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2. TOPIC:-MOLECULAR
MECHANISM OF LIGHT
PERCEPTION, SIGNAL
TRANSDUCTION AND
GENE REGULATION
Presented by :ZUBY GOHAR
ANSARI
TAM-2014-026

3. 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:
1. Quality (wavelength)
2. Quantity ( fluence or photon per meter square)
3. Directionality ( unidirectional or diffuse) or,
4. Duration ( photoperiod or day length)
PHOTOPERIODISM: Measurement of duration of light
or darkness in a twenty four hour cycle

4.  PHOTORECEPTORS : These are the chemicals or
compounds which can receive the light signals.
 Three photoreceptors are recognized:
1. Phytochromes that absorb maximally in red
(660nm) and far red (730nm), also absorb in UV-
A/blue;
2. B/UV-A photoreceptors absorb in blue and UV-A
parts of spectrum (320-480nm); and
3. UV-B photoreceptors absorb (280-320nm).

5. PHYTOCHROMES
 DISCOVERY: 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.

8.  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.

12.  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.

13.  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.

14.  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.

16.  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.

17. CRYPTOCHROME
These 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.

18.  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.

19. PTERIN

21.  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.

22.  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.

23. SIGNAL TRANSDUCTION AND
GENE REGULATION
 PHYTOCHROME 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.

24. STEPS: A diagram
1.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.

26.  B diagram
 1. 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.

27. 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.

28.  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.

29.  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).

30.  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).

31. NEGATIVE REGULATOR
 A series of mutant in Arabidopsis have been isolated in
screens for de –etiolated phenotype in dark grown
seedlings

32.  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 cytoplasm
 COP9 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.

33. PROPOSED MODEL:

34. FROM SIGNALING COMPONENT TO
GENE EXPRESSION

36. MODEL FOR LIGHT SIGNALING