These are the slides from the class lecture on light sensory circuits in plants.

1. Wavelength
λ (meters)
10‐12 10‐10 10‐8 ~0.5x10‐6 10‐5 10‐2 102
Gamma
Rays
X‐ Rays
Ultra
violet
Visible Infrared Microwave Radio
Sub‐atomic
particles
Atoms Molecules Microbes Insects
10‐6 104 101
Humans Mountains
Relative sized
item
Wavelength
(nanometers) 260 320 380 420 480 540 600 660 720 780
UV‐C UV‐B UV‐A Blue Green‐Yellow Red Far Red
UVR8 Cryptochromes Cryptochromes* Phytochromes
Pr PfrPhototropins
LOV‐Domain
Sensors
Green
Sensor?
Plant
Photosensor
Human Vision
Plant Responses
Folta and Carvalho, 2015

2. Borthwick’s Experiment in Grand Rapids Lettuce (1954)

3. -synthesized as Pr in darkness
-converted by red light (max=
666 nm) to Pfr
-Pfr is biologically active form
-Far-red light (730 nm) converts
to Pr

4. Early Developmental Effects of Phytochromes
Phytochromes regulate (100’s of processes
described)
-- stem elongation
-- cotyledon expansion
-- chloroplast development (greening)
-- apical hook opening
-- gene expression
Microarray analyses show that the early
effects of phytochromes are to induce the
expression of transcription factors that then
alter the expression of genes involved in
photomorphogenic development
(Tepperman et al., 2001).

5. Shade avoidance responses
--- Induced under FR enriched circumstances
---EOD-FR(End of Day Far Red) can induce a
similar response
---Can be induced by shade or light reflected from
neighboring plants (“density sensor?”)
---Controlled by Type II phytochromes
---Plants possess long petioles, longer stems and
unusually elongated organs in general.
Regulation of Flowering time
---phyB mutant shows an early flowering in LD (long day 16H/8D) condition.
---phyA mutant shows an late flowering in Extended SD (8L/8 FR-enrich/8D) condition.
Long-term Roles for Phytochrome in Regulation of Plant Physiology

6. ENTER ARABIDOPSIS
Koornneef M., Rolff E. and Spruit C.J.P. (1980) Genetic control of light-
inhibited hypocotyl elongation in Arabidopsis thaliana (L.) Heynh. Z.
Pflanzenphysiol. 100: 147-160.
hy Mutants normal phy?
hy1 yes
hy2 yes
hy3 no
hy4 yes
hy5 yes
additional hy’s isolated later
hy8 no
Mutation in heme oxygenase (Davis et al., 2001)
mutation in heme oxygenase
mutation of phytochrome B
mutation of cryptochrome1, a photolyase-like protein
mutation in a B-zip transcription factor
mutation of phytochrome A (Dehesh et al, 1993)
Would turn out to encode:

7. Has topology similar to
histidine kinases, yet
exhibits ser-thr kinase
activity.
phytochrome binds ATP
diverged from prokaryotic
orthologs after duplication
of kinase transmitter domain
(Yeh and Lagarius, 1998).
BUT IS IT A KINASE?

8. Type I phytochrome :
photo-labile (phyA)
Type II phytochrome :
photo-stable (phyB,
phyC, phyD, phyE)
Two Major Types of Phytochrome:
B, R, FR
PrA PfrA
Dark reversion
Destruction
PrB PfrB
Dark reversion
R
FR
FR

9. The phy chromophore is a tetrapyrrole
that is reliant on heme oxygenase for its
synthesis; mutants in heme oxygenase
1 exhibit phy-like phenotypes.
The phenotypes can be rescued by
exogenous application of biliverdin
(Parks and Quail, 1991).
increasing BV
Brian Parks, circa 2000
and Brian’s homespun
Dobsonian mount telescope,
circa 2003.

10. Parks and Quail, 1993
Peter Quail
hy8 and hy1 mutants exhibit
specific inability to sense
far-red;
hy8 mutants sense red light
just fine…

11. Parks and Quail, 1993
The phenotypes of the phytochrome mutants
defined as hy loci.
Growth is unaffected in darkness.
hy3 and hy1 are impaired in red light
sensing
hy8 and hy1 are impaired in far-red light
sensing

12. Red – Import within minutes
Import of phyA to nucleus by red light also occurs within minutes; followed
by disappearance of the protein.
A phyB::GFP fusion protein is strongly
detectable in the nucleus of transgenic
tobacco cells beginning after three
hours (Gil et al., 2000).

13. Far-red– import after 1.5 h; retained
throughout time course (Hisada et
al, 2000).
phyA effects FR physiology with a
similar time course; protein constantly
detectable in WT background.
parks and spalding, 1999

14. Null phyB mutants are tested for complementation using the GR fusions.
The results indicate that both light and nuclear import are required for full
restoration of phyB activity.

15. Interaction between PIF3 and PHYB is dependent upon state of PHYB
PIF3 and PHYB
only interact if
phyB is in Pfr
state.
Ni et al., 1999

16. PIF3 Interaction with Promoter is Sequence Specific, and is Mediated
Through the DNA Binding Domains
Martinez-Garcia et al., 2000

17. Martinez-Garcia et al., 2000
Binding of phyB to the Promoter Requires PIF3, the phyB Chromoprotein
in the Pfr Form, and PIF3 Domains Outside of the DNA-Binding Domains

18. deLucas et al., Nature 2008

21. Hartweck, 2008 Planta

22. Claus Schwechheimera,
and Björn Christopher Willigea
Curr op plant bio, 2008

23. PKS, NDPK2

24. Cryptochromes
The sequence of Hy4 was reported in 1993, and it was similar to DNA
photolyase (Ahmad and Cashmore 1993, Sancar 1994) yet has no
photolyase activity.
Re-designated “Cryptochrome 1” (Lin et al., 1995)
Involvement in circadian rhythms; led to discovery of animal crys
(Cashmore, 2003)
Many plant responses were not R-FR
reversible and had action spectra with
peaks in the blue and near-UV. There
must be a BL receptor(s).
Due to their elusive nature, Gressel
(1977) described BL receptors as
“cryptochromes”.
hy4 mutants showed a lack of
hypocotyl growth inhibition under blue
light,but were normal under red and
far-red.
Dark BL
Wild-type
BL
cry1
adapted from Neff and Chory, 1998

25. Photoactivation of cryptochromes is (likely) similar to what
is observed in photolyases. Both a folate (MTHF) and a
flavin serve as chromophores in photolyase, and these
two compounds purify
with cry when it is expressed
in E.coli (Malorta et al.,
1995).
model of activity in photolyase
Cashmore, 2003.

26. cry2 mutants
The Cry2 gene was identified in the genome by homology to Cry1 (Jarillo et
al., 1996).
The cry2 mutants were isolated as seedlings not
expressing CRY2 protein. The mutants flowered late and
were shown to be allelic to other flowering-time genes
(below; Guo et al., 1998).
cry2 mutants lack a
conspicuous end-point
phenotype under high-
fluence blue light (right;
Mockler et al., 1998)
50 µmol m-2 s-1
5.5 µmol m-2 s-1
0.6 µmol m-2 s-1

27. CRY2 is phosphorylated in a light-dependent
manner (Shalitin et al., 2002)
1. Test for light-dependent phosphorylation
-- incubate with γ32P-ATP
2. Test to verify presence of CRY2 in same
conditions
3. Specific gel buffer/ionic
conditions allow resolution of the
phosphorylated form of the protein.
Again, the phosphorylated form (P)
is detectable only after BL
exposure.
P

28. The timing of CRY1 and CRY2
phosphorylation generally
coincides with CRY2’s influence
on early growth suppression.
Shalitin et al., 2002, 2003
Folta and Spalding, 2001
CRY2 phosphorylation
CRY1 phosphorylation

29. CRY2 accumulates in cop mutants
The phosphorylated
form accumulates in
cop1– and both the
phosphorylated and
unphosphorylated forms
disappear in WT. Propose
a mechanism.

30. Xing Wang Deng, Yale
COP Mutants
First isolated by Deng et al., 1991
--Constitutive Photomorphogenic
phenotype:
expanded cotyledons, short hypo-
cotyls, light-regulated gene
expression patterns in darkness
1996 Mayer et al show that COP1
mutation affects expression of
many genes
OLD SLIDE REVISITED!

31. The hy5 mutant – long hypocotyl under light conditions, particularly blue
(Koorrneef et al., 1980). Encodes a B-zip transcription factor that is
presumably a positive regulator of photomorphogenesis.
HY5 accumulates rapidly in light and is not
as detectable upon transfer of plants to
darkness
fluence rate
Plants grown for days in light show different
levels of HY5– HY5 level correlates with advanced
photomorphogenic development.
Osterlund et al., 2000
How does HY5 regulate photomorphogenesis?
Is it simply present only in light and acting as
a positive regulator? Is it more complex? It
seems to be acting in a manner that is
antagonistic to COP1….

32. HY5 mRNA accumulates in both
dark and light conditions, but the
protein is detected only in light.
Osterlund et al., 2000
Is this another example of ubiquitin-mediated proteolysis?
How can this be tested?

33. 1. Test using genetic tools – cop1 mutants
2. Test using
pharmacological tools
– proteosome inhibitors
Proteosome inhibitors allow
increased accumulation of
HY5.
mRNA levels are the same in cop and WT
plants in response to light treatment, yet
protein levels are very different.
This strongly suggests that COP1 has a role in
regulating the accumulation of HY5, a positive
regulator of photomorphogenesis.
Osterlund et al,. 2000

34. These results support a model where HY5, a positive regulator of
photomorphogenesis is degraded in darkness via COP1 and the
proteosome.
Osterlund et al., 2000

35. The C-Terminus of cry1 Regulates Photomorphogenesis
Ectopic overexpression of the CRY1
C-terminal extension results in a
constitutive-photomorphogenic
phenotype.
Cashmore, 2003
Is cry functioning through
a mechanism involving
COP1?

36. Does COP1 Interact with CRY?
Yeast 2-hybrid assay between COP1
truncations and CCT2 (CRY2 c-terminus)
Conclusions: The WD repeat domain is
necessary and sufficient for interaction,
yet binding is strongly enhanced by the
coiled-coil and Zn binding domains.
CCT2-GUS interacts similarly.
Coimmunoprecipitation –
Using anti-CRY2, COP1 can be
coimmunoprecipitated, demonstrating
likely interaction in vivo.
Wang et al., 2001

37. Hellmann and Estelle, 2002
Proposed Model for Cryptochrome Function – COP1, CRY, HY5
Interaction to Regulate Degradation of HY5

38. Phototropism
Noted by Darwin in observations of dark-grown canary grass
Colodny-Went model proposed that auxin would be secreted from the
tip and light would guide a lateral gradient of auxin to accumulate,
leading to differential growth.
A split in the plant biology community– phototropism just a phy response
vs a specialized BL receptor

39. Propose a Forward Genetic Screen - what might you find?
photoreceptor(s)– the actual molecule that receives the photon
signaling mutants– non-redundant genes required for transduction of the
signal from activated photoreceptor to target genes.
response mutants – those that have impaired cell wall expansion, auxin
transporters required for differential auxin redistribution… etc. ?
upstream elements – possibly molecules required for photoreceptor
synthesis and/or function.

40. phot1- BL-activated, membrane-associated, autophosphorylating ser/thr
kinase with two LOV domains (Christie and Briggs, 1998).
NPH3 - novel protein with possible BTB-POZ transcription activation
domains. NPH3 functions downstream of phot1, possibly participating as
a scaffold protein in the phosphorelay.
RPT2 – deficient in root phototropism, very similar to NPH3 .
NPH4- the same gene as ARF7 (Harper et al., 1999).
The phototropic mutant loci would be shown to encode:

41. Phototropins Regulate:
Phototropism (Huala et al., 1997; Christie et al., 1998)
Chloroplast accumulation and avoidance (Kagawa, 2001; Jarillo, 2001)
Primary hypocotyl growth inhibition (0-30 min; Folta and Spalding, 2001,
Folta et al., 2003)
Stomatal opening (Kinoshita et al., 2001)
Leaf expansion (Sakamoto and Briggs, 2002)
Blue-light destabilization of specific mRNA’s (Folta and Kaufman, 2003)
Solar tracking?

42. Phototropin – The Phototropism Photoreceptor
Mediates responses to blue and
near UV light.
Binds two flavin mononucleotide
molecules as chromophore
(Christie et al., 1998).
phosphorylation.. signaling or
desensitization?
Palmer et al., 1993

43. Phot Topology
Contains two specialized PAS domains called LOV domains (light, oxygen,
voltage) and a kinase domain (Christie and Briggs, 2001).
The protein can autophosphorylate on many sites (Salomon et al., 2003).
PHOT1 and PHOT2 share 60% sequence homology.
,

44. A Summary of Phot-Mediated Responses
phototropism chloroplast relocation stomatal aperture
low fluence-rate blue
high fluence-rate blue
Christie and Briggs, 2001
Sakai et al., 2001
accum avoid

45. Chloroplast accumulation and avoidance responses
LFR BL HFR BL
Treatment of leaf surfaces with low-fluence-rate blue light results in migration
of chloroplasts to the anticlinal surfaces of the leaf, perpendicular to incident
light. Irradiation with high-fluence-rate blue light results in migration of
chloroplasts to the periclinal cell surfaces, parallel to incident light.
This is important because it balances photosynthetic potential against
photoinhibition (Kasahara et al., 2002).

46. phot2 (aka. cav1 and npl1)
cav mutants obtained from forward
genetic screen for plants that failed
to show chloroplast avoidance.
Gene mapped to a region containing
NPL1, an NPH1 (phot1)- like gene.
A reverse genetic screen for NPL1
resulted in plants that failed to
relocate chloroplasts.

47. Photoreceptors of Adiantium
Adiantium contains 2 phototropins, and
shows normal phototropism and chloroplast
accumulation responses to BLUE LIGHT…
But the rap2 mutant does not exhibit
chloroplast accumulation or curvature in
response to red light.
-- Red light mediates phototropism and
chloroplast accumulation in Adiantium via a
hybrid phy-phot photoreceptor.

48. Cryptochrome mysteries to be resolved:
What is the mechanism of cry1 activation? How do redox changes affect
signaling?
cry1 and cry2 function within seconds of irradiation at the membrane, a
process that is required for inhibition of stem growth (Folta and Spalding,
2001). Do cry’s have a signaling mechanism independent of COP1?
cry interaction with phy, complete with cross phosphorylation has been
reported (Ahmad et al., 1997; Mas et al., 2000; Jarillo et al., 2000).

49. Integrated Scheme
of cry and phy function
Quail, April 2002
What has changed
already?
Don’t memorize this for
the exam! It may be very
different in a year!