View
314
Download
1
Category
Preview:
Citation preview
Wavelengthλ (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‐atomicparticles Atoms Molecules Microbes Insects
10‐6 104 101
Humans MountainsRelative 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* PhytochromesPr PfrPhototropins
LOV‐Domain Sensors
GreenSensor?
Plant Photosensor
Human Vision
Plant Responses
Folta and Carvalho, 2015
Borthwick’s Experiment in Grand Rapids Lettuce (1954)
-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
Early Developmental Effects of Phytochromes
Phytochromes regulate (100’s of processesdescribed)
-- 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).
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
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:
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?
Type I phytochrome : photo-labile (phyA)
Type II phytochrome : photo-stable (phyB, phyC, phyD, phyE)
Two Major Types of Phytochrome:
B, R, FRPrA PfrA
Dark reversion
Destruction
PrB PfrB
Dark reversion
R
FR
FR
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 2000and Brian’s homespun Dobsonian mount telescope,circa 2003.
Parks and Quail, 1993
Peter Quail
hy8 and hy1 mutants exhibit specific inability to sense far-red;
hy8 mutants sense red lightjust fine…
Parks and Quail, 1993
The phenotypes of the phytochrome mutantsdefined as hy loci.
Growth is unaffected in darkness.
hy3 and hy1 are impaired in red lightsensing
hy8 and hy1 are impaired in far-red light sensing
Red – Import within minutes
Import of phyA to nucleus by red light also occurs within minutes; followedby 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).
Far-red– import after 1.5 h; retainedthroughout time course (Hisada etal, 2000).
phyA effects FR physiology with a similar time course; protein constantly detectable in WT background.
parks and spalding, 1999
Null phyB mutants are tested for complementation using the GR fusions.
The results indicate that both light and nuclear import are required for fullrestoration of phyB activity.
Interaction between PIF3 and PHYB is dependent upon state of PHYB
PIF3 and PHYBonly interact ifphyB is in Pfrstate.
Ni et al., 1999
PIF3 Interaction with Promoter is Sequence Specific, and is MediatedThrough the DNA Binding Domains
Martinez-Garcia et al., 2000
Martinez-Garcia et al., 2000
Binding of phyB to the Promoter Requires PIF3, the phyB Chromoproteinin the Pfr Form, and PIF3 Domains Outside of the DNA-Binding Domains
deLucas et al., Nature 2008
Hartweck, 2008 Planta
Claus Schwechheimera,
and Björn Christopher Willigea
Curr op plant bio, 2008
PKS, NDPK2
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 BLWild-type
BLcry1
adapted from Neff and Chory, 1998
Photoactivation of cryptochromes is (likely) similar to whatis observed in photolyases. Both a folate (MTHF) and a flavin serve as chromophores in photolyase, and these
two compounds purifywith cry when it is expressed
in E.coli (Malorta et al.,1995).
model of activity in photolyaseCashmore, 2003.
cry2 mutantsThe 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
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
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
CRY2 accumulates in cop mutants
The phosphorylatedform accumulates in cop1– and both the phosphorylated and
unphosphorylated forms disappear in WT. Propose
a mechanism.
Xing Wang Deng, Yale
COP MutantsFirst isolated by Deng et al., 1991
--Constitutive Photomorphogenicphenotype:
expanded cotyledons, short hypo-cotyls, light-regulated geneexpression patterns in darkness
1996 Mayer et al show that COP1mutation affects expression of
many genes
OLD SLIDE REVISITED!
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 notas detectable upon transfer of plants to darkness
fluence rate
Plants grown for days in light show differentlevels of HY5– HY5 level correlates with advancedphotomorphogenic development.
Osterlund et al., 2000
How does HY5 regulate photomorphogenesis?
Is it simply present only in light and acting asa positive regulator? Is it more complex? Itseems to be acting in a manner that is antagonistic to COP1….
HY5 mRNA accumulates in bothdark 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?
1. Test using genetic tools – cop1 mutants
2. Test using pharmacological tools – proteosome inhibitors
Proteosome inhibitors allowincreased accumulation of HY5.
mRNA levels are the same in cop and WTplants in response to light treatment, yet protein levels are very different.
This strongly suggests that COP1 has a role inregulating the accumulation of HY5, a positiveregulator of photomorphogenesis.
Osterlund et al,. 2000
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
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 througha mechanism involving COP1?
Does COP1 Interact with CRY?
Yeast 2-hybrid assay between COP1truncations and CCT2 (CRY2 c-terminus)
Conclusions: The WD repeat domain isnecessary 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, demonstratinglikely interaction in vivo.
Wang et al., 2001
Hellmann and Estelle, 2002
Proposed Model for Cryptochrome Function – COP1, CRY, HY5 Interaction to Regulate Degradation of HY5
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
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 thesignal 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.
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:
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?
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
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.
,
A Summary of Phot-Mediated Responses
phototropism chloroplast relocation stomatal aperture
low fluence-rate bluehigh fluence-rate blue
Christie and Briggs, 2001
Sakai et al., 2001
accum avoid
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).
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 NPL1resulted in plants that failed to relocate chloroplasts.
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.
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).
Integrated Schemeof cry and phy function
Quail, April 2002
What has changed already?
Don’t memorize this forthe exam! It may be verydifferent in a year!
Recommended