Plant Photoreceptors

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Plant Photoreceptors
Red/far-red light receptor phytochromes Blue/UV-A
light receptor cryptochromes Blue light receptor
phototropins
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Photosynthetic pigments absorb red light and blue
light
B
FR
R
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Phytochromes (phyA-phyE)
Cryptochromes (cry1, cry2)
Phototropins (phot1, phot2)
Photoperiodic responses
Movement responses
Growth responses
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I. Phytochromes
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Red/far-red light receptor phytochrome
COOH
Phytochrome structure
(H. Smith, Nature 407585)
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phytochrome
1936 Lewis Flint (USDA) found different effect of
red light and far red light on the germination of
lettuce seeds. He found that red light
stimulates germination but far red light inhibits
germination. 1958 Harry Borthwick, Sterling
Hendricks (USDA) did more detailed study of light
effect on lettuce seed germination. They proposed
that a single photoreversible pigment is
responsible for both red light and far red light
effects. 1959 Butler demonstrated that
phytochromes are photoreversible pigment proteins
in plant extract.
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Effects of different wavelengths of light on seed
germination
Red light (650-700 nm)
()
germination
(-)
Far red light (700-750 nm)
Red light stimulates germination, whereas far-red
(infrared) light inhibits germination.
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Partially purified phytochrome in solution
Pr red-light absorbing form of phytochrome, The
Pr solution is in blue color (because it absorbs
all the red light passing through it). Pr is
hypothesized to be inactive
Pfr far-red-light absorbing form of phytochrome
The Pfr pytochrome solution is of blue-green
color. Pfr form phytochromes are hypothesized to
be physiologically active
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About phytochrome
1. 120 kD protein covalently attached at a
cysteine residue to the linear tetraperole
chromophore (called phytochromobilin or P?B), the
chromophore-apoprotein linkage is catalyzed by
the phytochrome apoprotein itself independent of
light. 2. The difference absorption spectrum is
characteristic of phytochrome, which is caused by
the cis-trans isomerisation.
3. Phytochrome bound to the chromophore can be
visualized by its fluorescence in the presence of
zinc. 4. Phytochrome is thought to be
synthesized in the Pr form. Pfr is thought to be
the active form of phytochromes (I have doubt),
which can spontaneously covert to Pr form in the
dark.
Pr Pfr cis-trans isomerisation
C
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1. Molecular genetic studies of plant
photoreceptors
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Light-dependent development - photomorphogenesis
dark grown
light grown
Arabidopsis
Pea
Maize
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Isolation of Arabidopsis long hypocotyl mutations
by Martin Koornneef in 1980
hy1, hy2 impaired in genes encoding enzymes
synthesizing phytochrome chromophore hy3 phyB
mutation hy4 blue light receptor CRY1
mutation hy5 photoreceptor signaling mutation
hy1
hy2
hy3
hy4
hy5
wt
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About Koornneefs hy mutations
1. All the hy mutants have same length of
hypocotyl as wt in dark, but they grow taller
(with longer hypocotyl) than the wt in light.
2. hy1, hy2 tall in red and far red light,
but not in blue light. Mutations of genes
encoding enzymes required for the synthesis of
phytochrome chromophore 3. hy3 tall in red
light only, but not in blue or far red light A
mutation of the PHYB gene encoding phytochrome B
(phyB) 4. hy4 tall in blue light only, but not
in red or far red light. A mutation of the CRY1
gene encoding the blue light receptor cry1 5.
hy5 tall in red, far red, and blue
light. Mutation of the HY5 gene encoding a
transcription factor involving in the
photoreceptor signal transduction
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2. phytochrome are protein kinases
A cyanobacterial phytochrome two-component light
sensory system Yeh et al., Science, 1997,
2771505
Eukaryotic phytochromes light-regulated
serine/threonine protein kinases with histidine
kinase ancestry Yeh and Largarias, 1998 Proc.
Natl. Acad. Sci. USA, 95 13976
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Phytochromes are nuclear proteins with the
protein kinase activity
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Oat phyA is a protein kinase
A. Recombinant oat phyA (AsphyA-ST) expressed and
purified from yeast cell (and reconstituted with
PCB) can autophosphorylate and can
phosphorylated another protein H1 histone (may
not be the natural substrate although). The Pfr
form is more active than the Pr form in
autophosphorylation. B. Similar observations
found for a recombinant green algal phytochrome
(mcphy1b-ST). C. The phy autophosphorylation is
base-labile but acid-stable, suggesting the
phosphorylated residues are Ser and Thr.
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Arabidopsis phyA and phyB are nuclear proteins
A-D scanning section of every 1.5 ?m of a
trichome cell from a light-grown plant, here the
red color in the nucleus showing phyA-GFP. It
indicates that phyA accumulates as small speckles
E-H scanning for phyB-GFP, showing larger
speckles
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3. phyB, phyD, phyE play overlapping function
in promoting floral initiation in Arabidopsis
Devlin, P.F., Patel, S.R., and Whitelam, G.C.
(1998). Phytochrome E influence internode
elongation and flowering time in Arabidopsis.
Plant Cell 10, 1479-1487 Devlin, P.F., Robson,
P.R., Patel, S.R., Goosey, L., Sharrock, R.A.,
and Whitelam, G.C. (1999). Phytochrome D acts in
the shade-avoidance syndrome in Arabidopsis by
controlling elongation growth and flowering time.
Plant Physiol 119, 909-15 Devlin, P.F., Patel,
S.R., and Whitelama, G.C. (1999). Phytochrome E
Influences Internode Elongation and Flowering
Time in Arabidopsis. Plant Cell 10, 1479-1488
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4. Phytochromes also control photoperiodic
flowering in short day plants such as rice
Izawa et al., Plant J. 2000, 22391-399
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Se5 is a photoperiod-insensitive early flowering
mutant of rice, a SD plant
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Se5 exhibits long coleoptiles in light.
Se5 has a very low amount of phytochrome
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Cloning of the SE5 gene showed that it encode a
phytochrome B apoprotein
Overexpression of the wt PHYB gene restored the
photoperiodic flowering (results in d shows
segregation of the R1 plants).
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II. cryptochromes
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About Arabidopsis CRY1 gene
1. The CRY1 gene was cloned from a T-DNA tagged
hy4 mutant allele (HY4 CRY1, hy4 cry1). CRY1
encodes a protein that has sequence similarity to
microbial DNA photolyase 2. DNA photolyase is
enzyme that repairs UV-damaged DNA. Photolyase
activity is blue light-dependent. 3. CRY1
covalently bind FAD (flavin adenine dinucleotide)
as chromophore, FAD can absorb blue and UV-A
light
CRY1- cry1 mutant CRY1 CRY1-overexpresser
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Absorption spectrum of cry1
The fact of CRY1 being a flavoprotein ends a
longstanding debate of what pigment is the
blue/UV-A light receptor in plants
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Cryptochromes are photolyase-like flavoproteins
found in plants and animals
(Cashmore et al., Science (1999), 284 760-765)
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The cry1, cry2, and photolyase amino acid
sequences are similar, particularly in their
amino-terminal domains.
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It was known that - cry1 is related to
photolyase, an ancient protein widely found in
prokaryotes, - plant has many blue light
responses speculation Arabidopsis may have
more than one cryptochrome genes Test
screening Arabidopsis cDNA library at low
stringency using CRY1 gene as the hybridization
probe. Results Isolated the CRY2 gene.
Arabidopsis cry2 gene
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Cry2 is a nuclear protein
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Phenotypic changes associated with the
overexpression of cry1
Plants grown under continuous white light
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Transgenic plants overexpressing the CRY2 gene
Observation cry2-overexpressing seedlings are
shorter than wild type under blue light, but not
as short as cry1-overexpressing seedlings.
Hypothesis Cry2 may also participate in
mediating blue light inhibition of hypocotyl
elongation. Test Looking for cry2 mutant and
see if it is taller than the wt in blue light.
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Isolation of the Arabidopsis cry2 mutant
Primary screening Screening for plants that had
intermediate tall (but not extremely tall)
hypocotyl when grown under blue light.
Blue light
(400 putative cry2 mutant)
cry1 (unwanted)
Secondary screening Analysis of the CRY2 protein
of the putative mutant lines by immunoblots to
identify those that accumulate no CRY2 protein
(nonsense mutation due to a premature stop codon.
Result 2 lines were isolated that had no CRY2
protein detected, called cry2-1 and cry2-2.
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cry2 mutant showed a long hypocotyl phenotype in
blue light especially low intensity of blue
light, Suggesting cry2 plays a role in
de-etiolation
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cry2 is also a flowering-time mutant
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cry2 is a later-flowering mutant allelic to fha
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Cry2 mutant is impaired in photoperiodic
sensitivity