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At least 19 recordsLinked to original sources
PubMed1992

Phytochrome evolution: a phylogenetic tree with the first complete sequence of phytochrome from a cryptogamic plant (Selaginella martensii spring).

We have sequenced cDNA and genomic clones coding for phytochrome of the fern Selaginella. On the amino acid level, this phytochrome shares sequence homologies with phytochromes of higher plants which range between 62 (phytochrome B of Arabidopsis) and 55 (56)% [phytochrome C of Arabidopsis (Avena)]. Introns in the Selaginella gene are short and occupy positions known from phytochrome sequences of higher plants. A rooted phylogenetic tree based on mutation distances puts Selaginella phytochrome closest to the hypothetical ancestor. A similar tree arises if the tree is constructed with partial sequences (about 200 amino acids) around the chromophore attachment site. An extension of this tree by sequences of other cryptogamic plants (Mougeotia, Ceratodon, Psilotum) shows all these sequences including those of the phytochromes B and C of Arabidopsis on a branch, well separated from the branch formed by phytochromes known to accumulate in etiolated plants. The rooted phytochrome phylogenetic tree, however, is difficult to reconcile with the fossil record.

Amino Acid Sequence↗
PubMed1992

N-terminal domain of Avena phytochrome: interactions with sodium dodecyl sulfate micelles and N-terminal chain truncated phytochrome.

Phytochrome is the ubiquitous red light photoreceptor present in plants. Properties of the 6-kDa end terminal region of phytochrome A (PHYA from etiolated Avena) have been investigated by the use of synthetic polypeptide fragments corresponding to that region. This region of the phytochrome A protein has been viewed as a possible functional site due to the large differences in the sequence's conformation and exposure between the Pr (red light-absorbing form) and Pfr (far-red light-absorbing, gene-regulating form) species of phytochrome A. Hydrophobic moment calculations reveal amphiphilic helical potential in this section of the protein, consistent with the folding of the N-terminal region onto a hydrophobic chromophore/chromophore pocket. A large N-terminal synthetic peptide also demonstrated helical folding in the presence of SDS micelles. This experimental evidence indicates that the N-terminal alpha-helical folding upon conversion of the regulatorily inactive Pr to the active Pfr form of phytochrome A is likely driven at least in part by amphiphilic helix stabilization. Further, the large synthetic peptide was spectrally demonstrated to interact with phytochrome A lacking the N-terminal region. The formation of this nativelike complex may provide us with a tool for both biophysical and physiological studies on the mechanism of phytochrome A signal transduction.

Edible Grain↗
PubMed1992

Signal transduction by phytochrome: phytochromes have a module related to the transmitter modules of bacterial sensor proteins.

A C-terminal section of phytochromes turned out to share sequence homologies with the full length of the transmitter modules (about 250 amino acids) of bacterial sensor proteins. Coinciding hydrophobic clusters within the homologous domains imply that the overall folding of the two different types of peptides is similar. Hence, phytochromes appear to possess the structural prerequisites to transmit signals in a way bacterial sensor proteins do. The bacterial sensor proteins are known to be environmental stimuli-regulated kinases belonging to two-component systems. After sensing a stimulus by the N-terminal part of the sensor protein, conformational alterations confer the signal to its (mostly) C-terminal transmitter module which in turn is transitionally autophosphorylated at a conserved histidine. From the histidine the phosphate is transferred to the receiver module of a system-specific regulator protein which eventually acts on transcription or enzyme activity. The histidine is not conserved in phytochromes. Instead, a conserved tyrosine is found spatially very close to the histidine position. This tyrosine might play the role of histidine, and kinase function might be associated with this part of phytochrome. In spite of this divergence, the structural similarities point to a common evolutionary origin of the phytochrome and bacterial modules.

Amino Acid Sequence↗
PubMed1992

Phytochrome requires the 6-kDa N-terminal domain for full biological activity.

Phytochrome is a red/far-red-absorbing photoreceptor that controls many aspects of plant photomorphogenesis. Because proteolytic removal of approximately 6 kDa from the N terminus of 124-kDa oat phytochrome substantially alters many physicochemical properties of the chromoprotein, it has been proposed that the N terminus is required for biological activity. Here we test this hypothesis by comparing tobacco plants expressing full-length oat phytochrome (FL) with plants expressing a 118-kDa oat phytochrome lacking amino acids 7-69 (NA phytochrome). NA phytochrome, like its FL counterpart, exists as a homodimer in solution, is capable of covalently binding chromophore to form a red/far-red-photoreversible product, and is rapidly degraded in vivo after photoconversion to the far-red-absorbing form. However, like proteolytically degraded phytochrome missing the N terminus, the absorption maxima of the red- and far-red-light-absorbing forms of NA phytochrome are blue shifted relative to the maxima of the FL chromoprotein, and the rate of dark reversion of the far-red- to red-light-absorbing form is substantially increased. Tobacco plants producing high levels of NA phytochrome do not exhibit the light-exaggerated phenotype characteristic of FL phytochrome overexpression. By comparison of phytochrome-dose-phenotype-response curves generated by using a series of transgenic lines expressing various levels of FL or NA phytochrome, we demonstrate that NA phytochrome has less than 1/5th the biological activity of FL phytochrome expressed in tobacco. Furthermore, the shape of the dose-response curve for plants expressing FL phytochrome indicates that there is a sharp transition between phenotypically normal and abnormal plants over a relatively narrow range of phytochrome content, demonstrating that precise control of phytochrome levels is critical to photomorphogenesis.

Amino Acid Sequence↗
PubMed1979

Spectroscopic properties and chromophore conformations of the photomorphogenic receptor: phytochrome.

Fluorescence lifetimes of 'large (mol. wt. 120,000) and 'small' (mol. wt. 60,000) phytochromes isolated from oat and rye seedlings grown in the dark have been measured at 199 K and 298 K. Phytochrome model compounds have also been studied by phase modulation fluorometrically at 77 K for comparison with lifetime data for phytochrome. It was found that the fluorescence lifetime of 'large' phytochrome was significantly shorter than that of 'small' phytochrome and its chromophore models. The phytochrome chromophore of Pr form has been analyzed by fluorescence polarization, CD, and molecular orbital methods. The fluorescence excitation polarization of 'small' phytochrome and the chromophore model in buffer/glycerol mixture (3 : 1, v/v) at 77 K is very hight (0.4) at the main absorption band and is negative (--0.1) and close to 0 in the near ultraviolet band, respectively. Analyses of the spectroscopic data suggest that the chromophore conformation of Pr and Pfr forms of phytochrome are essentially identical. The induced ellipticity of 'large' rye phytochrome in the blue and near ultraviolet regions was found to be significantly higher than that of 'small' phytochrome, indicating that the binding interaction between the phytochrome chromophore and apoprotein is much tighter in the former than in the latter. In addition, the excitation energy transfer does occur from Trp residue(s) to the chromophore in 'large' phytochrome but not in 'small' Pr. This illustrates one feature of the role played by the large molecular weight apoprotein in the binding site interactions and primary photoprocesses of Pr. Finally, a plausible model for the primary photoprocesses and the mechanism of phytochrome interactions triggered by the Pr leads to Pfr phototransformation have been proposed on the basis of the above results.

Circular Dichroism↗
PubMed1992

The cucumber long hypocotyl mutant lacks a light-stable PHYB-like phytochrome.

A novel cDNA sequence homologous to a phytochrome B (phyB) gene that was isolated in a library from tobacco tissue has been used in an Escherichia coli expression system to raise anti-phytochrome B (anti-PHYB) polypeptide-specific monoclonal antibodies. The specificity of these antibodies has been tested by cross-reactivity against purified pea light-labile type 1 and light-stable type 2 phytochromes, with some antibodies reacting with the type 2 and none with the type 1 phytochromes. One such antibody, monoclonal mAT1, has been employed to analyze the phytochrome molecular species present in a photomorphogenic long hypocotyl (lh) mutant of cucumber. The results indicated that the mutant contains wild-type levels of the light-labile type 1 phytochrome polypeptide (PHYA), which has an apparent molecular mass of approximately 120 kD, but shows less than 1% (detection limit) of a light-stable polypeptide recognized by mAT1 in wild-type seedlings. This protein, not detectable in the lh mutant, has the properties of light-stable type 2 phytochrome, has an apparent molecular mass of 116 to 117 kD, and remains at constant levels under continuous low-fluence-rate red light. Therefore, we conclude that the lh mutant lacks at least one type 2 phytochrome-like polypeptide, most probably a phyB gene product. The correlation between the lack of this protein and the deficiency or absence of physiological responses to a light-stable phytochrome species in this mutant helps to identify the physiological roles played by the products of different subfamilies within the phytochrome gene family.

Antibodies, Monoclonal↗
PubMed2026

DNA-binding activity of PIF7 links phytochrome B signaling to plant responses to vegetation proximity.

PHYTOCHROME INTERACTING FACTORs (PIFs) are transcription factors that act as central signaling hubs in light-regulated processes. All PIFs contain an active phytochrome B-binding motif and a DNA-binding basic helix-loop-helix domain. In the shade-avoider Arabidopsis thaliana, PIF7 is a major promoter of hypocotyl elongation in response to vegetation proximity, becoming active when released from phytochrome B via its active phytochrome B-binding motif. Here we show that PIF7 promotes seedling elongation in other species, including the shade-avoider tomato and the shade-tolerant Cardamine hirsuta, suggesting that PIF7 has retained some of its key functional domains across diverse plants. Through complementation analyses using PIF7 variants lacking either the active phytochrome B-binding or basic helix-loop-helix domain, we demonstrate that, unlike PIF3, PIF7 versions unable to bind phytochrome B remain active regardless of light conditions, whereas loss of DNA-binding capacity fully disrupts PIF7 function. Our results further suggest that phytochrome B interaction imposes a dual regulatory control over PIF7, modulating both its abundance and its phosphorylation state (ie its ability to bind and regulate target genes).

Phytochrome B↗
PubMed1992

Detection and partial sequence of phytochrome genes in the ferns Anemia phyllitidis (L.)Sw (Schizaeaceae) and Dryopteris filix-mas L. (Polypodiaceae) by using polymerase-chain reaction technology.

Phytochrome controls several developmental steps during formation and differentiation of the fern gametophyte, including spore germination, morphogenesis of the gametophyte or differentiation of the sexual cells. To obtain information about the amino acid sequence and the regulation of phytochrome expression at the gene level, two degenerated oligonucleotides against well conserved amino acid regions were designed after an optimal alignment of the known phytochrome sequences. These primers were tested against DNA isolated from Arabidopsis thaliana, and the polymerase-chain reaction (PCR) products were cloned and sequenced. The DNA fragment produced with this method proved to be identical with a phytochrome-A-gene fragment from A. thaliana, and hence this fragment was used in further experiments to prove whether amplified DNA from fern species contains phytochrome-like DNA. With this procedure we successfully detected and cloned gene fragments both from gametophytes of Anemia phyllitidis and Dryopteris filix-mas, cultured for 7 days under vegetative conditions. In addition, poly(A)+ RNA was prepared from 7-day-old gametophytes of A. phyllitidis, induced to differentiate antheridia under generative conditions. This poly(A)+ RNA was transcribed into complementary DNA and used together with both phytochrome specific primers in a PCR experiment. We thereby obtained another DNA fragment. These data strongly suggest that A. phyllitidis has at least two phytochrome genes, and that at least one of them is expressed in light-grown gametophytes.

Amino Acid Sequence↗
PubMed1992

Partial nucleotide sequence of phytochrome from the zygnematophycean green alga Mougeotia.

Following polymerase chain reaction, a fragment of about 800 bp was amplified from genomic Mougeotia DNA using oligonucleotides directed to conserved regions of known phytochrome genes. The nucleotide sequence points to a different exon/intron structure in the neighborhood of the chromophore attachment site of this Mougeotia phytochrome gene, as compared to other phytochromes. Alignment of the derived amino acid sequence to phytochromes of higher and lower plants shows highest homology (> 60%) to type II (green type) phytochrome, while Northern blot analysis of total Mougeotia RNA indicates down-regulation of the phytochrome transcription in light. Signal pattern of hybridized genomic DNA after digestion reveals the presence of probably only one phytochrome gene in Mougeotia.

Amino Acid Sequence↗
PubMed1992

Isolation and characterization of a cDNA-clone coding for potato type A phytochrome.

We have isolated and sequenced a cDNA clone encoding the apoprotein of a potato phytochrome. Based on the deduced amino acid sequence, which shows 78% amino acid identity to the Arabidopsis phyA and 50% identity to the Arabidopsis phyB open reading frame, we have classified this cDNA clone as potato phyA phytochrome. The amino acid immediately preceding cysteine 323, which is the homologue of oat cystein 321, to which the chromophore has been shown to be attached, is a tyrosine residue. This contrasts with six other type A phytochrome sequences from both monocots and dicots that encode serine in this position. As already observed in three other cDNAs isolated from dicot species, the potato phyA clone encodes a short open reading frame (13 amino acids) preceding the phyA open reading frame (1123 amino acids), supporting the idea that this type of leader sequence might be involved in the regulated expression of the phytochrome apoprotein. Southern blot analysis revealed a single phyA gene as well as other related phytochrome sequences in the potato genome. phyA mRNA levels varied in different organs and were modulated by white light; in seedlings and sprouts, highest levels of mRNA were detected in the etiolated stage. Upon illumination with white light, mRNA levels decreased to the amount found in leaves of re-etiolated plants. Lowest expression was observed in leaves of plants grown in the light, in tubers irrespective of light treatment, and in roots of plants grown in the dark. In roots of plants grown in the light, elevated levels of phyA mRNA were detected. Using a monoclonal antibody generated against pea phytochrome as an immunochemical probe, the protein was only detectable in protein extracts from etiolated seedlings and sprouts.

Amino Acid Sequence↗
PubMed1992

Localization of protein-protein interactions between subunits of phytochrome.

We have used a novel assay based on protein fusions with lambda repressor to identify two small regions within phytochrome's carboxy-terminal domain that are capable of mediating dimerization. Using an in vivo assay, fusions between the DNA binding, amino-terminal domain of lambda repressor and fragments from oat PhyA phytochrome have been assayed for increased repressor activity, an indicator of dimerization. In this assay system, regions of oat phytochrome between amino acids V623-S673 and N1049-Q1129 have been shown to increase repressor activity. These short spans are highly conserved between proteins belonging to the phytochrome PhyA family. Embedded within these sequences are four segments that could potentially form amphipathic alpha helices. Two of the segments are well conserved between PhyA phytochrome and phytochromes encoded by the phyB and phyC genes, suggesting that heterodimers might form by way of subunit interaction at these sites.

Amino Acid Sequence↗
PubMed1991

Blue light is required for survival of the tomato phytochrome-deficient aurea mutant and the expression of four nuclear genes coding for plastidic proteins.

When dark-grown aurea mutant tomato seedlings which lack more than 95% of the phytochrome present in isogenic wild-type seedlings are kept in white or blue light, four nuclear-encoded transcripts coding for plastidic proteins (the light-harvesting chlorophyll a/b-binding protein of photosystem I and II [cab-PSII], plastocyanin and subunit 2 of photosystem I) are present in comparable amounts. These transcript levels in red light are strongly reduced in aurea seedlings when compared with those of wild type. Thus, blue light is required for normal expression of these genes in the mutant, while red light alone is not sufficient. Red light-grown aurea seedlings are very sensitive to blue light, even 10 minutes of blue light every day suffices to cause a measurable increase in cab-PSII transcript level. The action of blue light on the expression of cab-PSII in the mutant is under phytochrome control. After 8 days of blue light, phytochrome is almost as effective in inducing cab-PSII mRNA as in the isogenic wild type, whereas after 8 days of red light, only a small phytochrome response was observed in the mutant. It is concluded that blue light sensitizes the mutant to the residual phytochrome which allows normal gene expression and survival of the mutant under daylight conditions.

Gene Expression Regulation↗
PubMed1991

Resonance Raman study on intact pea phytochrome and its model compounds: evidence for proton migration during the phototransformation.

Resonance Raman (RR) scattering from intact pea phytochrome was observed in resonance with the blue band at ambient temperature. The relative populations of the red-light-absorbing form (Pr) and far-red-light-absorbing form (Pfr) under laser illumination were estimated from the absorption spectra. The most prominent RR band of Pr obtained by 364-nm excitation under 740-nm pumping exhibited a frequency shift between H2O and D2O solutions, but that of Pfr obtained by 407-nm excitation under 633-nm pumping did not, indicating a distinct difference in a protonation state of their chromophores. Since the protonation level of a whole molecule of intact phytochrome remains unchanged between Pr and Pfr, this observation indicates migration of a proton from the chromophore of Pr to the protein moiety of Pfr. As model compounds, octaethylbiliverdin (OEBV-h3), its deuterated and 15N derivatives, and their protonated forms were also studied with both RR and 1H and 15N NMR spectroscopies. The RR spectrum of the protonated form, for which the protonation site was determined to be C-ring pyrrole nitrogen by NMR, displayed a deuteration shift corresponding to that of Pr, suggesting a similar protonated structure for the pyrrolic rings of Pr. The RR spectral difference between OEBV-h3 and OEBV-d3 and that between H2O and D2O solutions of Pfr suggested that the N-H protons of the A-, B-, and D-rings of intact phytochrome are replaced with deuterons in D2O. A role of the 7-kDa segment of phytochrome is discussed on the basis of RR spectral differences between the intact and large phytochromes.

Biliverdine↗
PubMed1975

Reversible redistribution of phytochrome within the cell upon conversion to its physiologically active form.

The intracellular localization of phytochrome was seen in dark-grown oat (Avena sativa L., cv. Garry) and rice (Oryza sativa L., cv. unknown) shoots after various light treatments using an indirect peroxidase-antiperoxidase antibody labeling method. Phytochrome is generally distributed throughout the cytoplasm in cells of tissue that had not been exposed to light prior to fixation. Within, at most, 8 min after the onset of saturating red irradiation, phytochrome, now present in the far-red-absorbing form, becomes associated with discrete regions of the cell. These regions do not appear to be nuclei, plastids, or mitochondria. After phototransformation back to the red-absorbing form originally present, phytochrome slowly resumes its general distribution. It is possible that this discrete localization of the far-red-absorbing form of phytochrome represents a physiologically significant binding with a receptor site in the cell.

Cytoplasm↗
PubMed1992

Structural domains of phytochrome deduced from homologies in amino acid sequences.

A method of semiempirical identification of structural domains is proposed. The procedure is based on the comparison of amino acid sequences in groups of homologous proteins. This approach was tested using 32 known protein sequences from different cytochrome b5, cytochrome c, lysozyme, hemoglobin, and myoglobin proteins. The method presented was able to identify all structural domains of these reference proteins. A consensus secondary structure provided information on structural content of these domains predicting correctly 21 of 23 (91%) of alpha-helices. We applied this method to six homologous phytochrome sequences from Avena, Arabadopsis, Cucurbita, Maize, Oryza, and Pisum. Some of the identified domains can be assigned to the known tertiary structure categories. For example, an alpha/beta domain is localized in the region known to stabilize the phytochrome chromophore in the red light absorbing form (Pr). One alpha-helical and one alpha/beta domains are localized in regions important for the chromophore stabilization in the far-red absorbing form (Pfr). From an analysis of noncovalent interaction patterns in another domain it is proposed that a phytochrome dimer contact involves two segments localized between residues 730 and 821 (using numbering of aligned sequences). Also, a possible antiparallel beta-sheet structure of this region has been suggested. According to this model, the long axis of the interacting structures is perpendicular to a twofold symmetry axis of the phytochrome dimer.

Amino Acid Sequence↗
PubMed1975

Control over activation or synthesis of phenylalanine ammonia-lyase by phytochrome in mustard (Sinapis alba L.)? A contribution to eliminate some misconceptions.

1. Density labelling with 80 atom% of 2H2O has been used to examine the mode of action of phytochrome (continuous far-red light) in increasing levels of L-phenylalanine ammonia-lyase (EC 4.3.1.5) activity in cotyledons of developing mustard seedlings (Sinapis alba L.). 2. Bandwidths and density shifts of isopycnically banded enzyme show that in darkness the enzyme was synthesized de novo, continuously turning over (half-life approx. 3 h) and that maximum labelling achievable was reached at 12 h. 3. 2-fold (6h), 5-fold (12h) and 10-fold (24 h) light-mediated increases in enzyme activity were accompanied by a similar pattern of labelling as observed in darkness. 4. Experimental evidence and theoretical arguments are presented which make it unlikely that phytochrome increases enzyme activity by slowing down the rate of degradation or by activating preformed enzyme molecules. 5. The conclusion is drawn that the rate of turnover of phenylalanine ammonia-lyase in dark-grown mustard cotyledons is too rapid compared to the measured rise in enzyme activity for density labelling to reveal directly control over the rate of synthesis de novo by phytochrome. However, the elimination of other control mechanisms leads us to the conclusion that phytochrome most probably does control synthesis of this enzyme in mustard, which agrees with the previous findings for parsley cells.

Ammonia-Lyases↗