PARPhotosyntheticallyactive radiation
1
UV-A (390 nm)
BLUE (470 nm)
2
FAR-RED (740 nm)
RED (670 nm)
UV-A
BLUE
GREEN
RED
FAR-RED
3
Photoperiodism-control by day-length
4
Shade avoidance
5
Shade avoidance
Measures the ratio of red:far-red
RED
6
FAR-RED
Shade avoidance
Red Far-red
R > FR
7
Shade avoidance
Red Far-red
8
FR > R
Shade avoidance
Red Far-red
9
Germination
10
Germinate (Grand Rapids lettuce)
D R+FR+R+FR+R
Flint and McAlister 1935, 1937; Borthwick et al. 1954, Williams 2000
11
12
Foundations of molecular biology
Biochemistry
Structural
Biology
Molecular Biology
Genetics
13
Column separates proteins by size,
charge, hydrophobicity, or other
properties
Fractions are collected and assayed for
protein of interest
14
Purification of the “phytochrome” protein
Assay
Assay: spectral properties
Pr form
(red light absorbing)
Source material:
Oat coleoptile
Pfr form
(far-red light absorbing)
15
Electron micrographs of purified phytochrome (dimer)
16
Electron micrographs of purified phytochrome (dimer)
= small molecule chromophore
= apoprotein (protein component)
17
Linear tetra-pyrrole
18
Cloning of phytochrome genes
Known protein sequence
DNA sequence
PCR primers
Arabidopsis has 5 phytochrome apoprotein genes:
19
Plant photoreceptor families
red/far-red
blue and UV-A
blue and UV-A
20
Generic approach
(gene products)
Stimulus
Biological Response
(e.g. environment, hormones, etc.)
Response deficient
Stimulus
(e.g. environment, hormones, etc.)
Biological Response
X
Ectopic (biologically inappropriate) response
Stimulus
(e.g. environment, hormones, etc.)
X
Biological Response
21
Closed, undeveloped cotyledons
Apical hook
Apical meristem arrested
Open, expanded cotyledons
Rapid hypocotyl growth
Active apical meristem
(first true leaves)
Hypocotyl growth inhibited
Seedling deetiolation,
a.k.a. photomorphogenesis
22
Tomato
23
Blue/UV-A
UV-B
Red
Far-red
(Dark)
Etiolated
(Skotomorphogenetic)
24
Deetiolated
(Photomorphogenetic)
Screen up to 5,000 Arabidopsis
seedlings on a petri plate
25
Response deficient
Stimulus
(e.g. environment, hormones, etc.)
26
X
Biological Response
Maartin Koornneef
(1980s)
Mutagenize
long hypocotyl
‘hy’ mutants
27
A genetic screen for
long hypocotyl ‘hy’
mutants
28
Apical meristem
Apical meristem
29
M1
M2
30
mut1
mut2
mut3
mut4
mut5
31
WT
m1
32
Back-cross
Recessive
m2
Recessive
m3
Recessive
m4
Dominant
m5
Recessive
WT
m1
m2
m3
m4
m5
m1
m2
Self-cross
m3
m4
m5
33
WT
m1
m2
m3
m1
m2
m3
m4
m5
34
Out-cross
m4
m5
WT
m1
m2
m3
m4
m5
WT
m1
m2
m3
m4
m5
m1
m2
m3
m4
m5
35
m1
m3, m1
m2
m3
complementation
groups
m4
m5, m2
m5
36
WT
m1
m2
m3
m4
m5
m1
m3, m1
m2
These four mutants have identified
two loci m3
(two genes).
There are two alleles at each locus.
m4
At least two loci have been identified
in this screen.
m5
37
hy mutants were found in 8
complementation groups (loci)
hy1
hy2
hy3
h4
h5
h6
hy7
hy8
38
complementation
groups
m5, m2
None of hy mutants were
completely etiolated
39
Forward genetics
Forward genetics
Mutant
Gene
Mutant
Gene
Map-based cloning of EMS hy mutants
hy8
hy2
Genetic mapping
hy3
hy1
Candidate genes
40
hy4
hy5
Far-red
WT
hy8
Dark
WT
hy8
Red-light
WT
hy3
Arabidopsis has 5 phytochrome apoprotein genes:
= hy8
= hy3
41
42
Shade avoidance responses
43
hy1 mutant
X
Phytochrome
chromophore
44
hy mutants were found in 8
complementation groups (loci)
hy1 – phytochrome chromophore biosynthesis
hy2 – phytochrome chromophore biosynthesis
hy3 – phytochrome apoprotein B
hy4 ?
hy5 ?
hy6 – phytochrome chromophore biosynthesis
hy7 – phytochrome chromophore biosynthesis
hy8 – phytochrome apoprotein A
45
HY1
Blue/UV-A
Red
UV-B
HY3
(PHYB)
Deetiolation
46
Far-red
HY8
(PHYA)
hy4 mutant
WT
hy4
WT
WT
hy4
hy4
hy4 mutant (deficient in blue light perception)
47
HY1
Blue/UV-A
UV-B
Red
HY4
Deetiolation
48
HY3
(PHYB)
Far-red
HY8
(PHYA)
HY4 = Cryptochrome 1 (CRY1)
Discovery of T-DNA K.O. allele à plasmid rescue
T-DNA
(pterin)
PHR
(flavin)
(Photolyase
homology region)
(pterin)
CCT1
(CRY1-carboxy-terminal-domain)
E. coli Photolyase
(flavin)
49
Arabidopsis CRY1
PHR domain
50
E. coli photolyase
flavin
HY4 = Cryptochrome 1 (CRY1)
T-DNA
pterin
CRY1-C
(pterin)
FAD
PHR
(flavin)
(Photolyase
homology region)
CCT1
(CRY1-carboxy-terminal-domain)
Photolyase
(flavin)
(pterin)
51
HY4 = Cryptochrome 1 (CRY1)
T-DNA
CRY1-C
(pterin)
FAD
PHR
(flavin)
(Photolyase
homology region)
Photolyase
(pterin)
52
(flavin)
CCT1
(CRY1-carboxy-terminal-domain)
White light
HY5 b-zip transcription factor
WT
hy5
53
Hy mutants were found in 8
complementation groups (loci)
hy1 – phytochrome chromophore biosynthesis
hy2 – phytochrome chromophore biosynthesis
hy3 – phytochrome apoprotein B
hy4 – blue/UV-A photoreceptor (cryptochrome)?
hy5 – B-zip transcription factor
hy6 – phytochrome chromophore biosynthesis
hy7 – phytochrome chromophore biosynthesis
hy8 – phytochrome apoprotein A
54
A ‘generic’ signaling pathway
(animal and fungal systems)
HY5
Light
PHYA
PHYB
CRY1
Changes in
transcription
55
HY1, 2, 6, 7
Blue/UV-A
UV-B
Etiolated
growth program
HY4
HY5
Red
HY3
(PHYB)
Far-red
HY8
(PHYA)
De-etiolated
growth program
A model for photomorphogenesis
56
0