UVR8: a plant UV-B photoreceptor
Inaugural-Dissertation zur Erlangung der Doktorwürde der Fakultät für Biologie der Albert-Ludwigs-Universität Freiburg im Breisgau
Vorgelegt von Luca Rizzini
Freiburg, Dezember 2010
Dekan:
Prof. Dr. Gunther Neuhaus
Promotionsvorsitzender:
Prof. Dr. Samuel Rossel Prof. Dr. Stefan Rotter Prof. Dr. Karl-Friedrich Fischbach
Betreuer der Arbeit:
Prof. Roman Ulm
Referent:
Prof. Roman Ulm
Koreferent:
PD Gerhard Leubner
Tag der Verkündigung des Prüfungsergebnisses: 21-02-2011
TABLE OF CONTENTS SUMMARY ................................................................................................................. 5 LIST OF ABBREVIATIONS .................................................................................................. 7 1
INTRODUCTION ............................................................................................... 11 1.1 1.1.1 1.1.2 1.1.3 1.1.4 1.1.5 1.2 1.2.1 1.2.2 1.2.3 1.3 1.3.1 1.3.2 1.3.3 1.3.4 1.4 1.4.1 1.4.2 1.4.3 1.5
2
The Photoreceptors ............................................................................................... 12 Phytochromes ....................................................................................................... 13 Cryptochromes ..................................................................................................... 17 Phototropins ......................................................................................................... 22 LOV Domains and Zeitlupe Photoreceptors ........................................................ 24 Chimeric Photoreceptors ...................................................................................... 26 Photoreceptor Systems not Present in Plants ....................................................... 28 BLUF .................................................................................................................... 28 Rhodopsins ........................................................................................................... 28 The Aryl Hydrocarbon Receptor .......................................................................... 29 UV-B Radiation.................................................................................................... 29 UV-B Damage ...................................................................................................... 30 UV-B Damage Signaling ..................................................................................... 31 UV-B non-Damage Response .............................................................................. 32 UV-B Perception .................................................................................................. 35 Components of the Low-Fluence Rate UV-B Pathway ....................................... 36 HY5 ...................................................................................................................... 39 COP1 .................................................................................................................... 40 UVR8 ................................................................................................................... 42 Aim of This Work ................................................................................................ 45
MATERIALS AND METHODS .......................................................................... 46 2.1 2.1.1 2.1.2 2.1.3 2.1.4 2.1.5 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6 2.2.7 2.2.8 2.2.9 2.2.10 2.2.11 2.2.12
Materials ............................................................................................................... 46 Plant Material and Media ..................................................................................... 46 Bacterial Strains and Media ................................................................................. 46 Yeast Strains and Media ....................................................................................... 47 Plasmids, Oligonucleotides and Antibodies ......................................................... 47 Enzymes and Reagents ......................................................................................... 51 Methods ................................................................................................................ 52 Plant Growth ........................................................................................................ 52 Plant Protein Extraction ....................................................................................... 52 Cell-Free Degradation Assay ............................................................................... 53 Yeast Growth and Transformation ....................................................................... 53 Yeast Protein Extraction....................................................................................... 54 HEK293T Cells Growth and Transformation ...................................................... 54 Protein Extraction from Transfected HEK293T Cells ......................................... 55 UV-B Treatments ................................................................................................. 55 Agrobacterium Mediated Plant Transformation .................................................. 57 DNA Isolation ...................................................................................................... 57 PCR ...................................................................................................................... 58 Agarose Gel Electrophoresis ................................................................................ 58
2.2.13 2.2.14 2.2.15 2.2.16 2.2.17 2.2.18 2.2.19 3
RESULTS .......................................................................................................... 63 3.1 3.2 3.3 3.3.1 3.3.2 3.3.3 3.3.4 3.4 3.5 3.6 3.7 3.8 3.9 3.9.1 3.9.2
4
Site-Directed Mutagenesis ................................................................................... 58 CPRG Assay ......................................................................................................... 59 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) ..................................... 60 Immunoblot Analysis ........................................................................................... 60 BiFC ..................................................................................................................... 61 Luciferase Measurement ...................................................................................... 61 Bioinformatic Analysis ........................................................................................ 62
COP1-UVR8 Interaction in Yeast ........................................................................ 63 UVR8 Homodimer ............................................................................................... 69 UV-B-Dependent Monomerization of UVR8 ...................................................... 71 UV-B-Dependent Monomerization of UVR8 in HEK293T Cells ....................... 71 UV-B-Dependent Monomerization of UVR8 in Yeast ........................................ 75 UV-B Dependent Monomerization of UVR8 in Planta....................................... 78 UV-B-Dependent UVR8 Degradation ................................................................. 81 Evolutionary and Structural Considerations......................................................... 83 Site-Directed Mutagenesis ................................................................................... 88 Mixing-Extracts Experiment ................................................................................ 92 Physcomitrella UVR8 Ortholog ........................................................................... 93 UVR8 and HY5 Compete for COP1 Interaction .................................................. 95 RUPs..................................................................................................................... 96 RUPs UV-B-Dependent Interaction with UVR8 ................................................. 96 RUPs Mechanism ................................................................................................. 96
DISCUSSION .................................................................................................... 98 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8
UVR8 and COP1 Interaction in Heterologous System ........................................ 98 UVR8 Self-Interaction and UV-B Dependent Monomerization .......................... 99 UVR8 Protein Putative Conformational Change ............................................... 101 Phylogenetic and Structural Considerations....................................................... 103 UV-B Perception by UVR8................................................................................ 104 UVR8 Mechanism .............................................................................................. 106 RUPs Mechanism ............................................................................................... 109 Conclusions and Outlook ................................................................................... 110
ACKNOWLEDGEMENTS ...................................................................................... 112 5
REFERENCES ................................................................................................ 113
Summary Ultraviolet-B radiation is part of the sunlight spectrum reaching the Earth. The high energy per photon of this wavelength range can cause ROS production, lipid peroxidation, reduced photosynthetic activity, and DNA damage when absorbed by the genetic material. To counter these negative effects, plants have evolved a UV-B photoreceptor system which helps to minimize the UV-B-mediated damage through, e.g., “sunscreen’” pigment synthesis, induction of repair mechanisms and enhanced photomorphogenesis. It is known since over 30 years that plants are able to specifically perceive UV-B radiation, but the molecular identity of the photoreceptor remained elusive. The main difficulty in its identification is related to the property of UV-B to be absorbed by almost all organic compounds. Therefore, it is often not clear if the cellular UV-B signalling is due to a more general damage response or a damage-independent direct perception, i.e., a UV-B-photoreceptor-specific pathway. We approached the problem by working with very low fluence rate UV-B, to avoid damage responses but to activate the UV-B photoreceptor responses specifically. This approach led to the identification of the proteins UV-RESISTANCE LOCUS 8 (UVR8) and CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) as early components of the UV-B-specific signalling pathway in Arabidopsis thaliana. It has been shown that UVR8 and COP1 are able to interact in a UV-B-dependent manner in planta. The E3 ubiquitin ligase COP1 is a general regulator of light responses, whereas the β-propeller protein UVR8 seems to be a UV-B-specific plant signalling component. In this work I show that UVR8 is able to interact with COP1 in a UV-Bdependent manner in yeast. Moreover, I was able to show that UVR8 can homodimerize. Herein I further show that UVR8 is present almost exclusively as a homodimer in planta, and that the UV-B radiation is able to monomerize UVR8 in a human cells line, in yeast and in planta. Furthermore, I reproduced the UVR8 monomerization after protein purification, showing the same kinetics as in total extracts. UVR8 is able to monomerize in less than 5 seconds of UV-B treatment in plant protein extracts on ice, suggesting a direct perception of UV-B by UVR8. Moreover, the re-dimerization at room temperature takes much longer, as shown in human cells culture total protein extracts, possibly giving the time for signal transduction. In yeast, UVR8 also interacts UV-B-dependently with the WD40-repeat proteins REPRESSOR OF UV-B PHOTOMORHOGENESIS (RUP) 1 and 2, negative
regulators of the UV-B-specific signalling pathway which reduce the interaction of UVR8 with COP1 under UV-B in a negative feed-back loop. Moreover, we postulated that UVR8 could directly perceive UV-B photons through tryptophan residues. Indeed, a UVR8 protein mutant in one of the tryptophans is not capable anymore to monomerize under UV-B light, and the same UVR8 protein mutant is not capable to interact with COP1 in yeast anymore. Published data on UVR8, e.g., microarray analysis and phenotypic characterization, together with all the evidences reported in this work, strongly support the idea that UVR8 is a UV-B plant photoreceptor.
LIST OF ABBREVIATIONS 3D
Three dimensional
35S
promoter of the CaMV 35S RNS gene
6-4PP
6-4 photoproducts
AD
Activation Domain
BD
Binding Domain
bHLH
basic Helix-Loop-Helix motif
BiFC
Bimolecular Fluorescence Complementation assays
BLAST
Blast Local Alignment Search Tool
BLUF
Blue Light sensors Using FAD
bps
base pairs
BR
Brassinosteroid
bZIP
basic leucine-Zipper motif
CaMV
Cauliflower Mosaic Virus
CFP
Cyan Fluorescent Protein
cGMP
cyclic Guanosine MonoPhosphate
ChIP
Chromatin Immunoprecipitation
CHS
CHALCONE SYNTHASE
Col
Arabidopsis thaliana Columbia accession
COP1
CONSTITUTIVE PHOTOMORPHOGENIC 1
CPD
Cyclobutane Pyrimidine Dimers
cry1/2/3
Cryptochrome 1/2/3 (holoproteins)
CRY-DASH
Cryptochrome subfamily (Drosophila, Arabidopsis, Synechocystis, Humans)
CUL
CULLIN
DAS
DQXVP-Acid-STAES
DET1
DE-ETIOLATED 1
DSB
Double-Strand Break
EAL
Protein domain named after its conserved amino acids, also known as Domain of Unknown Function (DUF2)
ECFP
Enhanced Cyan Fluorescent Protein
eid6
empfindlicher im dunkelroten licht 6
EMS
Ethyl Methyl Sulfonate
EYFP
Enhanced Yellow Fluorescent Protein
FAD
Flavin Adenine Dinucleotide
FHL
FHY1-LIKE
FHY1
FAR-RED ELONGATED HYPOCOTYL1
FICZ
6-formylindolo[3,2-b]carbazole
FMN
Flavin MonoNucleotide
GAF
cGMP–specific and –stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA
GEF
Guanine nucleotide-exchange factor
GFP
GREEN FLUORESCENT PROTEIN
GR
Glucocorticoid Receptor
GW
Gateway
HFR1
LONG HYPOCOTYL IN FAR-RED LIGHT 1
HisKA
HisK-ATPase, Histidine Kinase ATPase Superfamily Domain
HKRD
Histidine Kinase–Related Domain
HR
Homologous Recombination
HY5
ELONGATED HYPOCOTYL 5
HYH
HY5-HOMOLOG
JA
Jasmonic Acid
kDa
kilodalton
LAF1
LONG AFTER FAR-RED LIGHT 1
LB
Luria-Bertani (medium)
Ler
Arabidopsis thaliana Landsberg erecta accession
LOV
Light, Oxygen or Voltage (domain)
MS
Murashige and Skoog (basal salt mixture)
MTHF
Methenyl Tetrahydrofolate
NER
Nucleotide Excision Repair
NES
Nuclear Export Signal
NHEJ
Non-homologous End Joining
NIS
Nuclear Import Signal
nm
nanometer
p
plasmid
P
Pterin
p53
tumour protein 53
PAH
Polycyclic Aromatic Hydrocarbon
PAL
PHENYLALANINE AMMONIUM LYASE
PAR
Photosynthetically Active Radiation
PAS
Per-ARNT-Sim domain
PBS
Phosphate Buffered Saline
phot1/2
Phototropin 1/2 (holoproteins)
PHR
Photolyase Homology Region
PHY
Phytochrome-specific domain related to PAS domain
phyA/B/C/D/E
Phytochrome A/B/C/D/E (holoproteins)
PIF3
Phytochrome Interacting Factor 3
PIN
Pinformed
PKD
Protein Kinase D (Serine-threonine protein kinase domain)
PLD
PAS-like domain
PR1/2/5
PATHOGENESIS-RELATED GENE 1
Pro
Promoter
PVDF
Polyvinylidene Difluoride (membrane)
Ran
Ras-related Nuclear protein
Ras
Rho family, small GTP binding protein
RCC1
REGULATOR OF CHROMATIN CONDENSATION 1
RING
REALLY INTERESTING NEW GENE
ROS
Reactive Oxygen Species
RT
Room Temperature
Rubisco
Ribulose-1, 5-bisphosphate carboxylase/oxygenase
RUP1/2
REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 and 2
RUS1/2
ROOT UV-B SENSITIVE 1 and 2
SA
Salicylic Acid
SAP
Sequestered Areas of Phytochromes
SCF
Skp, Cullin, F-box containing complex
SDM
Site-Directed Mutagenesis
SPA1/4
SUPPRESSOR OF PHYA 1 to 4
SPYNE/SPYCE
Split YFP N-terminal/C-terminal fragment expression
STH
STO HOMOLOGUE
STO
SALT TOLERANCE
TLP
Twin Love Protein 1
ULI3
UV-B LIGHT INSENSITIVE 1
UV-A/B/C
Ultraviolet-A/B/C
UVR8
UV-RESISTANCE LOCUS 8
WL
White Light
Ws
Arabidopsis thaliana Wassilewskija accession
YFP
YELLOW FLUORESCENT PROTEIN
Introduction
11
1 Introduction Light sensing is crucial for plant growth and survival because light is source of energy and environmental information. Light is an energy source for all photoautotrophic organisms (plants, algae and photosynthetic bacteria), bacteria) trapped by photosynthesis, and used to transform carbon dioxide into organic compounds. However, light is also a source of information and it is used to accomplish photomorphogenesis, a light-mediated mediated change in plant growth and development, as illustrated in Fig. I1 (Mohr, 1995; Taiz, 2002).
Figure I1:: Representation of different phenotypes of mustard seedlings exposed to light (left photomorphogenesis) or kept in the dark (right - skotomorphogenesis) (Mohr, 1995). 1995)
The signal transduction pathways that originate from the photomorphogenic processes are diverse and complex (Taiz, 2002).. In order to accomplish photomorphogenesis, plants have evolved evo perception systems to the different light wavelengths of the sunlight spectrum, spectrum which are able to assess quality, quantity, and duration of the radiation.. Photoreceptor proteins are able to perceive specific wavelengths and to initiate a signal transduction pathway. In plants, well known
Introduction
12
photoreceptors are phytochromes that perceive primarily red and far-red light and cryptochromes and phototropins that perceive UV-A and blue light. Light is not only source of energy and information but it is also causing damage. This is particularly noteworthy for plants which are characterized by a sessile lifestyle. Photomorphogenesis is an example of adaptation to the variable light qualities and quantities, i.e. plants are able to switch between two developmental programs, photomorphogenesis in the light and skotomorphogenesis in the dark (Fig. I1). Skotomorphogenesis allows plants to escape from the dark to reach the light (e.g. seed buried in soil). Once the seedling reaches appropriate light conditions, it switches to photomorphogenesis, starting energy capture and biomass production and, at the same time, avoiding higher light intensities able to cause damage (Buchanan, 2000). Not only high intensities of visible light are able to cause damage, but also UV-B radiation (Lumsden, 1997). UV-B radiation comprises a minor part of the solar spectrum thanks to the stratospheric ozone layer (Chapman, 1930), but the energy content per photon of such radiation, and its ability to interact with organic compounds, make it very harmful for living organisms. UV-B is damaging mainly DNA, but also RNA, protein and lipids. Plants show a specific response to UV-B wavelengths that result in, e.g., accumulation of “sunscreen” pigments (like flavonoids and anthocyanins) and enhanced photomorphogenesis. UV-B action spectra on anthocyanins and flavonoids synthesis are reviewed (Beggs and Wellmann, 1994). These data, together with other UV-B light specific responses, led scientists to postulate the presence of a UV-B photoreceptor (Tevini and Teramura, 1989; Cen and Bornman, 1990; Stapleton, 1992; Beggs and Wellmann, 1994).
1.1 The Photoreceptors Photoreceptors are proteins responsible for light perception, and they are able to initiate a signalling cascade which results in light specific responses in a variety of organisms. Usually the perception of light is mediated by a protein containing a prosthetic group, called chromophore. The protein together with the chromophore forms the photoreceptor holoprotein. The chromophore is able to perceive light through absorption. The energy of light causes photoisomerization or photoreduction of the chromophore, a physical change perceived by the apoprotein which initiates
Introduction
13
the light signal transduction. Different chromophores can bind to different apoproteins, like retinal chromophore (e.g. rhodopsin in animal), flavin chromophore (e.g. cryptochrome in plants and animal) and bilin chromophore (e.g. phytochrome in plants). In plants there are a variety of photoreceptors (Fig. I2), covering almost all the sunlight spectrum. The phytochromes absorb in the range of red and far-red light (phytochromes A, B, C, D and E), while cryptochromes (cryptochrome1 and chryptochrome2) and phototropins (phototropin1 and phototropin2) absorb in the range of UV-A and blue light.
Figure I2: Schematic representation of visible light spectrum and the main family of plant photoreceptors with their chromophores (above the photoreceptor models). Phytochromes, cryptochromes, and phototropins are shown (Jiao et al., 2007).
1.1.1 Phytochromes Garner and Allard (Garner and Allard, 1920) coined the word photoperiodism to describe that, e.g., flowering was induced by long days in some species and by short days in others. Later on, it was discovered that a unique pigment was responsible for photoperiodism and photomorphogenesis (Parker et al., 1946; Borthwick et al., 1948). Thanks to studies of the action spectra of plants, it was postulated that the chromophore responsible for this phenomena should absorb in the red/far-red range of the light with an additionally minor absorption in the blue light. Such action spectra were compatible with the chromophore phycobilin (Parker et al., 1946; Borthwick et al., 1948; Parker et al., 1950). Furthermore, it has been discovered the red/far-red reversibility of seed germination (Toole et al., 1953), which refers to the induction of germination by red light that can be reversed when followed by far-red light
Introduction
14
irradiation, leading to inhibition of germination. The photoreversibility photoreversibility was an amazing proof for photoperception, through a photoreversible chromophore, which results in an adaptative development to the physical surroundings. The final evidence that photoreversibility is achieved through the same photoreceptor was provided p by the isolation of the phytochrome (Butler et al., 1959).. The chromophore responsible for activation and inactivation of plant phytochromes is the phytochromobilin phytochromo (PΦB) (Butler et al., 1959; Siegelman and Firer, 1964). 1964) The non-active active Pr form of the phytochromes converts to active Pfr Pfr form upon red light illumination. The Pfr form can photorevert to the Pr form upon far-red far red light illumination or in the dark (Schmidt et al., 1973), as shown in Fig. I3a. In Fig. I3b the domain structure of plant phytochromes is presented. d. PLD, GAF and PHY are protein domains related to the PAS (Per-ARNT-Sim) Sim) domain. PAS domains have important roles as sensory modules for oxygen, tension, redox potential or light intensities (Ponting and Aravind, 1997). 1997) Moreover, PAS domains are involved in protein-protein protein protein interactions and they can bind to cofactors, like the GAF domain of phytochromes phyt which binds to PΦB P (Butler et al., 1959; Siegelman and Firer, 1964). 1964). HKRD is a histidine kinase related domain for which hich the kinase activity has not yet been unequivocally demonstrated.
a)
b)
Figure I3: a)) Photoreversible conversion of phytochromes from Pr to Pfr form in red light, light and vice versa in far-red light,, and dark reversion (Schafer and Bowler, 2002); b)) Phytochrome domain structure: NTE, N-amino-terminal terminal extension; PLD, PAS-like PAS like domain; GAF, a domain distantly related to PAS and found in phytochromes and cGMP-specific cGMP specific phosphodiesterases; PHY, a domain distantly related to PAS and specific to phytochromes; HKRD, histidine kinase related domain dom lacking a phosphoacceptor His residue and motifs characteristic of bona fide histidine kinases; HisKA, histidine kinase A domain-related; related; HisK-ATPase, HisK ATPase, histidine kinase ATPase superfamily domain (Sharrock, 2008).
Introduction
15
Phytochrome family of photoreceptor is widespread in living organisms, as shown in Fig. I4. In Arabidopsis thaliana this family is composed of five members named phytochrome A (phyA), phytochrome B (phyB), phytochrome C (phyC), phytochrome D (phyD), and phytochrome E (phyE) (Sharrock and Quail, 1989; Clack et al., 1994).
Figure I4: Plant phys (plant phytochromes), BphPs (Bacterio-phytochromes), Cphs (Cyanobacterial phytochromes), Fphs (Fungal phytochromes) and Phy-like (Phytochrome-like proteins) (Karniol et al., 2005).
Three distinct response modes of phytochrome action have been characterized in Arabidopsis thaliana, which differ for fluence requirement and red/far-red reversibility. These are the high irradiation response (HIR), the low fluence response (LFR), and the very low fluence response (VLFR). PhyB is responsible for the LFR together with, but to a lesser extent, the other light stable phytochromes, phyC, phyD and phyE. PhyA is responsible for the HIR response and VLFR response (Nagy and Schafer, 2002).
Introduction
16
PhyA and phyB localize to the cytoplasm and move to the nucleus upon light irradiation (Sakamoto and Nagatani, 1996; Kircher et al., 1999). Similar to phyA and phyB, also phyC, phyD and phyE localize to the cytosol in dark-grown seedlings and move to the nucleus upon light perception (Kircher et al., 2002). Because of the relationship between light perception and signalling, it is of relevance that phytochromes move into the nucleus upon light activation, where they can directly or indirectly activate transcription. Indeed, it has been discovered that nuclear phyA and phyB interact, in a light-dependent fashion, with Phytochrome Interacting Factor 3 (PIF3), a basic helix-loop-helix (bHLH) transcription factor (Ni et al., 1998; Bauer et al., 2004). The interaction between phyA and phyB with PIF3 corroborates the hypothesis that phytochromes move to the nucleus to activate light responses through transcriptional activation. Moreover, it has been shown that phyA and phyB move to the nucleus in their active Pfr form, underlying the link between photoperception and signal transduction (Kircher et al., 1999; Yamaguchi et al., 1999). However, the best proof that shows the nuclear import of phytochromes, as mandatory step for light signal transduction, comes from the fusion protein glucocorticoid receptor-phyB expressed in a phyB mutant background (Huq et al., 2003). The fusion of the protein to the glucocorticoid receptor (GR) caused its cytoplasmic retention; irrespective of the light condition, no signal transduction was taking place. When Dex was applied to the medium, the GR-phyB fusion protein was freed up from the cytoplasmic retention factors, allowing its translocation to the nucleus, and upon red light irradiation, to recover the phytochrome-mediated signalling. Using particle bombardment of onion cells it was demonstrated that the E3 ubiquitin ligase YFP-COP1, a central regulator of light signalling, co-localizes with phyA-CFP in the nucleus. PhyA has also been shown to interact with COP1 in vitro (Seo et al., 2004). Furthermore, COP1 is able to ubiquitinate phyA in vitro, and phyA shows higher stability in cop1 mutant lines (Seo et al., 2004), indicating that the interaction leads to proteasomal degradation of phyA. Despite the fact that there’s no definitive evidence for in vivo interaction between phyA and COP1, this set of data points to a possible pathway for light responses activated by phytochromes. Another link between perception and signalling is given by in vitro data which shows serine-threonine kinase activity of the histidine kinase related domain (HKRD) of phytochromes (Yeh and Lagarias, 1998).
Introduction
17
To conclude, it has also to be mentioned that phytochromes are present as homodimer and heterodimer in vivo (Brockmann et al., 1987; Sharrock and Clack, 2004). Recently, it has been shown that the homodimer is formed all along the phytochrome protein structure (Li et al., 2010). In summary, herein are presented the main properties of the oldest and most characterized plant photoreceptors, the phytochromes. These properties include light dependent activation, and translocation to the nucleus, which lead to transcriptional activation of light-responsive genes.
1.1.2 Cryptochromes The cryptochromes are UV-A/blue light photoreceptors. In Arabidopsis thaliana there are two cryptochromes, cryptochrome 1 (cry1) and cryptochrome 2 (cry2). Cryptochromes are widespread across the kingdoms of life (Fig. I5). Fig. I5 also includes the closest homologs of cryptochromes, namely 6-4 photolyases, CPD photolyases and CRY-DASH proteins.
Introduction
18
Figure I5: Phylogenetic tree of cryptochromes and related sequences. A, archaea; B, bacteria; F, fungi; I, insects; P, plants; S, sponges; V, vertebrates (Lin and Todo, 2005).
Maarten Koornneef and coworkers isolated the hy4 mutant in a screen for Arabidopsis thaliana mutants with an elongated hypocotyl in white light (Koornneef et al., 1980). Later on, the hy4 mutant was characterized for having elongated hypocotyl phenotype in white light and blue light, but not under red and far-red light, or in
Introduction
19
darkness (Ahmad and Cashmore, 1993). In the same work, HY4 was found to encode a photolyase-like protein. Nevertheless, HY4 was tested negative for photolyase activity that, together with other results, led to the conclusion that HY4 is a UV-A/blue light photoreceptor and it was renamed cryptochrome 1 (Lin et al., 1995a; Malhotra et al., 1995). In parallel to Arabidopsis thaliana, a photolyase-related gene was discovered in Synapis alba (Batschauer, 1993) that was later found to be a CRY1 ortholog. In this work an EST library was prepared from white mustard and screened to identify plant photolyase coding genes. For this reason HY4 was considered to be, in this work, a photolyase protein. It is interesting to note the similarity of cryptochromes to photolyases. Indeed, the chromophore of photolyase proteins was thought, even before the discovery of HY4, to be a candidate receptor for UV-A and blue light (Galland and Senger, 1988b, a). In Arabidopsis thaliana there are three cryptochromes, as shown in Fig. I6. cry2 is very similar to cry1 (Hoffman et al., 1996), while cryptochrome 3 (cry3 or A.t.CRY-DASH) is more divergent and its function is not clear yet.
Figure I6: Domain structure of plant cryptochromes. Cryptochromes cofactors are shown as well: MTHF (pterin) and FAD (flavin) (Batschauer et al., 2007).
The central domain of the cryptochromes in Arabidopsis thaliana is called the photolyase homology region (PHR). This region is highly conserved and similar to photolyases, and like in photolyases, it can bind to chromophores. Nonetheless, cryptochromes are divergent from photolyases in their C-terminal region, the so called DAS domain (DQXVP-acidic-STAES) (Lin, 2002). In cry3 the DAS domain is located in the N-terminal region. The Arabidopsis thaliana cryptochromes have two chromophores, a flavin (FAD) and a pterin (methenyltetrahydrofolate, MTHF) (Fig. I6). When CRY1, CRY2 and CRY3
Introduction
20
are expressed in Escherichia coli, they bind to FAD and to MTHF in a 1:1 stoichiometry (Lin et al., 1995b; Malhotra et al., 1995; Pokorny et al., 2005), indicating that the binding to both chromophores is required for function. The first crystallized full-length cryptochrome structure comes from Synechocystis (Brudler et al., 2003). This is a CRY-DASH cryptochrome which contains only FAD. It has been possible to decipher some properties of the cryptochrome from the crystallized structure, like the differences from photolyases, substrate recognition, and speculate on possible electron transfer events that are the basis for signal perception. The only Arabidopsis thaliana crystallized cryptochrome is cry3 (Pokorny et al., 2005). This crystal structure has shown a homodimeric conformation of cry3. Cryptochromes are phosphorylated upon blue-light irradiation. The phosphorylation has been found to be mandatory for signalling and to lead to degradation of cry2 (Shalitin et al., 2002; Bouly et al., 2003; Shalitin et al., 2003). These aspects of cry2 are reminiscent of phyA which, upon irradiation, move to the nucleus and it is subject to proteasomal dependent degradation. In the same studies it has also been shown that cry1 is able to autophosphorylate upon blue light irradiation. Because these studies were performed in vitro, it was possible to demonstrate that the presence of FAD is mandatory to achieve autophosphorylation of cry1. cry1 and cry2 are similar to class I CPD photolyases, while cry3 is similar to 6-4 photolyases. The crystal structure of many class I CPD photolyases has been solved (Park et al., 1995; Tamada et al., 1997; Komori et al., 2001). From this works it is possible to understand the importance of FAD for the catalytic activity of the enzyme and that MTFH is important to increase the DNA repair efficiency in low light. Photolyases are able to repair DNA damage caused mainly by UV-B radiation. UV-B can be absorbed by DNA causing the dimerization of pyrimidines. The main photoproduct of pyrimidin dimerization is the cyclobutane pyrimidine dimer (CPD). Photolyases are able to harness blue-light energy through the chromophore FAD reducing it to FADH-. The electron that is now in the chromophore can be used to destabilize CPD and break the bond to restore the integrity of DNA. Similarly to photolyase mode of action, cryptochromes are able to capture blue-light energy but using it for signalling rather than DNA repair (Malhotra et al., 1995; Cashmore et al., 1999). It has been possible to demonstrate this hypothesis for cry1 (Giovani et al., 2003). In this work, cry1 was synthesized in insect cells and subjected to laser excitation. From the excitation kinetics and the reduced state of FAD and additional
Introduction
21
data, it was possible to conclude that the electron transfer was taking advantage from a tyrosine radical and a tryptophan radical. Interestingly, in vitro and in vivo experiments with amino acid substitution led to a better understanding on the electron transfer that is taking place in cry1. Two substitutions of putative tryptophan electron donors with redox inactive phenylalanine, T400F and T324F, impaired cry1 signalling (Zeugner et al., 2005). These mutants were also impaired in autophosphorylation, and the characteristic phenotype with reduced hypocotyl growth under
blue
light,
showing
a
direct
correlation
between
light
absorption,
phosphorylation activity and downstream signalling. Domain swapping experiments between cry1 and cry2 lead to the hypothesis that cryptochromes are regulating their own degradation. The way by which cry2 gets degraded is not yet clear but its interaction with the C-terminal WD-40 domain of COP1 may suggest a proteasomal dependent degradation (Wang et al., 2001). Moreover, in cop1 mutant seedlings cry2 is stabilized under blue light and the phosphorylated cry2 is accumulating (Shalitin et al., 2002). It has to be noticed that cry1 is not blue-light dependent degraded but it is also interacting with COP1 (Yang et al., 2001). Unfortunately, there are still not enough data on COP1 mode of action to understand the meaning of the interaction of the photoreceptors with COP1. cry1 has a light dependent subcellular localization being nuclear in the dark and mainly cytoplasmic upon light irradiation (Cashmore et al., 1999; Yang et al., 2000). cry2 has a different behavior being constitutively localized in the nucleus (Guo et al., 1999; Kleiner et al., 1999). cry3 has been found to localize in chloroplast and mitochondria (Kleine et al., 2003). The different degradation and subcellular localization of cryptochromes in Arabidopsis thaliana is not enough to understand the function and mode of action of these photoreceptors; more comprehensive work will be needed to clarify their signal transduction. The mode of action of cryptochromes could be explained by the homodimerization of these photoreceptors. The overexpression of the C-terminus of cry1 and cry2 in wild type Arabidopsis thaliana seedlings is giving a constitutively photomorphogenic phenotype (Yang et al., 2000); while overexpression of the N-terminus of cry1 in wild type Arabidopsis thaliana seedlings has a cryptochrome mutant phenotype (Sang et al., 2005). In the same work the authors found that cryptochromes homodimerize in a light independent fashion at their N-terminus. It is now becoming clear that cryptochromes need to homodimerize to be functional and the overexpression of the
Introduction
22
C-terminus of cry2 was giving a constitutive response because of the GUS fusion. Indeed, GUS is known to oligomerize in a way that the GUS fusion to the C-terminal domain of cry2 was taking over the homodimerization function of the N-terminal domain.
1.1.3 Phototropins Phototropins are plasma membrane-associated UV-A/blue-light photoreceptors present in plants, which control phototropism, light-induced stomatal opening and chloroplast movements (Briggs and Christie, 2002; Kagawa, 2003; Celaya and Liscum, 2005). In Arabidopsis thaliana there are two phototropins, originally named NPL1 and NPH1, now known as phototropin 1 (phot1) and phot2, respectively (Huala et al., 1997; Briggs et al., 2001; Jarillo et al., 2001a; Kagawa et al., 2001) (Fig. I7).
Figure I7: Phylogram of the phototropin family of blue light photoreceptors (Briggs et al., 2001). Putative phototropins and the neochrome PHY3 in Adiantum capillus-veneris are shown as well.
Introduction
23
type kinases inactive in darkness and activated upon blue-light Phototropins are AGC-type irradiation (Bogre et al., 2003). 2003). The activation of the kinase domain of these photoreceptors is thought thought to be the starting point of their light responses. Phototropins perceive light through two N-terminal N terminal light, oxygen, voltage (LOV) domains, LOV1 and LOV2. LOV domains are PAS domains responsible for cofactor binding and protein-protein protein interaction (Taylor and Zhulin, 1999).. Phototropins bind to the chromophore flavin n mononucleotide (FMN) through the LOV domain, as illustrated in Fig. I8 (Christie et al., 1999; Salomon et al., 2000).
Figure I8:: Schematic illustration of phototropin protein domains; The three fused-hexagon fused represent the FMN bound to the LOV1 and LOV2 domains, PKD represent the serine-threonine serine threonine protein kinase domain (Tokutomi et al., 2008).
phot1 hot1 and phot2 undergo autophosphorylation upon blue light irradiation (Christie et al., 1998).. Mutations of a key amino acid in the phosphorylation domain of phot1 and phot2 prevents phosphorylation when expressed in insect cells, demonstrating the autophosphorylation property of this photoreceptor (Christie et al., 2002). 2002) The LOV1 domain of phot1 from Avena vena sativa is responsible for self-interaction, which could be important for autophosphorylation (Salomon et al., 2004). The autophosphorylation capability of phot1 and phot2 is dark-reversible dark (Short and Briggs, 1990; Hager et al., 1993; Salomon et al., 1997; Kinoshita et al., 2003). 2003). Domain swapping experiments of LOV1 and LOV2 in phot1 suggest that LOV2 acts as a repressor of the kinase activity of phot1 by intramolecular dimerization with LOV1. Indeed, in the absence of LOV2, the main in light sensor in phototropins (Christie et al., 2002),, LOV1 is constitutively active (Harper et al., 2004; Kaiserli et al., 2009). 2009) Upon irradiation, the LOV domain undergoes a conformational change as shown in spectroscopic studies (Swartz et al., 2002; Iwata et al., 2003; Nozaki et al., 2004). 2004) Altogether, these data allow postulating a model for which the light dependent conformational change activates the kinase activity in phototropins and subsequent downstream signalling.
Introduction
24
1.1.4 LOV Domains and Zeitlupe Photoreceptors The LOV domains are not only present in phototropins, but also in other blue light photoreceptors in plants, fungi and bacteria. The Zeitlupe (ZTL/ADO) family is composed of LOV domain photoreceptors, whose name derives from their influence on circadian clock. The Zeitlupe photoreceptors in Arabidopsis thaliana have only one LOV domain whereas phototropins have two LOV domains (Fig. I9). ZTL/ADO photoreceptors localize to the nucleus and the cytosol (Kiyosue and Wada, 2000; Yasuhara et al., 2004; Fukamatsu et al., 2005). The first ztl mutant was identified by different groups (Kiyosue and Wada, 2000; Nelson et al., 2000; Somers et al., 2000; Jarillo et al., 2001b). The ztl mutant phenotype is characterized by a lengthened circadian period; indeed it is influencing, e.g., circadian regulated gene expression and flowering time. The ZTL family of photoreceptors in Arabidopsis thaliana includes ZTL, FKF1, and LKP2. All these photoreceptors use a flavin (FMN) as chromophore (Nelson et al., 2000; Schultz et al., 2001). ZTL, FKF1 and LKP2 harbor a LOV domain followed by an F-box and a Kelch repeats (Fig. I9). The F-box domain is found in adaptor proteins of the modular E3 ubiquitin ligase SCF complex, which led to the assumption that ZTL and related proteins are involved in the turnover of circadian clock components in Arabidopsis thaliana. Indeed, it has been shown that Zeitlupe photoreceptors interact with the SCF complex thanks to their F-box protein domain (Mas et al., 2003; Han et al., 2004; Yasuhara et al., 2004). An additional LOV containing protein, not related to ZTL/ADO family, has been found in Arabidopsis thaliana (Crosson et al., 2003). This protein is not yet characterized and has been named Twin LOV Protein 1 (TLP1).
Introduction
25
Figure I9: Domain organization of a representative phototropin and a representative Zeitlupe-type Zeitlupe photoreceptor. In both classes of photoreceptors the LOV domain bound to an FMN molecule functions as the blue light sensor. Phototropins Phototropi harbor two FMN-binding binding LOV domains in their N-terminal region (LOV1 and LOV2) and a serine/threonine serine/threonine kinase domain in the C-terminal C part. Zeitlupe family photoreceptors harbor only one LOV domain at the N-terminus, N terminus, followed by an F-Box F motif and six Kelch repeats (KELCH) in the C-terminal C terminal region. By analogy with other proteins the Kelch repeats may serve as protein–protein protein interaction domain (Demarsy and Fankhauser, 2009). 2009)
As stated before, LOV domain containing proteins are not only present in planta but they are widespread across oss all kingdoms of life (Fig. I10).
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26
Figure I10: Phylogenetic tree reconstructed for LOV sequences from different taxa (Krauss et al., 2009).
1.1.5 Chimeric Photoreceptors Interesting examples of chimeric photoreceptors are present in different species, displaying different combinations of various chromophores and protein domains. A new class of photoreceptors that combines red light perception by a phytochrome-like domain and blue light perception by two phototropin-related domains has been found in the fern Adiantum capillus-veneris and in the green algae Mougeotia scalaris, and they have been named neochrome (Mougeotia) and PHY3 (Adiantum) (Nozue et al., 1998; Suetsugu et al., 2005). In Adiantum capillus-veneris red light spore germination can be reverted by blue light irradiation, indicating the dual specificity for red light and
Introduction
27
blue light of this photoreceptor (Furuya et al., 1997).. Moreover, the dual specificity is proven by heterologous expression of PHY3 in phot1/phot2 Arabidopsis rabidopsis thaliana mutant background, which confers hypocotyl curvature under red and blue light as well (Kanegae et al., 2006). 2006) Furthermore, more, phytochrome and blue light perception act cooperatively to mediate phototropism in Adiantum capillus-veneris veneris protonemata (Hayami et al., 1986).. A protein domains representation of the neochrome is shown in Fig. I11.
Figure I11:: The domain structure of Adiantum capillus-veneris neochrome; phytochrome-like phytochrome PAS domain (PHY), light, oxygen or voltage (LOV) domain, STKD, serine-threonine serine threonine kinase domain (Christie, 2007).
In the fungus Neurospora crassa,, blue light responses are driven by two photoreceptors: WHITE COLLAR 1 (WC-1) and WHITE COLLAR 2 (WC-2) (WC (Ballario et al., 1996; Linden and Macino, 1997). 1997) WC-1 1 contains a LOV domain and two PAS domains, while WC-2 2 contains a LOV LOV domain and a PAS domain. WC-1 WC and WC-2 heterodimerize in the nucleus and are able to activate the transcription of light regulated genes (Ballario et al., 1998; Talora et al., 1999; Schwerdtfeger and Linden, 2000). In the stramenopile algae Vaucheria frigida (Xanthophyceae) and Fucus distichus (Phaeophyceae) a light-activated activated transcription factor composed of a basic region/leucine zipper (bZIP) domain followed by a LOV domain has been identified and named AUREOCHROME (Takahashi et al., 2007). In bacteria, LOV domains have broad functions and mode of actions, being coupled to kinases, phosphodiesterases, response regulators, DNA-binding DNA binding motifs, and regulators ulators of stress sigma factors (Losi et al., 2004).
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28
The chimeric photoreceptors are astonishing example of evolutionary adaptation to different light conditions, and they underline the importance of light perception in photosynthetic and non-photosynthetic organisms.
1.2 Photoreceptor Systems not Present in Plants
1.2.1 BLUF BLUF (blue light sensors using FAD) domain photoreceptors are a novel class of blue light receptors which use FAD as chromophore, first described for the AppA protein from Rhodobacter sphaeroides (Gomelsky and Kaplan, 1998; Gomelsky and Klug, 2002). Next to prokaryotic members, BLUF photoreceptors have also been identified in eukaryotes like euglenozoa and fungi (Iseki et al., 2002). BLUF domains occur either in small proteins composed of a single BLUF domain or larger proteins where a BLUF domain is coupled to different effector domains, frequently involved in cyclic nucleotide metabolism, e.g., adenylate/guanylate cyclases and phosphodiesterases (Gomelsky and Klug, 2002; Barends et al., 2009). The AppA protein of Rhodobacter sphaeroides is a BLUF containing protein. AppA binds to the the transcription factor PpsR in low light, after light perception PpsR is released and it can act on photosynthetic gene expression (Metz et al., 2010). The BlrP1 protein in Klebsiella pneumoniae has a BLUF sensor domain and an EAL phosphodiesterase output domain. The light induced conformational changes could be thus propagated from the BLUF domain to the phosphodiesterase effector domain, modulating its enzymatic activity (Barends et al., 2009).
1.2.2 Rhodopsins The oldest photoreceptors found in animals are rhodopsins (Boll, 1876). They are membrane-bound photoreceptor (Kuehne, 1878b, a; Nathans, 1992), which use retinal as chromophore (Wald, 1933; Nathans, 1992). Different from the photoreceptors discussed before, rhodopsins strongly absorb green and blue light. Light induced isomerization of the chromophore results in a conformational change of
Introduction
29
rhodopsin that activates associated G proteins which initiate light responses (Hegemann et al., 1991; Lawson et al., 1991; Strader et al., 1994). Rhodopsins are widespread in animals, bacteria, algae and Fungi (Oesterhelt and Stoecken, 1973; Bogomolni and Spudich, 1982; Foster et al., 1984; Spudich et al., 2000; Hegemann, 2008).
1.2.3 The Aryl Hydrocarbon Receptor Recently, the aryl hydrocarbon receptor (AhR) has been described as a cytoplasmic target for UV-B in keratinocytes (Fritsche et al., 2007). AhR is a basic cytosolic helix-loop-helix transcription factor that belongs to the family of PAS proteins. AhR binds several chaperons in the cytoplasm, but, after ligand binding to polycyclic aromatic hydrocarbon (PAH), it moves to the nucleus and activates transcription of genes involved in PAH metabolism (Kahl et al., 1980; Knutson and Poland, 1980). A PAH ligand with very high affinity for AhR is the 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct of UV-B radiation (Rannug et al., 1995; Oberg et al., 2005). Production of FICZ upon UV-B radiation causes its binding to AhR, allowing the release of the AhR transcription factor from cytoplasmic retention factors, and the activation of responses in the nucleus.
1.3 UV-B Radiation The solar spectrum comprises wavelengths in the UV range. The stratospheric ozone layer is responsible for the decrease of UV wavelengths impinging on the earth surface, resulting in a complete depletion of UV-C and high reduction of UV-B radiation (Chapman, 1930). After studies on the accumulation of anthropogenic compounds in the atmosphere, namely chlorofluorocarbons (CFC) (Lovelock and Maggs, 1973), it has been found that the interaction between CFC and UV light results in ozone depletion. Indeed, this interaction leads to the formation of atomic Cl which is able to react with ozone (O3), reducing it to O2 (Molina and Rowland, 1974). The ozone depletion is of main concern causing the so called “ozone hole”, where
Introduction
30
UV rays can travel undisturbed, hitting the earth surface and harming living organisms (McKenzie et al., 2003).
1.3.1 UV-B Damage UV-B wavelengths impinging on earth are highly variable with spatial and time-dependent distribution (McKenzie et al., 2007). UV-B is a general damaging agent because of its high energy content per photon, which has damaging effects on biomolecules such as DNA, RNA, proteins and lipids, and the capability to induce the generation of reactive oxygen species (ROS) (Björn, 1996; Allan and Fluhr, 1997; Jansen et al., 1998; Hideg et al., 2002; Frohnmeyer and Staiger, 2003; Casati and Walbot, 2004). Of main concern is the DNA damage responsible for inhibition of replication and transcription, mutations, growth arrest and cell death. Plants are unavoidably exposed to UV-B radiation, because they need light for photosynthesis and have a sessile lifestyle. In plants, at physiological level, UV-B light causes altered flowering time, promotion of branching, reduced fertility and reduced biomass production (Tevini and Teramura, 1989; Rozema et al., 1997). Indeed, plants evolved UV-B light “sunscreen” protection pigments, sophisticated DNA repair processes, ROS scavenging systems and adaptive development. As DNA damaging agent, UV-B light can generate two photoproducts, pyrimidine pyrimidone photoproducts (6-4PP) and mainly cyclobutane pyrimidine dimers (CPDs) (Britt, 2004). In case DNA repair mechanisms fail, plants can cope with such photoproducts through dimer-bypass (Britt, 2004), which allows replication progression despite the lesion. In normal conditions, plants can repair the damage through different mechanisms. The main repair pathway for CPDs and 6-4PP in prokaryotes and eukaryotes, except placental mammals, is given by photolyases enzymes (Britt, 1999). Photolyases are able to use the energy of UV-A and blue light to break CPD and 6-4PP bonds thereby restoring the DNA sequence (Sancar, 2003). The Arabidopsis thaliana genome encodes two photolyases, namely PHR1 (also named UVR2) and UVR3 (Ahmad et al., 1997). Plants have also a light-independent DNA repair mechanism, the nucleotide excision repair (NER) mechanism (Shuck et al., 2008). In NER the DNA helices are
Introduction
31
completely opened, the damaged DNA is removed, new DNA is synthesized and the helices are closed again by ligation (Shuck et al., 2008). The third mechanism used upon DNA damage is the recombinational repair (Shinohara and Ogawa, 1995). Recombinational repair is involved in double strand breaks (DSBs) repair of the DNA helices and single-stranded gaps. DSBs repair relies on non-homologous end joining (NHEJ) and homologous recombination (HR) repair mechanisms (Bray and West, 2005; Schuermann et al., 2005). The integrity of the genetic information is crucial for living organisms’ survival and proliferation. The huge number of genotoxic agents in natural environment and the relevance of the genotoxic damage to organisms explain this plethora of DNA repair mechanisms.
1.3.2 UV-B Damage Signaling DNA repair mechanisms evolved in all kingdoms of life. In animals, damaged DNA acts as a signal through ATM and ATR protein kinases. ATM and ATR are able to recognize damaged DNA and to initiate a DNA damage response, which arrests cell cycle progression, giving time to the cell to repair the DNA before replication takes place (Sancar et al., 2004). Plant homologs of the ATR and ATM kinases were identified (Garcia et al., 2003; Culligan et al., 2004). Arabidopsis thaliana mutants lacking ATR are hypersensitive to UV-B light (Culligan et al., 2004), whereas Arabidopsis thaliana mutants lacking ATM are not (Garcia et al., 2003). It seems that ATR is specific for arresting cell cycle progression when there are DNA damages caused by UV-B radiation (Culligan et al., 2004). It has been demonstrated that ROS production increases under UV-B in plants (Hideg and Vass, 1996; Allan and Fluhr, 1997; Dai et al., 1997). The source of the ROS derived from UV-B irradiation of the plants is not clear yet, also because there are different sources of ROS production in plants, like photosynthesis and respiration. Nevertheless, it has been postulated that ROS production caused by UV-B radiation could come from inhibition of photosynthesis caused by UV-B light damage to protein, hence reduced ability to dissipate excitation energy (Barta et al., 2004). Plants counteract enhanced ROS production under UV-B light increasing
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ROS-scavenging systems (Casati and Walbot, 2004; Brown et al., 2005; Ulm and Nagy, 2005). DNA damage is thus a source of information, through which cells can undergo cell cycle arrest to prevent additional damage. On the other hand, ROS can also cause damage, and they are source of information, but they can also been used as defence in response to biotic and abiotic stresses (Apel and Hirt, 2004). Indeed, ROS influence gene expression, for example decreasing expression of LHCB1 which can be rescued by exogenous application of antioxidants (Surplus et al., 1998; Mackerness et al., 2001). Because DNA damage and ROS action can reprogram gene transcription, it is difficult to extrapolate the signalling component specific for UV-B irradiation. Moreover, most studies are performed under UV-B fluence rates well above the ones present in natural environments, increasing the signalling component of the damage response, and hiding, at the same time, a possible specific UV-B signalling component. Furthermore, UV-B irradiation activates genes normally involved in defence response and wounding (Mackerness, 2000; Brosche and Strid, 2003; Izaguirre et al., 2003), like pathogen related protein (PR-1, PR-2 and PR-5), and proteinase inhibitor genes. These genes are induced because UV-B light causes the production of signalling molecules, mainly jasmonic acid (JA), ethylene, salicylic acid (SA), brassinosteroids (BR) and ROS. In the mutants for these phytormones like NahG, etr1, jar1 and bri1, respectively impaired in the synthesis of SA, ethylene, JA, and brassinosteroids, the UV-B induction of genes involved in wounding and defence response was reduced or even absent (Surplus et al., 1998; Mackerness, 2000; Savenstrand et al., 2004). Moreover, ROS are signalling molecules in both defence and wounding responses (Surplus et al., 1998; Mackerness et al., 2001). The complex networks composed of cross-talking pathways complicates the isolation of the UV-B light specific signal responses and the identification of a putative UV-B light photoreceptor.
1.3.3 UV-B non-Damage Response UV-B light is not a mere source of damage but also an informational source for plants. Notwithstanding, it has to be noticed that information and damage are linked
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in a way that the information initiates a response, at lower UV-B fluence rate, which acclimates the plant to avoid damage at higher UV-B fluence rate (Brosche and Strid, 2003; Frohnmeyer and Staiger, 2003; Paul and Gwynn-Jones, 2003; Ulm and Nagy, 2005; Favory et al., 2009). Indeed, the information component of the UV-B light is a proactive defence response composed of transcriptional activation of genes encoding for pigment biosynthesis (flavonoids and hydroxycinnamic acid esters), which acts as “sunscreens” pigments absorbing UV-B radiation (Caldwell et al., 1983), genes encoding for photolyases, and genes encoding for proteins involved in ROS scavenging (Jenkins, 1997; Rozema et al., 1997; Jansen et al., 1998; Ulm and Nagy, 2005). Moreover, the impact of UV-B radiation on transcription is very broad modifying the expression of genes encoding enzymes, membrane and cytoskeletal proteins, transcription factors, signalling components and proteins involved in various processes like photosynthesis, primary and secondary metabolism, cell wall biosynthesis, stress protection, DNA-related processes, RNA processing, translation and proteolysis (Brosche and Strid, 2003; Izaguirre et al., 2003; Casati and Walbot, 2004; Ulm et al., 2004). As stated before, UV-B light responses are fluence rate dependent and can be divided in a stress response at damaging UV-B fluence rate, and an acclimation response at non-damaging UV-B (Kucera et al., 2003; Ulm et al., 2004; Favory et al., 2009).
Phenotypically,
non-damaging
UV-B
light
evokes
photomorphogenic
responses, including hypocotyl growth inhibition, cotyledon expansion, phototropic curvature, biosynthesis of anthocyanins and flavonoids, and stomatal opening (Beggs and Wellmann, 1994; Kim et al., 1998; Boccalandro et al., 2001; Eisinger et al., 2003; Suesslin and Frohnmeyer, 2003; Shinkle et al., 2004). It is tempting to distinguish between a UV-B light damage-mediated pathway and a UV-B light non-damage-mediated pathway, as shown in Fig. I12, ascribing the first one to a general stress response activated by e.g. DNA damage and ROS production, and the second one to a specific response activated by a putative UV-B light photoreceptor. There are already some evidence for a UV-B light specific pathway for photomorphogenesis and transcriptional induction, independent from the damage pathway. Indeed, UV-B light photomorphogenic responses can be separated from wounding response, defence response or, in general, stress responses. For instance, UV-B light fluence rate of 0,1 µmol m-2 s-1, well under UV-B light fluence rate present in sunlight, is causing hypocotyl growth inhibition (Kim et al., 1998; Boccalandro et
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34
UV B light pulses shorter than a second are enough to al., 2001).. Furthermore, UV-B induced transcript of marker genes like CHALCONE SYNTHASE (CHS), whereas under this condition CPDs formation is undetectable (Frohnmeyer et al., 1999). 1999) UV-B light pulses shorter than one second are not enough to start any damage pathway, endorsing the hypothesis of a specific damage-independent damage UV--B light pathway. CHS transcript is also not induced by ROS, and antioxidant are not repressing CHS induction under UV-B light (Jenkins et al., 2001). 2001). All these data suggest the presence of
a
specific
UV-B B
light
pathway
responsible
for
gene
induction
and
photomorphogenesis.
Figure I12:: Illustration of the UV-B UV B light damage response and the UV-B light non-damage response. The fluence rate dependence overlaps in planta (Whitelam G.C., 2007).
Another question arising from the analysis of plant responses to UV-B UV radiation is if there’s a unique UV-B B light photoreceptor or there are more photoreceptor systems at different UV-B B light fluence rate and/or wavelengths (Brosche and Strid, 2003; Frohnmeyer and Staiger, 2003; Casati and Walbot, 2004; Shinkle et al., 2004; Ulm et al., 2004). Indeed, UV-B B light specific responses could initiate from a multitude of factors ranging from DNA damage, lipid peroxidation, ROS production, phytormones or different combination of all these factors. It would would be really challenging to separate the sources of the UV-B B light signal in order to identify in which measure each component is contributing specifically to the UV-B UV B light response. Notwithstanding, the existence stence of a specific response at low fluence rate UV-B B (1,5 µmol µ m-2 s-1, i.e. damage independent) was recently demonstrated in Arabidopsis thaliana (Favory et
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35
al., 2009). The Fig. I13 shows the Venn diagram of a microarray analysis of Arabidopsis thaliana wild type seedlings versus mutant seedlings for the genes uvr8 and cop1-4 at low fluence UV-B radiation.
Figure I13: Venn diagram of gene up- and down-regulated under low fluence rate UV-B for the given time in hours, in wild type, and cop1-4 and uvr8-6 mutants (Favory et al., 2009).
This experiment shows the specific deregulation of about 850 genes at 6 hours after the UV-B light treatment in wild type seedlings, and almost no gene deregulation in the uvr8 and cop1 mutant seedlings. It seems that at this non-damaging UV-B light fluence rate only the UV-B light specific pathway is activated, which depends on the UVR8 and COP1 proteins. The question now is if this pathway is the UV-B light specific pathway and the other pathways described until now are general stress response pathways, or if there are more UV-B light pathways at different fluence rate and wavelengths. A possible indirect answer to this question could come from the identification of the UV-B light photoreceptor(s).
1.3.4 UV-B Perception How plants are able to perceive non-damaging UV-B light is unknown, but it doesn’t seem to happen through known photoreceptors. CHS transcript induction under UV-B light was unaffected in a cry1cry2 double mutant (Wade et al., 2001). Also single and combinatorial phytochrome mutants are not altered in their UV-B light induction of CHS transcript (Wade et al., 2001; Brosche and Strid, 2003; Ulm et al., 2004).
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Moreover, photomorphogenic UV-B light responses like cotyledon opening in phyB mutant or hypocotyl growth reduction in phyAphyB double mutant are not affected (Boccalandro et al., 2001; Suesslin and Frohnmeyer, 2003; Oravecz et al., 2006). Interestingly, the DNA repair mutants uvr1, uvr2 and uvr3 also show no altered hypocotyl growth inhibition under UV-B radiation (Kim et al., 1998; Boccalandro et al., 2001). Moreover, low fluence UV-B light gene induction is not altered in the uvr2 mutant background (Ulm et al., 2004). Action-spectra of UV-B radiation responses lead to postulate absorption maxima of 295-300 nm and 280-300 nm (Ensminger, 1993; Beggs and Wellmann, 1994; Brown et al., 2009). In this UV-B wavelength range, pterins or flavins could act as chromophore for a putative photoreceptor. Nevertheless, we don’t know if a chromophore is needed under UV-B light, given that most of the biomolecules are absorbing UV-B light. This is different for known photoreceptors because most of the biomolecules are blind to visible light. The maxima of the action-spectrum for UV-B light photomorphogenic response in planta is at longer wavelengths compared to the maxima action-spectrum for UV-B light damage response, the latter one corresponding to the maxima of UV-B absorption by DNA (Ensminger, 1993; Ballare et al., 1995).
1.4 Components of the Low-Fluence Rate UV-B Pathway A genetic screen for mutants with altered hypocotyl growth reduction under pulses of low fluence UV-B irradiation identified the uli3 mutant (UV-B light insensitive 1). The ULI3 gene encodes for a protein with limited similarity to a diacylglycerol kinase present in humans and is transcriptionally induced by UV-B and UV-A. This could explain why in the uli3 mutant CHS and PR-1 genes induction were impaired under both UV-A and UV-B irradiation (Suesslin and Frohnmeyer, 2003). It can thus be concluded that ULI3 is not specific for UV-B light signalling, excluding it as UV-B photoreceptor candidate. rus1 and rus2 mutants (root UVB sensitive 1 and root UVB sensitive 2) were identified in a screen of T-DNA-insertion lines for mutants showing root-growth defects (Tong et al., 2008; Leasure et al., 2009). Interestingly, the root of rus1 and rus2 mutant plants showed root hypersensitivity to UV-B light and this was
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37
independent from other known photoreceptors. The root was identified as the organ responsible for UV-B perception. Moreover RUS1 and RUS2 interact with each other in a yeast two-hybrid assay. The authors postulated that these proteins act as negative regulators of a UV-B signaling pathway. In a recent work, RUS2 was found in a screen for alteration in the auxin reporter construct DR5rev:GFP (Ge et al., 2010). In this work, RUS2 is shown to be involved in auxin transport and to maintain PIN FORMED (PIN) protein level. Moreover, RUS2 has been shown in all plant organs and in the plastids and, in this work, removal of UV-B light does not restore the wild type phenotype, suggesting that other factors than UV-B light cause this phenotype. The narrowband UV-B light pathway in Arabidopsis thaliana has been identified by weak UV-B irradiation at 1.5 µmol m-2 s-1 fluence rate. This fluence rate is enough to alter gene expression in Arabidopsis thaliana wild type plants, but not in cop1-4 or uvr8-6 mutant plants (Oravecz et al., 2006; Favory et al., 2009). Such UV-B light fluence rate is extremely low compared to environmental UV-B light, as shown in phytotron sunlight simulator experiments (Fig. I14). The mean UV-B biologically effective (UV-BBE) (Caldwell, 1971) quantity applied in this experiment was UVBE 400 mW m−2 (Favory et al., 2009).
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Figure I14: 25-day-old old Arabidopsis plants, wild type (WT), UVR8 overexpressor (Ox no. 2), and uvr8 mutant (uvr8-7)) grown in sunlight simulators under realistic conditions (+UV) or with the UV portion specifically filtered out (-UV) (Favory et al., 2009). 2009)
All these data point to a UV-B pathway started by plants at UV-B fluence rates causing negligible DNA damage or ROS production. Indeed, given the acclimation response of plants to UV-B radiation (Favory et al., 2009),, it is reasonable that the response starts before the occurrence of damage, and prepare the plants to cope with higher irradiation during the course of the day. Identification of UV-B B light response mutants mu using a HY5-promoter promoter driven luciferase reporter assay revealed two cop1 c mutant alleles and nine uvr8 mutant alleles, but no additional components of the UV-B photoregulatory pathway. This may indicate that there is high redundancy in other factors or that the low fluence rate UV-B pathway only uses a very limited number of upstream key players (Favory et al., 2009). 2009)
Introduction
39
1.4.1 HY5 The photomorphogenic UV-B light pathway has few defined upstream components. One of these components is HY5, a bZIP transcription factor involved in light induced morphogenesis under different light qualities (Oyama et al., 1997; Osterlund et al., 2000). HY5 localizes to the nucleus where it induces transcription of light responsive genes, and its action is modulated at the protein level through proteasomal dependent degradation mediated by the E3 ubiquitin ligase COP1 (Osterlund et al., 2000). It is thought that the regulation of the HY5 protein stability involves nucleocytoplasmic alterations in COP1 localization (Osterlund et al., 1999; Yi et al., 2002). In the dark COP1 is in the nucleus and HY5 is degraded, leading to skotomorphogenesis, while in the light COP1 localizes to the cytoplasm, leading to the stabilization of HY5, and induction of light responsive genes (Osterlund et al., 2000). Under UV-B irradiation HY5 transcript is induced and it activates photomorphogenesis (Ulm et al., 2004). The UV-B-dependent gene activation through HY5 is independent of phytochromes, cryptochromes and phototropins (Ulm et al., 2004). The UV-B-dependent activation of 127 genes requires HY5 (Fig. I15A), which correlates with a reduced tolerance to UV-B stress in hy5 mutant seedlings (Fig. I15B) (Oravecz et al., 2006).
Figure I15: A) Venn diagram of genes responding to UV-B radiation in Arabidopsis thaliana accession Landsberg erecta (Ler) and in the hy5-1 mutant in the same ecotype. The Venn diagram displays the number of UV-B light responding genes in either the wild type only (left; i.e., HY5-dependent genes), the wild type and mutant (centre; i.e., HY5-independent genes), or mutant only (right). B) Phenotypic characterization of wild type and hy5 mutant seedlings with and without UV-B irradiation (Oravecz et al., 2006).
Introduction
40
1.4.2 COP1 nother specific upstream component of the low fluence UV-B UV B light pathway is the E3 Another ubiquitin ligase COP1 (Oravecz et al., 2006). 2006). COP1 is a central component of light signal transduction in plants that is composed of a RING finger (a type of zinc finger domain) responsible for target protein ubiquitination (a signal for proteasomal degradation) and two protein-protein protein interaction domains, a coiled--coil domain, also responsible for self-dimerization, dimerization, and a WD-40 WD domain (Fig. I16) (Deng et al., 1991; Deng et al., 1992).
Figure I16:: Schematic representation of COP1 protein domains: RING finger domain (RING), coiled-coil domain (Coil), and β--propeller domain (WD-40) (Torii et al., 1998).
COP1 represses light-mediated mediated development through degradation of light-responsive light transcription factors like HY5, HYH, HFR1, and LAF1 (Osterlund et al., 2000; Holm et al., 2002; Seo et al., 2003; Jang et al., 2005; Yang et al., 2005). 2005). Moreover, COP1 is responsible for light-dependent dependent gene regulation for the majority of light-controlled light genome expression (Ma et al., 2002).. Is not known yet if COP1 itself is able to activate transcription, or the light-controlled light controlled genome expression is a consequence of the degradation activity of COP1 on light-responsive light responsive transcription factors. In white light, wild-type type seedlings show a photomorphogenic growth phenotype, including open and green cotyledons and reduced hypocotyl elongation. Wild-type Wild seedlings grown in darkness show the typical skotomorphogenic phenotype with pale and closed cotyledons forming an apical hook, and elongated elongated hypocotyls (Fig. I17). I1 In contrast, cop1 mutant seedlings show a constitutive photomorphogenic phenotype in darkness (hence the name constitutively photomorphogenic 1)) (Fig. I17). I1
Introduction
41
Figure I17: Wild-type type and cop1 mutant seedlings phenotype in the dark and in white light (Osterlund und et al., 1999). 1999)
As central regulator of light signaling, COP1 is interconnecting the photoreceptors signals and the light-responsive responsive transcription factors. Indeed, COP1 has been shown to interact with phyA, phyB, cry1 and cry2 and to ubiquitinate the light-labile photoreceptors phyA and cry2 (Yang et al., 2001; Shalitin et al., 2002; Seo et al., 2004; Chen et al., 2010; Jang et al., 2010). 2010). A schematic model with COP1 as central regulator between photoreceptor and transcription factors is shown in Fig. I18. I1 The figure indicates that the interaction of the photoreceptors with COP1 negatively regulates COP1. This regulation adds a level of complexity to the system which considers the nuclear-cytoplasmic cytoplasmic partitioning of COP1, under different light l conditions, as a level of regulation of COP1 activity (Yi and Deng, 2005). 2005)
Figure I18: COP1 interaction partners under different wavelengths (Yi and Deng, 2005). 2005) COP1 represses plant photomorphogenesis photomorphogenesis by direct interaction with photoreceptors (phyA, phyB, cry1 and cry2) and downstream transcription factors (HY5, HYH, HFR1 and LAF1).
Introduction
42
COP1 is not only present in higher plants but also in vertebrates, where it is involved in the degradation of transcription factors like the tumor-suppressor protein p53 and the protein c-Jun (Yi et al., 2002; Bianchi et al., 2003). Interestingly, human COP1 is also involved in UV-B-induced signaling in keratinocytes (Kinyo et al., 2010). In plants, in contrast to its role as a negative regulator of photomorphogenesis under visible
light,
COP1
is
considered
a
positive
regulator
of
UV-B-induced
photomorphogenesis (Oravecz et al., 2006). This idea is based on microarray analyses which show the lack of UV-B-induced gene activation in cop1-4 mutant seedlings (Favory et al., 2009). This includes HY5-dependent genes, and HY5 itself, indicating that COP1 is upstream of HY5 in the UV-B light signaling pathway (Fig. I13). Upon UV-B irradiation COP1 and HY5 protein levels are stabilized in the nucleus, two contradictory events if we consider that HY5 is a substrate of COP1 activity (Oravecz et al., 2006).
1.4.3 UVR8 Another upstream component of the UV-B light signaling in plants is the UV RESISTANCE LOCUS 8 (UVR8). uvr8 has been found and characterized to be hypersensitive to UV-B radiation and to lack CHS induction and flavonoids accumulation under UV-B irradiation (Kliebenstein et al., 2002). Differently from HY5 and COP1, which are also involved in visible light signaling pathways, UVR8 seems to be specific for the UV-B light response. Indeed, uvr8 mutant plants are unaltered in CHS gene activation by non-light stimuli like low temperature and sucrose, as well as red, far-red and blue light (Brown et al., 2005; Favory et al., 2009). The UVR8 protein sequence is related to the human protein regulator of chromatin condensation 1 (RCC1), a β-propeller protein with guanine nucleotide-exchange factor (GEF) activity for the small GTPase Ran (Ohtsubo et al., 1987; Bischoff and Ponstingl, 1991; Kliebenstein et al., 2002). RCC1 is constitutively localized to the nucleus where it binds to chromatin, and it generates a Ran-GTP/Ran-GDP gradient across the nuclear envelope, which is required to drive active nucleo-cytoplasmic protein transport and to regulate cell cycle and mitosis. There is no evidence that RCC1 is involved in UV-B light responses or transcriptional regulation. Differently, UVR8
Introduction
43
shows a cytoplasmic and nuclear localization (Brown et al., 2005) with nuclear enrichment under UV-B (Kaiserli and Jenkins, 2007). Moreover, UVR8 seems not be involved in nucleo-cytoplasmic transport and mitotic regulation indeed, the uvr8 mutant grows normally in standard conditions, whereas RCC1 mutations in Saccharomyces cerevisiae alter a wide variety of processes, including pre-mRNA processing and transport (Aebi et al., 1990; Kadowaki et al., 1993), mating behaviour (Clark and Sprague, 1989), initiation of mitosis (Matsumoto and Beach, 1991), and chromatin decondensation (Sazer and Nurse, 1994). UVR8 has been tested negative for Ran-GEF activity, and it is not able to interact with Arabidopsis thaliana Ran in yeast two-hybrid assay (Brown et al., 2005). However, UVR8, like RCC1, is able to bind chromatin (Brown et al., 2005; Cloix and Jenkins, 2008). The ability of UVR8 to bind chromatin seems to be independent from UV-B irradiation. It is possible to conclude that UVR8 and RCC1, despite the sequence homology, have different functions. RT-PCR analyses of uvr8 mutant versus wild type shows that plants lacking UVR8 are impaired in the UV-B-mediated induction of phenylpropanoid biosynthesis genes, and in genes involved in terpenoid and alkaloid biosynthesis, all UV-B light absorbing compounds, and in genes involved in protection against oxidative stress (like gluthatione peroxidase) and photooxidative damage (like ELIP proteins) (Kliebenstein et al., 2002; Brown et al., 2005). Microarray analyses of hy5 and uvr8 mutants demonstrate that UVR8 acts upstream of HY5 in the UV-B signalling pathway, and that approximately half of the genes regulated by UVR8 are regulated by HY5 (Brown et al., 2005). It has also been shown that UVR8 accumulates in the nucleus under UV-B irradiation and it binds to the HY5 promoter region, but the binding of UVR8 to the promoter region of HY5 was shown to be independent from UV-B irradiation (Brown
et
al.,
2005;
Kaiserli
and
Jenkins,
2007).
uvr8
mutants
lack
photomorphogenic response under low fluence rate, non-damaging UV-B irradiation, as shown in Fig. I19. Under the same experimental conditions both uvr8 and cop1 mutants lack completely UV-B light-dependent gene expression, as shown in Fig. I13 (Favory et al., 2009).
Introduction
44
Figure I19 : Wild type seedlings of Arabidopsis thaliana (Ws) and uvr8-7 mutant seedlings in the same ecotype under white light or white light supplemented with narrowband narro band UV-B UV (Favory et al., 2009).
UV B irradiation, irradiation and cop1-like UVR8 overexpressor lines show higher resistance to UV-B mutant phenotype under UV-B UV light (Fig. I14).. The enhanced resistance is associated with an enhanced UV-B B response, e.g. HY5 and CHS gene activation, anthocyanins accumulation and d hypocotyl growth inhibition (Favory et al., 2009).. In the same work UVR8 has been shown to homodimerize constitutively, and to interact with COP1 in a UV-B B light dependent manner. The UV-B UV B light dependent interaction of UVR8 and COP1 starts after five to ten minutes of UV-B UV B irradiation, defining an early event in the UV-B-induced induced photomorphogenic pathway.
Introduction
45
1.5 Aim of This Work The aim of a scientific work, in basic research fields, is in perpetual evolution. The result of each experiment is telling to a scientist which experiment to do in the following working days. The aim of our lab “UV-B perception and signaling in plants”, as from the title of the lab home page, was clearly the findings of the plant UV-B radiation photoreceptor. A luciferase-based genetic screen, after EMS mutagenesis of Arabidopsis thaliana plants, carried out in our laboratory by Dr. Agnieszka Brzezinska, didn’t identify any new component of the UV-B radiation pathway. Indeed, the genetic screen identified 2 alleles of cop1 and 9 alleles of uvr8. This result may indicate that the UV-B pathway in Arabidopsis has few upstream components, or redundancy of additional upstream components. Thanks to this result, the lab focused on UVR8 and COP1, which led to the finding of the UV-B dependent interaction of UVR8 with COP1. Is UVR8 and/or COP1 or something upstream to these proteins the UV-B photoreceptor in plants? With these questions I’ve started my PhD experience, checking the degradation of UVR8 under UV-B light, and complementation experiments, cloning COP1 homologs from the moss Physcomitrella patens, and transforming them in the cop1-4 mutant line of Arabidopsis thaliana. Later on, I tried to reproduce the UV-B light dependent interaction between UVR8 and COP1 in heterologous system, establishing the yeast two-hybrid analysis for this specific experiment.
Material and Methods
46
2 Materials and Methods 2.1 Materials 2.1.1 Plant Material and Media Two Arabidopsis thaliana wild-type accessions were used in this study: Columbia (Col), and Wassilewskija (Ws). Different mutants and transgenic lines in these backgrounds were used herein, as listed in Table M1.
Mutants
Background
Reference
cop1-4
Col
(McNellis et al., 1994)
uvr8-6
Col
(Alonso et al., 2003)
uvr8-9
Ws/ ProHY5:Luc+
(Favory et al., 2009)
hy5-215
Col
(Rubio and Deng, 2005)
uvr8-8
Ws/ ProHY5:Luc+
(Favory et al., 2009)
Transgenic lines Pro35S:A9RS92*
uvr8-8/ ProHY5:Luc+ (This work)
Pro35s:CFP-UVR8
Col
(Stec, unpublished)
Table M1: List of Arabidopsis thaliana mutants and transgenic lines used herein. (*) A9RS92, UVR8 ortholog in Physcomitrella patens.
2.1.2 Bacterial Strains and Media Escherichia coli Top10F’ (Invitrogen, Carlsbad, USA) was used for various cloning procedures, including Gateway-based cloning. Escherichia coli DB3.1 (Invitrogen, Carlsbad, USA) was used for the propagation of Gateway® plasmids containing the ccdB gene. Agrobacterium tumefaciens strain C58CIRifR containing the non-oncogenic Ti plasmid pGV3101 was used for stable plant transformations. Luria-Bertani (LB) medium consisting of 1% (w/v) Bacto-tryptone, 0.5% (w/v) Bacto-yeast-extracts and 0.5% (w/v) NaCl was used for growing liquid cultures of Escherichia coli and Agrobacterium tumefaciens. For solid media 1.5% (w/v) of Bacto-agar was added. Following antibiotics were used for plasmid selection:
Material and Methods
47
ampicillin (50 µg/ml), kanamycin (50 µg/ml), gentamycin (25 µg/ml), spectinomycin (100 µg/ml), chloramphenicol (60 µg/ml) or rifampicin (100 µg/ml).
2.1.3 Yeast Strains and Media Saccharomyces cerevisiae: strain PJ69-4A (MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4∆ gal80∆ LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ) was used for yeast two-hybrid and yeast three-hybrid analyses. Saccharomyces cerevisiae: strain L40 (MATa his3-∆200 trp1-901 leu2-3,112 ade2 LYS2::(LexAop)4-HIS3 URA3::(LexAop)8-lacZ) was used for yeast two-hybrid analysis. Yeast was grown at 30°C on either YPDA medium (20 g /l peptone, 20 g/l glucose, 10 g/l yeast-extract, 40 mg/l adenine-hemisulfate) or SD minimal medium (20 g/l glucose, 1.7 g/l yeast nitrogen base, 5 g/l ammonium sulfate, 10 g/l succinic acid, 6 g/l NaOH, 20 mg/l adenine, amino acids mix solution). The latter was supplemented as indicated by the manufacturer with premixed CSM dropout media (FORMEDIUM, Hunstanton, GB) lacking only specific amino acids as required. For solid media 16 g/l Bacto-agar was added.
2.1.4 Plasmids, Oligonucleotides and Antibodies Plasmids used in this study are listed in the following Table M2:
Name
Type
Reference/Manufacturer
pDONR207
Gateway entry
Invitrogen, Carlsbad, USA
pENTRY3C
Gateway entry
Invitrogen, Carlsbad, USA
General purpose
Agrobacterium pAlligator
Gateway destination http://www.isv.cnrs-gif.fr/jg/alligator/vectors.html
pB2GW7
Gateway destination (Karimi et al., 2002)
BiFC pE-SPYNE
(Walter et al., 2004)
pE-SPYCE
(Walter et al., 2004)
Material and Methods
48
Yeast pBTM116-D9
Gateway destination (Stelzl et al., 2005)
pGADT7_GW
Gateway destination (Marrocco et al., 2006)
pGBT9_GW
Gateway destination (Marrocco et al., 2006)
pAG-426GPD-ccdB
Gateway destination (Alberti et al., 2007)
HEK293T Cells pDEST27
Gateway destination Invitrogen, Carlsbad, USA
pcDNA-DEST40
Gateway destination Invitrogen, Carlsbad, USA
Table M2: List of plasmids used in this work.
All oligonucleotides used in this study were synthesized by Operon (Ebersberg, Germany) and salt-free purified, except for the site-directed mutagenesis, where the oligonucleotides were synthesized by Invitrogen and HPLC purified. Table M3 gives an overview:
Name
Sequence
Site-Directed Mutagenesis
W144F-gw
gaaggagaggtccagagtttcggccgcaaccag
W144F_antisense-gw
ctggttgcggccgaaactctggacctctccttc
Y194F-gw
cagaagatggtgacctctttggatggggctg
Y194F_antisense-gw
cagccccatccaaagaggtcaccatcttctg
Y246F-gw
cctactctggagcattgtttacttatggatggagcaa
Y246F_antisense-gw
ttgctccatccataagtaaacaatgctccagagtagg
Y248F-gw
cctactctggagcattgtatacttttggatggagcaaat
Y248F_antisense-gw
atttgctccatccaaaagtatacaatgctccagagtagg
W92F-gw
aggcatggaagtctacagtttcggatggggtgattttgg
W92F_antisense-gw
ccaaaatcaccccatccgaaactgtagacttccatgcct
W196L-gw
gatggtgacctctatggattgggctgggga
W196L_antisense-gw
tccccagcccaatccatagaggtcaccatc
W250L-gw
ctggagcattgtatacttatggattgagcaaatatggacag
W250L_antisense-gw
ctgtccatatttgctcaatccataagtatacaatgctccag
W300L-gw
cattgacttcagatggaaaactatatggattgggttggaataagttt
W300L_antisense-gw
aaacttattccaacccaatccatatagttttccatctgaagtcaatg
C127A-gw
cggtattcggatcaagcagattgctgctggggatagtcat
C127A_antisense-gw
atgactatccccagcagcaatctgcttgatccgaataccg
C132T-gw
tgcttgtggggatagtcatactttggctgtcactatggaa
Material and Methods
49
C132T_antisense-gw
ttccatagtgacagccaaagtatgactatccccacaagca
W94A-gw
gaagtctacagttggggagcgggtgattttgggag
W94A_antisense-gw
ctcccaaaatcacccgctccccaactgtagacttcc
W198A-gw
gacctctatggatggggcgcgggaagatacggaaattt
W198A_antisense-gw
aaatttccgtatcttcccgcgccccatccatagaggtc
W233A-gw
gaaaatgtcaatggttgcttgtggagcgcggcacacaata
W233A_antisense-gw
tattgtgtgccgcgctccacaagcaaccattgacattttc
W250A-gw
ggagcattgtatacttatggagcgagcaaatatggacagctagg
W250A_antisense-gw
cctagctgtccatatttgctcgctccataagtatacaatgctcc
W285A-gw
ctcccagatttcgggaggtgcgagacatacaatggcattg
W285A_antisense-gw
caatgccattgtatgtctcgcacctcccgaaatctgggag
W302A-gw
ggaaaactatatggatggggtgcgaataagtttggacaagtagg
W302A_antisense-gw
cctacttgtccaaacttattcgcaccccatccatatagttttcc
W337A-gw
agttcaagtctcatgtggagcgagacataccttggctgtc
W337A_antisense-gw
gacagccaaggtatgtctcgctccacatgagacttgaact
W233F-gw
gaaaatgtcaatggttgcttgtggattccggcacacaatatc
W233F_antisense-gw
gatattgtgtgccggaatccacaagcaaccattgacattttc
W285F-gw
ctcccagatttcgggaggtttcagacatacaatggcattg
W285F_antisense-gw
caatgccattgtatgtctgaaacctcccgaaatctgggag
W337F-gw
agttcaagtctcatgtggattcagacataccttggctgtc
W337F_antisense-gw
gacagccaaggtatgtctgaatccacatgagacttgaact
Cloning AT_COP1-WD-40-fw-gw ggggacaagtttgtacaaaaaagcaggcttaatgtatagcaacggccttgc AT_COP1-BP-rev-gw
ggggaccactttgtacaagaaagctgggtatcacgcagcgagtaccaga
AT_COP1-BP-fw-gw
ggggacaagtttgtacaaaaaagcaggcttaatggaagagatttcgacggatcc
Atcop1H69Y-fw
ctcacggcttgtggttatagtttctgc
Atcop1H69Y-rev
gcagaaactataaccacaagccgtgag
Atcop1-4_Stop
cggctcgagttacgaatctgacccactcagc
AtCOP1_EcoRI
cggaattcatggaagagatttcgacgg
At3g26100_fw-gw
ggggacaagtttgtacaaaaaagcaggcttcatgtgtaataaaagggtgatt
At3g26100_rev-gw
ggggaccactttgtacaagaaagctgggtcctagctgagatcgatatccac
At3g15430_fw-gw
ggggacaagtttgtacaaaaaagcaggcttcatggctgatcgaaactgtttg
At3g15430_rev-gw
ggggaccactttgtacaagaaagctgggtctcaacaaagccggctacgaga
At1g19880_fw-gw
ggggacaagtttgtacaaaaaagcaggcttcatggcggaagcgatgaattcg
At1g19880_rev-gw
ggggaccactttgtacaagaaagctgggtcctaagacttacgtggccttcc
At5g60870_fw-gw
ggggacaagtttgtacaaaaaagcaggcttcatggcagcgttaagccaccgc
At5g60870_rev-gw
ggggaccactttgtacaagaaagctgggtcttaaggtgatcttgagactaa
At5g12350_fw-gw
ggggacaagtttgtacaaaaaagcaggcttcatggcttcggatcttagtaga
Material and Methods
50
At5g12350_rev-gw
ggggaccactttgtacaagaaagctgggtctcagcgaggcaagtcctcact
rup1_nls-gw
cctaagaagaagagaaaggttggaggaatggaggctttgttc
rup2_nls-gw
cctaagaagaagagaaaggttggaggaatgaacactcttc
Fw_nls-gw
ggggacaagtttgtacaaaaaagcaggcttcatgcctaagaagaagag
RUP1_rev-gw
ggggaccactttgtacaagaaagctgggtcttagctttgtttgcccga
RUP2_rev-gw
ggggaccactttgtacaagaaagctgggtcctatggttttcttttgcc
UVR8-Cterm-nls
cgtgtacgaattcctaagaagaagagaaaggtttga
UVR8-Cterm-nls-gw
ggggaccactttgtacaagaaagctgggtatcaaacctttc
Table M3: List of oligonucleotides used herein.
Standard restriction and ligation methods were used according to standard methods (Sambrook and Russel, 2001). Nuclear localization signal was added by PCR extension primers. Gateway cloning was performed according to the Gateway manual (Invitrogen). After Escherichia coli transformation, the isolated DNA was sequenced at GATC (Konstanz, Germany).
All antibodies used in this study are presented in the following Table M4:
Name
Host
Peptide Sequence
Company
α-UVR8 (P60)
Rabbit
CGDISVPQTDVKRVRI
Eurogentec
α-UVR8 (FB4732)
Rabbit
a)CGDISVPQTDVKRVRI Eurogentec
Primary antibodies
b) MAEDMAADEVTAPPR α-UVR8
Rabbit
VPDETGLTDGSSKGN
Eurogentec on published epitope (Kaiserli and Jenkins, 2007)
α-UVR8
Guinea Pig CGDISVPQTDVKRVRI
Eurogentec
α-Actin
Mouse
Sigma
α-GST
Mouse
Sigma
α-LexA
Rabbit
Millipore
α-HY5
Rabbit
(Oravecz et al., 2006)
α-Plant-Ubiquitin
Rabbit
Bethyl Laboratories, Inc.
α-GFP
Rabbit
Invitrogen
Material and Methods
51
Secondary antibodies α-Mouse
Rabbit
Dako
α-Rabbit
Swine
Dako
α-Guinea Pig
Goat
Abcam
α-Mouse-Alexa 680
Goat
Invitrogen
α-Rabbit IRDye® 800CW Donkey
Li-COR biosciences
Table M4: List of antibodies used in this work.
2.1.5 Enzymes and Reagents Restriction enzymes were purchased from New England Biolabs (Beverly, USA) or Invitrogen (Carlsbad, USA). Buffers and enzymes for standard PCR were supplied by Genaxxon; for high-fidelity cloning PCR Herculase II enzyme and buffer (Stratagene) was used. GeneRulerTM 1kb DNA Ladder (Fermentas) was used as a size marker for DNA separation, whereas the pre-stained Precision Plus Protein Dual Colour Standards (Bio-Rad) was used as a size marker on protein gels. Following kits were used: RNeasy RNA extraction kit (RNeasy Plant Mini Kit, Qiagen), Plasmid Miniprep kit (E.Z.N.A.), Jetstar Plasmid Purification Kit (GENOMED), Nucleon PhytoPure Plant Genomic DNA Extraction Kit (Amersham) and ECL plus immunoblot detection kit (GE Healthcare). Chemicals of analytical grade were manufactured by Fluka (Buchs, USA), Sigma-Aldrich (St. Louis, USA), Merck (Darmstadt, Germany), Roth (Karlsruhe, Germany), Duchefa (Haarlem, NL), Becton Dickinson (Heidelberg, Germany), Bio-Rad (Hercules, USA) and Invitrogen (Carlsbad, USA).
Material and Methods
52
2.2 Methods 2.2.1 Plant Growth Arabidopsis thaliana seeds were surface-sterilized in 6% v/v sodiumhypochlorite with 0.1% (v/v) Tween 20, followed by 3 x washing with sterile distilled water. Seeds were sawn on MS plates (0.43% [w/v] Murashige and Skoog basal salt mixture [Duchefa], 0.05% [w/v] MES buffer [Roth] pH = 5.7) containing 1% (w/v) sucrose and 0.8% (w/v) agar, and were stratified for 2 days in the dark at 4°C. Seeds were germinated and grown aseptically at 25°C either in a standard grow th chamber (MLR-350, Sanyo Electric Co., Ltd.) under a 12 h dark / 12 h light cycle (fluence rate = 69 µmol m-2 sec-1) at 21°C/19°C, or under continuous irradiation in the narrowband UV-field at 23°C.
2.2.2 Plant Protein Extraction For protein extract experiments, 250 mg of Arabidopsis thaliana seedlings were harvested and snap frozen in liquid nitrogen. Seedling tissues were mixed with 5-7 glass beads and ground for 8 sec using a Silamat S5 mixer (Ivoclar Vivadent). 50 µl protein extraction buffer (0.05 M TRIS-HCl, pH7.5; 2 mM EDTA, pH8; 0.15 M NaCl; 1% [v/v] Igepal; 1xComplete EDTA-free Protease Inhibitor Cocktail [Roche], 20 µM MG132 [Sigma], and 20 µM ALLN [Sigma]) was added to each sample on ice. The samples were thoroughly mixed for three times using the Silamat and centrifuged for 10 min with 20800 g at 4°C (Centrifuge 5804R; Eppen dorf), and the supernatant was transferred to a pre-cooled eppendorf tube. Protein concentrations were measured with the amido-black method (Moser et al., 2000). Protein samples were diluted 1:50 in 200 µl distilled water, and a BSA (Bio-Rad) standard sample dilution series of 10-250 µg/ml was also prepared in 200 µl solutions. 800 µl precipitation solution (10% [v/v] acetic-acid; 90% [v/v] methanol; 0.01% [w/v] Naphtol Blue Black [Amidoblack]) was added. After mixing by vortexing samples were centrifuged for 15 min at RT at 20800 g. After washing with 1 ml washing solution (10% [v/v] acetic-acid; 90% [v/v] ethanol) and centrifugation for 15 min at RT with 20800 g, precipitates were air dried for ~15 min at RT, and dissolved
Material and Methods
53
in 250 µl 0.2 N NaOH. 200 µl of each sample were transferred to 96-well ELISA plates and extinctions were measured at 630 nm in a MRX Microplate Reader 630 (Dynex Technologies). Protein sample concentrations were determined according to the BSA calibration curve. The extract was heat treated at 100°C for 5 minutes or not, as described for each experiment. For immunoprecipitation assay protein were extracted as for total extract analysis. The extract was incubated with appropriate antibodies as indicated for 2 h at 4 °C. Then protein A-agarose beads (Roche Applied Science) were added for 1 hour and washed three times in extraction buffer spinning each time at 1 g at +4°C. The immunoprecipitate was stored at -20°C before analys is.
2.2.3 Cell-Free Degradation Assay Seven-day-old Arabidopsis thaliana seedlings were UV-B treated and harvested as described above for plant protein extraction. The protein extracts were then kept at room temperature adding 10 µmol proteasome inhibitors, or adding DMSO, as mock control, in the same volume of proteasome inhibitors (ALLN (Sigma), MG132 (Sigma), MG115 (Sigma), PS1 (Sigma)). After treatment the proteins were kept at -80°C before western analysis.
2.2.4 Yeast Growth and Transformation Yeast strains L40 and PJ69-4A were grown aseptically from glycerol stock on plates supplemented with amino acids complete media, YPDA. Yeast were picked from YPDA plates and grown in YPDA liquid culture to OD600 = 0.6, then yeast was transformed according to Gietz and Woods (Gietz and Woods, 2002). After transformation the yeast were grown on selective plates supplemented with drop-out media lacking amino acids as required.
Material and Methods
54
2.2.5 Yeast Protein Extraction Transformed yeast were transferred to liquid 2 ml miniculture in media lacking corresponding amino acids (-Leu and - Trp) and they were grown overnight at 30°C with 150 rpm. Fifty-ml liquid culture lacking corresponding amino acids was inoculated with the overnight pregrown miniculture, and it was grown at 30°C, 150 rpm until 0.6 OD600. Cells were then centrifuged and washed in distilled water. After, the cells were centrifuged and resuspended in extraction buffer as for plant protein extract supplemented with 0.1 M PMSF and 200 µl of 0.3 mm glass beads. The extraction was done at +4°C vortexing the samples f or 1 min and another 1 min on ice for a total of 10 min vortexing. Then the samples were centrifuged and +4 °C for 10 min and the supernatant was transferred in a new tube. The extract was heat treated at 90°C for 3 min or not, as described for each experiment. For immunoprecipitation assay protein were extracted as for total extract analysis. The extract was incubated with appropriate antibodies as indicated for 2 h at 4°C. Then protein A-agarose beads (Roche Applied Science) were added for 1 hour and washed three times in extraction buffer, spinning down each time at 1 g at +4°C. The immunoprecipitate was stored at -20°C before analys is.
2.2.6 HEK293T Cells Growth and Transformation Overexpression of target proteins in HEK293T cells was achieved through transient transfection. The day before cells’ transfection, HEK293T cells were split into 6 wells plates. Splitting was conducted from a 10 cm mother plate with 70% confluence rate. After removing the old medium the cells were treated for 2 minutes at 37°C with Trypsin/EDTA in order to detach the cells from the surface. Then, the cells were poured into a fresh well of a 6-well plate diluting 1:1 with new MEM +/+ medium. On the following day, for each single transfection, 100 µl of serum free MEM -/- medium was mixed with 3 µl of GeneJuice Transfection Reagent (Novagen, Darmstadt, Germany), agitated vigorously and kept at RT for 5 min. Then, 1 µg of DNA of interest (pDEST27-GST-UVR8, pcDNA-DEST40-UVR8 or both [0.5 µg each]) was added to the mixture, mixed by gently pipetting and kept at RT for 5-15 min. Finally the entire volume of GeneJuice reagent/DNA mixture was poured drop-wise on the
Material and Methods
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surface of the cell-containing medium. Transfected HEK293T cells were incubated for 72 hours at 37°C and 5% CO 2, and finally harvested. As positive control for successful transfection, a vector for expression of EGFP was used. The qualitative observation of GFP fluorescent cells accounted for the efficiency of transfection.
2.2.7 Protein Extraction from Transfected HEK293T Cells Cells in each well of a 6-well plate were washed once with 1x PBS. 300 µl CytoBuster extraction buffer (Novagen, Darmstadt, Germany) was added to each well and incubated for 5 min on ice. The cell layer was scraped off from the bottom of each well and the resulting lysate transferred to a reaction tube. After centrifugation at 16,000g for 15 min at 4°C to remove insoluble de bris and chromosomal DNA the remaining extract was stored at -20°C and used for subsequent experiments. For GST immunoprecipitation, 30 µl total extract was incubated overnight at 4°C with 50 µl GSH-Sepharose beads (Amersham Biosciences). Beads were sedimented by centrifugation at 100g, for 2 min at 4°C. After inc ubation, the beads were washed three times in extraction buffer, spinning each time at 100g at +4°C. The immunoprecipitate was stored at -20°C before analys is.
2.2.8 UV-B Treatments The UV-B treatments of Arabidopsis thaliana extracts, yeast extracts and HEK293T cell extracts were performed under the following UV-B fields: I) Short term irradiation was performed under a UV-B light field designated as the “broadband UV-B field” consisting of six broadband Philips TL 40W/12 RS UV fluorescent tubes (λ_max = 310 nm, half-bandwidth = 40 nm, fluence rate = 7 W/m2, or 18 µmol m-2 sec-1). The UV-B spectra was generated by filtering the emitted light through 3-mm transmission cut-off filter WG303 with half-maximal transmission at 303 (WG303 Schott, Germany), as shown in Fig. M1. Plastic filter served as the minus UV-B control, and WG303 as the weak UV-B treatment.
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7,00E-05 6,00E-05 5,00E-05
W/m2
4,00E-05 3,00E-05 2,00E-05 1,00E-05
0,00E+00 0,00
200,00
400,00
600,00
800,00 1000,00
Wavelenghts
Figure M1: Spectral irradiance in the broadband UV-B field under the 303 cut-off. Spectral energy distributions of UV-B sources were measured with an OL 754 UV-visible spectroradiometer (Optronic Laboratories, Orlando, FL).
Unless otherwise stated, the following irradiation protocol was used for UV-B treatments under this broadband UV-field: The protein extracts derived from plants or yeast were put on ice during irradiation and treated for the indicated time for each experiment, under plastic filter or WG303 filter. II) The continuous UV-B treatment was performed under a white light field supplemented with narrowband UV-B, designated as the “narrowband UV-B field” consisting of six dimmable light tubes. The white light is provided by three Osram L18W/30 tubes (3.6 µmol m-2 sec-1; measured with a LI-250 Light Meter, LI-COR Biosciences, Lincoln, NE) that is supplemented with UV-B irradiation provided by three Philips TL20W/01RS narrowband UV-B tubes (1.5 µmol m-2 sec-1; measured with a VLX-3W Ultraviolet Light Meter equipped with a CX-312 sensor, Vilber Lourmat, Marne-la-Vallée, France). The resulting spectrum is shown in Fig. M2. Plastic filters serves as the minus UV-B control, the UV-B range was modulated by the use of 3-mm transmission cut-off filters WG303 (WG303; Schott Glaswerke, Mainz, Germany). Spectral energy distributions of UV-B sources were measured as described above (Fig. M2).
Material and Methods
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W/m2
6,00E-06 4,00E-06 2,00E-06 2,90E-19
-2,00E-06
0,00
200,00 400,00 600,00 800,00 1000,00
Wavelenghts
Figure M2: Spectral irradiance in the narrowband UV-B field under the 303 cut-off. Spectral energy distributions of UV-B sources were measured with an OL 754 UV-visible spectroradiometer (Optronic Laboratories, Orlando, FL).
Unless otherwise stated, the following irradiation protocol was used for UV-B treatments under this narrowband UV-field: The protein extracts derived from HEK293T cells were put on ice during irradiation and treated for the indicated time for each experiment, under plastic filter or WG303 filter.
2.2.9 Agrobacterium Mediated Plant Transformation Arabidopsis thaliana plants were transformed using Agrobacterium tumefaciens harbouring the appropriate binary vector according to the ‘Floral dip’ method (Clough and Bent, 1998). Transformants were selected dependent on their selection marker: BASTA selection was performed on soil-grown plants by spraying at 7 DAG (day after germination), while GFP selection of seeds was performed under stereo microscope supplemented with UV light. After selection, the primary transformants were transferred to single pots and grown to maturity.
2.2.10
DNA Isolation
High quality plant DNA was isolated using the Nucleon PhytoPure Genomic DNA Extraction Kit (Amersham). Coding sequences were amplified from Col cDNA using primers listed in table M3.
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Plasmid DNA from Escherichia coli or Agrobacterium tumefaciens was extracted and purified with the E.Z.N.A. Plasmid Miniprep Kit according to the manual. For high quantities of plasmid DNA 100 ml liquid cultures of Escherichia coli were prepared and submitted to the extraction procedure provided with the Jetstar Plasmid Purification System (GENOMED).
2.2.11
PCR
High fidelity cloning PCR was performed according to the requirements for Herculase II fusion DNA-polymerase (Stratagene) as described in the manufacturers’ manual.
2.2.12
Agarose Gel Electrophoresis
For DNA gel electrophoresis, 1% (w/v) agarose gels were made in 1x TAE (Sambrook and Russel 2001) and 1 µl ethidium-bromide (1 mg/ml) was added directly to 50 ml of gel solution for visualization of nucleic acid under UV. DNA samples (PCR fragments or plasmid restrictions) were supplemented with 6x DNA-loading buffer (6x TAE, 30% (v/v) glycerol, 0.125% (w/v) bromophenol blue, 0.125% (w/v) xylene cyanol) and the GeneRuler 1kb DNA Ladder (Fermentas) was used as a size marker. The GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences) was used to purify DNA bands from agarose gels, if needed.
2.2.13
Site-Directed Mutagenesis
Site-Directed mutagenesis was done on UVR8 coding sequence recombined by Gateway technology in pDONR207. Specific primers were designed as listed in table M3. The primers are specific on UVR8 sequence and contain the desired mutations. The PCR amplification was done with Herculase II proof reading DNA polymerase, as described above. After amplification, DpnI was added to the PCR mix leading to degradation of parental plasmid. Then, the PCR mix was directly used for bacterial transformation leading to nick repair, and transformants were analyzed, after plasmid isolation, by sequencing at GATC (Konstanz, Germany).
Material and Methods
2.2.14
59
CPRG Assay
To quantify protein-protein interaction in yeast a β-galactosidase assay using CPRG as a substrate was performed. From the transformed mother plate, yeast cells were streaked on two plates with selective media as described above. Each plate was streaked three times, taking ten different colonies each time from the mother plate. The plates were then putted under a narrowband UV-B field at 30°C, with plastic filter or with WG303 filter respectively. The plates were incubated overnight. The following day each streak on the plate was independently harvested, resuspended in 1.5 ml YPDA, and OD600 was recorded. Yeast cultures were then spun down, and washed in distilled water, and spun down again. The pellet was resuspended in 500 µl of CPRG buffer (Per 100 ml solution: 2.38g HEPES, 0.9 g NaCl, 0.065 g L-aspartate hemi-Mg [Sigma], 1 g BSA, 50 µl Tween 20; dissolve in 75 ml, adjust to pH 7.3, adjust volume to 100 ml, filter sterilize). Then the pellet was spun down, and resuspended in 300 µl CPRG buffer, and 100 µl of this were transferred to a new tube and frozen in liquid nitrogen. The tube content was thaw in a termoblock at 37 °C and frozen, and this cycle was repeated three times before adding 0.7 ml CPRG substrate in each tube (27.1 mg CPRG [Roche] in 20 ml CPRG buffer). Then the tubes were put on a shaker at 37°C and the time was recorded until development of the colour was achieved, and the reaction was stopped by addition of 0.3 ml, ZnCl2 3 mM. The OD578 was measured and the β-galactosidase units were calculated according to the formula: β-galactosidase units = 1000 ⋅
OD 578 t ⋅ V ⋅ OD 600
t = elapsed time (in minutes) of incubation V = 0.1 x concentration factor Concentration factor = Volume of the starting culture for which OD600 was recorded (1.5 ml) / Final volume used to resuspended the pellet (0.3 ml) = 5
For yeast two-hybrid analyses, genes were cloned either in the GAL4 binding domain vector pGBT9_GW (for the strain PJ-69) or in the LexA DNA binding domain vector pBTM116-D9 (for the L40 strain). The activator domain was the same for both strains, pGADT7_GW vector. The yeast three-hybrid was done in the PJ-69 yeast strain, which allows additionally selection on the nutrient marker –URA. The plasmid used for the expression of the third protein was pAG-426GPD-ccdB (Alberti et al.,
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2007). Empty-Vector controls were performed with empty (i.e. ccdB gene removed) pBTM116-D9, pGBT9_GW and pGADT7_GW vectors (Bartels et al., 2009). In the yeast three-hybrid system, RCC1 has been used as control in the vector pAG-426GPD-ccdB.
2.2.15
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Approx. 20-30 µg of total cellular proteins were separated by sodium dodecyl sulfate polyacrylamid gel electrophoresis in 8% gels (12% for HY5 protein gel) according to standard methods (Sambrook and Russel, 2001) using the Mini Protean 3 Electrophoresis System (Bio-Rad). For protein size comparison Precision Plus Protein Dual Colour Standard (Bio-Rad) was used or, for HEK293T cells experiment, Pageruler Prestained Protein Ladder marker (Fermentas, St Leon-Rot, Germany) was used.
2.2.16
Immunoblot Analysis
Gel separated proteins were electrophoretically transferred at RT for 70 min with 100 V to polyvinylidene difluoride (PVDF) membranes (Roth) by wet transfer (transfer buffer: 3.03 g/l TRIS-HCl; 14.4 g/l glycine; 20% [v/v] ethanol; 0.015% SDS) in the Mini Trans-Blot Cell system (Bio-Rad) according to the manufacturer’s instructions. Membranes were blocked with 10% (w/v) non-fat dried milk and first antibodies were diluted and incubated overnight in 5% (w/v) non-fat dried milk (rabbit α-UVR8 polyclonal 1:5000 (Eurogentec), guinea pig α-UVR8 polyclonal 1:5000 Eurogentec, α-Actin monoclonal 1:20000 (Sigma), rabbit α-GST monoclonal 1:2000 (Sigma), rabbit α-LexA polyclonal 1:5000 (Millipore)). Secondary antibodies were diluted 1:20000 and incubated for 1 h at RT (α-Mouse (Dako), α-Rabbit (Dako), α-Guinea Pig (Abcam) α-Mouse-Alexa 680 (Invitrogen), α-Rabbit IRDye® 800CW (LI-COR biosciences)). Washing steps were done in TBS-T (200mM Tris-HCl pH 7.6, 80 g/l NaCl, 0,1% (v/v) Tween-20) and signal detection was performed as described in the ECL plus Western detection kit (GE healthcare) except in HEK293T cells experiments where detection was done through the fluorescent dye linked to the
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secondary antibody using the Odyssey LI-COR system (Licor, Bad Homburg, Germany). Before re-hybridisations with a different probe, the membranes were stripped by 30 min incubation at 65°C in stripping buffer (62.5 mM TRIS-HCl, pH = 6.7; 2% [v/v] SDS, 100 mM β-Mercaptoethanol).
2.2.17
BiFC
Sinapis alba–based BiFC assays were performed using the biolistic PDS-1000/He system (Bio-Rad) as described previously (Stolpe et al., 2005). A Pro35S:CFP control plasmid was always co-bombarded to identify transformed cells prior to the analysis of YFP fluorescence. UVR8 coding sequence was cloned with the Gateway system into the pE-SPYNE-GW and pE-SPYCE-GW destination vectors. The Empty-Vectors used as negative controls were generated by recombination with an empty pENTRY3C, where the ccdB gene was removed (Bartels et al., 2009).
2.2.18
Luciferase Measurement
Transgenic plants were grown for 6 days in a SANYO MLR-350 chamber (under 12h light / 12 h dark cycles) and then transferred to Top Count microtiter plates filled with MS medium containing 1% sucrose and 1% agar using sterile forceps. For luminescence detection 20 µl of sterile 0.5 mM luciferin (Biosynth AG; diluted in sterile 0.01% Triton-X solution from the filter sterilized 50 mM luciferin stock solution) was applied on the top of the the seedlings. The plates were then wrapped in foil, and put back into the growth chamber for at least 16 hours. On the following day the seedlings were irradiated using the “broadband UV field” under WG303 cut-off for 15 min. Afterwards plates were covered with sticky transparent foil and then the foil was perforated creating 96 needle-size holes allowing air exchange. The parameters of the measurements on the Top Count (PACKARD TOPCOUNT NXT 96 WELL PLATE SCINTILLATION COUNTER) were the following: each well was measured for 1 sec with 12 wells measured in parallel, cycle number was infinite, each plate and well were measured once per cycle.
Material and Methods
2.2.19
62
Bioinformatic Analysis
The accession numbers of the protein used in the multiple sequence alignment and in the phylogenetic tree are given: Arabidopsis thaliana Q9FN03, Vitis vinifera D1HKC1, Ricinus communis B9SAB0, Populus tremula B9HF48, Glycine max C6TC00, Oryza sativa B8AJF0, Sorghum bicolor C5Y8Q8, Oryza sativa A2XTP3, Physcomitrella patens A9RS92, Physcomitrella patens A9TJL3, Chlamydomonas reinhardtii A8JGB3, Arabidopsis thaliana Q9LU80, Arabidopsis thaliana Q9LDU3, Arabidopsis thaliana Q94CK7, Arabidopsis thaliana Q8L7B6, Arabidopsis thaliana Q9FJG9, Homo sapiens P18754, Saccharomyces cerevisiae YGL097W, Selaginella moellendorffii EFJ17371, Selaginella moellendorffii EFJ35396, Volvox carteri EFJ39741. UVR8 multiple sequence alignment is generated from the input by running ClustalW as option of Jalview version 2.4.0.b2 (Thompson et al., 1994). Secondary structure prediction of UVR8 and RCC1 was carried out with Jpred 3 (Cole et al., 2008). The secondary structure and the multiple alignment were edited with Jalview (Waterhouse et al., 2009). The tertiary structure prediction was done with the automated homology modeling server (PS)2 using RCC1, PDB entry 1A12, chain A, as template (Chen et al., 2006). The predicted model was edited with PyMOL v1.1 (The PyMOL Molecular Graphics System, Version 1.1, Schrödinger, LLC). The phylogenetic tree was inferred with MEGA4 (Tamura et al., 2007) on the multiple alignment shown herein, and with the sequences presented in this section. The tree was inferred with the Neighbor-Joining method (Saitou and Nei, 1987). A bootstrap test on 500 replicates was conducted (Felsenstein, 1985). The evolutionary distances were computed using the Poisson correction method (Zuckerkandl and Pauling, 1965).
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3 Results
3.1 COP1-UVR8 Interaction in Yeast In order to understand if COP1 and/or or UVR8 were possible candidate for UV-B perception we tried successfully to reproduce their the UV-B-dependent dependent interaction in yeast, using a yeast two-hybrid hybrid assay system. system Fig. R1 shows a quantitative assay as a histogram on β-galactosidase osidase activity, and a qualitative assay as nutritional growth on plates lacking histidine. Empty-Vector Empty ector controls are shown as well. Yeast was grown at 30 °C under narrowband UV-B light , applying on the top p of the petri dish a plastic cut-off or a WG303 filter. In the yeast growth assay a background signal was detected for the COP1 control. However, this background growth was wa not apparent when co-expressing UVR8 in white light without UV-B (plastic cut-off), cut and it was weaker than the signal given by the interaction between UVR8 and COP1 in white light supplemented with UV-B (WG303 cut-off). Moreover, no background activity was detectable in the corresponding β-galactosidase assay.
Figure R1: Yeast two-hybrid hybrid analysis on the interaction between UVR8 (fused to the LexA DNA binding domain, BD) and COP1 (fused to the GAL4 activation domain, AD).. The protein interaction is shown as β-galactosidase -galactosidase activity in Miller Units (CPRG assay), and yeast growth assay (-His). The non-UV-B -UV-B treatment and the Empty-Vector Emp controls display levels of β-galactosidase activity <1.0 Miller Units.
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We checked in which measure the narrowband UV-B irradiation was affecting yeast growth treating untransformed yeast with or without UV-B. After UV-B treatment we counted the number of colonies on plates grown under UV-B, and compared it to colony growth under conditions devoid of UV-B. Yeast was grown for two days at 30°C under broadband UV-B (Fig. R2, left panel), an d under narrowband UV-B (Fig. R2, right panel). Narrowband UV-B did not cause cell death. Notwithstanding, colony growth was slightly affected, while broadband UV-B treatment was completely killing the yeast cells.
Figure R2: Yeast viability assay. Colonies were counted after 2 days of continuous UV-B light treatment, shown as number of colonies per plate, and picture of a representative section of the plate. Left panel: broadband UV-B. Right panel: narrowband UV-B.
Moreover, the UV-B fluence rate dependence of the UV-B-dependent interaction between COP1 and UVR8 in yeast was tested, under narrowband UV-B, and under broadband UV-B respectively. Yeast was UV-B treated for the indicated time, as shown in Fig. R3. Then, one hour without supplemental UV-B was given for transcription and translation of the β-galactosidase reporter, followed by CPRG assay. Under broadband UV-B the β-galactosidase activity was increasing faster compared to narrowband UV-B radiation. Under broadband UV-B the signal was decreasing after 30 min, probably because of cell-death, as shown by reduced cell number after the UV-B treatment.
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Figure R3: Time-course of yeast two-hybrid interaction between UVR8 and COP1 under different UV-B irradiation conditions. Interaction strengths are displayed as β-galactosidase activities in Miller Units. Upper panel: time-course of the UVR8 interaction with COP1 under narrowband UV-B. Lower panel: time-course of the UVR8 interaction with COP1 under broadband UV-B. Under broadband UV-B the yeast cell growth was tested. Drops of media containing yeast were distributed on the plates and UV-B treated as for the time-course analysis. After broadband UV-B treatment, the yeast was grown for 2 days in the dark, and representative pictures were taken. After 30 min broadband UV-B irradiation, the yeast proliferation was affected compared to the non-UV-B control. At 60 min broadband UV-B irradiation yeast colonies are not detectable anymore.
Strong UV-B radiation causes cross-linking among proteins. We checked known interactions in the yeast two-hybrid system to test if the UV-B treatment employed in the COP1-UVR8 interaction assay was affecting the experiment (Fig. R4). These interactions did not increase under UV-B light, but rather they showed a decrease of the interaction strength compared to the non-UV-B control. Such decrease is likely associated with the reduced yeast proliferation under narrowband UV-B irradiation.
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Figure R4: Yeast two-hybrid analysis of previously published protein-protein interactions. Upper left histogram: HY5 interaction with COP1 (Ang et al., 1998). Upper right histogram: SPA1 interaction with COP1 (Hoecker and Quail, 2001). Lower histogram: MAP kinases interactions with the phosphatase MKP1 (Ulm et al., 2002).
As control for the specificity of the interaction of COP1 with UVR8 we checked the interaction among COP1 and plant UVR8 homologs, chosen by the BLAST algorithm, with standard parameters, at the TAIR database (Fig. R5). Because of the negative result, we additionally checked by western if the plant UVR8 homologs were expressed in yeast.
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Figure R5: Yeast two-hybrid hybrid analysis of the interaction among COP1 and UVR8 plant homologs under narrowband UV-B. Upper panel: COP1 interaction with UVR8 homologs shown as β-Galactosidase -Galactosidase activity and yeast growth assay (-His).. Lower panel: Protein level of UVR8 and UVR8 homologs in yeast.
Because uvr8 mutants uvr8-9 (UVR8G202R) and uvr8-15 (UVR8G145S) were shown not to be functional in planta and to lack interaction with COP1 (Favory et al., 2009), 2009) we checked if the respective spective protein mutants were able to interact with COP1 in yeast two-hybrid assay. Indeed, UVR8G202R and UVR8G145S mutants were not able to interact UV-B-dependently with COP1 in yeast two-hybrid assay (Fig. Fig. R6). R6
Figure R6: Yeast two-hybrid two analysis, shown as β-galactosidase activity in Miller Units and yeast growth assay (-His),, of the interaction between UVR8 mutant proteins and COP1. The UVR8 mutants UVR8G145S (uvr8-15) and UVR8G202R (uvr8-9) were analyzed.
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We additionally checked if cop1 protein mutants were able to interact with UVR8. The result in Fig. R7 shows that UVR8 is specifically interacting with the WD-40 WD domain of COP1. Indeed, UVR8 is interacting with COP1C340 and with COP1H69Y (cop1eid6), the latter has been shown to have a functional UV-B response (Oravecz et al., 2006). 2006) Moreover, UVR8 is not interacting inte with cop1-4 (COP1N282) and with cop1-19 (COP1G608R) which are respectively missing completely the WD-40 40 domain of COP1, COP1 and have a point mutation in the WD-40 WD domain of COP1. The corresponding two cop1 mutants have been shown to lack UV-B UV responses (Oravecz et al., 2006; Favory et al., 2009).
Figure R7: Yeast two-hybrid hybrid analysis, shown as β-galactosidase activity of the interaction between UVR8 and COP1 mutants. The right panel shows, in a schematic illustration, the domains of COP1 that are analyzed in each assay, and point mutation are indicated by arrows. Schematic representation of the COP1 domains form the upper wild type protein to the bottom: full length protein pr (COP1), N-terminal domain with zinc-finger, -finger, and coiled-coil domains (COP1N282, cop1-4), cop1-4 point mutation in the WD-40 domain (COP1G608R, cop1-19), point mutation in the zinc-finger domain (COP1H69Y, C340 cop1eid6), C-terminal WD-40 domain of COP1 (COP1 ( ).
The data shown in this section demonstrate the UV-B-dependent dependent interaction of UVR8 with COP1 in the heterologous yeast system, where no UV-B photoreceptor pathway is known, and neither UVR8 nor COP1 homologs are present. We can reasonably speculate that these proteins may be sufficient for UV-B perception. Moreover, the WD-40 C-terminal domain of COP1 is necessary and sufficient for the interaction with UVR8.
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3.2 UVR8 Homodimer omodimer UVR8 has an overall identity of 30.6% 3 .6% compared to the human homolog RCC1. This identity value resides in the “twilight zone” (20%-35%) (20% 35%) of protein structure-function prediction. Despite the fact that UVR8 seems not to have Ran-GEF activity, which characterizes RCC1, these proteins are structurally similar, as identified by secondary and nd tertiary structure prediction (Fig. R8a, Fig. R8b).. In the secondary structure prediction it is possible to see the blades of RCC1 and UVR8, each blade is composed of four β-strandss,, except for the first blade which has only three β-strand. Indeed, the first blade is completed by the C-terminal β-strand allowing the β-propeller -propeller structure to close on its own. The tertiary structure prediction of UVR8 has been inferred,, as in material and methods, using RCC1 crystal structure as template. If the template option was not used and the three dimensional structure was inferred from protein multiple alignment, the result was similar (data not shown).
Figure R8a:: Secondary structure prediction of RCC1 (left panel) and UVR8 (right panel). The secondary structure prediction has been inferred with Jpred 3 (Cole et al., 2008).
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Figure R8b:: a and b: three dimensional structure of RCC1 (Renault et al., 1998). 1998) c and d: three dimensional structure prediction of UVR8 (Chen et al., 2006). The tertiary structure prediction was inferred with the automated homology modeling server s (PS)2 using RCC1, PDB entry 1A12, chain A, as template. Three hree central tryptophans tryptophan are highlighted in the predicted 3D structure of UVR8. UVR8
ic conformation (Kim et al., RCC1 is hypothesized to be functional in its homodimeric 2006), even though ough the homodimerization of this protein has not yet been demonstrated.. Because of the homology h between UVR8 and RCC1, RCC1 I’ve decided to test if UVR8 was able to homodimerize. Indeed, Indeed this turned out to be true, true as shown in Fig. R9.
Figure R9: Left panel: yeast two-hybrid two analysis of UVR8 dimerization,, shown as β-galactosidase activity and yeast growth assay. Right panel: UVR8 homodimerization in Sinapis inapis alba using transient expression and bimolecular fluorescence complementation (BiFC) assay (Favory et al., 2009). 2009) Empty-Vector controls are shown as well. well
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3.3 UV-B-Dependent Monomerization of UVR8
3.3.1 UV-B-Dependent Monomerization of UVR8 in HEK293T Cells After the finding of the UV-B dependent UVR8 and COP1 interaction in yeast, I have started collaboration with Davide Faggionato (University of Freiburg, Laboratory of Prof. Baumeister). Expressing GST-UVR8 in HEK293T cells, we noticed, on western basis, a higher band compared to the expected molecular size of the GST-UVR8 monomer, when the protein extract was not boiled. We reasoned that the GST-UVR8 protein was mainly present as a homodimer if the total protein extract was not denatured by boiling. However, when the total protein extract was treated with UV-B radiation or boiled, the GST-UVR8 protein was mainly running at a molecular size comparable
to
its
monomeric
conformation,
indicating
a
UV-B-dependent
monomerization of GST-UVR8 (Fig. R10). The UV-B-dependent monomerization was detected also after GST pull-down, where the purified protein, and not the total extract, was UV-B treated. This let us to speculate that the UVR8 protein was sufficient for UV-B perception, and maybe for UV-B-induced conformational change of UVR8 leading to monomerization.
Figure R10: Western blot analysis of GST-UVR8 monomerization with or without 60 min narrowband UV-B light treatment. The HEK293T cells lysate was treated at 42°C or 95°C as shown. Left panel: total protein extract. Right panel: GST pull-down. Cross-reacting bands are marked (*).
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The GST protein is known to multimerize. To exclude that pDEST27-GST-UVR8 dimerization in HEK293T cells was an artefact due to the GST-tag, a non-tagged pcDNA-DEST40-UVR8 was tested as well (Fig. R11). Moreover, to exclude that UVR8 was not binding to different human proteins, pDEST27-GST-UVR8 and pcDNA-DEST40-UVR8 were co-expressed in HEK293T cells and detected with the respective antibodies coupled to different fluorescent dyes. As result, the GST-UVR8 homodimer and the UVR8 homodimer are visible, as well as the heterodimer between GST-UVR8 and UVR8. The GST tag has a molecular weight of 26 kDa. UVR8 has a molecular weight of 47 kDa. Expected molecular weight: UVR8 monomer = 47 kDa UVR8 homodimer = 94 kDa GST-UVR8 monomer = 73 kDa GST-UVR8 homodimer = 146 kDa GST-UVR8 / UVR8 heterodimer = 120 kDa The UVR8 monomer and the GST-UVR8 monomer are running at the expected molecular weight. The homodimers of GST-UVR8 and UVR8 and the heterodimer GST-UVR8 with UVR8 are running lower than the expected molecular weight. It has to be considered that the protein were not denatured and the three dimensional conformation can influence the running of the protein in the gel. Interestingly, the monomers are running at the proper size, as if their conformations are different from the conformations of the respective dimers. Moreover, it is clear that UVR8 is binding only to UVR8 and not aspecifically to other human proteins. Indeed, the shift of the GST-UVR8 homodimer and the shift of the UVR8 homodimer are proportional respectively to the presence and absence of the GST tag, i.e. higher shift for the GST-UVR8 tagged version. The proof that UVR8 is self-binding is given by the co-expression of GST-UVR8 with UVR8 which runs at an intermediate molecular weight between the UVR8 homodimer and the GST-UVR8 homodimer. This band is not present if only GST-UVR8 or only UVR8 are expressed in the human cells culture.
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Figure R11: UVR8 monomerization in human cells culture. Western blot analysis of human cells culture protein extract expressing different combination of UVR8, GST-UVR8, and GST Empty-Vector. Upper panel: fluorescent dye bound to the secondary antibody for the detection of UVR8. Lower panel: fluorescent dye bound to the secondary antibody for the detection of the GST tag. Ladder (1st lane), GST-UVR8 (2nd-3rd lanes), GST-UVR8 and UVR8 (4th-5th lanes), UVR8 (6th-7th lanes), GST-UVR8 and GST-Empty-Vector (8th-9th lanes). Cross-reacting bands are shown as well (*). Molecular marker is shown in kDa.
In order to have a better resolution of the UVR8 UV-B-dependent monomerization, time-course experiments were carried out. GST-UVR8 from total extract or purified protein clearly shows a time dependent switch between the dimeric and monomeric conformations under narrowband UV-B (Fig. R12).
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Figure R12: Western blot analysis of pDEST27-GST-UVR8 monomerization time-course in HEK293T cells. Upper panel: monomerization time-course in protein total extract, under narrowband UV-B, on ice. Lower panel: time-course of UVR8 UV-B-dependent monomerization of purified GSTUVR8 protein, under narrowband UV-B, on ice. The protein detection has been achieved with the P60 antibody in the upper panel, and with the anti-GST antibody in the lower panel.
Is UVR8 monomerization caused by UV-B dependent denaturation of the protein? To address this question we tried to recover the UVR8 dimer after UV-B-dependent monomerization in HEK293T cells protein total extract. Initially we treated the extract under narrowband UV-B light for 60 min, which resulted in the complete monomerization of UVR8. After the treatment, the extract was split and moved to a dark box at room temperature. At different times aliquots of the extract were moved to -20°C. Fig. R13 shows the monomerization of UVR8 in the protein extract (upper panel) and the time dependent recovery of the dimer (lower panel). We cannot exclude transcription and translation in the total extract, but it is unlikely that these processes are taking place after more than 24 hours from protein extraction. For this reason we conclude that UVR8 was not denatured after UV-B treatment and it was able to recover to its dimeric conformation.
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Figure R13: Western blot analysis of pDEST27-GST-UVR8 re-dimerization, expressed in human cells culture. Upper panel: time-course of UVR8 monomerization under narrowband UV-B. Samples were heat treated at 42 °C for 30 min or at 95 °C for 5 min. Lower panel: subsequent recovery of the dimeric UVR8 protein in the dark at room temperature. No recovery of the UVR8 dimeric conformation for samples treated at 95 °C (data not shown). A cross-reacting band is shown as well (*).
With the experiments presented in this section, we were able to show that UVR8 is able to undergo monomerization after UV-B exposure. The UVR8 monomerization is taking place with the same kinetics irradiating purified proteins, indicating that UVR8 is able to perceive the UV-B light.
3.3.2 UV-B-Dependent Monomerization of UVR8 in Yeast After demonstration of UV-B-dependent UVR8 monomerization in HEK293T cells, we reasoned to confirm it with another system, especially in yeast were we could link the monomerization to the interaction with COP1. To do this, UVR8 was expressed with the same plasmid used for yeast two-hybrid analyses, pBTM116-D9, in the L40 yeast strain. The plasmid contains the LexA DNA binding domain fused to the protein. In this system it was possible to reproduce the UV-B-dependent monomerization, irradiating yeast extract for 5 min with broadband UV-B on ice (Fig. R14). Similarly, the UVR8 mutant proteins UVR8G145S and UVR8G202R were tested in yeast. These mutant proteins were lacking the dimeric conformation, suggesting that the glycine
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mutations cause misfolding of the protein, which could be also the reason for the lack of interaction with COP1 (Fig. R6). Similarly to the expression of UVR8 in human cells culture, also in yeast UVR8 dimer is running lower than the expected molecular weight. Expected molecular weight: LexA-UVR8 monomer = 72 kDa LexA-UVR8 homodimer = 144 kDa
Figure R14: Western blot analysis of pBTM116-UVR8 monomerization in yeast total extract. LexA-UVR8 UV-B-dependent monomerization is shown, as well as UVR8 mutant proteins LexA-UVR8G145S and LexA-UVR8G202R. Degradation bands relative to LexA-UVR8 are marked (*). Molecular size is shown in kDa. Coomassie staining is shown for loading control.
We tried to reproduce the monomerization with a non-tagged UVR8 protein in yeast. Astonishingly, the non-tagged UVR8 proteins under non-UV-B conditions were not visible. Nevertheless, it was possible to see the accumulation of the monomeric UVR8 protein after UV-B irradiation (Fig. R15). Considering that the work was carried out on ice with the same amount of protein extract in each well of the protein gel, there should be no difference in protein amount among the samples, and no degradation of the protein. I have reasoned that the UVR8 dimer was not detectable, possibly because the epitope was not accessible to the antibody in the dimeric conformation of the protein.
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Figure R15: Western blot analysis of tagged and non-tagged UVR8 monomerization in yeast total protein extract. LexA-UVR8 monomerization and UV-B-dependent UVR8 accumulation are shown. Degradation products of LexA-UVR8 are marked (**). Proteins were detected with the P60 antibody. Molecular size is shown in kDa. Protein detection with P60 antibody.
Following the hypothesis that detection of the UVR8 homodimer was not possible because of an inaccessibility of its epitope, I’ve attempted an in-gel monomerization of UVR8. To do this, the protein samples were electrophoretically separated by SDSPAGE and the protein gel was then treated under broadband UV-B for 10 min before transfer of the proteins to a PVDF membrane. According to the hypothesis, the dimeric conformation of UVR8 may monomerize in the gel, revealing the epitope for antibody-mediated protein detection. Indeed, a band at higher molecular weight now became detectable, which could correspond to the UVR8 dimer (Fig. R16). Moreover, it is possible to notice that, when UVR8 is expressed without any tag, no protein degradation bands are visible.
Figure R16: Western blot analysis of non-tagged-UVR8 monomerization in yeast protein total extract. Acrylamide gel non-UV-B and broadband UV-B treated for 10 min are shown in the left and the right panels, respectively. Proteins were detected with the P60 antibody.
Non-tagged-UVR8 purified from yeast total extract was checked for monomerization. The UVR8 protein was pulled-down with α-UVR8 P60 (host: rabbit) antibody and
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detected with the α-UVR8 (host: guinea pig) antibody. Also in this experiment the gel was irradiated with broadband UV-B to see the dimer in non-UV-B samples (Fig. R17).
Figure R17: Western blot analysis of non-tagged-UVR8 monomerization after protein purification from yeast total extract. Acrylamide gel non-UV-B and broadband UV-B treated are shown respectively in the left and in the right panel. Proteins were detected with P60 antibody.
3.3.3 UV-B Dependent Monomerization of UVR8 in Planta In plant protein extract, as in yeast protein extract, it was possible to detect the homodimeric conformation of UVR8 only in tagged proteins (Fig. R18).
Figure R18: Western blot analysis of CFP-UVR8 / UVR8 monomerization in plant total protein extract under broadband UV-B. CFP-UVR8 protein construct was inserted by Agrobacterium tumefaciens mediated transformation in an Arabidopsis thaliana wild type background. Proteins were detected with anti-GFP antibody. Molecular size is shown in kDa.
Also in plant protein extract there was the need of UV-B gel irradiation for non-tagged UVR8 protein detection in non-UV-B treated samples. Nevertheless, in non-UV-B treated samples the dimer was very weak even after UV-B irradiation of the gel. To
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confirm the presence of protein in non-UV-B treated samples the extract was denatured at 95°C for 3 min (Fig. R19). Similarly t o the expression of UVR8 in human cells culture and in yeast, also in planta the UVR8 dimer is running lower than the expected molecular weight.
Figure R19: Western blot analysis of UV-B-dependent UVR8 monomerization under 5 min broadband UV-B in plant protein extracts. Samples heat treated at 95 °C for 3 min and samples not heat treated are shown. Protein detection with P60 antibody and anti-actin antibody. Molecular weight is shown in kDa.
The time-course of the protein total extract showed a clear accumulation of monomeric UVR8 under UV-B irradiation (Fig. R20). Monomeric UVR8 accumulated in less than 5 sec after UV-B irradiation of protein extract on ice. Such a fast reaction on ice seems unlikely to be associated with post translational protein modifications, and reinforces the hypothesis that UVR8 is able to directly perceive UV-B light.
Figure R20: Western blot analysis of the time-course of UVR8 monomerization in plant protein extract under broadband UV-B. Loading controls are shown with boiled extract after time-course irradiation of UVR8 and Actin. Detection with P60 and anti-actin antibodies.
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However, the protein amount of UVR8 monomer did not match the protein amount of UVR8 dimer. Given that the protein extract was splitted among the wells of the protein gel, this can be explained as i) UVR8 is in a higher molecular weight complex and it is not transferred from the protein gel to the PVDF membrane, or ii) the irradiation treatment of the gel is not completely unmasking the epitope for the antibody. I tried then to detect the protein with an antibody generated against a different C-terminal epitope (Kaiserli and Jenkins, 2007). This turned out to be successful, and it was possible to visualize an amount of UVR8 dimer very close to the amount of UVR8 monomer after irradiation (Fig. R21).
Figure R21: Western blot analysis of UVR8 monomerization in plant protein extract under broadband UV-B for 5 min. Denatured samples have been heat treated at 95°C for 3 min. A cross-reacting band just above UVR8 monomer, present also in the uvr8-6 mutant, is indicated (*). Detection with UVR8 specific antibody (Kaiserli and Jenkins, 2007) and anti-actin antibody.
The same C-terminal α-UVR8 antibody (Kaiserli and Jenkins, 2007) was used to repeat the time-course of the UV-B-dependent UVR8 monomerization (Fig. R22). A higher band at about 100 kDa was detected in this blot, and it was marked as “UVR8?”. This band was not present in the mutant line and it appeared UV-Bdependently. The band at 100 kDa was not present after UVR8 protein purification and gel irradiation (data not shown). For these reasons, we can reasonably exclude the possibility of multimerization of the monomeric UVR8. It is tempting to speculate that UV-B activates UVR8 through monomerization, and that the UVR8 monomeric conformation is able to interact with other partner proteins, resulting in the plant UV-B response.
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Figure R22: Western blot analysis of the time-course of UVR8 monomerization in plant protein extract under broadband UV-B. Loading control with boiled samples after UV-B time-course treatment and Actin are shown. A cross-reacting band just above UVR8 monomer, present also in uvr8-6, is indicated (*). Detections with UVR8 specific antibody (Kaiserli and Jenkins, 2007) and anti-actin antibody.
3.3.4 UV-B-Dependent UVR8 Degradation The level of UVR8 protein was checked under broadband UV-B radiation. A slight decrease in UVR8 protein amount after broadband UV-B treatment of seven days old seedlings was detected, which was partially recovered after removal of UV-B irradiation (Fig. R23).
Figure R23: Western blot analysis of seven days old seedlings under broadband UV-B for the given time in minutes. In the 5th lane, after 15 min of broadband UV-B treatment, a recovery time of 60 min in white light was given.
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Considering the UV-B-dependent interaction of UVR8 with the E3 ubiquitin ligase COP1, it was of interest to analyse whether UVR8 was susceptible to proteasomedependent degradation. For this purpose, a cell-free degradation experiment was carried out. Interestingly, the two antibodies shown in Fig. R24 detected UVR8 protein levels differently. While the FB4732 revealed a slight decrease in UVR8 protein amount in DMSO (mock) treated samples, the P60 antibody showed a strong reduction.
Figure R24: Cell-free degradation assay on plant total protein extract for the given time in min. Upper panel: detection with the final bleed FB4732 UVR8-specific antibody, after stripping the membrane presented in the lower panel, which was probed with the C-terminal P60 antibody.
Then, the UVR8 protein levels were analysed in wild type and in cop1-4 mutant seedlings. As shown in Fig. R25, the protein level of UVR8 in 5-day-old seedlings was lower in wild type compared to cop1-4, indicating that COP1 was responsible for the degradation of UVR8. At the same time, proteasome inhibitors can stabilize the UVR8 protein in wild-type seedlings but not in cop1-4 mutants. An anti-ubiquitin antibody detected an accumulation of ubiquitinated proteins showing the efficacy of the proteasome inhibitors treatment. It should be noted that also the actin protein control was stabilized by the proteasome inhibitors treatment. The same protein extract was probed with the antibody FB4732 giving the same result (data not shown).
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Figure R25: UVR8 protein levels in wild type and in cop1-4 seedlings. The seedlings were soaked for 4 hours in liquid MS media, or MS plus DMSO as mock control, or MS plus proteasome inhibitors. Detection with anti-ubiquitin antibody, P60 antibody and anti-actin antibody.
The level of UVR8 protein thus seems to be dependent on COP1. Nevertheless, the functional relevance of UVR8 degradation for the plant UV-B pathway remains to be determined.
3.4 Evolutionary and Structural Considerations RCC1 seems not to be functionally related to UVR8 (Brown et al., 2005). Heterologous expression of UVR8 in yeast shows a dimeric conformation while RCC1 is monomeric (data not shown). Moreover, there’s no experimental evidence showing self interaction of RCC1. UVR8 is conserved in the plant kingdom and it is also present in the unicellular green algae like Chlamydomonas reinhardtii and Volvox carteri, as shown in the phylogenetic tree (Fig. R26). The phylogenetic tree clearly shows that the UVR8 orthologs are clustering together, and they are less related to the closest homologs RCC1 in human and the RCC1-related protein in yeast. Arabidopsis UVR8 homologs are closer related each other than to Arabidopsis UVR8, its orthologs or RCC1.
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Figure R26: Evolutionary relationships among UVR8, UVR8 orthologs, UVR8 plant homologs, human RCC1 and the RCC1-like protein in yeast. The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The optimal tree with the sum of branch length = 6.45 is shown. The percentage of replicate trees in which the associated clades clustered together in the bootstrap test (500 replicates) are shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method (Zuckerkandl and Pauling, 1965) and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 317 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007).
The multiple alignment of the protein sequences from which the phylogenetic tree was generated, is shown in Fig. R27a. In the multiple alignment tryptophans are underlined. The tryptophans cluster in conserved patterns among the UVR8 orthologs; seven tryptophans reside in four patterns which can be summarized as [YF]-X-[WYF]-G-W-X(2)-[YF], while other three tryptophans reside in three patterns G-W-R-H-T. The percentage identity table presented in Fig. R27b shows that the identity among UVR8 orthologs resides above the “twilight zone” (20%-35%) of protein structure-function prediction.
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Figure R27a: Multiple alignment of UVR8, UVR8 orthologs, RCC1, RCC1-like like in yeast, and UVR8 homologs in Arabidopsis. Accession numbers are reported in Material and Methods. The multiple alignment was done with TCOFFEE (Notredame et al., 2000). The alignment ment was edited with Jalview (Waterhouse et al., 2009). Tryptophans ryptophans are marked. marked
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Figure R27b: Percentage identity among UVR8, UVR8 orthologs, RCC1, RCC1-like protein in yeast, and UVR8 homologs. Percentage identity calculated with MatGAT (Campanella et al., 2003).
The predicted three dimensional surface structure of UVR8 is shown (Fig. R28), and the tryptophans are highlighted. The three tryptophans W233, W285, and W337 in the G-W-R-H-T patterns (W233, W285 and W337), as well as the four tryptophans conserved in the patterns [YF]-X-[WYF]-G-W-X(2)-[YF] (W94, W198, W250 and W302) reside on the predicted protein surface structure (Fig. R28, left panel). The other tryptophans are regularly distributed on the lateral surface of the predicted structure (Fig. R28, right panel).
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Figure R28:: In the left panel the predicted three dimensional surface structure of UVR8 is shown. Surface tryptophans highly conserved in the amino acids clusters are highlighted. In the right panel a cartoon structure of UVR8 is shown. The tryptophans in the cluster G-W-R-H-T -R-H-T are highlighted as blue spheres, the conserved tryptophan of the four clusters [YF]-X-[WYF]-G-W-X(2)-[YF] [YF]-X-[WYF]-Gare highlited ghlited as magenta spheres, and all the other tryptophans are highlited as red spheres. The image has been edited with PyMOL (The PyMOL Molecular Graphics System, Version 1.1, 1. , Schrödinger, LLC).
3.5 Site-Directed Mutagenesis M If we assume that the UVR8 protein protei is intrinsically able to perceive UV-B, the perception could be achieved directly by its aromatic amino acids (Creed, 1984). 1984) To address this question, we chose site-directed site directed mutagenesis (SDM) to selectively mutate single amino acid.. I started a first series of SDMs with the amino acid substitutions based on homology with RCC1 and plant homolog proteins, to avoid protein misfolding. This didn’t lead to any significant significa result because the UVR8 protein mutants were still able to monomerize and to interact interact with COP1 or, in the case of UVR8W300L, it was not homodimerizing homo and it was not interacting with COP1, COP1 suggesting protein misfolding (Fig. R29).
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Figure R29: Analysis of the UVR8 mutant proteins derived from SDM. In the upper panel, the western blot shows the monomerization of the different mutant versions. Protein degradation (**) and cross-reacting bands (*) are shown. In the lower panel, the β-galactosidase activity shows the level of interaction of the indicated UVR8 mutant proteins (BD-fusions) with COP1 fused to the activation domain.
Then, I’ve decided to focus on the analysis of the three dimensional structure of UVR8, i.e. on the tryptophans present in the clusters, and to mutate the three thryptophans in the pattern G-W-R-H-T, (W233, W285 and W337), to alanine and to phenylalanine. I’ve chosen phenylalanine because it has a closer structure to tryptophan, it has relatively low absorption in the UV-B wavelengths, and because it is redox inert (unable to donate/accept electrons). These changes can be summarized as: UVR8W233A/F, UVR8W285A/F, and UVR8W337A/F. Moreover, the four
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tryptophans conserved in the four clusters [YF]-X-[WYF]-G-W-X(2)-[YF], and residing on the predicted protein surface, were changed to alanine (i.e., UVR8W94A, UVR8W198A, UVR8W250A, UVR8W302A) (Fig. R30). These last four amino acid mutations to alanine were compromising the dimerization at different levels, but they were still responsive to the UV-B radiation, as shown by their interaction with COP1. This was striking in the mutant UVR8W198A, where no dimer was visible, and the intensity of the interaction with COP1 was comparable with the wild type UVR8 protein. All the amino acid substitutions of the central tryptophans in the clusters G-W-R-H-T were impaired in their interaction with COP1. Two amino acid substitutions were particularly interesting, UVR8W285A/F. The change to alanine of this tryptophan, UVR8W285A, led to a constitutive monomeric conformation, which was slightly interacting with COP1 under non-UV-B conditions, and the interaction was still slightly responsive to UV-B, even if not at the level of the wild type UVR8 protein (Fig. R31). The same tryptophan, but mutagenized to phenylalanine, UVR8W285F, was completely “blind” to UV-B radiation being unable to monomerize under UV-B light, and unable to interact with COP1 (Fig. 30 and Fig. 31). Moreover, the constitutive dimeric conformation of UVR8W285F demonstrates that the amino acid substitution is not significantly compromising the three dimensional structure of UVR8. Furthermore, it can be noticed that the mutation UVR8W233A is monomeric without UV-B and it dimerizes under UV-B. The same behavior, but with a weaker dimerization under UV-B light, was also detected in the mutant UVR8W337A. This indicated that UVR8W233A and UVR8W337A were still able to perceive UV-B radiation. Without a crystal structure of the UVR8 protein it is presently not possible to interpret these results. Given the needs of the tryptophan W285 for UV-B-dependent monomerization of UVR8, which can be abrogated by its substitution to phenylalanine, and the range of behaviors of the other UVR8 mutant proteins, I would expect that they are due to the intrinsic property of the protein to directly perceive the UV-B radiation.
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Figure R30: SDM Analysis of the UVR8 mutant proteins derived from SDM. In the upper panel the western blot shows the monomerization of the different UVR8 mutant versions. Protein degradation bands are shown (**). In the lower panel, the β-galactosidase activity shows the level of interaction of the different UVR8 protein mutant versions with COP1 fused to the activation domain.
A repetition of the yeast two-hybrid analysis for the mutants UVR8W285A and UVR8W285F is shown in Fig. R31. Empty-Vector controls are shown as well.
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Figure R31: Yeast two-hybrid analysis of the UVR8 mutant proteins derived from SDM. Empty-Vector controls are shown as well. The β-galactosidase activity shows the level of interaction of UVR8 with COP1 compared to the interaction of the UVR8 mutant proteins with COP1.
3.6 Mixing-Extracts Experiment We addressed the question if the UV-B irradiation of UVR8 was necessary and sufficient for the interaction with COP1 to occur. I proposed to Dr. Favory to UV-Birradiate separately extracts containing either YFP-COP1 but not UVR8 (cop1 uvr8/Pro35S:YFP-COP1 line) or UVR8 but not YFP-COP1/COP1 (cop1 mutant) and then mixed them with non-irradiated extracts containing the partner protein prior to co-immunoprecipitation assays of UVR8 using anti-YFP antibodies (Fig. R32). The results clearly showed that UV-B irradiation of extracts containing UVR8 was both required and sufficient for the interaction with YFP-COP1, indicating a primary function of UVR8 in UV-B signal perception. Moreover, 5 min broadband UV-B light irradiation of protein total extract causes the complete monomerization of UVR8 (e.g. Fig. R21). This implicates that the monomeric form of UVR8 interacts with COP1, excluding any involvement of the monomerization process in the interaction with COP1.
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Figure R32: Co-IP of UVR8 with anti-GFP-pulled-down YFP-COP1. Protein extracts were independently irradiated and then mixed before immunoprecipitation with GFP antibody.
3.7 Physcomitrella UVR8 Ortholog As shown in the phylogenetic tree (Fig. R26), we postulated different orthologs of UVR8 based on protein homology. To demonstrate the functional conservation of UVR8 orthologs, we tested genetic complementation. Two UVR8 orthologs were cloned from the moss Physcomitrella patens. The accession numbers for these proteins are A9RS92 and A9TJL3. In western blot analysis, it was not possible to detect any dimeric conformation of the Physcomitrella patens UVR8 othologs expressing them in yeast (Fig. R33), even after longer exposure of the film (data not shown).
Figure R33: Western blot of Physcomitrella UVR8 orthologs expressed in the L40 yeast strain with the plasmid pBTM116-D9 and subjected to white light or broadband UV-B irradiation. Protein detection with the anti-LexA antibody.
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Despite the lack of monomerization in yeast, the ortholog A9RS92 was able to rescue the uvr8-8/ProHY5:Luc+ transgenic line (Fig. R34). The line uvr8-8/Pro ProHY5:Luc+ has the luciferase reporter driven by the HY5 promoter, a gene known to be strongly induced under UV-B irradiation (Ulm et al., 2004). 2004) The UV-B-dependent nt induction of the luciferase reporter is impaired in this line because of the uvr8-8 mutant background (data not shown). Tranformation of the UVR8 ortholog A9RS92 into this line led to the restoration of UV-B-responsiveness responsiveness of the luciferase reporter. Control Co plants with + ProHY5:Luc+ in wild type background and non-transformed non uvr8 uvr8-8/Pro HY5:Luc
seedlings (data not shown) were measured in parallel as positive and negative controls, respectively.
Figure R34:: TopCount data with luminescence kinetics of the luciferase reporter illustrating transcriptional activation of HY5 gene promoter after broadband UV-B B irradiation. The curve which connect the crosses (X) shows normalized luminescence averaged from control plants (Pro ( HY5:Luc+) measured in a TopCount microplate icroplate reader. reader Each of the other curves show normalized luminescence from an individual T2 plant measured in a TopCount microplate reader.
Similarly to the SDM for the mutant protein UVR8W198A, also A9RS92 lack the dimeric conformation in yeast,, confirming that the dimeric conformation is not needed to perceive UV-B light, and that the monomeric conformation is not sufficient to activate the response, but it needs UV-B irradiation to get active. Hence, the dimerization is a read out of a putative e conformational change of the UVR8 protein after UV-B perception.
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3.8 UVR8 and HY5 Compete for COP1 Interaction HY5 is known to interact with the WD-40 domain of COP1 (Holm et al., 2001), like UVR8 (Fig. R7). Moreover, HY5 and COP1 proteins are accumulating under UV-B radiation in the nucleus (Oravecz et al., 2006), which may be due to inactivation of the E3 ligase activity of COP1. I’ve postulated the hypothesis of competition for COP1 binding between UVR8 and HY5 under UV-B radiation. A yeast three-hybrid assay (Fig. R35) showed that the HY5 and COP1 interaction was reduced by co-expression of UVR8 under UV-B, reflecting potential competition for the binding between the proteins. The control RCC1 did not interact with HY5 or COP1 (data not shown). It has to be pointed out that the result was very difficult to reproduce, with only one out of ten experiments showing a comparable result.
Figure R35: Yeast three-hybrid assay showing competition for the binding between HY5 and UVR8 for COP1. The interactions are shown with the β-galacosidase reporter. The RCC1 protein is used as control.
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3.9 RUPs
3.9.1 RUPs UV-B-Dependent Interaction with UVR8 The Repressor of UV-B Photomorphogenesis 1 and 2 (RUPs) are WD-40 β-propeller proteins which negatively regulate the UV-B pathway in plant (Gruber et al.). These proteins were shown to interact with UVR8 in plants. In this work, the interaction between UVR8 and RUPs was also reproducibly detected in yeast (Fig. R36). Additionally, in yeast these interactions were enhanced by UV-B radiation, reinforcing the role of UVR8 as UV-B photoreceptor (Fig. R36).
Figure R36: Yeast two-hybrid analysis of the interaction between UVR8 and RUP1 and RUP2 proteins. The interactions are shown with the β-galacosidase reporter, Empty-Vector controls are shown as well.
3.9.2 RUPs Mechanism RUP proteins interact UV-B-dependently with UVR8, similar to COP1. Then I thought to the possibility of a competition between RUPs and COP1 for the binding of UVR8 under UV-B. This possibility was tested in a yeast three-hybrid competitive assay.
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Indeed, there was detectable competition for UVR8 binding in this assay (Fig. R37); the UV-B-dependent interaction between UVR8 and COP1 was reduced by the co-expression of RUP1 or RUP2 proteins. The control RCC1 did not interact with COP1 or UVR8 (data not shown). The interaction between UVR8 and COP1 was particularly affected by co-expression of RUP2. This is in agreement with the stronger phenotype of rup2 versus rup1 single mutant plants under UV-B (Gruber et al.).
Figure R37: Yeast three-hybrid analysis of the interaction between UVR8 and COP1, co-expressing RUPs proteins or the RCC1 control in the plasmid pAG-426GPD-ccdB. The interaction is shown as quantitative assay with the β-galacosidase reporter.
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4 DISCUSSION
4.1 UVR8 and COP1 Interaction in Heterologous System In yeast, UVR8 interacted with COP1 under UV-B radiation specifically (Fig. R1). It should be noted that yeast does not contain a COP1 homolog (Yi et al., 2002), and its closest UVR8 homolog is the RCC1-like protein YGL097W (Fig. R26) (Fleischmann et al., 1991). There is also no evidence available for the presence of a UV-B photoreceptor pathway in yeast. This strongly indicates that UVR8 and COP1 are sufficient for UV-B perception and heterodimerization. This is very similar to the red and blue light-specific interactions of phytochrome and cryptochrome photoreceptors with their early targets in yeast (Shimizu-Sato et al., 2002; Hiltbrunner et al., 2005; Hiltbrunner et al., 2006; Liu et al., 2008). Moreover, the interaction in yeast was specific for the UVR8 protein, as five Arabidopsis β-propeller proteins with sequence similarity to UVR8 did not interact with COP1 under UV-B light (Fig. R5). UV-B, a known cross-linking agent, also did not have a general influence on yeast two-hybrid interactions as, for example, the established interaction of COP1 and HY5 (Ang et al., 1998), the established interactions of MAP kinases MPK3, MPK4 and MPK6 with the MAP kinase phosphatase MKP1 (Ulm et al., 2002), and the established interaction of SPA1 with COP1 (Hoecker and Quail, 2001) were not affected under narrowband UV-B radiation (Fig. R4). Similarly to the evidence in Arabidopsis (Favory et al., 2009), the UVR8 protein mutants UVR8G145S (uvr8-15) and UVR8G202R (uvr8-9), impairing UV-B light-induced photomorphogenesis in vivo, did not interact with COP1 in yeast (Fig. R6). Vice versa, non-functional mutant COP1 versions representing cop1-4 (COP1N282) and cop1-19 (COP1G608R) were not capable to interact with UVR8 (Fig. R7). In contrast, COP1H69Y still interacts with UVR8 in a UV-B light-specific manner (Fig. R7), in agreement with the ability of the corresponding mutant, cop1eid6, to respond to UV-B light (Oravecz et al., 2006). The absence of UVR8 interaction with the N-terminal 282 amino acids (COP1N282) without the C-terminal WD-40 repeats indicated a requirement of this domain for the interaction. In agreement, expression of the C-terminal 340 amino acids (COP1C340) comprising of the WD-40 repeats only, demonstrated that the WD-40 domain of COP1 is sufficient for UV-B dependent interaction with UVR8 (Fig. R7). The UV-B
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dependent interaction of COP1 and UVR8 in yeast is in line with a direct role of UVR8
and/or COP1 in
UV-B
perception.
Complementary “mixing-extracts”
experiments were carried out to further dissect the roles of UVR8 and COP1. Extracts containing either only YFP-COP1 (cop1 uvr8/Pro35S:YFP-COP1 line) or only UVR8 (cop1 mutant) were irradiated with UV-B and then mixed with non-irradiated extracts containing the partner protein before co-immunoprecipitation assay of UVR8 using anti-YFP antibody (Fig. R32). The data clearly showed that the UV-B irradiation of extracts containing UVR8 was necessary and sufficient for the interaction with YFP-COP1 to occur (Fig. R32), indicating UVR8 as primary candidate in UV-B photoperception. Together, the “mixing-extracts” experiment and the UV-B specific interaction of UVR8 with COP1 in yeast, support the idea that UVR8 may constitute the plant UV-B photoreceptor. In Fig. R3, the time-course of the interaction in yeast two-hybrid system between UVR8 and COP1 under narrowband and broadband UV-B light is shown. These results are interesting because they indicate that the interaction between UVR8 and COP1 increases proportionally under increasing doses of UV-B. Moreover, it was noted before that other properties of UVR8 are reminiscent of known photoreceptors (Favory et al., 2009).
4.2 UVR8 Self-Interaction and UV-B Dependent Monomerization The UVR8 protein homodimerizes as shown in BiFC experiments and in yeast two-hybrid assay (Fig. R9) (Favory et al., 2009). Moreover, the dimeric conformation of UVR8 is shown in HEK293T cells extract (Fig. R10) and in yeast extract (Fig. R14). Particularly, in Fig. R11 it is possible to see the heterodimer between GST-UVR8 and UVR8 which confirms that the interaction is indeed the dimerization among UVR8 proteins and not an aspecific interaction between UVR8 and a human protein. In HEK293T cells, in yeast and in Arabidopsis, it was possible to detect the dimeric conformation of UVR8 in tagged proteins, if protein were not denatured by boiling. The Laemmli buffer containing reducing agents (5% DTT or 5% β-mercaptoethanol) and gel electrophoresis did not monomerize the UVR8 protein. The analysis of the three dimensional surface structure let us speculate on the possibility that a strong ionic interaction is responsible for the homodimerization of
Discussion
100
teins. Indeed, a strong ionic interaction could explain the persistence of the UVR8 proteins. dimer in the Laemmli buffer and during gel electrophoresis (Gentile et al., 2002). 2002) In Fig. D1 is shown an antiparallel distribution of positively and negatively charged amino acids. The antiparallel distribution distribution of charged amino acids is crucial to achieve an ionic homodimerization. Moreover, it could be noticed that UVR8 is a soluble protein and the charged amino acids thought to be responsible for the ionic interaction cannot give a strong interaction, because because they are solvated and neutralized by counterions of the salts in solution. On the other hand, hand it has to be considered that these positively and negatively charged amino acids are surrounded by aromatic amino acids and nonpolar amino acids, which could contribute c to originate a hydrophobic environment favorable for the ionic interaction (Fig. D1).
a)
b)
Figure D1:: Predicted three dimensional surface structure of UVR8. a) Surface urface aromatic and non-polar aliphatic amino acids are shown in black. b) Surface positively charged amino acids lysine and arginine, and surface negatively charged amino acids aspartate and glutamate are shown. Image edited with PyMOL (The PyMOL Molecular Graphics System, Version 1.1, 1. , Schrödinger, LLC).
The UVR8 homodimer undergoes monomerization monomerization after UV-B irradiation, and this was initially found in HEK293T cells (Fig. R11). It was possible to reproduce the UV-B dependent monomerization also in yeast (Fig. R14). In HEK293T cells culture (Fig. R10) and in yeast (Fig. R17), it was possible possible to reproduce the monomerization, with the same kinetics, UV-B irradiating the UVR8 proteins after purification, suggesting that no other proteins are involved involved in the monomerization of UVR8; this indicates that the monomerization is i an intrinsic property of the UVR8 protein under
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101
UV-B radiation. A difference between HEK293T cells culture and yeast resided in the UV-B irradiation conditions. Indeed, while in HEK293T cells it was possible to work with narrowband UV-B, yeast required broadband UV-B treatment. The difference was due to the strong protein degradation that was taking place in yeast, which required fast manipulation and fast treatment of protein extracts. It was possible to reproduce the UVR8 UV-B dependent monomerization also in plant extract (Fig. R21). Fig. R21 shows UVR8 mainly homodimeric in the absence of UV-B radiation. The result was achieved, as in HEK293T cells protein extract and in yeast, by avoiding heat denaturation of the proteins before loading. Time-course experiments on the UV-B-dependent UVR8 monomerization were conducted in HEK293T cells protein extract under narrowband UV-B (Fig. R12), and in Arabidopsis protein extract under broadband UV-B (Fig. R20 and Fig. R22). The result in Fig. R20 and Fig. R22 is particularly intriguing. Indeed, in a plant protein total extract on ice, it is possible to see the monomerization after 5 sec of UV-B irradiation. Also phytochromes are found to react to red light after few seconds of irradiation aggregating in the so called sequestered areas of phytochromes (SAPs) (Speth et al., 1986). In such a short time, and in this experimental set-up, it can be excluded that any post-translational protein modification is taking place, underpinning the concept that UVR8 is able per se to perceive UV-B radiation. An example of time needed for protein modification of a photoreceptor is given by the cryptochromes. Indeed, autophosphorylation of CRY2 starts after 2.5 min to 5 min of blue light irradiation at RT (Shalitin et al., 2002), well above the time needed for UVR8 monomerization.
4.3 UVR8 Protein Putative Conformational Change I’ve postulated a putative UV-B-dependent conformational change of UVR8 from the UV-B dependent monomerization of UVR8, and from the difference in antibody’ epitope availability in UV-B and non-UV-B treatment of the protein. In protein extracts from HEK293T cells, the dimeric conformation of non-tagged UVR8 was clearly detectable (data not shown). In Arabidopsis (Fig. R19), and in yeast (Fig. R15) non-tagged UVR8 was not detectable in non-UV-B condition (i.e. dimeric conformation), but it was only detectable after irradiation of the extract with UV-B or heat denaturation of the protein extract, in the monomeric conformation.
Discussion
102
Then, I’ve tried to irradiate the gel just after running the protein, and before protein transfer to the membrane. This experiment allowed the detection of the dimeric UVR8 in yeast (Fig. R16), and in Arabidopsis protein extracts (Fig. R19), probably causing an in-gel monomerization of the dimeric UVR8. It has to be said that the dimeric conformation of UVR8 migrated faster than predicted by molecular weight in HEK293T cells culture, in yeast and in planta, but this is not unexpected given that the proteins were not heat-denatured, and they conserved their three dimensional structure. Moreover, the UVR8 dimerization was demonstrated by BiFC experiments, yeast two-hybrid analysis and in HEK293T cell protein extracts, where the homodimeric conformation is supported by co-expression of GST-UVR8 and UVR8, which are shown to heterodimerize (Fig. R11). Nevertheless, the result in Arabidopsis protein extracts (Fig. R19 and Fig. R20) was unsatisfactory, because the amount of visible UVR8 dimer after UV-B gel irradiation was much less than the UVR8 monomer after UV-B irradiation. For this reason, another UVR8 specific antibody synthesized against a different C-terminal epitope was tested (Fig. R21 and Fig. R22) (Kaiserli and Jenkins, 2007), revealing an amount of UVR8 dimer in non-UV-B treated samples very close to the amount of UVR8 monomer after UV-B irradiation of the samples, underpinning the hypothesis of a conformational change of the protein under UV-B radiation. Moreover, if the conformation of the protein can be changed irradiating the gel with UV-B light, i.e. after protein separation, and after protein purification (Fig. R17), we can reasonably exclude that other proteins are involved in the monomerization process. The UVR8 protein needs UV-B or heat denaturation to be detected (Fig. R21). It could be possible to explain the in-gel UV-B-dependent protein detection assuming that irradiation by UV-B leads to conformational change or protein denaturation, thus resolving the dimer. We can exclude protein denaturation because, in protein extract from HEK293T cells, after UV-B-dependent monomerization, it is possible to rescue the dimeric conformation removing the UV-B radiation (Fig. R13). Interestingly, the dimeric regulatory protein NPR1 shows a similar behavior. Indeed, when the protein extract is not boiled, the tagged-NPR1 dimer is visible on western basis, as it is for the tagged-UVR8 dimer (Mou et al., 2003). NPR1 dimer is not detectable, on western basis, if the protein does not have a tag (Mou et al., 2003). An interesting difference between NPR1 and UVR8 is that NPR1, a redox response regulator, monomerizes under reducing agents like DTT or GSH. UVR8 is not
Discussion
103
monomerizing under reducing agents, indeed the application of Laemmli buffer containing 5% β-mercaptoethanol or 5% DTT has no effect on the UVR8 dimer. This is in agreement with the UV-B perception function of UVR8, which would be expected to be independent from the redox state of the cell.
4.4 Phylogenetic and Structural Considerations The phylogenetic analysis (Fig. R26) shows the evolutionary relationship among Arabidopsis thaliana UVR8, representative orthologs, the UVR8 plant homologs, which have been analyzed for lack of interaction with COP1 (Fig. R5), and the closest homologs in human beings and Saccharomyces cerevisiae. The UVR8 orthologs presented in the phylogenetic tree are clustering together, showing evolutionary conservation among these proteins, which goes evolutionarily back to the unicellular green algae, e.g., Volvox carteri and Chlamydomonas reinhardtii. Moreover, the multiple alignment in Fig. R27a underpins the tryptophans conservation among UVR8 orthologs, while the Fig.27b shows the high percentage identity among these proteins. In the orthologs, four clusters of aromatic amino acids are characterized by the following pattern: [YF]-X-[WYF]-G-W-X(2)-[YF]. The third aromatic amino acid in this pattern is always a tryptophan and in the predicted three dimensional surface structure, these tryptophans are always located on the same side of the protein (W94, W198, W250, W302) (Fig. R28). Moreover, the pattern G-W-R-H-T, which contains a tryptophan, is present three times in the primary protein structure of UVR8 and its orthologs. The three tryptophans in this second pattern are clustering together on the surface, in the central part of the predicted three dimensional structure (W233, W285, W337) (Fig. R8 and Fig. R28). These patterns are not present in RCC1, in the RCC1-like protein in yeast, and in the UVR8 protein homologs. Furthermore, in the UVR8 orthologs there are fourteen tryptophans, while RCC1 has only four tryptophans. None of the ten tryptophans present in clusters in the UVR8 predicted structure are conserved in RCC1. Three tryptophans outside the clusters are conserved among some of the homologs and RCC1, and the last one is specific for UVR8 orthologs but in a C-terminal extension that is absent in the green algae (Fig. R27b). The geometrical distribution of aromatic amino acids in these clusters let me speculate on a putative antenna complex for UV-B perception.
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104
To confirm the structure-function evolutionary conservation of UVR8 orthologs I cloned the moss ortholog A9RS92 and transformed it into an uvr8 mutant line of Arabidopsis thaliana. The complementation experiment was successful, (Fig. R34), and it indicates that the orthologs are structurally and functionally conserved proteins. UV-B radiation reaches from 1 to 20 meters under water surfaces (Booth and Morrow, 1997). This means that only superficial algae and terrestrial plants are exposed to this radiation. Indeed, we found that the UVR8 orthologs, with the characteristic clusters of aromatic amino acids previously described, are present in green algae, mosses, and in lower and higher plants. The distribution of UVR8 orthologs evolutionarily starting in green algae, and conserved in lower and higher plants, hints to the necessity of UV-B perception for water to land transition. On the other hand, we didn’t find UVR8 orthologs in fungi, insects and animals, which live in the shadow or can escape from direct sunlight exposure. Adaptation to UV-B light in the green algae Chlamydomonas reinhardtii and Chlorella species has been reported (Danilov and Ekelund, 2000; Estevez et al., 2001). Historically, the best characterized effect of UV-B radiation in living organisms is the production of “sunscreen” pigments (Rozema et al., 2002). Mosses have flavonoids, the “sunscreen” pigments for UV-B light widespread in gymnosperm and angiosperm (Melchert and Alston, 1965). Moreover, the distribution of “sunscreen” pigments for UV-B (Rozema et al., 2002) is overlapping with the taxa containing UVR8 orthologs. Recently, it has been found that the moss Physcomitrella patens posses the secondary metabolite pathway for flavonoid biosynthesis, as well as UVR8 and COP1 (Wolf et al., 2010).
4.5 UV-B Perception by UVR8 Many evidences support a role of UVR8 as UV-B photoreceptor. UVR8 has been shown to be an early component in the UV-B pathway in a microarray analysis between wild type and uvr8 mutant seedlings under UV-B light (Fig. I13) (Favory et al., 2009). Additionally, the uvr8 mutant does not show a photomorphogenic phenotype under UV-B light, as shown in Fig. I19 (Favory et al., 2009). Moreover, the rapid UVR8 and COP1 UV-B-dependent interaction in planta (Favory et al., 2009) and in heterologous system (Fig. R1), reinforce this idea. Nevertheless, the strongest
Discussion molecular
105 evidence
for
direct
UV-B
perception
is
the
UV-B-dependent
monomerization (e.g. Fig. R11). The UV-B-dependent monomerization was detectably in heterologous systems and after pull-down of the protein, underlying a specific role in UV-B perception by UVR8. A possible question is how the UVR8 protein can perceive the UV-B light. While the known photoreceptors need a chromophore, as described in the introduction, the short wave length UV-B radiation is able to interact with organic compounds. If most of the organic compounds are able to absorb the UV-B light, which one is the photoreceptor? This is also the reason which makes the discovery of the UV-B receptor so difficult. It is possible that a protein is able per se to absorb the UV-B light through its amino acids, like the GFP protein (Shimomura et al., 1962; Chalfie et al., 1994; Heim et al., 1994). The aromatic amino acids are particularly suited for UV-B light absorption, and above all tryptophans (Creed, 1984). Indeed, in the case of the known UV-B aryl hydrocarbon photoreceptor in human keratinocytes, the chromophore is a UV-B light photoproduct of the free amino acid tryptophan, the 6-formylindolo[3,2-b]carbazole (FICZ) (Rannug et al., 1995; Oberg et al., 2005). We’ve already seen that UVR8 orthologs have specific clusters of aromatic amino acids which contain tryptophans. Through site-directed mutagenesis I’ve mutated the tryptophan W285 to phenylalanine. UVR8W285F was completely “blind” to UV-B radiation. Indeed, this protein was not able to monomerize under UV-B light, and it was also not able to interact with COP1 (Fig. R30 and Fig. R31). The dimeric conformation was however intact, indicating that the amino acid substitution was not compromising the three dimensional structure of the protein. Interestingly, all the amino acid substitutions in the clusters G-W-R-H-T impair interaction with COP1. A question that arises from this result is if the three central tryptophans of these clusters are actually the antenna of UVR8, or if these tryptophans are the binding domain for a putative chromophore. It is difficult to draw a conclusion on all the other mutants because they are missing the dimeric conformation in non-UV-B condition, which could indicate misfolding of the UVR8 protein. The only additional evidence is given by the mutant proteins UVR8W198A, and UVR8W250A which have no dimeric conformation but they are still interacting UV-B-dependently with COP1. This suggests that the monomeric conformation of UVR8 is not enough for interaction with COP1, and that the dimeric conformation of UVR8 is not needed for UV-B perception. Hence, the monomerization may only be a read out of a UV-B dependent putative
Discussion
106
conformational change. Indeed, also A9RS92, A9RS92, the UVR8 ortholog in Physcomitrella patens seems not to form dimers in yeast,, but it is able to rescue the UV-B dependent HY5 induction in the uvr8-8/ProHY5:Luc+ Arabidopsis line (Fig. R34).
4.6 UVR8 Mechanism echanism COP1 is a repressor of photomorphogenesis, which degrades photomorphogenesis promoting transcription factors, acting as a switch between light perception and downstream signaling. A genetic model of COP1 mode of action is shown in Fig. D2.
Figure D2: Genetic model indicating that COP1 is a repressor repressor of photomorphogenic development, whereas light signals, perceived by photoreceptors, abrogate its repressive action (Osterlund et al., 1999).
As already mentioned, HY5 is a known transcription factor involved in photomorphogenesis and it is a substrate of COP1. In Fig. D3 the HY5 protein levels under different light conditions are shown, in different photoreceptor mutant and photoreceptors overexpressor xpressor lines. From this published data it is clear that the HY5 protein level depends on the protein level of the photoreceptor responsible for the specific light condition.
Discussion
107
Figure D3 : Anti-HY5 western blots of seedlings grown in continuous red light (a), continuous far-red light (b) and continuous blue light (c). The seedlings include wild type (three ecotypes), cry1, cry2, cry1/cry2 double mutant, phyA, phyB, phyA/phyB double mutant, a PHYB overexpression line (PHYBOE, red light only), and a PHYA overexpression line (PHYAOE, far-red light only) (Osterlund et al., 2000).
Similarly to white light irradiation, HY5 is stabilized under UV-B light, and it accumulates in the nucleus together with COP1 (Oravecz et al., 2006). HY5 accumulation under UV-B is impaired in uvr8 mutants, and UVR8 overexpressor lines show a cop1-like mutant phenotype under UV-B in sun simulator experiments (Favory et al., 2009). Moreover, under red light, a phytochromes quintuple mutant shows a skotomorphogenic-like phenotype, but not under blue light (Strasser et al., 2010). Similarly, cryptochrome mutant seedlings shows reduced hypochotyl shortening under blue light but not under red light (Lin et al., 1996). It’s interesting that, under UV-B, COP1 and its substrate HY5 are stabilized and accumulate in the same subcellular compartment. It has been proposed that the interaction of CRY1 and CRY2 with COP1 inhibit the E3 ligase activity of COP1 (Wang et al., 2001; Yang et al., 2001). The question is if the UV-B-dependent binding of UVR8 to COP1 may inhibit HY5 degradation. The photoreceptors CRY1, CRY2, phyA and phyB bind to
Discussion
108
the WD-40 domain off COP1 (Yi and Deng, 2005).. Interestingly, also the transcription factors responsible for photomorphogenesis bind to the WD-40 40 domain of COP1 (Ang et al., 1998; Holm et al., 2001; Wang et al., 2001; Yang et al., 2001; Holm et al., 2002; Seo et al., 2004; Jang et al., al. 2005).. UVR8 binds also to the WD-40 domain of COP1 (Fig. R7). The Fig. R35 suggests competition for the binding, between UVR8 and HY5 for COP1, which could result in the stabilization of of HY5, HY5 under UV-B, in Arabidopsis. Moreover, COP1 is able to homodimerize and to autoubiquitinate (Torii et al., 1998; Saijo aijo et al., 2003). 2003). The UV-B dependent UVR8 binding to COP1 could inhibit also the autoubiquitination activity of COP1, COP1, resulting in COP1 protein stabilization.. I would like to postulate a model where COP1 is inactivated under UV-B by UVR8, which causes COP1 and HY5 protein stabilization in the nucleus. Moreover, I would like to speculate that such model could also be applied to other photoreceptors, which also bind to COP1, as shown in Fig. D4.
Figure D4: Schematic model indicating the excitation of UVR8, cry1, cry2, phyA and phyB by the sunlight spectrum impinging on earth, earth indicated as light wavelength in nm.. The activation of the photoreceptors causes their interaction with the WD-40 domain of COP1 (WD-40), shown schematically in its domains: the RING RING finger domain (R), the coiled-coil domain (Coil), and the WD-40 domain (WD-40).. The photomorphogenesis - promoting transcription factors, which also bind to the WD-40 domain of COP1, are competing with the photoreceptors in their interaction with COP1, which results in the stabilisation of these transcription factors, factors, and induction of photomorphogenesis. photomorphogenesis
Discussion
109
The model presented in Fig. D4 is very schematic and it is thought to postulate a possible mechanism for COP1 function in light response. This model is i very similar to Fig. I18.. The concepts here underpinned are: are the UVR8 protein as photoreceptor for the UV-B radiation,, and the competition for the binding among photoreceptors and light responsive transcription factors for the interaction with the WD-40 domain of COP1. Of course, each photoreceptor has its specificity in signaling. In this model, the commonalities in light response refer exclusively to the control of the photomorphogenic
development
through
differential
degradation
of
photomorphogenesis - promoting transcription factors.
4.7 RUPs Mechanism echanism RUPs are negative regulators of the UV-B pathway in Arabidopsis, and their transcript is induced under UV-B light (Gruber et al.).. RUPs bind UV-B dependently to UVR8 (Fig. R36). If UVR8 is UV-B-dependently UV-B dependently binding to RUPs and to COP1, then there could be competition for the binding among these proteins. Indeed, Fig. R37 shows UV-B dependent competition for the interaction between RUPs and COP1 with UVR8. This competition for the interaction could could modulate the plant UV-B response (Fig. D5).
Figure D5: Schematic model depicting the competition for the binding between the WD-40 domain of COP1 and RUP2 with UVR8. If UVR8 bind to COP1 (solid line pathway), then HY5 cannot bind COP1, it is stabilized,, and activates light responses. RUP2, transcriptionally induced under UV-B light, binds UVR8 (dashed line pathway) and HY5 can bind to COP1, leading to its proteasomal dependent degradation,, and to repression of photomorphogenesis. photomorphogenesis
Discussion
110
4.8 Conclusions and Outlook This work addressed the question if UVR8 is the plant UV-B photoreceptor. A series of compelling evidences support this hypothesis. Nevertheless, many aspects related to photoperception are still elusive. UVR8 and COP1 have been shown to be upstream factors in the plant UV-B pathway thanks to microarray analysis and phenotypic characterization under narrowband UV-B. In the same work UVR8 and COP1 have been found to interact UV-B dependently in the first minutes of UV-B irradiation, showing an early event in the UV-B signal transduction pathway. At this point, a main question was still open. Is UVR8 and/or COP1 or something upstream to these proteins the UV-B photoreceptor? The reproduction of the UV-B dependent interaction between UVR8 and COP1 in yeast has been the first indication that UVR8 and/or COP1 could be the UV-B photoreceptor,
pointing
to
the
absence
of
upstream
factors
involved
in
photoperception. This finding has been followed by the finding of the UV-B dependent monomerization of UVR8 in human cells culture, strongly indicating that UVR8 was able to undergo conformational change upon UV-B perception. Later on, the monomerization has been reproduced in yeast and in Arabidopsis protein extracts. The necessity of protein gel irradiation for UVR8’ visualization, as dimer in non-UV-B condition, and the monomerization after protein purification were additional compelling evidences for the ability of UVR8 to direct UV-B perception. Moreover, the “mixing-extracts” experiment exclude COP1 from photoperception, given that the merely UV-B irradiation of UVR8 is necessary and sufficient for the UVR8 and COP1 interaction to occur. The mutant protein UVR8W285F blind to UV-B radiation, is additional evidence that the UVR8 protein is able to perceive UV-B, even if the action of a cofactor cannot be excluded. Also the UV-B dependent interaction of UVR8 with the RUPs protein in yeast, which hinders the UV-B dependent interaction of UVR8 with COP1, is adding value to the hypothesis. Indeed, the positive UVR8 and COP1 interaction for the UV-B plant response can be hindered by interaction of UVR8 with these negative regulators of the UV-B pathway.
Discussion
111
Other indirect evidences presented in this work suggest photoreceptor properties of UVR8. The monomerization of UVR8 on ice, in less than 5 seconds, and its re-dimerization at room temperature in HEK cells protein extract with a slow kinetic, hints to the ability of fast light detection, which later give time to start a plant response. The evolutionary distribution of the protein, from green algae to mosses, and in lower and higher plants, partially confirmed by the complementation experiment with the UVR8 ortholog A9RS92 of Physcomitrella patens in Arabidopsis, could reflect the necessity of UV-B perception for water to land transition. In this way, the UVR8 pathway and the “sunscreen” pigment production could have been a competitive advantage for conquer of the land. All this evidence strongly suggest UVR8 being UV-B photoreceptor. Many questions are still open. How can UVR8 perceive UV-B light? Does UVR8 need a cofactor? What’s causing the UV-B light dependent monomerization of the UVR8 protein? Is there a conformational change of the UVR8 protein upon UV-B light irradiation? Is the competition for the binding among proteins sufficient to explain the UVR8 and COP1 mode of action and the plant response?
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Acknowledgements A would like to thank Davide Faggionato for our funny time in the laboratory of Prof. Baumeister, which led to the discovery of the UV-B light dependent monomerization of UVR8, hence to the main hint of photoperception by UVR8, between 22:00 and 3:00 in the morning, when we were used to work on our “secret experiments”. The next day we were working in our respective labs, waiting for the following night to continue to play with science. I would also like to thank Dr. Tim Kunkel which many useful discussions helped to achieve the detection of the dimeric conformation of non-tagged UVR8 proteins in yeast and in Arabidopsis. A special thanks to Prof. Eberhard Schäfer, which has been a mentor in my PhD experience. Moreover, I want to thank all the co-workers which would be too many to mention, who helped during the course of my PhD work.
References
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