MUTATIONAL ANALYSIS OF PHYTOCHROME B IDENTIFIES A SMALL COOH-TERMINAL-DOMAIN REGION CRITICAL FOR REGULATORY ACTIVITY

Citation:

Wagner, D, Quail PH.  1995.  Mutational analysis of phytochrome B identifies a small COOH-terminal-domain region critical for regulatory activity, Sep 12. Proceedings of the National Academy of Sciences of the United States of America. 92:8596-600., Number 19

Abstract:

Overexpression of phytochrome B (phyB) in transgenic Arabidopsis results in enhanced deetiolation in red light. To define domains of phyB functionally important for its regulatory activity, we performed chemical mutagenesis of a phyB-overexpressing line and screened for phenotypic revertants in red light. Four phyB-transgene-linked revertants that retain parental levels of full-length, dimeric, and spectrally normal overexpressed phyB were identified among 101 red-light-specific revertants. All carry single amino acid substitutions in the transgene-encoded phyB that reduce activity by 40- to 1000-fold compared to the nonmutagenized parent. The data indicate that the mutant molecules are fully active in photosignal perception but defective in the regulatory activity responsible for signal transfer to downstream components. All four mutations fall within a 62-residue region in the COOH-terminal domain of phyB, with two independent mutations occurring in a single amino acid, Gly-767. Accumulating evidence indicates that the identified region is a critical determinant in the regulatory function of both phyB and phyA.

Notes:

Wagner, DQuail, P HProc Natl Acad Sci U S A. 1995 Sep 12;92(19):8596-600.

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FLOWERING RESPONSES TO ALTERED EXPRESSION OF PHYTOCHROME IN MUTANTS AND TRANSGENIC LINES OF ARABIDOPSIS THALIANA (L.) HEYNH

Citation:

Bagnall, DJ, King RW, Whitelam GC, Boylan MT, Wagner D, Quail PH.  1995.  Flowering responses to altered expression of phytochrome in mutants and transgenic lines of Arabidopsis thaliana (L.) Heynh, Aug. Plant physiology. 108:1495-503., Number 4

Abstract:

The long-day plant Arabidopsis thaliana (L.) Heynh. flowers early in response to brief end-of-day (EOD) exposures to far-red light (FR) following a fluorescent short day of 8 h. FR promotion of flowering was nullified by subsequent brief red light (R) EOD exposure, indicating phytochrome involvement. The EOD response to R or FR is a robust measure of phytochrome action. Along with their wild-type (WT) parents, mutants deficient in either phytochrome A or B responded similarly to the EOD treatments. Thus, neither phytochrome A nor B exclusively regulated flowering, although phytochrome B controlled hypocotyl elongation. Perhaps a third phytochrome species is important for the EOD responses of the mutants and/or their flowering is regulated by the amount of the FR-absorbing form of phytochrome, irrespective of the phytochrome species. Overexpression of phytochrome A or phytochrome B resulted in differing photoperiod and EOD responses among the genotypes. The day-neutral overexpressor of phytochrome A had an EOD response similar to all of the mutants and WTs, whereas R EOD exposure promoted flowering in the overexpressor of phytochrome B and FR EOD exposure inhibited this promotion. The comparisons between relative flowering times and leaf numbers at flowering of the over-expressors and their WTs were not consistent across photoperiods and light treatments, although both phytochromes A and B contributed to regulating flowering of the transgenic plants.

Notes:

Bagnall, D JKing, R WWhitelam, G CBoylan, M TWagner, DQuail, P HGM 47475/GM/NIGMS NIH HHS/Plant Physiol. 1995 Aug;108(4):1495-503.

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PHYTOCHROMES: PHOTOSENSORY PERCEPTION AND SIGNAL TRANSDUCTION

Citation:

Quail, PH, Boylan MT, Parks BM, Short TW, Xu Y, Wagner D.  1995.  Phytochromes: photosensory perception and signal transduction, May 5. Science. 268:675-80., Number 5211

Abstract:

The phytochrome family of photoreceptors monitors the light environment and dictates patterns of gene expression that enable the plant to optimize growth and development in accordance with prevailing conditions. The enduring challenge is to define the biochemical mechanism of phytochrome action and to dissect the signaling circuitry by which the photoreceptor molecules relay sensory information to the genes they regulate. Evidence indicates that individual phytochromes have specialized photosensory functions. The amino-terminal domain of the molecule determines this photosensory specificity, whereas a short segment in the carboxyl-terminal domain is critical for signal transfer to downstream components. Heterotrimeric GTP-binding proteins, calcium-calmodulin, cyclic guanosine 5′-phosphate, and the COP-DET-FUS class of master regulators are implicated as signaling intermediates in phototransduction.

Notes:

Quail, P HBoylan, M TParks, B MShort, T WXu, YWagner, DGM47475/GM/NIGMS NIH HHS/New York, N.Y.Science. 1995 May 5;268(5211):675-80.

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COP1, AN ARABIDOPSIS REGULATORY GENE, ENCODES A PROTEIN WITH BOTH A ZINC-BINDING MOTIF AND A G BETA HOMOLOGOUS DOMAIN

Citation:

Deng, XW, Matsui M, Wei N, Wagner D, Chu AM, Feldmann KA, Quail PH.  1992.  COP1, an Arabidopsis regulatory gene, encodes a protein with both a zinc-binding motif and a G beta homologous domain, Nov 27. Cell. 71:791-801., Number 5

Abstract:

Plant seedling development is capable of following 1 of 2 distinct morphogenic pathways: skotomorphogenesis in darkness and photomorphogenesis in light. Dark-grown Arabidopsis seedlings with recessive mutations at the constitutively photomorphogenic (COP1) locus indicate that the wild-type COP1 protein represses photomorphogenesis in darkness and that light reverses this repressive activity. Using a T-DNA-tagged mutant, we have cloned the COP1 locus. The amino-terminal half of the encoded protein contains a conserved zinc-binding motif, whereas the carboxyl-terminal half contains a domain homologous to the WD-40 repeat motif of G beta proteins. The presence of both a putative DNA-binding motif and a G protein-related domain in a single polypeptide suggests that COP1 may be the first of a new class of regulatory molecules. This novel structure could endow COP1 with the capacity to function as a negative transcriptional regulator capable of direct interaction with components of the G protein signaling pathway.

Notes:

Deng, X WMatsui, MWei, NWagner, DChu, A MFeldmann, K AQuail, P H1-R29-GM47850-01/GM/NIGMS NIH HHS/Cell. 1992 Nov 27;71(5):791-801.

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OVEREXPRESSION OF PHYTOCHROME B INDUCES A SHORT HYPOCOTYL PHENOTYPE IN TRANSGENIC ARABIDOPSIS

Citation:

Wagner, D, Tepperman JM, Quail PH.  1991.  Overexpression of Phytochrome B Induces a Short Hypocotyl Phenotype in Transgenic Arabidopsis, Dec. The Plant cell. 3:1275-1288., Number 12

Abstract:

The photoreceptor phytochrome is encoded by a small multigene family in higher plants. phyA encodes the well-characterized etiolated-tissue phytochrome. The product of the phyB gene, which has properties resembling those of “green tissue” phytochrome, is as yet poorly characterized. We have developed a phytochrome B overexpression system for analysis of the structure and function of this protein. Using newly generated polyclonal and monoclonal antibodies that are selective for phytochrome B, we have demonstrated high levels of expression of full-length rice and Arabidopsis phytochrome B under the control of the cauliflower mosaic virus 35S promoter in transgenic Arabidopsis. The overexpressed phytochrome is spectrally active, undergoes red/far-red-light-dependent conformational changes, is synthesized in its inactive red light-absorbing form, and is stable in the light. Overexpression of phytochrome B is tightly correlated with a short hypocotyl phenotype in transgenic seedlings. This phenotype is strictly light dependent, thus providing direct evidence that phytochrome B is a biologically functional photoreceptor. Based on similarities to phenotypes obtained by overexpression of phytochrome A, it appears that phytochromes A and B can control similar responses in the plant.

Notes:

Wagner, D.Tepperman, J. M.Quail, P. H.Plant Cell. 1991 Dec;3(12):1275-1288.

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THE CALCIUM REQUIREMENT FOR STABILITY AND ENZYMATIC ACTIVITY OF TWO ISOFORMS OF BARLEY ALEURONE ALPHA-AMYLASE

Citation:

Bush, DS, Sticher L, van Huystee R, Wagner D, Jones RL.  1989.  The calcium requirement for stability and enzymatic activity of two isoforms of barley aleurone alpha-amylase, Nov 15. The Journal of biological chemistry. 264:19392-8., Number 32

Abstract:

alpha-Amylases (EC 3.2.1.1) secreted by the aleurone layer of barley grains are Ca2+-containing metalloenzymes. We studied the effect of Ca2+ on the activity and structure of the two major groups of aleurone alpha-amylase by incubating affinity purified enzyme in solutions containing Ca2+ from pCa 4 to 7. Both groups of isoforms required one atom of Ca2+/molecule of enzyme as determined by isotope exchange, but the two groups differed by more than 10-fold in their affinity for Ca2+. Both groups of alpha-amylase were irreversibly inactivated by incubation in low Ca2+ (pCa 7). This inactivation was not due to changes in primary structure, as measured by molecular weight, but appeared to be the result of changes in secondary and tertiary structure as indicated by circular dichroism spectra, serology, lability in the presence of protease, and fluorescence spectra. Analysis of the predicted secondary structure of barley aleurone alpha-amylase indicates that the Ca2+-binding region of barley amylases is structurally similar to that of mammalian alpha-amylases. Our data indicate that micromolar levels of Ca2+ are required to stabilize the structure of barley alpha-amylases in the endoplasmic reticulum of the aleurone layer where these enzymes are synthesized.

Notes:

Bush, D SSticher, Lvan Huystee, RWagner, DJones, R LJ Biol Chem. 1989 Nov 15;264(32):19392-8.

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