability in squash roots

Red light enhancement of phytochrome pelletability is commonly explained by the association of phytochrome with particulate material. Evidence is presented that squash roots contain some substance(s) able to partially inhibit this phenomenon. The inhibitory activity is lost by dialysis and desalting in Sephadex G-50 of the squash root extract, suggesting that it has a small molecular size.

Intracellular Phytochrome Distribution as a Function of its Molecular form and of its Destruction

The immunocytochemically observed intracellular redistribution of phytochrome as a function of its molecular form is described by utilizing color photomicrography. The reversible change from a diffuse to a discretely localized distribution following photoconversion of the red-absorbing Pr form to the far-red-absorbing Pfr form observed with etiolated oat (Avena sativa L., cv. Garry) coleoptile parenchyma cells is not seen with etiolated wheat (Triticum sativum L., cv. unknown), barley (Hordeum vulgare L., cv. Harrison), or rye (Secale cereale L., cv. Balbo). Whether redistribution in these latter cases does not occur or is below the limit of detection is not known. Upon continuous actinic irradiation, phytochrome, which is discretely localized as Pfr, rapidly disappears by both immunocytochemical and spectral assay. However, after about 90 min irradiation, a new association of phytochrome with nuclei is evident which is more pronounced after 4 or 8 h of irradiation. With longer irradiation times there is a total loss of antigenically detectable phytochrome at the resolution employed in these experiments.

INTRACELLULAR PHYTOCHROME DISTRIBUTION AS A FUNCTION OF ITS MOLECULAR FORM AND OF ITS DESTRUCTION

The immunocytochemically observed intracellular redistribution of phytochrome as a function of its molecular form is described by utilizing color photomicrography. The reversible change from a diffuse to a discretely localized distribution following photoconversion of the red‐absorbing Pr form to the far‐red‐absorbing Pfr form observed with etiolated oat (Avena sativa L., cv. Garry) coleoptile parenchyma cells is not seen with etiolated wheat (Triticum sativum L., cv. unknown), barley (Hordeum vulgare L., cv. Harrison), or rye (Secale cereale L., cv. Balbo). Whether redistribution in these latter cases does not occur or is below the limit of detection is not known. Upon continuous actinic irradiation, phytochrome, which is discretely localized as Pfr, rapidly disappears by both immunocytochemical and spectral assay. However, after about 90 min irradiation, a new association of phytochrome with nuclei is evident which is more pronounced after 4 or 8 h of irradiation. With longer irradiation times there is a total loss of antigenically detectable phytochrome at the resolution employed in these experiments.

Gel filtration of particle-bound phytochrome

Pelletable phytochrome from hypocotyl hooks of Cucurbita pepo L. seedlings has been separated into two fractions by gel filtration on Sepharose CL-2B. One fraction with a K av of 0.7 was detected only after red irradiation (in vivo or in vitro). This separated from a ribonucleoprotein fraction during gel filtration. The weak interaction with ribonucleo-protein which required magnesium (optimal at 10 mM) was overcome by high salt concentrations and prevented by ribonuclease treatment. The second phytochrome fraction was strongly associated with a high molecular weight material with a K av of less than 0.1. Low levels of this complex were detected in extracts from dark grown tissue but were increased by red irradiation of excised hooks or crude extracts. The binding of phytochrome to the high molecular weight material did not require magnesium, was unaffected by ribonuclease treatment, and was much more resistant to high salt concentrations than was the phytochrome-ribonucleoprotein association. These results suggest that the association of phytochrome with this membrane-containing fraction is not electrostatic.The separation by agarose-gel filtration offers a useful technique for the preparation of membraneassociated phytochrome for physiological studies.

PHYTOCHROME DISTRIBUTION IN ETIOLATED GRASS SEEDLINGS AS ASSAYED BY AN INDIRECT ANTIBODY‐LABELLING METHOD

The distribution of phytochrome in several etiolated grass seedlings (Avena saliva L., cvs. Garry and Newton; Secale cereale L., cv. Balbo; Hordeum vulgare L., cv. Harrison; Oryza sativa L; Zea mays L., cv. Golden Cross) was determined, by an indirect antibody‐labelling method employing peroxidase as the ultimate label. Although the pattern of phytochrome distribution in etiolated shoots varies widely, it is nevertheless clear that, with the exception of corn, in which phytochrome is relatively uniformly distributed, the distribution of phytochrome is highly specific with respect both to organs and to cell types within an organ for a given species. Oat, rye, barley, and rice shoots all have high concentrations of phytochrome near the tips of their coleoptiles, as well as near the shoot apex itself. Rice, barley, and rye also have high concentrations of phytochrome in their leaf bases, but oat leaves are almost totally devoid of measurable phytochrome. An association of phytochrome with vascular tissue often occurs and is most pronounced in the rice shoot. Dark‐grown roots were found to have high levels of phytochrome only in the root caps, with lesser amounts, if any, observed in other parts of the root.

ACTION AND INTERACTION OF MONOCHROMATIC LIGHT ON PHOSPHATE METABOLISM OF GERMINATING LETTUCE SEEDS

A previously described photoresponse of lettuce seed germination to red and far-red light is shown to be paralleled by a response of phosphate metabolic activity: (1) When seeds were continuously irradiated, red light accelerated and far-red suppressed their phosphate uptake and esterification. (2) The influence of monochromatic light on phosphate metabolism of seeds, determined after 36 and 64 hours of germination, respectively, indicated maximum potentiation between 550 and 650 mμ, maximum suppression beyond 700 mμ, and partial suppression at 475 mμ. Stimulation was encountered at 400 mμ, but with shorter wavelengths of the ultraviolet spectrum, suppression appeared again. (3) Photoactivation of phosphate metabolism in response to each of the three loci (550, 600, and 650 mμ, i.e., green, orange, and red light, respectively) of the potentiating spectral band was reversed by far-red (750 mμ) light. These activations and inhibitions could be reversed several times in an alternating sequence. Complete reversibility depended entirely upon the magnitudes of the radiant flux for the two counteracting wavelengths, and this was characteristic for each pair of antagonistic wavelengths. In view of the association of phytochrome with the isolated mitochondria and of the specific manner in which their phosphorylation activity is influenced by light, it is suggested that a part of the energy flow required for cellular development may be channeled through the mitochondrial–phytochrome system.