CHARACTERIZATION OF THE STABILITY OF PRR7, A CLOCK PROTEIN IN
ARABIDOPSIS THALIANA
By
Saundra Lynn Mason

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Biochemistry and Molecular Biology
2012




ABSTRACT
CHARACTERIZATION OF THE STABILITY OF PRR7, A CLOCK PROTEIN IN
ARABIDOPSIS THALIANA
By
Saundra Lynn Mason
The circadian clock in Arabidopsis is comprised of a series of
transcriptional/translational feedback loops. The pseudo-response regulators (PRRs)
play a central role in the Arabidopsis circadian network. Members of the PRR family
have been shown to be post-translationally regulated by light. PRR5 and TOC1 are
targeted for degradation by ZTL, an F-box protein with a blue light absorbing LOV
domain. Blue light has been shown to stabilize PRR5. PRR7 protein expression
increases throughout the day and peaks near dusk before being degraded in darkness.
Previous work has shown that this protein is stabilized by white light relative to
darkness. In the present study, PRR7 protein stability was characterized under different
light conditions. PRR7 degradation under white light, blue light, and darkness was
similar during the subjective day and night. Under red light, the half-life of PRR7 protein
was extended by almost an hour in the subjective evening as measured by Western
blotting. PRR7 protein is stabilized by red light relative to darkness in the subjective
evening. Blue light did not stabilize PRR7. ZTL does not affect PRR7 protein
abundance; as blue light does not seem to affect PRR7 stability, it is unlikely that other
members of the ZTL family regulate PRR7.




ACKNOWLEDGEMENTS
I would first like to thank my advisor, Dr. Eva Farre. She has continued to support
me despite the difficult decision to transfer to the Master of Science program. She has
been more compassionate and understanding than I probably deserve. Her mentorship
and encouragement have helped me to come this far when I was not sure that I could.
I would also like to thank the members of my committee, Dr. David Arnosti and
Dr. Beronda Montgomery. I would particularly like to thank Dr. Montgomery and her lab
for participating in a special topics seminar I gave over the Summer semester to
complete my course requirements.
My lab mates have been a terrific resource to me as someone who was new to
plant science at the beginning of my studies. They have helped me in any way they
were able. They have also stepped in to help whenever I had been called away abruptly
to care for my daughter. I am truly grateful to have had the chance to work with such
wonderful people.
Finally, I would like to thank my husband. He has supported my decisions and
been an exemplary spouse and parent. Thank you is insufficient to describe how glad I
am to have such an amazing partner.




iii


TABLE OF CONTENTS
LIST OF TABLES.. ……………...……………………………………………………………...v
LIST OF FIGURES…..…………………………………………………...………………..…..vi
LIST OF ABBREVIATIONS ……………………………………………...………………......vii
INTRODUCTION …………………………………………………………………………...…..1
Overview of Circadian Oscillators…………..…………………………………………………1
The Circadian Clock in Arabidopsis thaliana……….…….…...……..……………...…....…2
The Circadian Regulated Pseduo-Response Regulators…..………………………...…….4
Post-Translational Regulation of PRR Proteins………..…………………………………….6
Photoreceptors and Light Input to the Circadian Clock….………………………...………..7
Photoreceptor Mediated Protein Stability…………………………………………………...11
RESULTS …………………..………………………………………………………………….13
Cycloheximide Treatment Optimization …………………………………………………….16
Degradation of PRR7 Under Different Light Conditions….………………………………..18
DISCUSSION …………………………….………………………………………………...…21
MATERIAL AND METHODS ……………………...…………………………………………46
REFERENCES ….……………………………………………………………………….……52




iv


LIST OF TABLES
Table 1. Half-life of PRR7 Protein Under Different Light Conditions………………….45




v


LIST OF FIGURES
Figure 1. Overview of the Circadian Oscillator in A. thaliana….……………….…………28
Figure 2. Expression of the Circadian PRRs, The Conserved Domains of the Circadian
PRRs………..…………………………………………………………………………………..29
Figure 3. Examples of Light-Regulated Proteolysis in A. thaliana ……………………….31
Figure 4. Line Optimization of Cycloheximide Treatment in the Light …………………..32
Figure 5. PRR7-LUC Protein Stability Under Different Light Conditions..…...…….……35
Figure 6. Luciferase Activity of the prr7-3 35S::PRR7-LUC-HA… ………………………37
Figure 7. Optimization of Cycloheximide Treatment in Darkness at ZT4, ZT12.….…....38
Figure 8. Comparison of DB7.1 Staining and TOC75 Detection as Loading Control…..39
Figure 9. Comparisons of HA-PRR7 Protein Degradation Rates During the Subjective
Day and Subjective Night ……………………………………………………………….……40
Figure 10. Analysis of the Stability of HA-PRR7 Protein Evaluated by Western Blot
Under Red Light…………………………………………………………………………….....41
Figure 11. Analysis of the Stability of HA-PRR7 Protein Evaluated by Western Blot
Under Blue Light at ZT4, ZT12……...……………………………………………………... 43




vi


LIST OF ABBREVIATIONS
ANOVA
CCA1
CCT
CHE
CIB
CO
COP1
CRY
ELF3
FAD
FHY1
FKF1
FMN
GI
HA
HFR1
HRT
HY5
LAF1
LD
LHY
LKP2
LL
LUC
LUX
PHY
PIF
PR
PRR
RLU
SCF
SEM
SPA1
TCP
TOC75
ZT
ZTL




Analysis of Variance
CIRCADIAN CLOCK-ASSOCIATED 1
CONSTANS, CONSTANS-LIKE, TOC1
CCA1 HIKING EXPEDITION
CRYPTOCHROME-INTERACTING FACTOR
CONSTANS
CONSTITUTIVE PHOTOMORPHOGENEIC 1
Cryptochrome
EARLY FLOWERING 3
FLAVIN ADENINE DINUCLEOTIDE
FAR-RED ELONGATED HYPOCOTYL1
FLAVIN-BINDING KELCH REPEAT F-BOX1
FLAVIN MONONUCLEOTIDE
GIGANTEA
Hemagglutinin
LONG HYPOCOTYL IN FAR-RED1
HYPERSENSITIVE RESPONSE TO TCV
ELONGATED HYPOCOTYL5
LATE ELONGATED HYPOCOTYL
Light/Dark
LONG AFTER FAR-RED LIGHT1
LOVE KELCH PROTEIN2
Constant Light
Luciferase
LUX ARRHYTHMO
Phytochrome
PHYTOCHROME-INTERACTING FACTOR
PSEUDO-RECIEVER
PSEUDO-RESPONSE REGULATOR
Relative Light Units
Skp, Cullin, F-box
Standard Error for the Mean
SUPPRESSOR OF PHYA-105
TEOSINTE BRANCHED1, CYCLOIDEA, and PCF
TRANSLOCON OUTER MEMBRANE COMPLEX
Zeitgiber
ZEITLUPE

vii





Introduction
Overview of Circadian Oscillators
The endogenous circadian oscillator generates rhythmic outputs of biological
processes (Harmer, 2009). The circadian clocks of different organisms share several
defining characteristics. First, they are endogenous and self-sustained. The biological
rhythms can be observed under constant environmental conditions, such as constant
light or temperature and have a period of about 24 hours. Second, they are entrainable;
the oscillation of daily biological rhythms receives input from the surrounding
environment, predominantly in the form of light/dark or thermal cycles (Millar, 2003;
McClung & Salome, 2005). The phenomenon of entrainment allows the organism to
adjust its daily rhythms to match the surrounding environment. Third, they are
temperature compensated so that the period remains consistent over a range of
physiologically relevant temperatures. In plants, different clock mutants display changes
in temperature compensation (Salomé, et al., 2010). Alternative splicing has been
proposed to be a mechanism by which temperature compensation is achieved in plants
(James et al., 2012). This has also been demonstrated to be the case in Drosophila
(Majercack, et al., 1999).

Circadian clocks have evolved independently among algae, plants, insects, and
mammals (Bell-Pedersen, et al., 2005). In eukaryotes the molecular components of the
oscillator are not conserved, although the structure is similar among different taxa.
Eukaryotic clocks are based on a network of transcriptional and translational feedback
loops (Robash, 2009). However, in eukaryotes there is also evidence to support non-




1




transcriptional oscillators. Circadian rhythms in Cyanobacteria are based on a
transcriptional-less oscillator. Here the oscillator is comprised of three proteins, KaiA,
KaiB, and KaiC. KaiC is an ATPase that also exhibits autokinase activity and its
phosphorylation status oscillates with a period of 24 hours. KaiA activates the
autokinase and ATPase activity of KaiC (Iwasaki, et al., 2002), while KaiB serves to
inhibit the activity of KaiA (Kitayama, et al., 2003). This system has been reconstituted
in vitro (Nakajima, et al., 2005). Rhythmic outputs have been observed in the absence
of new transcription. Peroxiredoxin proteins in human red blood cells, as well as those
in the unicellular algae Osterococcus tauri, undergo rhythmic oscillations in redox state
with a period of 24 hours in the absence of transcription (O’Neill & Reddy, 2011; O’Neill,
et al., 2011).

In plants, processes such as de-etiolation, flowering time, photosynthesis,
immune responses, and light sensitivity are regulated by the circadian clock (Harmer
2009). Transcriptional regulation is a primary mechanism by which this is achieved in
Arabidopsis (Doherty & Kay 2010). Upward of 30% of the Arabidopsis transcriptome is
circadian regulated, with the organism showing oscillations in gene expression under
constant environmental conditions (Harmer, et al., 2000; Covington, et al., 2008).

The circadian clock in Arabidopsis thaliana

Relative to other plants, the circadian clock of Arabidopsis is the best
characterized to date. In Arabidopsis, the oscillator is comprised of at least three




2




transcriptional/translational feed-back loops (Figure 1). The first loop described consists
of the morning expressed Myb transcription factors CIRCADIAN CLOCK ASSOCIATED
1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) that repress transcription of the
evening expressed TOC1 (TIMING OF CAB EXPRESSION) gene during the day by
binding a sequence in its promoter known as the evening element (AAATATCT)
(Alabadí et al., 2001, Perales & Más, 2007) (Figure 1). TOC1 had initially been thought
to promote expression of CCA1 and LHY in the morning by antagonizing the binding of
a TCP (TEOSINTE BRANCHED1, CYCLOIDEA, and PCF) transcriptional repressor
CCA1 HIKING EXPEDITION 1 (CHE1) (Pruneda-Paz et al., 2009), although recent
evidence suggests it functions as a repressor of morning genes, including CCA1 and
LHY during the night (Huang et al., 2012) (Figure 1).

TOC1 is a member of the small gene family in Arabidopsis known as the pseudo
response regulators (PRRs) (Matsushika, et al., 2000). There are four other members of
this family involved in the Arabidopsis circadian clock: PRR3, PRR5, PRR7, and PRR9.
PRRs 5, 7 and 9, together with CCA1 and LHY form an additional regulatory loop (Farre
et al., 2005; Nakamichi, et al, 2005) (Figure 1). During the day, these PRRs sequentially
bind the promoters of CCA1 and LHY to inhibit transcription (Nakamichi, et. al, 2005).
PRR9 binds early in the day, followed by PRR7 through the afternoon, and PRR5
through the evening. In turn, CCA1 and LHY activate the transcription of PRR7 and
PRR9 by directly binding their promoters (Farre et al., 2005).




3




A third transcriptional loop exists within the Arabidopsis circadian network. CCA1
and LHY bind the evening element in the promoter of LUX ARRHYTHMO (LUX) to
prevent its transcription (Hazen et al., 2005); in turn, LUX specifically binds to the LUX
binding site within its own promoter as well as the promoter of PRR9 (Helfer et al.,
2011). LUX binding to the PRR9 promoter has been suggested to be a mechanism for
indirect regulation of CCA1 and LHY transcription by the evening-phased LUX. EARLY
FLOWERING 3 (ELF3) is another evening-expressed gene whose protein product
associates with the PRR9 promoter to repress its expression in a time-dependent
manner (Dixon et al., 2011). LUX has been shown to be necessary for ELF3 binding to
DNA (Chow et al., 2012).

Additional components of the oscillator include GIGANTEA (GI), which interacts
with ZEITLUPE (ZTL), a component of the SCF-E3 ubiquitin ligase complex that targets
TOC1 and PRR5 proteins for degradation during the night (Fujiwara et al., 2008)
(Figure 1). The interaction between TOC1 and ZTL is inhibited by PRR3 (Para et al.,
2007) promoting stabilization of the TOC1 protein at the beginning of the night.

The circadian regulated pseudo-response regulators

The PRR genes implicated in the clock mechanism also undergo circadian
regulation. These genes are expressed sequentially throughout the day, beginning with
PRR9 in the morning, followed by PRR7, PRR5, PRR3, and ending with PRR1 (TOC1)
in the evening (Figure 2A, Matsushika et al., 2000). Peak protein expression of these




4




PRRs is delayed 4-8 hours relative to the maximum mRNA accumulation (Fujiwara et
al., 2008). PRR 9, 7, and 5 each repress the transcription of CCA1 and LHY. In
Arabidopsis, mutations in either PRR7 or PRR9 cause period lengthening in constant
light of up to two hours, although no effect on period length is observed in constant
darkness. This result indicates that these genes play a role in transmitting light signals
to the central oscillator (Farre, et al., 2005). prr9 mutants show period lengthening
defects under a broad range of red and blue light fluences, while the prr7 mutant period
length is more dramatically affected by red light. All prr7 mutant lines are hyposensitive
to red light, as determined by hypocotyl measurements (Nakamichi, et al., 2005). The
prr7-3 prr9-1 double mutant has a long period phenotype, which is stronger under red
than blue light relative to the wild type. These mutant-specific light defects indicate
specific functions of each of the PRRs , or may point to their differential regulation by
light, in addition to their overlapping roles as transcriptional repressors within the
circadian network.

The circadian-regulated pseudo-response regulators share a conserved domain
structure. The C-terminus contains a CCT (CONSTANTS, CONSTANTS LIKE, TOC1)
motif that is conserved among all circadian-regulated PRR proteins (Figure 2B). The
CCT domain resembles the DNA binding motif of the HAP2/3/5 complex in the budding
yeast Saccharomyces cerevisiae (Wenkel et al., 2006). Recently it has been shown that
this domain is able to bind DNA directly in vitro, although the degree of sequence
specificity remains to be demonstrated (Gendron et al., 2012).




5




At the N-terminus, the pseudo-receiver (PR) domain resembles the receiver
domain found in bacterial and plant response regulators (Figure 2B; Mizuno &
Nakamichi, 2005). The PR domain lacks the conserved aspartate that is phosphorylated
in the signaling pathway mediated by response regulators (Mizuno & Nakamichi, 2005).
The receiver domain of PRR5 has been shown to mediate the degradation of the
protein, as well as its interaction with ZTL (Kiba, et al., 2007). The PR domain of PRR5
also interacts with TOC1 (Wang, et al., 2010). In some organisms, such as the
cyanobacterium Synechococcus elongatus, the pseudo-reciever domain has been
suggested to mediate protein-protein interactions important for regulating the pace of
the oscillator (Gao, et al., 2007). The cyanobacterial protein lacks the CCT motif found
in the circadian PRRs of Arabidopsis.

Post-translational Regulation of PRR Proteins

The circadian PRR proteins are post-translationally modified; each PRR protein
becomes increasingly phophorylated through its daily expression cycle, reaching
maximal phosphorylation prior to being degraded (Fujiwara et al., 2008). Neither the
location of the phosphorylation site(s) or the kinase(s) responsible for the modification
are known. In addition, the role of phosphorylation has not been fully described for the
PRR proteins in Arabidopsis. PRR5 and TOC1 phosphorylation enhances their
interaction with ZTL, a blue-light absorbing F-box protein that targets them for
degradation by the proteasome (Fujiwara et al., 2008). This agrees with new insights
suggesting that variation in phosphorylation state can induce conformational changes




6




generated by charge repulsion within a protein. These conformational changes mediate
the protein’s stability by affecting its ability to interact with other proteins as has been
proposed for some mammalian clock proteins (Menet & Rosbash, 2011). Experiments
with proteasomal inhibitors have shown that all circadian PRR proteins are degraded by
the 26S proteasome (Farre & Kay, 2007; Fujiwara et al., 2008), although the regulatory
partners for PRR3, PRR7, and PRR9 have not been identified. Phosphorylation status
has also been shown to facilitate the interaction of TOC1 with PRR3 to prevent TOC1
interaction with ZTL and stabilize the protein, although the interaction of TOC1 with ZTL
also depends on its phosphorylation state (Fujiwara et al., 2008).

Post-translational modifications might mediate the sub-cellular localization of the
PRRs. TOC1 nuclear accumulation is mediated by PRR5 (Wang, et al., 2010). Nuclear
localized TOC1 appears to be more highly phosphorylated than that which remains in
the cytosol, although it is unclear whether this enhanced phosphorylation precedes
nuclear import. It has been demonstrated that phosphorylation status regulates the
sub-cellular distribution for clock proteins in other organisms (Diernfellner, et al., 2009).

Photoreceptors and Light Input to the Circadian Clock

In plants, the phytochromes, cryptochromes, and ZTL family of proteins have
been implicated as the major light receptors for generating rhythmic oscillations
(Somers, 1998, Baudry et al., 2010; Yanovsky, et al., 2000). These plant photoreceptors
bind a chromophore that induces an activating conformational change within the protein




7




upon absorbing a photon of light.

There are five red/far-red sensing phytochromes (phyA-phyE) in Arabidopsis.
Phytochromes can be divided in two classes: light labile (phyA) and light stable (phyBphyE). The phytochromes are localized to the cytosol in their biologically inactive red
light absorbing state, or Pr form. After irradiation with red light, they undergo a
conformational change to their biologically active far-red light absorbing Pfr form.

The UV-A/blue light perceiving cryptochromes (CRY) were first identified in
plants, and have since been identified in other multicellular eukaryotes (Lin & Shalitin,
2003). In mammals, the cryptochromes are molecular components of the central
oscillator (Cashmore, 2003), though it has been shown that this is not true in
Arabidopsis where they are involved in light input to the clock (Yanovsky, et al., 2000).
The cryptochromes structurally resemble DNA photolyases; at the amino terminus is a
photolyase-like domain, a central chromophore binding region, and a carboxy-terminal
effector region known as the cryptochrome carboxy-terminal extension (Lin, et al.,
2003). Only CRY3 has been shown to exhibit DNA repair activity in Arabidopsis
organelles and does not appear to have any blue light signaling activity (Kleine, et al.,
2003). Upon blue light perception, the FAD (Flavin Adenine Dinucleotide) cofactor of
cryptochrome becomes reduced, and the protein is subsequently phosphorylated, a
modification considered important for its signal transducing activity. Following
phosphorylation, it is proposed that the proteins undergo a conformational change that
results in dissociation of its amino and carboxy terminal domains to facilitate




8




subsequent signaling events (Yu et al., 2007). Phosphorylation has also been shown to
be important for degradation of CRY proteins (Liu et al., 2011; Shalitin, et al, 2003).

ZTL, as well as FLAVIN-BINDING, KELCH REPEAT, F-BOX1 (FKF1) and LOV
KELCH PROTEIN 2 (LKP2), are a family of F-box proteins that function predominantly
in the circadian clock and confer time-specific changes in protein abundance (Baudry, et
al., 2010). Members of this family contain a LIGHT OXYGEN VOLTAGE (LOV) domain
at their amino terminus that perceives blue light. This domain binds flavin
mononucleotide (FMN) non-covalently where it serves as a chromophore (Möglich, et
al., 2010). These proteins also possess a Kelch repeat domain that seems to be
important for mediating protein-protein interactions (Ito, et al., 2012).

The cryptochromes and phytochromes mediate circadian entrainment by light
(Sommers, et al., 1998). The photoreceptors have specific and overlapping roles in
transmitting light signals to the clock that in turn affect circadian period. phyA and phyB
act as the main mediators of red light signaling to the clock. phyB has been
demonstrated to be the principal high-fluence red light receptor for circadian light input
(Somers, et al., 1998), and phyB mutants have long periods under high fluences of red
light (Sommers, et al., 1998; Devlin & Kay, 2000). phyA is responsible for far-red
response of the clock (Kolmos, et al., 2011), as well as low-fluence red light input
(Sommers, et al., 1998; Devlin & Kay, 2000). phyA mutants exhibit long periods under
low-fluence red light (Sommers, et al., 1998; Devlin & Kay, 2000) . phyA is also able to
absorb blue light, and acts with CRY1 to transduce low fluence blue light to the clock




9




(Sommers, et al., 1998; Devlin & Kay, 2000). In accordance with its role in perceiving
blue light, phyA mutants have long periods under low fluence blue light (Sommers, et
al., 1998). CRY1 is also responsible for providing high-intensity blue light information to
the oscillator (Sommers, et al., 1998). cry1 mutants exhibit long periods under low
fluences of blue light (Sommers, et al., 1998). cry2 single mutants do not exhibit a
fluence-dependent period phenotype relative to wild type in blue light (Devlin & Kay,
2000). The cry1cry2 double mutant exhibits a long period phenotype over a range of
blue light fluences that is longer than the period defects observed in the cry1 single
mutant. Thus, the cryptochromes may have partially redundant roles in mediating blue
light entrainment (Devlin & Kay, 2000). Taken together, these results point to a complex
and interwoven network of signaling from the photoreceptors to convey light information
to the clock.

There are several mechanisms through which the photoreceptors transduce light
signals to the clock. The transcription of some clock genes is activated in a manner
dependent on phytochromes. PRR9 transcription has been shown to be rapidly induced
by light in a phytochrome-dependent manner (Ito, et al., 2007). ELF4 is regulated by
both red and far-red light in a phyB and phyA dependent manner respectively (Khanna
et al.; Li, et al., 2011). FHY3, HY5, and FAR1 are all positive regulators of phyA
signaling that bind the promoter of ELF4 and activate its transcription during the day (Li,
et al., 2011). In turn, ELF4 has been shown to mediate the transcriptional activation of
CCA1 and LHY in response to red light (Kikis, et al., 2005).




10




Light signaling to the clock is also regulated through post-transcriptional
mechanisms. The translation of LHY mRNA is induced by light (Kim, et al., 2003).
Recently it has been shown that some red-light irradiated phytochrome is retained in the
cytosol where it can act to regulate translation (Paik, et al., 2012). This may be how
LHY mRNA translation is regulated by light.

The post-translational regulation of several clock components by light is another
mechanism by which light information is conveyed to the clock. PRR9 and PRR7
proteins are degraded in the dark by the proteosome (Ito, et al., 2007; Farre & Kay,
2007). How these proteins are targeted for degradation in the dark remains to be
elucidated. PRR5 is stabilized by blue light and targeted for degradation in the dark by
ZTL (Kiba, et al., 2007; Fujiwara, et al., 2008). ZTL, which perceives blue light through
its LOV domain, is also stabilized by light in a manner that depends on GI (Kim, et al.,
2007). In the dark, ZTL is able to interact with PRR5 and target it for degradation
(Fujiwara, et al., 2008).

Photoreceptor Mediated Protein Stability

Phytochromes have been shown to regulate protein levels in a light-dependent
manner, particularly proteins that act downstream in light signaling. phyA regulates
FHY1 protein levels through direct interaction and causes phosphorylation by the
phytochrome to regulate FHY1 protein stability (Shen, et al., 2009). FHY1 mediates
phyA nuclear import (Genoud, et al., 2008). phyA regulation of FHY1 protein levels




11




serves as a mechanism through which phyA can refine red light transcriptional
responses (Shen, et al., 2009). PIF3 protein levels have also been demonstrated to be
regulated in a phytochrome-dependent manner, mainly by phyB (Soy, et al., 2012).

COP1 mediates the dark dependent degradation of several proteins. For
example the red light signaling components LONG AFTER FAR-RED LIGHT 1 (LAF1)
and LONG HYPOCOTYL IN FAR –RED (HFR1) are also regulated at the level of
protein abundance by different qualities of light. The differential regulation of these
proteins by light depends on the phytochromes. These proteins are targeted for
degradation by the E3-ubiquitin protein ligase COP1 (CONSTITUTIVELY
PHOTOMORPHOGENIC1) (Jang, et al., 2007). The HFR1 protein is degraded in the
dark by COP1 and stabilized by light, regardless of light quality (Yang, et al., 2005).
LAF1, another positive regulator in phyA signaling, is similarly degraded by COP1 in the
dark. In the light, COP1 becomes excluded from the nucleus, although residual COP1 is
able to interact with and degrade LAF1 through its interaction with SPA1
(SUPPRESSOR OF PHYA-105) to attenuate light signaling (Seo, et al., 2003). LAF1
and HFR1 positively regulate different aspects of red light signaling; however, these two
proteins interact to promote the stability of each other by preventing their interactions
with COP1 (Jang, et al., 2001). phyA protein itself is regulated by light. The Pfr form
interacts with the COP1/SPA1 complex. This interaction is dependent on the
phosphorylation state of the protein, as hypophosphorylated phyA interacts
preferentially with FHY1, presumably to prevent premature interaction with the
COP1/SPA1 complex (Saijo, et al., 2008).




12




Cryptochromes also regulate protein stability at the post-transcriptional level in
response to blue light. An example of this is the regulation of CO protein, a floral
inducer, by CRY2. The interaction of CRY2 with SPA1 facilitates the interaction of
CRY2 with COP1. CRY2 interaction with COP1 sequesters COP1 away from CO
protein, which is then able induce flowering in long days. (Zuo, et al., 2011). CRY2
protein is phosphorylated, polyubiquitinated, and subsequently targeted for degradation
by COP1 in response to blue light. The interaction of CRY2 with COP1, and thus its
stability, is regulated by phyA and SPA1 (Weidler et al., 2012). CRY1 also interacts with
the COP1/SPA complex to disrupt the interaction of COP1 with SPA1 (Lian, et al.,
2011). CRY1 interaction with COP1 prevents the degradation of HY5 in response to
blue light and allows HY5 to initiate light induced transcription (Liu, et al., 2011). The
cryptochromes also mediate the stability of proteins involved in pathways other than
light signaling. An example of this is the blue light stabilization of viral resistance R
protein HRT (HYPERSENSITIVE RESPONSE TO TCV) mediated in part by CRY2.
CRY2 promotes the stabilization of HRT in blue light by interacting with COP1 to
prevent HRT degradation by the proteasome (Jeong, et al., 2010).

The ZTL familiy of proteins mediates protein degradation in a manner that is
regulated by blue light. For example, FKF1 targets the CO repressor CDF1 for
degradation. Blue light induces the interaction of FKF1 with GIGANTEA (GI) through
the LOV domain of FKF1 (Sawa, et al., 2007). The light-dependent interaction of FKF1
with GI allows FKF1 to interact with CDF1. This enables FKF1 to target CDF1 for




13




degradation (Sawa, et al., 2007). CDF1 degradation results in the expression of CO.
CO is then able to go on to induce FT expression and cause subsequent flowering in
long days. FKF1 also stabilizes CO protein through interaction with its LOV domain to
promote flowering (Song, et al., 2012). ZTL interaction with GI under blue light stabilizes
ZTL and confers oscillations in the relative abundance of ZTL protein, as ZTL mRNA is
constitutively expressed (Kim, et al., 2007). The regulation of ZTL protein abundance by
light in addition to the role of ZTL regulating other proteins is likely a mechanism for
fine-tuning clock protein expression. ZTL targets PRR5 and TOC1 proteins for
degradation in the dark (Kiba, et al., 2007b; Más, et al., 2003, Fujiwara et al). Blue light
absorption by the LOV domain of ZTL prevents its interaction with these PRR5 and
TOC1, and thus results in their stabilization in blue light (Kiba, et al., 2007b; Fujiwara et
al., 2008). FKF1 and LKP2 share some of the targets of ZTL since TOC1 and PRR5
proteins are more stable in a ztlfkf1lkp2 triple mutant than in the ztl single mutant
background (Ito, et al., 2011).

Light regulated post-transcriptional control is known to be an important
mechanism for regulating the pace of the circadian clock in all organisms (Kojima, et al,
2011)⁠. Protein modification and regulated proteolysis are processes that govern the
peak of expression for circadian proteins. PRR7 protein levels have been shown to be
post-transcriptionally regulated by light and the circadian clock (Farré & Kay, 2007). The
protein expression peaks at the end of the light period and is degraded in the dark by
the proteasome. This dark-mediated degradation occurs regardless of the time of day,
although PRR7 protein is more stable earlier in the day in both light and dark than




14




during the subjective night. The question of how the protein is degraded remains
unanswered, as it does not interact with ZTL (Kiba, et al., 2007)⁠. COP1 is another
candidate E3 ligase mediating PRR7 protein degradation, although the interaction
between PRR7 and COP1 remains to be tested. To further investigate the lightmediated stability of PRR7, I measured the protein levels in seedlings expressing PRR7
under control of its endogenous promoter fused to an amino-terminal HA tag under
different light conditions. The 35S::PRR7-LUC+ line was also used to characterize
PRR7 degradation during the subjective evening.




15




Results
Cycloheximide Treatment Optimization
PRR7 protein abundance has previously been demonstrated to be regulated
post-translationally by light and the circadian clock (Farre & Kay, 2007). Other circadian
PRR proteins have been shown to be regulated by specific qualities of light (Fujiwara, et
al., 2008; Kiba, et al., 2007). As part of this study, an Arabidopsis line expressing the
PRR7 coding region fused to Luciferase under the control of the constitutive 35S
promoter in the prr7-3 mutant background (prr7-3 35S::PRR7-LUC-HA) was used to
further elucidate the qualities of light that mediate the stability of PRR7 protein. The
relative abundance of this fusion protein cycles independently of the mRNA, as
measured by Western blot (Farre & Kay, 2007), providing further evidence that PRR7
protein is subject to strong post-transcriptional regulation.

Cycloheximide can be used as a translational inhibitor in eukaryotes to study
protein degradation to determine protein half-life in the absence of additional translation.
The measurement of luciferase activity has been used by other groups to study the
stability of proteins using Luciferase translational fusions (Shen, et al., 2005). To
determine the stability of PRR7 in the prr7-3 35S::PRR7-LUC-HA line, seedlings were
incubated in MS media supplemented with 500 µM cycloheximide and 0.01%
Triton X-100. Other groups have used similar methods for performing cycloheximide
treatments (Fujiwara, et al., 2008). A line expressing firefly Luciferase under the control
of a light-inducible PRR9 promoter (Para et al., 2007) was used to test the effectiveness




16




of this treatment. Seedlings were transferred in darkness to MS media supplemented
with different concentrations of cycloheximide before putting them in the light for four
hours. As shown in Figure 4, this treatment is able to inhibit translation over
concentrations of cycloheximide ranging from 200 µM to 1 mM relative to no treatment.
Free Luciferase protein is relatively stable in plants (Millar et al., 1992). Thus, luciferase
activity measured from samples collected at the beginning of the treatment is presumed
to be residual Luciferase from the previous day(s) light exposure.

This method of cycloheximide treatment was used to measure the degradation of
the PRR7-LUC fusion protein under white and red light as well as darkness. In
treatments beginning at ZT12, the stability of the protein in red light increased relative to
darkness up to four hours (Figure 5). Despite this trend, these two points are not
statistically significant as determined by the student’s t-test. At ZT4, when the protein
begins to accumulate there was no apparent difference in the degradation rates
between the different light conditions and darkness (data not shown). Farre & Kay
(2007) had previously reported PRR7 protein is stabilized by light relative to darkness.
The luciferase activity in samples collected over one diurnal period does cycle similarly
to the protein as detected by Western blotting (Figure 6, this study; Farre & Kay, 2007).
However, the activity shows large variability, and this variability likely masks the
differences in degradation between experimental conditions. Therefore I decided to
analyze PRR7 stability using the prr7-3 PRR7::HA-PRR7 line that had been previously
used (Farre & Kay, 2007) and the protein levels were measured by Western blotting to
confirm the Luciferase results.




17





Performing cycloheximide treatments using a method that relies on diffusion
instead of vacuum infiltration poses a problem, particularly in samples transferred to
darkness. Light mediates stomatal opening, and stomata close in the dark (Chen, et al.,
2011), which would prevent the cycloheximide from quickly entering the cells and
inhibiting translation at the start of the treatment. When measuring the relative
abundance of PRR7 in the prr7-3 PRR7::HA-PRR7 line in darkness, an increase in
PRR7 protein abundance was observed up to two hours after beginning the treatment,
indicating the treatment was not immediately inhibiting protein synthesis. This increase
was also observed in some samples transferred to different light conditions, although to
a lesser extent under white light. Pre-treating the samples with cycloheximide in white
light for 10-30 minutes at room temperature prior to transfer to darkness was sufficient
to allow cycloheximide to enter the cells and resulted in a decrease in HA-PRR7 protein
abundance at two hours (Figure 7).

Degradation of PRR7 Under Different Light Conditions

To study the effect of different qualities of light on PRR7 protein stability, one
week old seedlings were grown under 12:12 light:dark cycles. Seedlings were
transferred to MS media supplemented with 2 mM cycloheximide and 0.01% Triton X100 before being transferred to different light conditions (70 µmol/m2/sec white and red,
30 µmol/m2/sec for blue) or darkness at ZT4 or ZT12. Samples were collected 2, 4, and
7 or 8 hours after beginning the cycloheximide treatment. I determined HA-PRR7




18




protein abundance by Western blotting using anti-HA antibody. The signal at each timepoint was normalized to Direct Blue 7.1 total protein staining of the membrane (Hong, et
al., 2000), and to the amount of protein at the starting time of the experiment. A
chloroplast import protein TOC75 was used as an additional loading control for several
experiments and found to be similar to the results obtained using Direct Blue staining
(Figure 8).

Under these experimental conditions, there was not a difference in the protein
half-life at ZT4 relative to ZT12 in white light as had been reported in Farre & Kay
(2007) (Figure 9, Table 1). Similar experiments were performed under red light (70
µmol/m-2/sec-1) and blue light (30 µmol/m-2/sec-1) as well as darkness at ZT4 and ZT12.
Under blue light the rate of HA-PRR7 protein degradation was similar between ZT4 and
ZT12 (Figure 9, Table 1). Under red light, the half-life of PRR7 is longer at ZT12 than
ZT4 (Table 1). The relative abundance at each timepoint for both subjective day and
night, however, are not statistically different (Figure 9).

At ZT4, there was not a difference in the degradation of PRR7 under red and
blue light relative to each other or to white light. At ZT12, red light treatment appeared to
stabilize the protein relative to blue light. The relative abundance four hours after the
beginning of the treatment under red light is statistically higher than the abundance
under blue light (student’s t-test, p = 0.0234). Red light did have a statistically significant
effect on the stability of PRR7 relative to darkness four hours after treatment with
cycloheximide (Figure 10, student’s t-test, p = 0.047). In similar experiments, the PRR7-




19




LUC line did show a tendency for this effect at ZT12, although the points are not
statistically different (Figure 5). Red light did not have a statistically significant effect on
the degradation relative to white light as measured by Western blotting.




20




Discussion
This study describes an attempt to determine if red or blue light promotes the
stability of PRR7 protein. It has been previously shown that PRR7 protein is stabilized
by white light (Farre & Kay, 2007). The mechanism for this stabilization has not yet been
defined. In the previous study the prr7-3 PRR7::HA-PRR7 line was used to determine
the stability of the protein during the subjective day and night under white light and
darkness. White light was reported to promote the stability of the protein relative to
darkness regardless of the time of day although the PRR7 protein was more stable
during the subjective day than during the subjective night (Farre & Kay, 2007). In
accordance with these results the levels of a PRR7-LUCIFERASE translational fusion
expressed under the control of the constitutive 35S promoter cycled under constant
white light independently of the mRNA abundance (Farre & Kay, 2007).

In this study, I first used the prr7-3 35S::PRR7-LUCIFERASE-HA Arabidopsis
line (PRR7-LUC) to determine the stability of the protein under different light conditions.
Relative abundance of PRR7-LUC was measured by luciferase activity. The in vitro
luciferase activity of this line oscillates over one diurnal cycle (Figure 6) similarly to the
protein as measured by Western blot (Farre & Kay, 2007). This indicates that the
expression of this fusion protein is similar to the expression of PRR7 under its native
promoter. Thus, the luciferase activity was expected to reflect the relative abundance of
PRR7 under different light conditions.




21




In experiments beginning at the start of the subjective night, PRR7-LUC under 70
µmol/m2/sec1 red light showed a tendency to be stabilized relative to darkness four
hours after beginning the treatment (Figure 5), although the difference is not statistically
different. In experiments beginning at ZT4, or early in the day when PRR7 protein
abundance is lower, no difference between white light, red light and darkness was
observed (data not shown). This results contrasts with previous experiments that
showed that PRR7 expressed under its native promoter was stabilized by white light
relative to darkness during the subjective day (Farre & Kay, 2007). The extractable
luciferase activity of the prr7-3 35S::PRR7-LUCIFERASE-HA line oscillates over one
diurnal cycle with a peak at ZT12, but the variability is large (Figure 6). This may explain
why I am unable to distinguish between the protein abundance under different light
conditions in treatments beginning at ZT4 using this line. These results prompted the
use of the prr7-3 PRR7::HA-PRR7 line that was used in the previous studies (Farre &
Kay 2007).

Using the prr7-3 PRR7::HA-PRR7 line I observed differences in the ability of
cychloheximide treatment to block translation, especially in the dark at ZT12 (Figures
10B, 11B). The relative abundance of HA-PRR7 would frequently increase two hours
into the cycloheximide treatment before degradation was observed. This indicated that
cycloheximide was unable to quickly enter the tissue in the dark. One explanation may
be dark induced stomatal closure, which would prevent the cycloheximide from entering
the tissue to inhibit translation. Stomata are closed in the dark, whereas both red and
blue light regulate stomatal opening, as does the circadian clock (Chen, et al., 2012). At




22




ZT12, the plants may have closed their stomata as a result of entrainment, expecting
the onset of darkness. Under white light at ZT12, cycloheximide seemed to act more
quickly at inhibiting translation than under red or blue light as well as darkness at
(Figures 10, 11). Therefore, I pretreated seedling in white light with cycloheximide to
test if the reagent was able to enter the tissue faster before being transferred to
darkness. Figure 7 shows that pre-incubating the seedlings with cycloheximide in the
presence of Triton X-100 in the light results in a decrease in PRR7 protein two hours
after beginning the treatment in the dark. These results may suggest that the
degradation of PRR7 in the dark at ZT12 as measured in this study is artificially slower
due to insufficient translational inhibition. Pre-treating samples with cycloheximide under
a uniform white light regime before treatment under various light conditions might
remove some of the variability from these measurements.

The results of the experiment using PRR9::LUC expressing seedlings (Figure 4)
indicate that under white light, the cycloheximide treatment used should be sufficient to
inhibit translation and prevent new protein synthesis. This permits the measurement of
the degradation of existing protein under these conditions. Previously it had been shown
that PRR7 protein is degraded faster during the subjective night than during the
subjective day under white light (Farre & Kay, 2007). Table (1) shows the half-life of
PRR7 calculated in this study. Under white light, I did not measure a significant
difference in the half-life of PRR7 during the subjective day and night. Seedlings used in
the previous study were older than those used presently, and so the differences in the
stabilization may be an effect of the developmental stage of the seedlings. The half-life
under blue light was also the same during the subjective day and night. In the dark, the



23




half-life of PRR7 was calculated to be 2.7 and 2.2 hours at ZT4 and ZT12, respectively.
The half-life at ZT12 in the dark is similar to that previously reported of 2.4 hours (Farre
& Kay, 2007). Red light in the subjective night extended the half-life by approximately
one hour relative to the subjective day (4.2 hours and 3.4 hours, respectively).
Therefore under the red light conditions used, PRR7 half-life was calculated to be nearly
double the half-life in darkness at ZT12 (Figure 10B). Therefore my results suggest that
PRR7 may be stabilized by red light in the evening prior to sun down. This effect could
be a way to convey timing information to the oscillator.

PRR7 does not appear to be targeted for degradation by ZTL (Fujiwara, et al.,
2008). Blue light stabilizes TOC1 and PRR5 by inhibiting their interaction with ZTL
(Fujiwara et al., 2008; Kim, et al., 2003). Other members of the ZTL family include
FKF1 and LKP2. These proteins have been shown to also contribute to the regulation of
TOC1 and PRR5, particularly in the absence of ZTL (Baudry, et al., 2010). FKF1 also
mediates blue light dependent protein interactions indicating that blue light might
stabilize all the targets of this protein family. Since blue light did not stabilize PRR7
protein (Figure 11), it suggests neither ZTL or other members of the ZTL family are
likely candidates for mediating the stability of PRR7. In contrast, the present study
suggests it may instead be regulated by red light. The effect of red light on PRR7
stability could be mediated by the phytochromes.




24




The phytochromes have been shown to mediate the light-dependent stability of
other proteins, particularly proteins that function in red light signaling. Positive regulators
of light signaling, including HY5, HFR1, FHY1, and LAF1, are down regulated in the
dark in a phytochrome-dependent manner (Osterlund, et al., 2000; Jang, et al., 2005;
Shen, et al., 2005; Seo, et al., 2003). The exact mechanism for the phytochromedependent stabilization of proteins in the light is unknown. Each of these proteins is
targeted for degradation by COP1, an E3-ubiquitin ligase that functions prominently in
light signaling (Subramanian, et al., 2004). In the dark, COP1 is localized to the nucleus.
Most of the COP1 pool is exported to the cytoplasm in the light (von Arnim, et al., 1998).
Upon blue light absorption cryptochromes interact with COP1 in the nucleus and the
sequestered COP1 is no longer able to target proteins for degradation (Zuo, et al.,
2011; Liu, et al., 2011). The phytochromes may regulate protein stability in a similar
manner by repressing COP1 activity in the light.

Since COP1 targets proteins for degradation in the dark it could be a potential
PRR7 regulator. However, no interaction between COP1 protein and PRR7 protein has
been demonstrated to date. PRR7 protein levels have also not been examined in cop1
mutants. COP1 does not have an effect on PRR9 protein levels (Ito, et al., 2007); PRR7
and PRR9 are very similar proteins, and so COP1 may be an unlikely candidate in
mediating PRR7 degradation. To test if COP1 is involved in the degradation of PRR7
protein would require a line expressing a tagged PRR7 protein in a cop1 mutant
background. There is currently no PRR7-specific antibody available, and thus a tagged
protein is required for PRR7 protein detection. If COP1 were responsible for targeting




25




PRR7 for degradation, PRR7 would be expected to accumulate in the cop1 mutant.

To confirm whether different qualities of light affect the stability and abundance of
PRR7 protein, it would be informative to test how different photoreceptors affect protein
oscillation. Photoreceptors mediate the stability of several proteins in a light-dependent
manner (Kim, et al., 2003; Shen, et al., 2005; Shen, et al., 2007; Shen, et al., 2008).
Under constant white light, PRR7 protein cycles robustly under the control of the native
promoter as well as the constitutive 35S promoter. I have crossed the prr7-3
35S::PRR7-LUC line with phytochrome and cryptochrome mutants and homozygous
lines are currently being selected. These lines can then be used to test if the PRR7-LUC
protein oscillations change in different photoreceptor mutant backgrounds by
determining protein levels by Western blot. It may also be possible to use these lines to
measure the stability of the PRR7-LUC protein by luciferase activity by further refining
the luciferase assay to improve reproducibility.

Post-transcriptional regulation of the circadian clock contributes to the robustness
of the oscillator by defining the peak of the expression of clock proteins. Studying how
these proteins are regulated is important for understanding how the clock functions and
regulates subsequent output processes. Unlike PRR5 and TOC1, PRR7 does not
appear to be regulated by blue light. Instead, it appears that red light may play a
specific role in mediating PRR7 stability. Additional experiments to further characterize
the effect of red light on PRR7 protein levels will need to be performed. These include




26




identifying which specific photoreceptor(s) play a role in regulating PRR7 abundance
and how PRR7 is targeted for degradation by the proteasome.




27





Figure 1. Overview of the circadian oscillator in A. thaliana. The central loop is
comprised of the MYB-domain transcription factors CCA1 and LHY that activate
transcription of PRR9 and PRR7. PRR9 and PRR7, as well as PRR5, PRR3, and TOC1
sequentially repress the transcription of CCA1 and LHY. CCA1 and LHY repress the
transcription of TOC1 and other evening genes such as ELF3 and LUX during the day.
ZTL, an F-box protein that functions as part of the SCF-E3-ubiquitin ligase, targets
PRR5 and TOC1 for degradation. PRR3 prevents ZTL binding to TOC1 and stabilizes
TOC1 protein. ZTL protein stability is dependent on GI. For interpretation of the
references to color in this and all other figures, the reader is referred to the electronic
version of this thesis.




28




Figure 2. A, B
A

B




29





Figure 2. A, Expression of the circadian PRRs. The expression of the circadian PRR
quintet cycles with a period of 24 hours under light/dark cycles (LD) as well as constant
light (LL). Data were obtained from the Diurnal Project (www.diurnal.mockerlab.org),
(Mockler, et al., 2007, Michael, et al., 2008). PRR9 expression peaks early in the
morning, followed by PRR7, PRR5, PRR3, and TOC1/PRR1 in the evening. B, The
conserved domains of the circadian PRR proteins. At the amino terminus, the
pseudo-receiver domain resembles the receiver domain from canonical two-component
response regulator proteins. The pseudo-receiver domain lacks the conserved
aspartate residue that functions in the phospho-relay system of the response regulators.
The carboxy-terminal CONSTANTS/CONSTANTSLIKE/TOC1 CCT motif is conserved
among the PRRs, as well as the floral regulatory protein CONSTANTS. The carboxyterminus also contains a putative nuclear localiztion signal (NLS).




30





Figure 3. Examples of light-regulated proteolysis in A. thaliana. The photoreceptors
phytochromes and cryptochromes are activated by red and blue light, respectively.
Activated photoreceptors undergo a conformational change before being translocated
into the nucleus, except CRY2, which is constitutively nuclear. phyA requires FHY1 and
related proteins to enable it to enter the nucleus. phyB seems to require PIF3 for
nuclear import. In the nucleus, activated photoreceptors interact with COP1, an E3ubiquitin ligase that targets proteins for protesomal degradation. The interaction of
photoreceptors with complexes of COP1 stabilize other proteins that function




31




downstream in light signaling. For example cryptochromes interact with COP1 to help
stabilize CO protein and induce flowering in response to blue light. This process is also
governed by FKF1, which interacts with CO in a blue light-dependent manner to
stabilize the protein. Activated phytochromes and cryptochromes are targeted for
degradadation by COP1.




32





Figure 4. Optimization of cycloheximide treatment in the light. The PRR9::LUC line
was used to test the effectiveness of different cycloheximide treatments for inhibiting
translation in the light. The PRR9 promoter is light-inducible. Seedlings were grown for
one week in 12:12 light/dark cycles. At ZT0, seedlings were transferred to media with
Triton X-100 and varying concentrations of cycloheximide in darkness before being
transferred to light for four hours. Luciferase activity was compared to samples not
treated with cycloheximide. * Indicates statistically significant by the student’s t-test




33




(p = 0.0212, 0.0476, 0.0311 for 200 µM, 500 µM, and 1 mM respectively). Each time
point represents the average of three biological replicates and error bars represent the
standard error for the mean (SEM).




34





Figure 5. PRR7-LUC protein stability under different light conditions. 35S::PRR7LUC-HA seedlings were treated with 500 µM cycloheximide and 0.01% Triton X-100
and transferred to white light (70 µmol/m2/sec1), red light (70 µmol/m2/sec1) and
darkness at ZT 12 for the times indicated. The black bar represents darkness and the
gray bar represents white or red light. Time indicates hours since the start of the




35




cycloheximide treatment at the indicated ZT time. Luciferase activity was measured
using the Bright Glo kit from Promega. Relative light units were normalized to protein
concentration as determined by Bradford Assay. The data represent the average of 5
experiments under each light conditions and the error bars represent the standard error
for the mean (SEM).




36





Figure 6. Luciferase activity of the prr7-3 35S::PRR7-LUC-HA line. Seedlings were
grown for one week under 12:12 light/dark cycles. Samples were collected in triplicate
at each time point. Day and night are represented by the white and black bars,
respectively. Luciferase activity was assayed using the Bright Glo kit from Promega.
Relative Light Units (RLU) were normalized to the protein concentration in each sample
as determined by Bradford Assay. Error bars represent the standard error for the mean
(SEM).




37





Figure 7. Optimization of cycloheximide treatment in darkness. The PRR7::HAPRR7 line was used to test if treating seedlings briefly in light before transfer to
darkness affected the ability of cycloheximide to inhibit translation due to stomata
closing. Seedlings were transferred to media with Triton X-100 and cycloheximide and
directly transferred to darkness (-) or subjected to a 15 minute light treatment prior to
being transferred to darkness (+). Samples were taken at 2 and 4 hours after the
beginning of the treatment to evaluate the ability of cycloheximide to enter tissue and
inhibit translation in the dark. Panel A and B show degradation at ZT4 and ZT12,
respectively. For comparison, samples not pre-treated in the light ((-), n = 8) are shown
(dashed line). Error bars represent the standard error for the mean (SEM). The
experiment with the light pre-treatment was performed once.




38





Figure 8. Comparison of DB7.1 staining and TOC75 detection as loading controls.
A, blot for PRR7 degradation at ZT12, comparison of DB7.1 staining and TOC75
detection for protein quantitation. Membranes probed with anti-HA antibody were
stripped and re-probed with anti-TOC75 antibody. The same membranes were stained
with DB7.1 stain for total protein. B,panel, quantification of western blot signals using
Metamorph image analysis software. Similar results were obtained in other
experiments.




39





Figure 9. Comparisons of HA-PRR7 protein degradation rates during the
subjective day and subjective night. A, White light; B, dark; C, red light; D, blue light.
Data are from Figures 10 and 11. The values represent the average of 8 for dark, 9 for
white light, 4 for red light, 3 for blue light independent experiments Error bars represent
the standard error for the mean (SEM).




40




Figure 10. A, B




41





Figure 10. Analysis of the stability of HA-PRR7 protein evaluated by Western blot
under red light. One week-old prr7-3 PRR7::HA-PRR7 seedlings entrained under
12:12 light/dark cycles were transferred to MS media with 2 mM cycloheximide and
0.01% Triton X-100 before being transferred to white light (70 µmol/m2/sec1), red light
(70 µmol/m2/sec1) or darkness at ZT4 (A) or ZT12 (B). Time indicates hours since the
start of the cycloheximide treatment at the indicated ZT time. Protein levels were
quantified by western blot using an anti-HA antibody. Graphs represent the average of 4
for red light, 9 for dark, and 10 for white light independent experiments and error bars
represent the standard error for the mean (SEM). * Indicates statistically significant
(student’s t-test, p = 0.047). The blots are from one representative experiment.




42




Figure 11. A, B




43




Figure 11. Analysis of the stability of HA-PRR7 protein evaluated by Western blot
under blue light . Western blot of seedlings treated with white light (70 µmol/m2/sec1),
blue light (30 µmol/m2/sec1), and darkness. One week-old prr7-3 PRR7::HA-PRR7
seedlings entrained under 12:12 light/dark cycles were transferred to MS media with 2
mM cycloheximide and 0.01% Triton X-100 before being transferred to different light
conditions at ZT4 (A) or ZT12 (B). Time indicates hours since the start of the
cycloheximide treatment at the indicated ZT time. Graphs represent the average of 3 for
blue light, 10 for white light, and 9 for dark independent experiments. – Indicates a
misloaded negative control; the four hour time-point for blue light is the last lane labeled
“19”. Error bars represent the standard error for the mean (SEM).




44





White Light

Red Light

Blue Light

Darkness

ZT4

3.5 hrs

3.4 hrs

2.4 hrs

2.8 hrs

ZT12

3.1 hrs

4.2 hrs

2.4 hrs

2.2 hrs

Table 1. Half-life of PRR7 Under Different Light Conditions. Half-life (in hours) of
PRR7 under white light (70 µmol/m2/sec1), red light (70 µmol/m2/sec1), blue light
(30µ mol/m2/sec1), and darkness. Degradation curves in Figures 10 and 11 were fit with
the first order decay function. Half-life was calculated from the decay constant k using
ln(k)/2 = t ½.




45




Materials and Methods

Plant material

The Arabidopsis prr7-3 T-DNA line in the Col-0 accession has been described
previously (Farre et al., 2005). The lines PRR7::HAPRR7 and 35S::PRR7LUC-HA in
the prr7-3 background have been described previously (Farre & Kay, 2007). The
PRR9::LUC construct has been described previously (Para et al., 2007). Plants were
grown on Murashige and Skoog media (Murashige & Skoog, 1962) supplemented with
0.8% agar and 2% sucrose. Seedlings were sterilized by gas sterilization and stratified
at 4°C in darkness for 3-7 days before being grown for 7-9 days under 12:12 white light
(70 µmol m-2 sec-2)/dark cycles at 22°C in Percival Environmental Chambers (Percival
Scientific).

Cycloheximide Treatment Optimization

For determining the effectiveness of the cycloheximide treatment in white light,
PRR9::LUC seedlings were grown as described above. Murashige and Skoog media
supplemented with 0.01% Triton X-100 and either no cycloheximide, 20 µM, 200 µM,
500 µM, and 1 mM cycloheximide were prepared and aliquoted into Costar 24 well
culture dishes (Fisher Scientific). Seven-day old PRR9::LUC seedlings grown under
12L:12D cycles were transferred to the media in the dark at ZT0 and immediately
transferred to white light (70 µmol m-2 sec-2). Seedlings were collected at ZT4, patted




46




dry, and flash frozen in liquid nitrogen. Luciferase activity was measured in these
samples using the Bright Glo Luciferase Assay Kit from Promega as described below.

To test the effectiveness of cycloheximide treatment in the dark, seven-day-old
PRR7::HAPRR7 seedlings were transferred at ZT4 and ZT12 to Murashige and Skoog
media supplemented with 0.01% Triton X-100 and 500 µM or 2 mM cycloheximide.
Seedlings were incubated in the light for 10-15 minutes in white light before being
transferred to darkness. Effectiveness of the treatment was determined by quantifying
relative HA-PRR7 protein levels in the dark two and four hours after transfer (see below
for extraction and blotting procedure).

Cycloheximide Treatment Under Different Light Conditions

Seedlings were transferred to Murashige and Skoog liquid media supplemented
with 2 mM cycloheximide (Sigma) and 0.01% Triton X-100 (Sigma) in Costar 6-well
culture dishes (Fisher Scientific) before being transferred to their respective light
conditions. Light intensity for red and blue light was determined using a LI-COR 250A
light meter (LI-COR Biosciences). 397 Pale Grey Roscolux filters (Premier Lighting)
were used to adjust light intensity (Mazzoni, et al., 2005). Seedlings (15-20) treated with
cycloheximide under different light conditions were collected at 2 hours, 4 hours, and 7
or 8 hours after the beginning of the light treatment. Plant material was patted dry and
flash frozen in liquid nitrogen.




47




Protein Analysis by Western Blot

To prepare samples for analysis by Western blotting, frozen plant tissue was
ground for 2.5 minutes at 30 Hz using a ball mill (Qiagen). Plant extracts were prepared
by adding 50-75 µL of extraction buffer [100 mM sodium phosphate buffer, 150 mM
sodium chloride, 5 mM EDTA, 5 mM EGTA, 0.1% Triton X-100, 10% glycerol, 3 µM
dithithreitol, 50 uM MG132, Roche Protease Inhibitor, 5 mM Benzamidine, 1 mM PMSF,
0.5% Deoxycholate, pH 8.0, 0.1% sodiumdodecylsulfate] to each sample and
immediately vortexing. Extracts were clarified by centrifugation at 14,000 x g,
transferring the supernatant to a new tube, and repeating the centrifugation. Protein
concentrations were determined using the BioRad Bradford Assay (BioRad). Protein
standards were prepared in a 1:10 dilution of extraction buffer to account for the
background signal caused by reducing agents in the extraction buffer in the samples. All
extracts were diluted 1:10 in water before determining their protein concentration.
Samples were mixed with sodium dodecyl sulfate loading buffer (final concentrations 25
mM Tris-HCl, pH 6.8, 2% SDS, 0.12% Bromophenolblue, 10% Glycerol) before
incubating at 37°C for 2 minutes prior to loading. 80-150 µg of protein were loaded per
lane on a 7% acrylamide/bisacrylamide gel run in the BioRad Mini-PROTEAN Tetra Cell
System (Biorad).

Acrylamide gels were transferred to 0.22 µm NitroPure nitrocellulose membrane
(GE Healthsciences, WP2HY00010) at 18 V overnight using the BioRad Criterion
Blotter. Membranes were blocked with 5% dry milk dissolved in TBST [50 mM Tris, pH




48




7.5, 150 mM sodium chloride, 0.05% Tween-20 (Sigma)] for 30 minutes at room
temperature, then treated with a 1:1,000 α-HA-HRP antibody (Anti-HA-Peroxidase from
Mouse IgG, Roche) diluted in 5% milk in TBST at room temperature for one hour.
Membranes were washed briefly 3 times, then three times for 5 minutes, and twice for
10 minutes with TBST. Peroxidase activity was detected using SuperSignal West Femto
Chemiluminescent Substrate or SuperSignal West Pico Chemiluminescent Substrate
(Pierce) at different ratios as needed for adequate detection. Blots were visualized using
the BioRad VersaDoc 4000 MP (BioRad) Chemiluminescent Ultra Sensitivity in the
Quantity One image acquisition software. Direct-Blue 71 (Sigma) staining was used for
normalization to loading controls (Hong, et al., 2000). Images were quantified using the
Metamorph image analysis software (Molecular Devices, LLC.). The HA-PRR7 signal
was normalized to the corresponding region on the DB7.1 stained membrane before
normalizing all time-points to the signal measured at the beginning of the treatment.

TOC75 detection
Membranes previously probed with α-HA-HRP antibody were first stripped using
the Restore Western Stripping Buffer (Thermo Scientific) and subsequently blocked with
5% BSA dissolved in TBST. The membranes were probed with rabbit α-TOC75
(1:2,500) in TBST-BSA for 1 hour at room temperature or overnight at 4°C. Membranes
were then washed with TBST and blocked with dry milk in TBST as described above.
Goat α-Rabbit-HRP (Roche) was diluted 1:10,000 in TBST -5% dry milk and
membranes were incubated at room temperature for one hour. Blots were visualized as
described above




49





Analysis of Luciferase Activity
For Luciferase assays, frozen seedlings were ground as described for Western
Blotting. 1X Cell Culture Lysis Reagent (CCLR, Promega) was prepared at room
temperature supplemented with 3 µM dithiothreitol, 50 µM MG132, Roche Protease
Inhibitor, 5 mM Benzamidine, 1 mM PMSF, 180 nM Epoxomycin, and 1X Phosphatase
Inhibitor Cocktails I and III (Sigma). 150 µL of modified CCLR was added to each
ground sample and vortexed. Samples were centrifuged for 10 minutes at 14,000 X g,
the supernatant transferred, and centrifuged a second time. Clarified extracts were
diluted in modified 1X CCLR. 10 µL of diluted extracts were transferred to a 96-well
plate. Room temperature Bright Glo Assay Buffer (Promega) was added to each well
and luminescence was measured for 2-8 seconds. Relative Light Units (RLU) were
normalized to the protein content in the diluted extract as determined by Bradford Assay
(Biorad) using an identical dilution of modified CCLR for diluting the standards to
account for buffer background signal.




50





References




51





References
[1]
Alabadi D., Oyama T., Yanovsky M.J., Harmon F.G., Mas P., Kay S.A.,
Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian
clock, Science 293 (2001) 880-883.
[2]
Ang L.H., Chattopadhyay S., Wei N., Oyama T., Okada K., Batschauer A., Deng
X.W., Molecular interaction between COP1 and HY5 defines a regulatory switch for light
control of Arabidopsis development, Mol Cell 1 (1998) 213-222.
[3]
Baudry A., Ito S., Song Y.H., Strait A.A., Kiba T., Lu S., Henriques R., PrunedaPaz J.L., Chua N.H., Tobin E.M., Kay S.A., Imaizumi T., F-box proteins FKF1 and LKP2
act in concert with ZEITLUPE to control Arabidopsis clock progression, Plant Cell 22
(2010) 606-622.
[4]
Bell-Pedersen D., Cassone V.M., Earnest D.J., Golden S.S., Hardin P.E.,
Thomas T.L., Zoran M.J., Circadian rhythms from multiple oscillators: lessons from
diverse organisms, Nat Rev Genet 6 (2005) 544-556.
[5]
Chen C., Xiao Y.G., Li X., Ni M., Light-regulated stomatal aperture in
Arabidopsis, Mol Plant 5 (2012) 566-572.
[6]
Chen M., Tao Y., Lim J., Shaw A., Chory J., Regulation of phytochrome B
nuclear localization through light-dependent unmasking of nuclear-localization signals,
Curr Biol 15 (2005) 637-642.
[7]
Chow B.Y., Helfer A., Nusinow D.A., Kay S.A., ELF3 recruitment to the PRR9
promoter requires other Evening Complex members in the Arabidopsis circadian clock,
Plant Signal Behav 7 (2012) 170-173.
[8]
Covington M.F., Maloof J.N., Straume M., Kay S.A., Harmer S.L., Global
transcriptome analysis reveals circadian regulation of key pathways in plant growth and
development, Genome Biol 9 (2008) R130.
[9]
Diernfellner A.C., Querfurth C., Salazar C., Hofer T., Brunner M.,
Phosphorylation modulates rapid nucleocytoplasmic shuttling and cytoplasmic
accumulation of Neurospora clock protein FRQ on a circadian time scale, Genes Dev
23 (2009) 2192-2200.
[10] Dixon L.E., Knox K., Kozma-Bognar L., Southern M.M., Pokhilko A., Millar A.J.,
Temporal repression of core circadian genes is mediated through EARLY FLOWERING
3 in Arabidopsis, Curr Biol 21 (2011) 120-125.




52




[11] Doherty C.J., Kay S.A., Circadian control of global gene expression patterns,
Annu Rev Genet 44 (2010) 419-444.
[12] Farre E.M., Harmer S.L., Harmon F.G., Yanovsky M.J., Kay S.A., Overlapping
and distinct roles of PRR7 and PRR9 in the Arabidopsis circadian clock, Curr Biol 15
(2005) 47-54.
[13] Farre E.M., Kay S.A., PRR7 protein levels are regulated by light and the
circadian clock in Arabidopsis, Plant J 52 (2007) 548-560.
[14] Fujiwara S., Wang L., Han L., Suh S.S., Salome P.A., McClung C.R., Somers
D.E., Post-translational regulation of the Arabidopsis circadian clock through selective
proteolysis and phosphorylation of pseudo-response regulator proteins, J Biol Chem
283 (2008) 23073-23083.
[15] Gao T., Zhang X., Ivleva N.B., Golden S.S., LiWang A., NMR structure of the
pseudo-receiver domain of CikA, Protein Sci 16 (2007) 465-475.
[16] Gendron J.M., Pruneda-Paz J.L., Doherty C.J., Gross A.M., Kang S.E., Kay S.A.,
Arabidopsis circadian clock protein, TOC1, is a DNA-binding transcription factor, Proc
Natl Acad Sci U S A 109 (2012) 3167-3172.
[17] Genoud T., Schweizer F., Tscheuschler A., Debrieux D., Casal J.J., Schafer E.,
Hiltbrunner A., Fankhauser C., FHY1 mediates nuclear import of the light-activated
phytochrome A photoreceptor, PLoS Genet 4 (2008) e1000143.
[18] Harmer S.L., The circadian system in higher plants, Annu Rev Plant Biol 60
(2009) 357-377.
[19] Harmer S.L., Hogenesch J.B., Straume M., Chang H.S., Han B., Zhu T., Wang
X., Kreps J.A., Kay S.A., Orchestrated transcription of key pathways in Arabidopsis by
the circadian clock, Science 290 (2000) 2110-2113.
[20] Hazen S.P., Schultz T.F., Pruneda-Paz J.L., Borevitz J.O., Ecker J.R., Kay S.A.,
LUX ARRHYTHMO encodes a Myb domain protein essential for circadian rhythms,
Proc Natl Acad Sci U S A 102 (2005) 10387-10392.
[21] Helfer A., Nusinow D.A., Chow B.Y., Gehrke A.R., Bulyk M.L., Kay S.A., LUX
ARRHYTHMO encodes a nighttime repressor of circadian gene expression in the
Arabidopsis core clock, Curr Biol 21 (2011) 126-133.
[22] Hiltbrunner A., Tscheuschler A., Viczian A., Kunkel T., Kircher S., Schafer E.,
FHY1 and FHL act together to mediate nuclear accumulation of the phytochrome A
photoreceptor, Plant Cell Physiol 47 (2006) 1023-1034.




53




[23] Hong H.Y., Yoo G.S., Choi J.K., Direct Blue 71 staining of proteins bound to
blotting membranes, Electrophoresis 21 (2000) 841-845.
[24] Huang W., Perez-Garcia P., Pokhilko A., Millar A.J., Antoshechkin I., Riechmann
J.L., Mas P., Mapping the core of the Arabidopsis circadian clock defines the network
structure of the oscillator, Science 336 (2012) 75-79.
[25] Iwasaki H., Nishiwaki T., Kitayama Y., Nakajima M., Kondo T., KaiA-stimulated
KaiC phosphorylation in circadian timing loops in cyanobacteria, Proc Natl Acad Sci U S
A 99 (2002) 15788-15793.
[26] James A.B., Syed N.H., Bordage S., Marshall J., Nimmo G.A., Jenkins G.I.,
Herzyk P., Brown J.W., Nimmo H.G., Alternative splicing mediates responses of the
Arabidopsis circadian clock to temperature changes, Plant Cell 24 (2012) 961-981.
[27] Jang I.C., Henriques R., Seo H.S., Nagatani A., Chua N.H., Arabidopsis
PHYTOCHROME INTERACTING FACTOR proteins promote phytochrome B
polyubiquitination by COP1 E3 ligase in the nucleus, Plant Cell 22 (2010) 2370-2383.
[28] Jang I.C., Yang J.Y., Seo H.S., Chua N.H., HFR1 is targeted by COP1 E3 ligase
for post-translational proteolysis during phytochrome A signaling, Genes Dev 19 (2005)
593-602.
[29] Jang S., Marchal V., Panigrahi K.C., Wenkel S., Soppe W., Deng X.W., Valverde
F., Coupland G., Arabidopsis COP1 shapes the temporal pattern of CO accumulation
conferring a photoperiodic flowering response, EMBO J 27 (2008) 1277-1288.
[30] Kaczorowski K.A., Quail P.H., Arabidopsis PSEUDO-RESPONSE
REGULATOR7 is a signaling intermediate in phytochrome-regulated seedling
deetiolation and phasing of the circadian clock, Plant Cell 15 (2003) 2654-2665.
[31] Kiba T., Henriques R., Sakakibara H., Chua N.H., Targeted degradation of
PSEUDO-RESPONSE REGULATOR5 by an SCFZTL complex regulates clock function
and photomorphogenesis in Arabidopsis thaliana, Plant Cell 19 (2007) 2516-2530.
[32] Kikis E.A., Khanna R., Quail P.H., ELF4 is a phytochrome-regulated component
of a negative-feedback loop involving the central oscillator components CCA1 and LHY,
Plant J 44 (2005) 300-313.
[33] Kim W.Y., Fujiwara S., Suh S.S., Kim J., Kim Y., Han L., David K., Putterill J.,
Nam H.G., Somers D.E., ZEITLUPE is a circadian photoreceptor stabilized by
GIGANTEA in blue light, Nature 449 (2007) 356-360.
[34] Kim W.Y., Geng R., Somers D.E., Circadian phase-specific degradation of the Fbox protein ZTL is mediated by the proteasome, Proc Natl Acad Sci U S A 100 (2003)
4933-4938.




54




[35] Kitayama Y., Iwasaki H., Nishiwaki T., Kondo T., KaiB functions as an attenuator
of KaiC phosphorylation in the cyanobacterial circadian clock system, EMBO J 22
(2003) 2127-2134.
[36] Kolmos E., Herrero E., Bujdoso N., Millar A.J., Toth R., Gyula P., Nagy F., Davis
S.J., A reduced-function allele reveals that EARLY FLOWERING3 repressive action on
the circadian clock is modulated by phytochrome signals in Arabidopsis, Plant Cell 23
(2011) 3230-3246.
[37] Laubinger S., Marchal V., Le Gourrierec J., Wenkel S., Adrian J., Jang S., Kulajta
C., Braun H., Coupland G., Hoecker U., Arabidopsis SPA proteins regulate
photoperiodic flowering and interact with the floral inducer CONSTANS to regulate its
stability, Development 133 (2006) 3213-3222.
[38] Leivar P., Quail P.H., PIFs: pivotal components in a cellular signaling hub,
Trends Plant Sci 16 (2011) 19-28.
[39] Lian H.L., He S.B., Zhang Y.C., Zhu D.M., Zhang J.Y., Jia K.P., Sun S.X., Li L.,
Yang H.Q., Blue-light-dependent interaction of cryptochrome 1 with SPA1 defines a
dynamic signaling mechanism, Genes Dev 25 (2011) 1023-1028.
[40] Liu B., Zuo Z., Liu H., Liu X., Lin C., Arabidopsis cryptochrome 1 interacts with
SPA1 to suppress COP1 activity in response to blue light, Genes Dev 25 (2011) 10291034.
[41] Liu L.J., Zhang Y.C., Li Q.H., Sang Y., Mao J., Lian H.L., Wang L., Yang H.Q.,
COP1-mediated ubiquitination of CONSTANS is implicated in cryptochrome regulation
of flowering in Arabidopsis, Plant Cell 20 (2008) 292-306.
[42] Lu S.X., Knowles S.M., Andronis C., Ong M.S., Tobin E.M., CIRCADIAN CLOCK
ASSOCIATED1 and LATE ELONGATED HYPOCOTYL function synergistically in the
circadian clock of Arabidopsis, Plant Physiol 150 (2009) 834-843.
[43] Majercak J., Sidote D., Hardin P.E., Edery I., How a circadian clock adapts to
seasonal decreases in temperature and day length, Neuron 24 (1999) 219-230.
[44] Mas P., Kim W.Y., Somers D.E., Kay S.A., Targeted degradation of TOC1 by
ZTL modulates circadian function in Arabidopsis thaliana, Nature 426 (2003) 567-570.
[45] Matsushika A., Makino S., Kojima M., Mizuno T., Circadian waves of expression
of the APRR1/TOC1 family of pseudo-response regulators in Arabidopsis thaliana:
insight into the plant circadian clock, Plant Cell Physiol 41 (2000) 1002-1012.
[46] McWatters H.G., Bastow R.M., Hall A., Millar A.J., The ELF3 zeitnehmer
regulates light signalling to the circadian clock, Nature 408 (2000) 716-720.




55




[47] Menet J.S., Rosbash M., A new twist on clock protein phosphorylation: a
conformational change leads to protein degradation, Mol Cell 43 (2012) 695-697.
[48] Michael T.P., Mockler T.C., Breton G., McEntee C., Byer A., Trout J.D., Hazen
S.P., Shen R., Priest H.D., Sullivan C.M., Givan S.A., Yanovsky M., Hong F., Kay S.A.,
Chory J., Network discovery pipeline elucidates conserved time-of-day-specific cisregulatory modules, PLoS Genet 4 (2008) e14.
[49]
283.

Millar A.J., Input signals to the plant circadian clock, J Exp Bot 55 (2004) 277-

[50] Mizuno T., Nakamichi N., Pseudo-Response Regulators (PRRs) or True
Oscillator Components (TOCs), Plant Cell Physiol 46 (2005) 677-685.
[51] Mockler T.C., Michael T.P., Priest H.D., Shen R., Sullivan C.M., Givan S.A.,
McEntee C., Kay S.A., Chory J., The DIURNAL project: DIURNAL and circadian
expression profiling, model-based pattern matching, and promoter analysis, Cold Spring
Harb Symp Quant Biol 72 (2007) 353-363.
[52] Nakajima M., Imai K., Ito H., Nishiwaki T., Murayama Y., Iwasaki H., Oyama T.,
Kondo T., Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation
in vitro, Science 308 (2005) 414-415.
[53] Nakamichi N., Kita M., Ito S., Sato E., Yamashino T., Mizuno T., The Arabidopsis
pseudo-response regulators, PRR5 and PRR7, coordinately play essential roles for
circadian clock function, Plant Cell Physiol 46 (2005) 609-619.
[54] O'Neill J.S., Reddy A.B., Circadian clocks in human red blood cells, Nature 469
(2011) 498-503.
[55] O'Neill J.S., van Ooijen G., Dixon L.E., Troein C., Corellou F., Bouget F.Y.,
Reddy A.B., Millar A.J., Circadian rhythms persist without transcription in a eukaryote,
Nature 469 (2011) 554-558.
[56] Osterlund M.T., Wei N., Deng X.W., The roles of photoreceptor systems and the
COP1-targeted destabilization of HY5 in light control of Arabidopsis seedling
development, Plant Physiol 124 (2000) 1520-1524.
[57] Paik I., Yang S., Choi G., Phytochrome regulates translation of mRNA in the
cytosol, Proc Natl Acad Sci U S A 109 (2012) 1335-1340.
[58] Para A., Farre E.M., Imaizumi T., Pruneda-Paz J.L., Harmon F.G., Kay S.A.,
PRR3 Is a vascular regulator of TOC1 stability in the Arabidopsis circadian clock, Plant
Cell 19 (2007) 3462-3473.




56




[59] Perales M., Mas P., A functional link between rhythmic changes in chromatin
structure and the Arabidopsis biological clock, Plant Cell 19 (2007) 2111-2123.
[60] Pfeiffer A., Nagel M.K., Popp C., Wust F., Bindics J., Viczian A., Hiltbrunner A.,
Nagy F., Kunkel T., Schafer E., Interaction with plant transcription factors can mediate
nuclear import of phytochrome B, Proc Natl Acad Sci U S A 109 (2012) 5892-5897.
[61] Pruneda-Paz J.L., Breton G., Para A., Kay S.A., A functional genomics approach
reveals CHE as a component of the Arabidopsis circadian clock, Science 323 (2009)
1481-1485.
[62] Rosbash M., The implications of multiple circadian clock origins, PLoS Biol 7
(2009) e62.
[63] Salome P.A., Weigel D., McClung C.R., The role of the Arabidopsis morning loop
components CCA1, LHY, PRR7, and PRR9 in temperature compensation, Plant Cell 22
(2010) 3650-3661.
[64] Salome P.A.a.M., C. R. , What makes the Arabidopsis clock tick on time? A
review on entrainment, Plant, Cell & Environment 28 (2005).
[65] Sawa M., Kay S.A., GIGANTEA directly activates Flowering Locus T in
Arabidopsis thaliana, Proc Natl Acad Sci U S A 108 (2011) 11698-11703.
[66] Sawa M., Nusinow D.A., Kay S.A., Imaizumi T., FKF1 and GIGANTEA complex
formation is required for day-length measurement in Arabidopsis, Science 318 (2007)
261-265.
[67] Seo H.S., Yang J.Y., Ishikawa M., Bolle C., Ballesteros M.L., Chua N.H., LAF1
ubiquitination by COP1 controls photomorphogenesis and is stimulated by SPA1,
Nature 423 (2003) 995-999.
[68] Shen H., Moon J., Huq E., PIF1 is regulated by light-mediated degradation
through the ubiquitin-26S proteasome pathway to optimize photomorphogenesis of
seedlings in Arabidopsis, Plant J 44 (2005) 1023-1035.
[69] Shen H., Zhu L., Castillon A., Majee M., Downie B., Huq E., Light-induced
phosphorylation and degradation of the negative regulator PHYTOCHROMEINTERACTING FACTOR1 from Arabidopsis depend upon its direct physical interactions
with photoactivated phytochromes, Plant Cell 20 (2008) 1586-1602.
[70] Shen Y., Khanna R., Carle C.M., Quail P.H., Phytochrome induces rapid PIF5
phosphorylation and degradation in response to red-light activation, Plant Physiol 145
(2007) 1043-1051.




57




[72] Somers D.E., Devlin P.F., Kay S.A., Phytochromes and cryptochromes in the
entrainment of the Arabidopsis circadian clock, Science 282 (1998) 1488-1490.
[73] Song Y.H., Smith R.W., To B.J., Millar A.J., Imaizumi T., FKF1 conveys timing
information for CONSTANS stabilization in photoperiodic flowering, Science 336 (2012)
1045-1049.
[74] Soy J., Leivar P., Gonzalez-Schain N., Sentandreu M., Prat S., Quail P.H., Monte
E., Phytochrome-imposed oscillations in PIF3 protein abundance regulate hypocotyl
growth under diurnal light/dark conditions in Arabidopsis, Plant J 71 (2012) 390-401.
[75] Subramanian C., Kim B.H., Lyssenko N.N., Xu X., Johnson C.H., von Arnim
A.G., The Arabidopsis repressor of light signaling, COP1, is regulated by nuclear
exclusion: mutational analysis by bioluminescence resonance energy transfer, Proc Natl
Acad Sci U S A 101 (2004) 6798-6802.
[76] Trupkin S.A., Debrieux D., Hiltbrunner A., Fankhauser C., Casal J.J., The serinerich N-terminal region of Arabidopsis phytochrome A is required for protein stability,
Plant Mol Biol 63 (2007) 669-678.
[77] van Ooijen G., Millar A.J., Non-transcriptional oscillators in circadian
timekeeping, Trends Biochem Sci 37 (2012) 484-492.
[78] Viczian A., Adam E., Wolf I., Bindics J., Kircher S., Heijde M., Ulm R., Schafer E.,
Nagy F., A short amino-terminal part of Arabidopsis phytochrome A induces constitutive
photomorphogenic response, Mol Plant 5 (2011) 629-641.
[79] von Arnim A.G., Deng X.W., Light inactivation of Arabidopsis photomorphogenic
repressor COP1 involves a cell-specific regulation of its nucleocytoplasmic partitioning,
Cell 79 (1994) 1035-1045.
[80] Wang L., Fujiwara S., Somers D.E., PRR5 regulates phosphorylation, nuclear
import and subnuclear localization of TOC1 in the Arabidopsis circadian clock, EMBO J
29 (2010) 1903-1915.
[81] Wang X., Li W., Piqueras R., Cao K., Deng X.W., Wei N., Regulation of COP1
nuclear localization by the COP9 signalosome via direct interaction with CSN1, Plant J
58 (2009) 655-667.
[82] Wenden B., Kozma-Bognar L., Edwards K.D., Hall A.J., Locke J.C., Millar A.J.,
Light inputs shape the Arabidopsis circadian system, Plant J 66 (2011) 480-491.
[83] Wenkel S., Turck F., Singer K., Gissot L., Le Gourrierec J., Samach A., Coupland
G., CONSTANS and the CCAAT box binding complex share a functionally important
domain and interact to regulate flowering of Arabidopsis, Plant Cell 18 (2006) 29712984.




58




[84] Yanovsky M.J., Izaguirre M., Wagmaister J.A., Gatz C., Jackson S.D., Thomas
B., Casal J.J., Phytochrome A resets the circadian clock and delays tuber formation
under long days in potato, Plant J 23 (2000) 223-232.
[85] Yanovsky M.J., Mazzella M.A., Casal J.J., A quadruple photoreceptor mutant still
keeps track of time, Curr Biol 10 (2000) 1013-1015.
[86] Zhu D., Maier A., Lee J.H., Laubinger S., Saijo Y., Wang H., Qu L.J., Hoecker U.,
Deng X.W., Biochemical characterization of Arabidopsis complexes containing
CONSTITUTIVELY PHOTOMORPHOGENIC1 and SUPPRESSOR OF PHYA proteins
in light control of plant development, Plant Cell 20 (2008) 2307-2323.
[87] Zuo Z., Liu H., Liu B., Liu X., Lin C., Blue light-dependent interaction of CRY2
with SPA1 regulates COP1 activity and floral initiation in Arabidopsis, Curr Biol 21
(2011) 841-847.




59