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TOPIC:-MOLECULAR MECHANISM OF LIGHT PERCEPTION, SIGNAL TRANSDUCTION ANDGENE REGULATIONPresented by :ZUBY GOHAR ANSARI TAM-2014-026

INTRODUCTION:LIGHT PERCEPTION: It is the process by which an organism or man made device perceives and interprets light from the environment.Light signal is vary in four parameters:Quality (wavelength)Quantity ( fluence or photon per meter square)Directionality ( unidirectional or diffuse) or,Duration ( photoperiod or day length)PHOTOPERIODISM: Measurement of duration of lightor darkness in a twenty four hour cycle

PHOTORECEPTORS : These are the chemicals or compounds which can receive the light signals.

Three photoreceptors are recognized:

Phytochromes that absorb maximally in red (660nm) and far red (730nm), also absorb in UV-A/blue;

B/UV-A photoreceptors absorb in blue and UV-A parts of spectrum (320-480nm); and

UV-B photoreceptors absorb (280-320nm).

PHYTOCHROMESDISCOVERY: In 1930 Flint and McAlister at the U.S.D.A. Seed Testing Laboratory observed that the seed germination in a certain variety of lettuce was promoted by irradiation with red light and inhibited by far red.In 1959 phytochrome discovered (ab. In fungi) .Two approaches used : 1. the criterion for red and far red reversibility.2.extraction method become more refined, monoclonal antibodies against specific epitope of phytochrome could be prepared and used immunologically.

STRUCTURE AND SYNTHESIS: Phytochromes molecules are soluble chromoproteins . In Arabidopsis ,the apoprotein moiety of phytochrome is coded for five distinct genes PHYA,P YB,PHYC,PHYD, and PHYE which occur in single copies.Phytochrome molecule has 2 component: a protein part ( apo protein) and a chromophore.Molecular mass of PHYA apoprotein in different species about 125 kDa ( range 118 to 130).The apoprotein is folded into 2 structural domains: a slightly larger N-terminal domain, which carries the chromophore, and a smaller C-terminal domain.These 2 domains are linked by hinge like segment, susceptible to proteolysis during extraction.

Comparisons among phytochrome apoproteins indicate that the N- terminal domain with the chromophore is highly conserved among different PHY gene families, where as the C-terminal domain is variable.N-terminal domain of approx 600 amino acid including the chromophore, involved in photo perception as well as R/FR reversal where as C-terminal domain involved in dimerization of monomers and in signal transduction.CHROMOPHORE: It is an open chain tetrapyrrole , known as phytochromobilin and is attached to the apoprotein at a conserved cysteine residue.Phytochromobilin chromophore is similar to phycocyanobilin chromophore present in BGA.

Apoprotein encoded by its genes (PHYA,PHYB) and synthesized in cytoplasm from its m-RNA , the chromophores is synthesized in plastid.On the export into the cytoplasm , chromophores ligated covalently to apoprotein .Apoprotein contain amino acids sequences that are able to auto catalyze this covalent attachment, it is possible to express a cDNA encoding the apoprotein in transgenic plants, yeast, Escherichia coli etc and, on, an exogenous supply of chromophore precursors , obtain a spectrally functional phytochrome.This technique has proven very useful in the dissection of phytochrome signaling.

Psi : Refers to the ratio of Pfr to total phytochrome in a light environment.LIGHT STABILITY AND DEGRADATION OF PHYA:PHYA is unstable in light , it is synthesized in dark in Pr form (PrA) and is relatively stable in dark, half life approx 100 hr.On conversion of Pfr form in red or white light , it is much stable and is degraded rapidly with half life 60 min or 1/100 of Pfr. These drop due to 3 factors:1.phy A in the Pfr form down regulates the transcription of its gene.2.PHYA mRNA degraded in the light environment.3.certain conformational changes occur in Pfr A that predispose it to selective destruction by the proteolytic machinery in the cell.

Another motif with short half lives is PEST sequence (named after amino acid Pro,Glu,Ser,Thr) .This sequence highly conserved in PHY A molecules but is absent in apoprotein sequences in PHYB, PHY C.Except phyA , phy B- phyE in Arabidopsis and their homologues are studied ,are light stable, type 2 phytochromes.In etiolated plants , phyA is more as compare to others but in light its proportion decreases. In dark 10:1 and in light 1:1 phyA to phyB proportion.In light it is substantially decreases , still it is major phytochrome in plant during light.

CRYPTOCHROMEThese are blue light receptors that mediate various light- induced responses in plants and animals. They share sequence similarity to photolyases, flavoproteins that catalyze the repair of UV light-damaged DNA , but do not have photolyase activity.eg: include phototropic curvature in response to unidirectional light, de- etiolation response in etiolated seedling, induction of chalcone synthase (CHS) and other flavonoid synthesis gene (DFR)and promotion of stomatal opening.

STRUCTURE OF CRYPTOCHROME:Cry1 andCry2 are structurally similar proteins although cry2 is smaller.Both protein have N-terminal domain , involved in light perception and C-terminal domain in signaling.N-terminal bear some similarity to bacterial photolyases.Photolyases are enzyme that catalyze the repair of pyrimidine dimer in DNA caused by exposure to UV.N-terminal having pterin which absorb in UV-A or blue and pass energy to chromophore,FAD.FAD reduced and bring about DNA repair by cleavage of the pyrimidine dimer.Both cry1 and 2 participate in blue light induced inhibition of hypocotyl growth and in the expansion of cotyledons.

PTERIN

Cry1 apoprotein consist N-terminal having chromophore binding site (pterin)and C- terminal having FAD binding site.N-terminal consist 505 amino acid.C-terminal consist of 581 amino acid.

Cry1 is stable in light , cry2 is decline rapidly in green, blue and UV-A light( wavelength that activate the receptor , although not in dark or R light).

Cry1 is activated by the UV-B light and play role in biological clock, cry2 activated by blue light involved in flowering response.

Cry1, cry2 (for cryptochromes 1 and 2) its response is de- etiolation , anthocyanin synthesis, flowering.

Nph1 ,npl1 (for non phototropic hypocotyl, and nph like)and its response in phototropism.

SIGNAL TRANSDUCTION AND GENE REGULATIONPHYTOCHROME AS A KINASE: The idea was reinforced by subsequent discovery of gene sequences in two bacteria one higher plant phytochromes and other histidine kinase domain of bacterial sensor protein.Cyanobacterium fremyella diplosiphon has sensor kinase which enables it to adapt to different height conditions. N-terminal show similarity to the N-terminal chromophore bearing domain of higher plant phytochromes.

STEPS: A diagram1.The CPH1 sequence from synechocystis expressed in E.coli ,gives a recombinant protein that binds to phycocyanobillin(PCB) or phytochromobilin chromophores to yield a functional phytochrome with red/ far red reversibility.2.The recombinant CPH1 supplied radiolabeled ATP is autophosphorylated on the conserved histidine , which in, turn transfer the phosphates to aspartate on RCP1. Autophosphorylation and photo transfer to RCP1 are mediated by FR light or in the dark, in the Pr form, unlike higher plant phytochromes.

B diagram1. Oat phyA expressed in yeast and supplied PCB or phytochromobilin yields functional cytochrome.2.The recombinant protein, supplied radiolabeled ATP, is able to phosphorylate and to transfer the labeled phosphate group to RCP1 from synechocystis.SUBSTRATE FOR PHYTOCHROME KINASE:Carboxy terminal of phyA is used for signaling.Two protein used phytochrome kinase substrate 1 (PKS1) and nucleoside diphosphate protein kinase 2 (NDPK2 but not NDPK1) from Arabidopsis have been identified that interact with PHYA as well as PHYB.

MODEL FOR HIGHER PLANT PHYTOCHROME SIGNALING AND COMPARISON OF OAT PHY A AND SYNECHOCYSTIS CPH1.The model shows that red light stimulates phytochrome autophosphorylation at a serine residue (ser598) and transfer of a phosphate group to some substrate . A candidate substrate PK1. Also, it is possible that a phospho specific interactions occur with a down stream element in the signaling cascade. Phosphorylation of serine rich amino terminal region of phytochrome (ser7) is thought to down regulate the response.CPH1 is smaller protein and lacks the ser rich domain at the N-terminal and the PAS related domain in the C-terminal half of the higher plant phytochromes.

ACTIVATED PHYA AND PHYB ARE LOCALIZED TO NUCLEUS:Phytochromes are present in cytoplasm but they are translocated to nucleus when activated by respective irradiating wavelength.Immunocytochemical staining with a phytochrome specific (PHY A or PHYB) fluorescent antibody has been used for many to localize individual phytochromes intracellularly.This method used in combination with phy A null mutant as a control to provide greater specificity for phyA localization.Cry1 translocates to nucleus in response to light, cry2 seems to be constitutively nuclear localized.

MOLECULARLY CHARACTERIZED INTERMEDIATES IN PHYTOCHROME SIGNALING:PIF3 positive regulator of phyB bHLH (beta helix loop helix)transcription factor (nucleus).PKS1 - negatively regulates phy B substrate for phytochrome kinase activity ( cytoplasm).NDPK2 positive regulator of phy A and phyB in cotyledon opening and hook straightening- substrate for phytochrome kinase activity (cytoplasm and nucleus).FAR1- positively regulates phy A signaling protein with a coiled coil domain (nucleus).SPA1 negative regulator of phy A WD repeat protein (nucleus).

PAT1 positive regulator of phy A signaling- GRAS type transcription factor (cytoplasm).HY5 downstream regulator for phyA, phyB,and cry 1-Bzip transcription factor (nucleus).

NEGATIVE REGULATORA series of mutant in Arabidopsis have been isolated in screens for de etiolated phenotype in dark grown seedlings

These mutants, such as de-etiolated (det), or constitutively photomorhogenic (cop),similar phenotype to a group of mutants known as fusca (fus), identified through a screen for purple seed color.COP1 has Zn binding domain involved in DNA and 2 other motif , coiled coil helix and WD40 repeat. Present in nucleus in darkness and in light cytoplasmCOP9 SIGNALOSOME: several other COP/DET/FUS genes encode nuclear localized proteins that occur as subunits of a large multimeric complex, known as the COP9 signalosome.

PROPOSED MODEL:

FROM SIGNALING COMPONENT TO GENE EXPRESSION

MODEL FOR LIGHT SIGNALING