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A Comparative Expresssional Analysis of a Family of CCA-Like MYB Transcription Factors in Two Higher Plant Species

Permanent Link: http://ufdc.ufl.edu/UFE0021436/00001

Material Information

Title: A Comparative Expresssional Analysis of a Family of CCA-Like MYB Transcription Factors in Two Higher Plant Species
Physical Description: 1 online resource (57 p.)
Language: english
Creator: Sullivan, Meredith L
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: circadian, clock, phylogenetics, poppy, rhythms
Botany -- Dissertations, Academic -- UF
Genre: Botany thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Circadian clocks are ubiquitous among most living species. Since life on Earth originated in the presence of light/dark cycles, organisms had to evolve mechanisms to cope with such environmental fluctuations. An accurate timekeeping apparatus affords an organism with temporal organization of crucial molecular and cellular processes. The components that constitute the central oscillator of the clock vary greatly among plants and animals but the basic architecture appears similar. This resemblance serves as a foundation on which evolutionary-based investigations on the conservation of such machinery can be conducted. In the plant Arabidopsis thaliana, two genes involved in circadian regulation were identified and characterized as members of a family of Myb transcription factors characterized by only one Myb repeat sequence. These genes, CCA1 and LHY, are necessary to maintain rhythmicity in the plant and have been shown to have a role in floral induction. Using the Floral Genome Project (FGP) database of known flowering genes, two EST homologs of CCA1 and LHY were identified in Eschscholzia californica, eca_4_183384 and eca_4_184056 (E1 and E2), based on sequence similarity. These orthologs demonstrate transcript oscillations over a 24hr period with peak levels of expression occurring just prior to dawn. In situ analyses revealed similar patterns of expression in young and older floral tissue of both plant species. In this paper I present molecular evidence that the transcription- translation- based feedback loop mechanism of the circadian oscillator is conserved in these higher plant species and suggest that this mechanism can also be observed in other higher plants.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Meredith L Sullivan.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Oppenheimer, David.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021436:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021436/00001

Material Information

Title: A Comparative Expresssional Analysis of a Family of CCA-Like MYB Transcription Factors in Two Higher Plant Species
Physical Description: 1 online resource (57 p.)
Language: english
Creator: Sullivan, Meredith L
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: circadian, clock, phylogenetics, poppy, rhythms
Botany -- Dissertations, Academic -- UF
Genre: Botany thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Circadian clocks are ubiquitous among most living species. Since life on Earth originated in the presence of light/dark cycles, organisms had to evolve mechanisms to cope with such environmental fluctuations. An accurate timekeeping apparatus affords an organism with temporal organization of crucial molecular and cellular processes. The components that constitute the central oscillator of the clock vary greatly among plants and animals but the basic architecture appears similar. This resemblance serves as a foundation on which evolutionary-based investigations on the conservation of such machinery can be conducted. In the plant Arabidopsis thaliana, two genes involved in circadian regulation were identified and characterized as members of a family of Myb transcription factors characterized by only one Myb repeat sequence. These genes, CCA1 and LHY, are necessary to maintain rhythmicity in the plant and have been shown to have a role in floral induction. Using the Floral Genome Project (FGP) database of known flowering genes, two EST homologs of CCA1 and LHY were identified in Eschscholzia californica, eca_4_183384 and eca_4_184056 (E1 and E2), based on sequence similarity. These orthologs demonstrate transcript oscillations over a 24hr period with peak levels of expression occurring just prior to dawn. In situ analyses revealed similar patterns of expression in young and older floral tissue of both plant species. In this paper I present molecular evidence that the transcription- translation- based feedback loop mechanism of the circadian oscillator is conserved in these higher plant species and suggest that this mechanism can also be observed in other higher plants.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Meredith L Sullivan.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Oppenheimer, David.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021436:00001


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b08a7b7a69f8e4180b9a731c34aa17a8
e662a2e6aabe94b73137736e09034b6109496fcb







A COMPARATIVE EXPRESSIONAL ANALYSIS OF A FAMILY OF CCA-LIKE MYB
TRANSCRIPTION FACTORS INT TWO HIGHER PLANT SPECIES



















by

MEREDITH L. SULLIVAN


A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF
FLORIDA INT PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2007
































O 2007 Meredith L. Sullivan

































To my parents with love and respect









ACKNOWLEDGMENTS

Most of all, I thank my family for their endless love and support. They are the pillars of my

strength. I also thank my advisor, Dr. David Oppenheimer for affording me the opportunity to

attend the University of Florida. I also express gratitude to Dr. Bernard Hauser for his support of

this work. A special thanks to Zhengui Zheng for his time and assistance with this proj ect. Also I

thank Xiaoguo Zhang and Stacey Jeffries for the invaluable advice they offered and the support

they provided me throughout my time at UF. Finally I extend special appreciation to the love of

my life, Brad for his continued encouragement.












TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. ...............4.....


LI ST OF FIGURE S .............. ...............6.....


AB S TRAC T ......_ ................. ............_........7


CHAPTER


1 LITERATURE REVIEW ................. ...............9...............


Biological Rhythms .............. ...............9.....
The Circadian Clock ................. ...............10...............
Clock- Controlled Genes .............. ...............13....
The Arabidopsis Central Oscillator ................. ...............18.......... ....
Evolution of Core Clock Components ................. ...............21........... ...

2 TWO CCA-LIKE MYB TRANSCRIPTION FACTORS ARE PRESENT INT
CALIFORNIA POPPY ................. ...............27.................


Summary ................. ...............27.................
Introducti on ................. ...............28.................
Materials and Methods .............. ...............28....
Plant Growth Conditions .............. ...............28....
In situ Hybridizations .............. ...............30....
R e sults................ ....... .. .... ................ ........ ... .. .........3
RISE and SHINE Encode CCAl-like Myb Transcription Factors ................. ...............32
RISE and SHINE Expression is Under Circadian Control ................. ......................32
D discussion ................... ........ ... ..... ....... .. ....... .. .. ...........3
Feedback Loop Mechanism as the Basis of the Circadian Oscillator .................. ...........34
The RISE and SHINE Genes Encode MYB Transcription Factors That Are Similar
to LHY and CCAl ................ .. .............. ..........3
The Biological Importance of Circadian Clock Genes............... ...............38.

3 CONCLU SION................ ..............4


LI ST OF REFERENCE S ................. ...............50................


BIOGRAPHICAL SKETCH .............. ...............57....










LIST OF FIGURES


Figure page

1-1 The three basic components of the circadian clock. ........................ ........_.......25

1-2 Positive and negative factors act upon the circadian clock. ........._. ...... .._._...........25

1-3 Comparisons of three conserved regions of LHY and CCAl ................. .....................26

1-4 The Arabidopsis circadian oscillator. ............. ...............26.....

2-1 Alignment of the LHY/CCAl genes in Arabidopsis thaliana and Eschscholzia
californica. .............. ...............41....

2-2 A phylogenetic analysis of CCA-like family of genes. ................ ......... ...............42

2-3 Alignment of the RISE (eca_4_183 384) and SHINE (eca_4_184056) EST sequences
using the BLASTN program from the FGP database (Albert et al, 2005; Carlson et
al., 2006). ............. ...............43.....

2-4 Analysis of RISE and SHINE mRNA expression. ............. ...............44.....

2-5 Analysi s of LHY and CCA 1 mRNA expression ................. ...............45.............

2-6 Expression of LHY and CCA1 in Arabidopsis tissue .................... ............... 4

2-7 RISE and SHINE transcripts are expressed in both young and mature floral tissue of
the California poppy plant. ............. ...............47.....

2-8 Proposed mechanism of the central oscillator of Eschscholzia californica. ................... ...48









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

A COMPARATIVE EXPRESSIONAL ANALYSIS OF A FAMILY OF CCA-LIKE MYB
TRANSCRIPTION FACTORS INT TWO HIGHER PLANT SPECIES

By

Meredith L. Sullivan

August 2007

Chair: David Oppenheimer
Major: Botany

Circadian clocks are ubiquitous among most living species. Since life on Earth originated

in the presence of light/dark cycles, organisms had to evolve mechanisms to cope with such

environmental fluctuations. An accurate timekeeping apparatus affords an organism with

temporal organization of crucial molecular and cellular processes. The components that

constitute the central oscillator of the clock vary greatly among plants and animals but the basic

architecture appears similar. This resemblance serves as a foundation on which evolutionary-

based investigations on the conservation of such machinery can be conducted. In the plant

Arabidopsis thaliana, two genes involved in circadian regulation were identified and

characterized as members of a family of Myb transcription factors characterized by only one

Myb repeat sequence. These genes, CCA1 and LHY, are necessary to maintain rhythmicity in the

plant and have been shown to have a role in floral induction. Using the Floral Genome Proj ect

(FGP) database of known flowering genes, two EST homologs of CCAl and LHY were

identified in Eschscholzia californica, eca_4_183384 and eca_4_184056 (El and E2), based on

sequence similarity. These orthologs demonstrate transcript oscillations over a 24hr period with

peak levels of expression occurring just prior to dawn. hz situ analyses revealed similar patterns

of expression in young and older floral tissue of both plant species. In this paper I present









molecular evidence that the transcription- translation- based feedback loop mechanism of the

circadian oscillator is conserved in these higher plant species and suggest that this mechanism

can also be observe din other higher plants.









CHAPTER 1
LITERATURE REVIEW

Biological Rhythms

The daily rotation of the earth leads to periodic fluctuations in environmental conditions.

Because life on Earth originated in the presence of a cyclical environment, many organisms have

evolved timing mechanisms to organize important events. In order to adapt to the changes

presented by the environment, organisms must modulate their behaviors with the daily cycles of

light and temperature variation, and establish an effective method for tracking time. Since the

environment imposes a period of approximately 24 hours, organisms with rhythmic behavior that

matches these oscillations have higher fitness than those that do not.

The first noted rhythmic behavior occurred in the fourth century BC when the sleep

movements of the tamarind tree were noted by Androsthenes, a Greek philosopher

(Chandrashekaran, 1998). In the mid-1700s, a French astronomer named Jean- Jacques d'Ortous

de Mairan recorded the daily leaf movements of the M~imosa pudica plant and demonstrated that

the rhythms persisted for several days when the plant was subj ected to complete darkness

(Golden and Strayer, 2001; Sweeney, 1987). This was the first evidence that the rhythmic

behavior must be endogenous and that the rhythmicity, once established, continues in the

absence of environmental cues. Erwiin Bunning's work during the 1930s, in which he

demonstrated a rhythm in the eclosion behavior of the fruit fly Drosophila, further supported the

idea of an endogenous timekeeping mechanism. Since the rhythms occurred with a 24 hour

period, the term circadian was coined from the Latin words circa and dias, meaning "about a

day" (Chandrashekaran, 1998; Halberg, 1959).

Circadian rhythms are variations in physiological and behavioral activities that occur over

a period of about 24 hours (Hardin, 2000). In the context of a biological process, the time









interval between two successive events is described as a biological 'rhythm' (Kumar, 2002).

Biological rhythms are found in almost all living organisms. They have been described

extensively in mammals, insects, fungi, plants and bacteria (Dunlap et al., 1999; McClung, 2001;

Ouyang et al., 1998). These rhythms occur over a large range of time scales: from millisecond

oscillations to seasonal changes. The ubiquity of circadian rhythmicity across a broad taxonomic

spectrum suggests that adaptive fitness is enhanced by the synchronization of certain events with

the diurnal cycle imposed by the environment (McClung 2000).

In a number of different species, a plethora of activities are regulated by circadian rhythms.

Cyanobacteria demonstrate daily oscillations in nitrogenase activity, photosynthesis and

metabolic activities (Kondo and Ishiura, 1999). In vertebrates, a number of behavioral and life

processes, ranging from the molecular level to the cellular and systemic levels, are driven by

circadian rhythms including eating, sleeping, seasonal migration and cell proliferation (Gillette

and Sejnowski, 2005). Similarly, a variety of events such as leaf movement, stomatal opening

and regulation of flowering time and fragrance emission are tightly regulated by the plant

timekeeper. In other organisms, such as Neurospora and Drosophila, development, cell

signaling and stress responses with self-sustaining rhythms can be regulated by the circadian

clock. The role that diurnal rhythms play in a number of different activities suggest that an

endogenous timekeeping system provides an adaptive advantage, enabling the anticipation of

environmental change and the coordination of crucial events to occur at specific phase

relationships with the environment (Mas, 2005).

The Circadian Clock

Circadian or biological rhythms, although they parallel the environmental cycles of light

and dark, are generated within an organism by a complex timekeeping system (Hardin, 2000).

Many organisms have evolved an endogenous 'chronometer', the circadian clock, to temporally









coordinate important processes with the daily variations in the environment. Circadian rhythm of

gene expression has been shown to function as the underlying mechanism of the clock (Wang

and Tobin, 1998).

Two important attributes of the circadian clock, entrainment and temperature

compensation, ensure synchrony between important rhythmic activities and the surroundings of

an organism. The first, entrainment, is the manner in which the clocks are set to 'local' time by

environmental cues such as the light / dark cycles. The second, temperature compensation,

describes the ability of the clock to run at the same rate independent of temperature changes

(Hardin 2000).

The notion that biological rhythms are created within an organism stimulated interest in the

mechanism that maintains these oscillatory patterns. In theory, numerous regulatory schemes

could achieve such fluctuations. The most common method involves a regulatory circuit with a

positive and negative product (Fig. 1-2). The negative element feeds back to slow down the rate

of the process itself and creates a delay in the execution of the feedback. A positive element is

required to activate the clock and prevent it from winding down (Dunlap et al., 1999). The

generally conserved clock mechanism consists of an autoregulatory feedback loop in which

positive factors act on genes encoding negative factors that in turn feedback to inhibit their own

expression (Strayer et al., 2000; Dunlap, 1999). A circadian system often consists of one or more

interconnected feedback loops.

The circadian system is divided into three main components: input pathways, a central

oscillator and output pathways (Fig. 1-1). Input pathways, also known as entrainment pathways,

transmit environmental signals to the central timekeeping apparatus. In plants, this signal

transduction pathway adjusts the clock in response to external cues most frequently through the









action of cryptochromes and phytochromes. The entrainment of circadian clocks to the light/dark

cycles is usually mediated by light-induced changes in the level of a component of the oscillatory

feedback loop. The ability to re-entrain the clock ensures synchrony with the environment and

allows the anticipation of dawn and dusk (Devlin, 2002).

The core timekeeping component of the circadian clock is the central oscillator (Hardin,

2000). It serves as the system's pacemaker and is responsible for generating circadian rhythms.

In order for the oscillator to function in the absence of environmental cues it must be

synchronized with the external time via the input pathways. Environmental transitions between

dawn and dusk help to adjust the endogenous period created by the oscillator to precisely match

the 24- hour period found in nature (Mas, 2005).

Completing the circadian clock model are the output pathways which provide a link

between the central oscillator and the rhythmic physiological responses they control. These

pathways are activated at specific times of the circadian cycle and the outputs are in phase with

the oscillation of light cycles (Dodd et al., 2005). In both animals and plants, there are a

substantial number of physiological and metabolic processes that are regulated by the circadian

outputs. These events include such varied responses such as olfactory responses in Drosophila,

leaf movements and hypocotyl growth in plants, and enzyme activity. The rhythmic behaviors

that are generated in response to day length are known as photoperiodic responses (Schultz and

Kay, 2003).

The discovery of components of the circadian oscillator has enabled scientists to

concentrate on the mechanisms that interconnect the three components to form an effective

timekeeping system. Recent studies of several model organisms including Drosophila,

Neurospora and mice have revealed a common molecular mechanism at the center of the









circadian oscillator (Barak et al., 2000). Clock proteins, encoded by clock genes, serve as

negative elements that repress their own expression by blocking their transcriptional activators,

or positive elements (Fig. 1-2). A decrease in clock transcripts and proteins results in the de-

repression of the transcriptional activators and thus reinitiates the cycle.

Clock- Controlled Genes

The identification of genes which function at the center of the clock machinery has rapidly

increased over the last decades. These clock-controlled genes, or ccgs, encode pieces of the

central oscillator. Their products produce and maintain the oscillations that drive other circadian

rhythms. A substantial number of clock-associated genes whose expression relies on the rhythms

generated by the oscillator have been identified in the genomes of many organisms. Recent

microarray analyses of the model plant Arabidopsis indicated that up to 6% of the genes are

rhythmically expressed (Harmer et al., 2000; Schaffer et al., 2001).

Components of the biochemical feedback loop whose rhythmic activity is required for

oscillator function are referred to as state variables. A number of criteria have been defined to

identify and characterize state variables of the circadian system (Aronson et al., 1994; Kay and

Millar, 1995). First, the component itself must demonstrate circadian oscillations in its activity

and expression. The second criterion requires the component to control its own levels by

feedback inhibition of its synthesis. Additionally, clamping of the amount of the putative

oscillator component at any level from null to high stops the clock and thus rhythmicity. The

final criterion states that induced transient perturbations in the abundance of the component

should cause a phase shift in the clock output. These criteria are applied in the investigations of

putative oscillatory components to assign positive and negative roles to the elements. Knowledge

of interconnected loops is obtained from the identification through classical genetics of genes

that are within one of the core loops of the oscillator (Roenneberg and Merrow, 1998).









The molecular basis of circadian clocks is best understood in Neurospora and Drosophila.

The genetics of rhythms originated in these two organisms and much of what scientists have

learned about how mammalian clocks operate closely parallels the behavior of one or both of

these species. In each of these model systems, genetic screens for rhythm mutants that affect the

period length of the clock or abolish its activities were found (Hardin and Siwicki, 1995; Dunlap,

1996).

The first clock mutations to be discovered were the period (per) mutant from Drosophila

and the frequency (~frq) mutant from Neurospora (Dunlap, 1993; Konopka and Benzer, 1971;

Feldman and Hoyle, 1973). The PER gene product, as well as its transcript, oscillates with a

circadian rhythm and the PER protein was required for a feedback regulation of its own gene

products (Williams and Sehgal, 2001). Oscillations of PER transcripts and the protein they

encode persisted in continuous dark conditions, suggesting that the gene must be under the

control of the circadian clock. The phases of the per messenger RNA (mRNA) and protein

rhythms were noticed to be quite distinct; PER protein level peaked in abundance approximately

six hours after the peak in PER mRNA levels. This difference accounts for the delay or lag time

in oscillations required by the central timekeeper. In addition, mutant per fruit flies exhibited

altered rhythmic behavior, which suggests a role for this gene in the oscillator. The tint mutant

was identified in a screen for recessive mutants that affected the eclosion behavior of the fly.

Similar to the per mutant, the tint mutants exhibited arrhythmic behaviors, periods that were

shortened or lengthened, and in some mutant alleles, PER expression was dampened (Allada et

al., 2001).

PER contains a protein interaction domain known as a PAS domain, which enables the

protein to interact with the second Drosophila circadian gene identified, timeless (tint). PER and









TIM form a heterodimer that serves as part of a feedback loop to inhibit per and tim

transcription. In return, the PER-TIM heterodimer activates a gene called Drosophila Clock

(dClk), a transcription factor that is also rhythmically expressed (Scully and Kay, 2000). dCLK

interacts with another transcription factor, CYCLE (CYC) and together the two form a

heterodimer complex that is required for the activation of PER and TI2~transcription. The

dCLK-CYC complex binds to a specific sequence in PER and TI2~promoters known as the E-

box motif, a consensus hexanucleotide sequence found in basic helix-loop-helix (bHLH)

transcriptional factors (Fairman et al., 1993). Binding of the complex to the E-box motif allows

transcription of PER and TIM~ and results in a increase in PER-TIM heterodimers in the

cytoplasm. When expression is high, usually in the evening, these complexes move into the

nucleus where PER binds to the dCLK-CYC complexes. This releases the dCLK and CYC

proteins from the promoters and results in the shutting off of PER and TI2~transcription. As the

levels of PER and TIM decline, the dCLK-CYC dimers are released and thus the dCLK-CYC-

dependent repression of dClk expression is lifted. As the levels of dCLK begin to rise, usually in

the morning, an increased number of dCLK-CYC complexes are formed and per and tim

transcription is re-activated (Glossop et al., 1999). Since the products of the per gene and the

dClk gene inhibit their own synthesis it appears that the Drosophila oscillator consists of two

interconnected negative feedback loops: a per-tim loop and a dClk loop (Glossop et al., 1999). In

this model PER and TIM serve as the negative elements while dCLK and CYC serve as the

positive elements (Dunlap et al., 1999).

The second timekeeping component identified through forward genetic screens for clock

mutants was the FREQUENCY (FRQ) gene in the fungus Neurospora cra~ssa. Mutations of this

gene resulted in arrhythmic expressions, altered periodicity and deficiencies in temperature









compensation (Aronson et al., 1994). The FRQ RNA and protein levels cycle with a circadian

rhythm and the protein negatively regulates its own transcript resulting in a feedback loop similar

to observed in Drosophila (Dunlap, 1996). The expression of FRQ is activated by two PAS-

domain-containing transcription factors, WHITE COLLAR 1 (WC-1) and WHITE COLLAR-2

(WC-2) that form the white collar complex (WCC), which bind to circadian photoreceptor

connecting light signals and the oscillator (He et al., 2002). After the accumulation of FRQ, the

proteins begin to dimerize, enter the nucleus and interact with the WCC diminishing its activity

and dampening FRQ expression (Froelich et al., 2003). FRQ also promotes the synthesis of WC-

1 increasing the level of WCC. This results in a mass of WCC that is held inactive by FRQ until

the protein is phosphorylated and targeted for ubiquitination, accounting for the delay that is

required by the circadi an oscillator. Therefore Neurospora has a transcript onal/tran sl ati onal

negative feedback loop at the core of its oscillator with WC-1 and WC-2 acting as the positive

elements and FRQ as the negative (Dunlap et al., 1999). Recent evidence suggests that this FRQ-

based oscillator might work in cooperation with other oscillators within the organism as well

(Correa et al., 2003).

Over recent decades, numerous advances in understanding the mechanisms underlying the

biological oscillator in mammals have been made. The first cloned mammalian clock component

identified by forward genetics was the CLOCK (CLK) gene of2~us musculus, the mouse (Antoch

et al., 1997). Like transcription factor proteins that are central to the clock in other organisms, the

mouse Clock gene contains 1) a PAS domain, 2) its levels of mRNA and proteins oscillate, and

3) in clk mutant mice, the cyclic expression the mPeriod (mPer) homolog is reduced. These data

suggest that CLOCK, as a member of the oscillator, controls the transcription of circadian genes

(Gekakis et al., 1998). It was shown that CLOCK binds to another transcription factor, BMAL1









(Brain and Muscle Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT)-Likel) and this

complex activates the transcription of the PER and CRYPTOCHROM~E (CRY) genes. The mPER

and CRY proteins form heterodimers and homodimers and upon translocation to the nucleus,

they inhibit the activity of the CLOCK-BMAL 1 complex, which in turn suppresses PER and

CRY transcription (Panda et al., 2002). Once the mPER and CRY proteins are phosphorylated,

they are targeted for degradation and transcriptional repression is relieved. Although other

oscillators might be present in the mouse, central to its core oscillator is the negative feedback

loop in which the negative elements, the PER homologs repress the activation of the positive

elements CLOCK and BMAL1 (Dunlap, 1999).

Circadian rhythms, once only thought to be a feature of eukaryotic organisms, have

recently been identified in some prokaryotes. The cyanobacteria Synechococcus elongatus serves

as the model system for molecular investigations of this group. Approximately one hundred

clock mutants identified from an ethylmethansulfonate (EMS) mutagenesis screen that were

characterized by arrhythmia, atypical periods and some mutants that could be rescued by the

introduction of wild-type DNA from a Synechococcus genomic library (Lorne et al., 2000). One

cluster of DNA fragments that could be rescued represented the kai cluster of genes: kaiA, kaiB

and kaiC. Transcribed from two different promoters, PkaiA and PkaiBC, a monocistronic kaiA

mRNA and a dicistronic kaiBC mRNA are produced and both transcripts cycle in abundance.

Overexpression studies revealed that KaiC represses the activation of the PkaiBC promoter while

KaiA enhances PKaiBC transcription (Ishiura et al., 1998). Since KaiC represses its own

transcription it functions as the negative element of the negative feedback loop at the core of the

Synechococcus elongatus oscillator while KaiA, which helps drive expression from PkaiBC,

functions as the positive element. A role for KaiB has yet to be determined.









Although circadian rhythms were first observed in photosynthetic organisms, the

molecular mechanisms underlying the circadian oscillator in plants have been difficult to

elucidate. Much of what we know about circadian rhythms has come from the studies of animal

systems. Over the last decade there has been substantial effort put into identifying and

understanding the roles of oscillator genes in the model plant Arabidopsis and in a few other

plant species. Plant researchers are working to determine if a mechanism similar to those

observed in other organisms is conserved among members of the plant family.

The Arabidopsis Central Oscillator

The molecular basis of circadian rhythms has been thoroughly studied in Drosophila,

Neurospora, mice and cyanobacteria model systems. The common denominator among these

organisms is a biological clock based on a central oscillator that uses transcriptional feedback

loops to generate a circadian oscillator with a 24 hour period that regulates circadian outputs

(Dunlap, 1999). The oscillator responds to environmental signals through input pathways, which

entrain the clock, and controls output pathways that generate a rhythm in phenotype or

biochemical pathway Mizoguchi et al., 2002). Although some of the first recorded circadian

rhythms were identified in plants, the molecular mechanisms underlying these rhythms have

remained unclear until the last decade. Advances in the identification and characterization of

plant circadian components have been made primarily through genetic studies of Arabidopsis

thaliana.

The first Arabidopsis clock mutant was identified by fusing a luciferase marker gene to the

CAB2 (chloropyhlla/b binding) promoter to investigate clock-regulated gene expression in

different populations. The result was the identification of a mutant, timing of CAB (toc) that

altered the period of the clock (Millar et al., 1995). The TOC1 protein was shown to contain a

pseudo-response regulator motif, similar to those in two-component signal transduction









pathways, at its amino terminus and a CONSTANS (CO)-like motif at its carboxyl terminus

(Strayer et al., 2000). Interestingly the CO family represents a group of plant transcription factors

that are involved in flowering response (Putterill et al., 1995). Since the TOC1 protein shows

similarity to the CO family, TOC1 might play a role in flowering as well. The levels of TOC1

mRNA cycled in light-dark conditions and peak levels of transcript were observed late in the day

while minimal levels were observed at dawn. In addition, since the tocl-1 mutant was

characterized by a circadian oscillator with a shortened period which demonstrated that TOC1

products reduce their own expression (Strayer et al., 2000). Based on the observed data, TOC1

appears to be a component of the central circadian oscillator of Arabidopsis.

A second potential Arabidopsis clock component was identified as a result of the

identification of a day length-insensitive flowering mutant. The mutant late elongated hypocotyl

(lhy) caused an elongated hypocotyl and reduced chlorophyll, as well as an altered flowering

phenotype (Schaffer, 1997). lhy mutants were also arrhythmic for leaf movements and for the

expression of several other clock-regulated genes. Rhythmic expression of LHY was observed

with levels of the transcript peaking at dawn.The sequence of the LHY protein was used to

screen the GenBank database using the TBLASTN program to identify any potential homologs

in the plant. The Arabidopsis DNA-binding protein CIRCADIAN CLOCK AS SOCIATED-1

(CCAl) was most closely related to LHY (Schaffer et al., 1998) CCAl was first identified as a

factor that binds to the promoter of the Chlorophyll a b-binding light-harvesting complex

(LHCB) gene in Arabidopsis and functions in the phytochrome signaling pathway to induce the

transcription of LHCB (Wang et al., 1997). Later studies revealed that ccal mutants display a

shorter period of circadian rhythms. Overexpression of this protein disrupted rhythmicity in

several clock outputs including hypocotyl elongation, leaf movements and circadian gene









expression (Green and Tobin, 1999; Wang and Tobin, 1998). CCA1 transcripts also oscillate

with peak levels of expression early in the morning and in constant conditions the rhythms

persist suggesting this gene is under circadian control (Wang and Tobin, 1998). In lhy ccal

double mutants circadian rhythms were observed with an abnormal phase and oscillations of

transcripts were dampened. Early expression ofLHY and CCA1 (morning genes) and some

evening genes were also observed in the double mutants suggesting that these two genes function

as components of a negative feedback loop (Schaffer et al., 1998).

LHY and CCA1 genes function redundantly and are required for the maintenance of

circadian rhythms in Arabidopsis (Alabadi et al., 2002). Both genes are closely related MYB-like

transcription factors but are unique in that they only possess a single MYB repeat sequence

whereas other myb transcription factors usually contain two to three of the motifs. LHY and

CCAl are also related outside of the MYB domain sharing other regions that exhibit at least 80%

identity (Fig. 1-3). Overall, the two genes are 46% identical to one another (Schaffer et al.,

1998). This sequence analysis suggests that LHY and CCAl encode related DNA-binding

proteins with a single MYB repeat that function as transcription factors. This notion is supported

by evidence that shows LHY and CCAl bind specifically to a sequence known as the 'evening

element' (EE) in the promoter of many genes whose expression peaks nears dusk (Alabadi et al.,

2001; Harmer et al., 2000).

In Arabidopsis, the model for the plant clock components is based on the regulation of the

three plant genes described above, CCA1, LHY and TOC1. These components appear to operate

in a transcriptional/translational-based negative feedback loop similar to that observed in other

studied systems. Light activation of LHY and CCA1 expression results in transcript levels that

peak at dawn followed by a peak in proteins approximately two hours later. Both of the proteins









bind to the EE motif located in the promoter of the TOC1 gene, a positive element, and repress

TOC1 expression during the day (Alabadi et al., 2001). A drop in TOC1 protein results in the

reduction of LHY and CCA1 transcript and protein levels. The low levels of expression of the

two genes results in the derepression of TOC1 transcription. In return, levels of TOC1 protein

peak during the late evening resulting in the activation of LHY and CCA1 transcription just prior

to dawn (Carre and Kim, 2002). This cross-regulation between LHY, CCAl and TOC1 is

proposed to function as the central oscillator of the Arabidopsis clockwork where LHY and

CCAl function as the negative elements and TOC1 serves as the positive element (Fig. 1-4).

This central clockwork regulates numerous genes in Arabidopsis responsible for photosynthesis,

nitrogen assimilation, biosynthesis of photo-protective pigments, lipid modification, hypocotyl

elongation and flowering (Harmer et al., 2000; Schaffer et al., 2001; Mas, 2005).

Evolution of Core Clock Components

The circadian clock has been well characterized in organisms from cyanobacteria to fungi,

mice and plants. In these organisms, the central oscillator measures time with a molecular

feedback loop or loops that cycle with a 24-hr period (Dunlap, 1999). The central timekeeper

generates rhythms by controlling transcription of numerous clock genes. Regulation of the

feedback loop is based on negative elements, which repress their own expression and positive

elements that stimulate transcription. The negative feedback along with a delay is sufficient to

produce oscillations. Although the basic architecture of the circadian oscillator appears

conserved among different species, the mechanisms at the core of the feedback loop differ.

Biological clocks have either evolved multiple times to perform similar tasks thus they are an

example of convergent evolution.

The use of positive and negative elements to regulate transcriptional and translational

activity in a feedback loop is common among the well-studied circadian systems (Fig. 1-6). The










positive elements serve as the transcriptional activators in the loop and they have been found in

Synecohcoccus (kaiA), Neurospora (WC-1 and WC-2), Drosophila (CLK and CYCLE),

mammals (CLOCK and BMAL 1) and Arabidopsis (TOC1). Similarly, negative elements also

compose a portion of the feedback mechanism by inhibiting the action of the positive elements

and these include kaiC, FRQ, PER and TIM, PER and CRY and CCAl and LHY in

cyanobacteria, fungi, fruit flies, mice and plants, respectively (Dunlap et al., 1999). Yet despite

their similarities, the time at which these elements are expressed, late in the evening or early

morning, differs among the organisms.

Transcription factor proteins serve important roles in the circadian oscillatory system. The

type of transcriptional inducer varies among model systems. In mammals and Drosophila, the

activators are basic helix loop helix (bHLH) proteins which contain a specific region which binds

to DNA (Gekakis et al., 1998; Darlington et al., 1998). The Neurospora positive elements are

similar to the Drosophila complex but they contain an additional zinc Einger binding domain thus

they are categorized as zinc finger factors (Loros and Dunlap, 2001). In the plant circadian

oscillator, the activation of transcription is induced by MYB-like transcription factors (Carre and

Kim, 2002). The oscillatory mechanism of cyanobacteria is still under investigation.

Another difference between the circadian machinery of different organisms is the number

and location of oscillators. Mammals have a master circadian pacemaker that is localized to the

suprachiasmatic nucleus (SCN) located within the hypothalamus of the brain. The SCN entrains

multiple clocks that are located in the periphery of the organism (Yamazaki et al., 2000). In

contrast, plants contain at least one oscillator in each cell and these oscillators function

autonomously and independently of any central pacemaker (Thain et al., 2000; Barak et al.,










2000). This organization makes it is possible to set different rhythms of gene expression to

different phases in varying parts of a single plant or organ.

The general organization of the circadian apparatus suggests a selective advantage in the

rhythmic control of physiological and behavioral processes. The ubiquity of the system implies

that the endogenous circadian programs enhance fitness. Evidence from cyanobacteria suggests

that an organism with a circadian rhythm close to that of its external environment is favored

under competition as a result of soft selection (Futuyama, 1998). Circadian clocks are also

important since they provide a timing mechanism required for the response of organisms to daily

and seasonal changes in light. Temporal organization of processes such as those involving photo-

labile enzymes in plants is crucial for the optimization of important endogenous events. The

close connection between the clock and light signaling pathways allow an organism to predict

environmental changes even in their absence. Thus, the circadian clock provides an adaptive

advantage by enabling the anticipation of the external transitions and the temporal

synchronization of physiological events with specific phases of the environment (Johnson,

2001).

Since a number of discrepancies exist between the circadian components of different

systems, the next phase of chronobiology concentrates on elucidating the molecular mechanisms

that underlie the oscillator in other species. The identification of similar elements allows insight

into the evolutionary lineage of the clock apparatus as well as the resulting rhythmic outputs

while the differences between systems provide relevant information on species-specific

adaptations. From this data, it is possible to create evolutionary relationships between specific

clock-controlled genes across numerous taxa. One such proj ect, the Floral Genome Proj ect

(FGP) examines gene families in a number of different plant species that play a role in the









evolution of flowering (Albert et al., 2005). One identified family of genes involved in floral

initiation was shown to be similar to the LHY and CCAl transcription factors of the Arabidopsis

central oscillator.

Genetic and molecular analyses have proven valuable tools in the elucidating the central

oscillatory mechanism of circadian clocks. In this study, two EST homologs to LHY and CCA1,

the MYB-like family of transcription factors in Arabidopsis, are identified in Eschscholzia

californica, the California poppy plant. Expressional analyses suggest that these genes are

rhythmic components of the circadian oscillator that participate in the initiation of flowering.

This suggests that the feedback loop mechanism of the plant circadian oscillator is conserved in

these two species.






















INPUT OSCILLAsTOR


Th central
pacmaar that
genra~tes
ascillations.


pathwvay
that
rest the
clock.


OUTPUT

The pathwvay
responsble
for
beh~avioral
and
physio~~loil
rhy~thms.


Figure 1-1. The three basic components of the circadian clock. Numerous input and output
pathways function within the system.



NEGATIVE


CG


POSITIVE|


Figure 1-2. Positive and negative factors act upon the circadian clock. Negative elements block
the activation of the positive elements (blunted arrow) which in turn promotes the
expression of the negative element (pointed arrow).




















Figure 1-3. Comparisons of three conserved regions of LHY and CCAl. The number at the end
of each row corresponds to the last amino acid shown within the original protein.
Conserved amino acids are highlighted.


DAY Meiong~ cenes


Figure 1-4. The Arabidopsis circadian oscillator. Light activates expression of LHY and CCA1
inducing their transcription near dawn. Their gene product binds to the TOC1
promoter and inhibits its expression during the early day. As LHY and CCAl
expression dwindles, the repression of TOC1 is lifted and TOC1 protein accumulates
in the evening. TOC1 then induces the expression of LHY and CCA1 starting the
cycle over again.









CHAPTER 2
TWO CCA-LIKE MYB TRANSCRIPTION FACTORS ARE PRESENT INT CALIFORNIA
POPPY

Summary

Biological clocks play an important role in the lives of many organisms. This machinery

allows species to coordinate important activities or behaviors in relation to their environment.

The clock generates rhythms over which these events operate with a period of approximately

twenty- four hours, thus the term circadian rhythms. The clock is primarily composed of three

main components: input pathways, a central oscillator and output pathways.

An analysis was conducted to determine if two circadian clock genes found in the

California poppy plant, RISE and SHINE, are related to two transcription factors involved in

maintaining circadian rhythms in Arabidopsis, LHY and CCAl. In Arabidopsis, these two genes

function in a feedback loop of the central circadian oscillator and are crucial for maintaining

rhythms within the organism. A comparison of the genomic sequences revealed that there was an

acceptable degree of homology between the two sequences. In addition, an expressional analysis

revealed that the levels of messenger RNA (mRNA) of the genes oscillated over a twenty- four

hour period which suggests circadian control. The location of expression was also similar

between the two plant species. In young tissue, the transcripts were localized to the meristematic

regions as well as the premature leaves. In older tissue, expression was highest in the

reproductive organs and pollen grains. Since the circadian clock plays a role in promoting

flowering and the release of pollen, it is no surprise the transcripts were localized to these

various regions.

Due to these similarities this paper proposes that homologs of LHY and CCAl exist in the

California poppy plant and function as critical components of the negative feedback loop at the

center of the circadian oscillator. Additional potential homologs of LHY and CCAl have been










identified in other species based on sequence similarity. This discovery suggests that the

circadian machinery is conserved among higher plants. In addition, temporal organization of

important events seems to confer a selective advantage for the organisms.

Introduction

Biological clocks are important in maintaining the rhythmicity of crucial events in

different species. In Arabidopsis thaliana, two genes, LHY and CCA1 play important roles in

generating and maintaining the rhythms within the organism (Schaffer, 1997; Schaffer et al.,

1998; Wang et al., 1997). They have been identified as components of the central oscillator of

the clock, one of its three core components. LHY and CCAl function in a feedback loop, along

with the TOC1 gene, in which they negatively regulate their own expression (Alabadi et al.,

2001; Carre and Kim, 2002). The pauses that occur as a result of this feedback loop are efficient

for generating the observed oscillations. These oscillations, in turn, are conveyed as changes in

the organisms' physiological or behavioral changes via the output pathway.

Two potential homologs of the LHY and CCA1 genes were identified in Eschscholzia

californica on the FGP database (Albert et al., 2005). These two EST sequences could

potentially serve as components of a central oscillator in California poppy. Designated as RISE

and SHINE, these genes might be the functional equivalents of LHY and CCA1. This paper

reveals that these genes show an acceptable degree of sequence homology and share similar

expressional patterns suggesting they are components of the central oscillator in Eschscholzia

californica.

Materials and Methods

Plant Growth Conditions

Arabidopsis thaliana and Eschscholzia californica plants were grown under ideal

temperate conditions in the University of Florida Department of Botany greenhouse in









Gainesville, Florida between March and July 2004 and June through September of 2005.

California poppy was grown on sterile soil under normal light conditions with a period that

matched the exogenous environment. Arabidopsis was sowed on autoclaved soil at an irradiance

of 100 Clmol m 2 S 1 as recommended by Kranz and Kirchheim (1987), and the day length was set

so that it matched the period of the environment. Tissue for in situ hybridizations was harvested

mid-morning and included floral meristems and floral buds. The collected material was placed in

4% paraformaldehyde to prepare for fixation. For RT-PCR analysis, plant tissue consisting of

small leaves, floral meristems and buds was collected from each species every four hours for

three days. Each sample was placed in liquid nitrogen and stored in a -800C freezer.

Sequence Analysis and DNA Isolation

The CCA1 and LHY genomic sequences, identified as genes At2g46830O and Atl g01060,

were identified in the Floral Genome Proj ect (FGP) database (Albert et al, 2005; Carlson et al.,

2006) and recognized as a distinct family of transcription genes associated with the circadian

clock. The sequences were used to search for homologs in other plant species associated with the

FGP database using the site's BLAST program and a number of candidate genes were identified.

In Eschscholzia californica (California poppy) two ESTs or expressed sequence tags, which are

small fragments of genes that have been cloned, demonstrated a notable level of similarity to the

Arabidopsis genes. The FGP identification numbers for these two sequences are eca_4_183384

and eca 4 184056 which I will refer to as RISE and SHINE respectively. Alignments of the

cDNA and protein sequences of CCA1, LHY, RISE and SHINE were constructed using the

GenomeNet database program CLUSTALW (Thompson et al., 1994)

Clones of the RISE and SHINE sequences were received from Penn State University, a

participant in the FGP grant. Their preparation has been described previously (Carlson et al.,









2006). Luria-Bertani (LB) media was prepared and 2.5 mL cultures were prepared with

ampicillin at a concentration of 50plg/mL. The cultures were grown for 16 hrs in a shaking

incubator at 370C. It was noted that the cultures grew slowly due to their low turn-over rate. The

alkaline lysis mini-prep protocol (Morelle, 1989) was used to purify plasmid DNA from 1 mL of

culture.

Arabidopsis genetic material was obtained from wild type plants as previously has been

described (Edwards et al., 1991).

In situ Hybridizations

Except for the modifications noted below, previously described methods were used for in

situ hybridization (Jackson, 1991; Drews et al., 1991). To generate templates for probe synthesis,

DNA from plasmids containing the RISE and SHINE EST sequences as well as genomic DNA

isolated from Arabidopsis was PCR amplified. The T7 RNA polymerase promoter sequence

(TAATACGAGTCACTATAGGG) was placed in front of each reverse primer which allowed

direct synthesis of digoxigenin-labeled antisense probes from PCR products. The sense control

probes were designed with the T7 promoter sequence in front of the forward primers. In addition,

probes were designed within the exons of the genomic sequences of each species in order to

hybridize to corresponding messenger RNAs (mRNAs) in situ. Since Myb transcription factors

contain a similar conserved motif in their amino terminus or 5' region, it was important to design

primers in the carboxyl or 3' region that would be unique to each sequence. The following

primers were used to amplify RISE templates for probe synthesis:

TCTCTTTCGCCTCTACCGAACA and

TAATACGACTCACTATAGGGAAGCACTCTTCAGGGAACCTCA The primers used to

amplify SHINE DNA were ACCACCACCAACTGCAACTCCTAT and

TAATACGACTCACTATAGGGTGTACGGCGATTACTGAAGGGT. Amplification of LHY









DNA used the following primers: CAGTTCCAACTCCAGCAATGAC and

TAATACGACTCACTATAGGGCTGAAACGCTATACGACCCTCT The primers for CCAl

(TCTGGTTATTAAGACTCGGAAGCCAT and

TAATACGACTCACTATAGGGTTCATTGGCCATCTCAGGATGC were used to amplify its

PCR product. RNA probes were synthesized using the Dig-RNA labeling kit (Roche Applied

Science, Indianapolis, IN). The cRNA products, 263bp for RISE, 266bp for SHINE, 476bp for

LHY and 361bp for CCA1, were synthesized and added to the hybridization buffer a so the final

concentration was 500 ng mL '. Slides were hybridized at 450C overnight and washed at 500C.

For signal detection, a few grains of tetramisole hyrochloride (Sigma, St. Louis, MO) was added

to the Western Blue substrate (Promega, Madison, WI). Slides were evaluated using a Zeiss

Axiostar Plus Microscope (Carl Zeiss, Inc, Thornwood, NY) and images were photographed

with an Axiocam MRc5 camera (Carl Zeiss, Inc., Thornwood, NY).

Quantitative RT-PCR

For each plant species, fresh tissue including leaves, floral buds and meristematic tissue

was collected every four hours for three days and placed immediately in liquid nitrogen and

stored at -800C. Total RNA was isolated from the tissues using the RNeasy plant RNA isolation

kit (Qiagen, Valencia, CA). RNA concentration was measured using Ribogreen dye (Molecular

Probes, Eugene, OR) and a TBS-380 Mini Fluorometer (Turner BioSystems, Sunnyvale, CA).

The RNA templates were transcribed using the Reverse Transcriptase product protocol (Roche ,

Applied Science, Indianapolis, IN). PCR was used in order to determine the differences in

transcript expression using the same primers described previously for probe construction.

Differences in the 18S ribosomal RNA (rRNA) positive control transcripts were determined by

using the following primers: TTGTGTTGGCTTCGGGATCGGAGTAAT and









TGCACCACCACCCATAGAATCAAGAA (Cho and Cosgrove, 2000). PCR products were

separated by size on agarose gels stained with ethidium bromide and visualized under a UV light.

Images of the gels were captured using a Chemlmager 4400 (Alpha Innotech Corp., San

Leandro, CA) and the relative sizes of bands were determined by comparison to a standard 1kb

plus DNA ladder (Invitrogen, Carlsbad, CA).

Results

RISE and SHINE Encode CCAl-like Myb Transcription Factors

Sequence similarity among genes of different species can provide relevant information

about the evolution of particular gene families and the conservation of important mechanisms.

RISE and SHINE share significant sequence identity with the CCA1 and LHY genes of

Arabidopsis. In particular, RISE was shown to be comparable to LHY with over 40% identity in

a region at the C-terminus. SHINE displayed similarity to the CCAl sequence with 30% identity

in the C-terminal region (Fig. 2-1). These regions located at the carboxyl or 5' end of the genes

and ESTs corresponds to a DNA-binding domain that is found in plant Myb transcription factors.

This motif is highly conserved among the Myb gene family and provides evidence that RISE and

SHINE are indeed part of the family (Fig. 2-2). Additionally, RISE and SHINE share 40%

identity in the region investigated which demonstrates the redundancy between the two

components (Fig. 2-3).

RISE and SHINE Expression is Under Circadian Control

Circadian clock genes (ccgs) show a rhythmic pattern of transcript and protein expression.

In Eschscholzia californica, this is no exception. The RISE and SHINE transcripts oscillate over

a 24 hr period. Both RISE and SHINE oscillate in a pattern analogous to CCAl and LHY in

Arabidopsis (Fig. 2-4). The transcripts abundance varies during the day. Peak levels of

transcription occur just prior to dawn and decrease throughout the day. By evening, the levels of









RISE and SHINTE transcripts are greatly reduced but begin to rise in the early hours of the

morning. This evidence supports the notion that RISE and SHINTE are activated by a light signal

similar to the mechanisms of CCAl and LHY (Fig. 2-5). The transcripts also oscillate with a

period of approximately 24 hrs, which matches the external environment of the organism, further

supporting their role in the circadian clock.

Similar Expression Patterns in a CCAl- like Family of Myb Transcription Factors

In situ hybridizations are ideal for determining the location of transcript expression within

an organism. This method was used to analyze the expression pattern of CCAl and LHY in

Arabidopsis thaliana and RISE and SHINTE in Eschschohzia cahifornica. For both species, two

stages of development were analyzed: a younger stage characterized by premature inflorescence

meristems and a later stage which is exemplified by floral buds.

In the young Arabidopsis tissue, a strong LHY signal is detected in the meristematic region

and in the stamen and carpel primordia (Fig 2-6A). In older tissue, LHY is expressed in the

gynoecium, ovules, and anthers and to a lesser degree in pollen grains (Fig. 2-6B). The

expression of the LHY homolog, CCA1, is similar to its counterpart. High transcript levels are

detected in the young developing floral meristem and include the premature reproductive organs

(Fig. 2-6C). Expression of CCAl in older tissue is limited to the reproductive tissues (Fig. 2-6D).

The sense probe does not have a signal (Fig. 2-6E).

The expression patterns of the CCA-like Myb transcription factors in Eschschohzia

cahifornica is similar to the patterns observed in Arabidopsis. No signal could be detected on the

sense probe control (Fig. 3-7E). High levels of RISE transcript were detected in the young

developing tissue in the meristematic region, premature leaves and axillary buds (Fig. 2-7A). In

older tissue, expression is highest in the carpel, ovules, anthers and pollen grains but is still









detected in the developing petals (Fig. 2-7B). Like its Arabidopsis counterpart, SHINE

transcripts are detected in both the young and older stages of the poppy plant. Transcripts are

detected in the sepal primordia, cauline leaves, and the floral meristem (Fig. 2-7C). SHINE

expression in older tissue is confined to the reproductive organs and petals (Fig. 2-7D).

However, it should be noted that a lower expression level can be detected for both genes

throughout the specimen (Fig. 2-7A-D), showing that RISE and SHINE transcripts are located

within a variety of tissue types..

Discussion

Feedback Loop Mechanism as the Basis of the Circadian Oscillator

An autoregulatory feedback loop involving both positive and negative elements is central

to the circadian oscillator. Circadian systems are often composed of one or more interconnected

loops. Knowledge of these interlocked loops results from the identification of genes that function

within the core loop of the oscillator. In Arabidopsis thaliana, three genes with required roles in

maintaining rhythmicity have been identified: LHY, CCA1 and TOC1 (Schaffer, 1997; Wang et

al., 1997; Millar et al., 1995). The two Myb transcription factors LHY and CCAl serve as the

negative elements of the core loop and function to block the activation of the positive element

TOC1. The positive regulator, TOC 1 activates expression of LHY and CCAl. In this study, we

have identified two potential homologs to LHY and CCAl in Eschscholzia californica. These

genes are hypothesized to serve similar roles in the core oscillator of the poppy plant, thus

providing a conserved mechanism for maintaining rhythmicity in higher plants.

The RISE and SHINE Genes Encode MYB Transcription Factors That Are Similar to
LHY and CCA1

Sequence analyses revealed that two California poppy EST sequences located in the FGP

database (Albert et al, 2005; Carlson et al., 2006) share sequence identity with known










components of the central circadian oscillator from Arabidopsis (Fig. 2-1). The Myb domain,

which functions as a DNA-binding domain, shares the most sequence similarity with RISE and

SHINE. Research shows that LHY and CCAl bind to an evening element (EE) located within

the promoter of TOC1, an evening gene (Alabadi et al., 2001; Harmer et al., 2000). Because of

the similarity between the components of these two circadian systems, I wanted to determine if

RISE and SHINE function in a similar manner to CCAl and LHY in the Arabidopsis central

oscillator.

A prerequisite for a protein to function as a negative element in the circadian clock is that

its expression and activity must oscillate in synchrony with the environmental oscillations. In

addition, this component regulates its own transcription by negative feedback which creates a

delay in the rhythmic cycle. In the Arabidopsis model plant, LHY and CCA transcripts were

shown to oscillate over a 24 hr period with peak levels accumulating just prior to dawn

(Mizoguchi et al., 2002). Similar results for this species were obtained (Fig. 2-5). The RISE and

SHINE transcripts displayed a similar pattern of expression with minimal levels of mRNA

detected in the evening (Fig.2-4). The accumulation of transcripts just prior to dawn shows that

LHY and CCA1, as well as RISE and SHINE, are regulated by a light signal and are entrained to

anticipate dawn.

The genes at the center of the Arabidopsis circadian oscillator serve as either positive or

negative factors to influence the rate of transcription. The activation and inhibition of ccgs

occurs at particular points within the circadian cycle and when coupled, form a loop in which the

components serve crucial roles in generating and maintaining rhythmicity within an organism.

The LHY and CCAlgene products in Arabidopsis function in a manner that is antagonistic to

TOC1. In Eschscholzia californica, putative homologs for LHY and CCAl have been identified









but other components of the oscillator remain unknown. Based on the previously described

similarities between the two systems, it is reasonable to hypothesize that a TOC1- like gene also

functions in the poppy oscillator (Fig. 2-8.).

The spatial expression patterns of the Myb transcription factors in Arabidopsis and

California poppy provide relative information about their functional similarities. In both species,

the young tissue contained a high level of expression in the meristem and sepal primordia (Fig.

2-6A, C; Fig. 2-7A, C). The older tissues were characterized by high levels of transcript in the

reproductive organs and petals (Fig. 2-6B, D; Fig. 2-7B, D). The similarity in the expression

pattern suggests that these genes might be true orthologs stemming from a common ancestor. In

addition, the location of expression provides relevant information on the processes regulated in

that particular region. The high level of transcript expression in pollen grains in the older tissue

of Arabidopsis and poppy could control the timed release of pollen, a mechanism that evolved

for maximizing reproductive success (Subba et al., 1998). The fact that the circadian clock

regulates expression of floral pathway genes that in turn activate floral meristem identity genes

(Vij ayraghavan et al., 2005) seems logical to explain the high level of expression of the Myb

transcription factors in the meristematic regions of the young tissue.

Together these sequence comparison and mRNA expression data suggest that RISE and

SHINE encode Myb transcription factors that could function as the negative elements in the

oscillator of the California poppy plant similar to the manner of LHY and CCAl in Arabidopsis.

An Evolutionary Conserved Clock Mechanism in Higher Plants

Although the above data suggests similarities exist among the circadian systems of

Arabidopsis thaliana and Eschscholzia californica, little is known about the elements and

mechanisms underlying the clocks of other higher plants. It is possible that the molecular









components that form the clock machinery are unique to higher plants. In this case it is

important to determine whether other plant species have homologs for each of the Arabidopsis

clock components and whether they share similar functions. This paper demonstrates that

homologs for two Arabidopsis clock genes exist in the California poppy plant and that they

appear to be expressed in a similar manner to their counterparts. In other plant species,

components of the central oscillator remain unknown however, recent evidence has identified

several clock-associated genes that are involved in the input pathway to the clock. Studies of

Pisum sativum, peas, have revealed circadian clock gene homologs of TOC1, CCA and LHY

referred to as TOC1 and MYB1 (a CCA/LHY homolog) respectively (Hecht et al., 2007). Two

additional Arabidopsis orthologs, EARL YFLO WERING4 (ELF4) and LA TE BLOOM~ER1

(LA TRISE), were characterized in pea plants and their diurnal rhythm expression conformed

closely to those associated with their counterparts, ELF4 and GIGAN7EA (GI). In Arabidopsis,

ELF4 promotes clock entrainment and is required for sustained rhythms in the absence of

environmental cues (McWatters et al., 2007). The GI gene regulates flowering in long day (LD)

conditions in a clock-controlled pathway, where it acts as an intermediate between the central

oscillator and the FLOWERING LOCUS T(FLT) gene (Mizoguchi et al., 2005). Investigations in

the clock components in other species continue, including Oryza sativa (rice), M~edicago

trunculata( a legume) and Lycopersicon esculentum (tomato). In addition, sequence analysis of

the Myb family of transcription factors using the FGP database (Albert et al, 2005; Carlson et al.,

2006) revealed one CCAl/LHY homolog in Cucumis sativus (cucmber), Asparagus ofjicinalis,

Liriodendron tulipifera (tuliptree) and Salruma henryi (standing ginger) and two homologs in

Acorus amnericanus (the American Sweet Flag) and Nuphar advena (water lily). The presence of

similar sequences across a wide variety of species suggests that the oscillator mechanism that









involves CCAl and LHY in Arabidopsis is conserved in higher plants. The conservation of this

mechanism and its components implies that such organization is beneficial for the organism.

The ubiquity of the feedback loop mechanism of plant circadian oscillators suggests that an

adaptive advantage results from the spatial and temporal organization of important rhythmic

activities. A recent experiment compared the performance of wild type Arabidopsis plants with

lines having mutations that alter period length in a range of environmental period lengths that

were either matched or mismatched to the endogenous clock. The results showed that a

photosynthetic advantage was conferred by matching the endogenous clock period with the

light/dark period (Dodd et al., 2005). Incorrect matching of the periods resulted in reduced leaf

chlorophyll, reduced assimilation, reduced growth and increased mortality (Dodd et al., 2005).

Optimization of physiological parameters by the circadian clock probably has been selected

during plant evolution. Similar results have been described in the cyanobacteria Synechococcus

as well (Ouyang et al., 1998)

The Biological Importance of Circadian Clock Genes

Although this paper addresses the circadian oscillator and its key mechanisms in higher

plants, the importance of the circadian machinery also resonates throughout the animal kingdom.

In addition, elucidating the components underlying the feedback loops of the oscillator in either

plants or animals provides relative information on the general architecture of the mechanisms.

Both plants and animals use circadian clocks to temporally organize important processes

involving reproduction and development which are crucial in the evolution of every species. In

humans, many behaviors are regulated by the circadian clock including the sleep/wake cycle,

feeding patterns, hormone production and cell regeneration (Edgar et al., 1993; Stokkan et al.,

2001; Czeisler and Klerman, 1999; Shibata, 2004).









A number of human illnesses are attributed to a dysrhythmia in a behavioral or

physiological process. Abnormal circadian rhythms have been associated with affective disorders

like A number of human illnesses are attributed to a dysrhythmia in a behavioral or physiological

process. and the existing therapy drugs used to treat these disorders such as lithium act upon the

circadian cycle (Hallonquist et al., 1986). Insomnia and sleep problems also result from

abnormal circadian rhythmicity and usually are characterized by an endogenous clock that runs

faster or slower than the norm (Zisapel, 2001). Individuals that suffer from attention-defieit

hyperactivity disorder (ADHD) are often plagued by sleep disturbances which result from a

dysrhythmic clock (Owens, 2005). In women who suffer from menopause, hot flashes disrupt the

clock' s rhythm resulting in a clock that is misentrained. This abnormal entrainment results in

sudden awakenings during the sleep cycle (Freedman et al., 1995). Recently a role for the

circadian clock has been identified in cancer studies. Research suggests that at least eight central

clock genes coordinate many basic functions, including cell proliferation, tumor growth and

apoptosis in circadian time. This work indicates that circadian clock genes and their products

potentially represent novel targets for the control of cancer growth (Wood et al., 2006).

Elucidating the mechanisms that lie beneath the circadian oscillator has become the

primary focus of chronobiologists. A wealth of knowledge stands to be gained since nearly all

processes crucial for species survival involve rhythmicity of one or more elements. The

availability of technologies to analyze global gene expression should become a powerful tool in

clock research. This advancement should aid in the identification of new genes affected by the

timekeeping apparatus and help characterize the interactions of those clock proteins that have

been previously identified. However, the question of how the genes involved in the clocks are

regulated is just starting to be addressed.









In this study, two homologs of the Arabidopsis Myb transcription factors CCAl and LHY

were identified in Eschscholzia californica. Sequence analyses suggest that these genes are true

orthologs and are similar in their temporal and spatial expression. This information provides

evidence that there is a conserved transcriptional- translational feedback loop at the center of the

circadian oscillator in higher plants. Based on this congruence, other circadian clock genes

involved in the maintenance of rhythms in California poppy should resemble those described in

Arabidopsis (Fig. 2-8).













































Figure 2-1. Alignment of the LHY/CCAl genes in Arabidopsis thaliana and Eschscholzia
californica. The nucleotide sequences of LHY and CCAl were used to search the
FGP database (Albert et al., 2005; Carlson et al., 2006) using BLASTN in order to
form an alignment with the SHINE and RISE ESTs. The residues shaded in gray
represent the bases that are conserved among all four genes. Those highlighted in
yellow represent the nucleotides that are conserved between LHY and RISE. The
residues in blue illustrate the conserved bases among CCAl and SHINE.












C CA












Figure 2-2. A phylogenetic analysis of CCA-like family of genes. This tree illustrates the
relationships among the CCA-like family of Myb transcription factors in Arabidopsis
thaliana (CCA, LHY), Eschscholzia californica (SHINTE, RISE) and Nuphar advena
(NAD), a basal angiosperm. Here, the NAD gene was identified in the FGP EST
database and serves as the outgroup for this analysis. Multiple sequence alignment
and tree construction were produced using the MAFFT program (Katoh et al., 2002;
Katoh et al., 2005).











































Figure 2-3. Alignment of the RISE (eca_4_183 384) and SHINE (eca_4_184056) EST sequences
using the BLASTN program from the FGP database (Albert et al, 2005; Carlson et
al., 2006). Conserved residues are shaded in gray.












0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60


Hours


RISE


Figure 2-4. Analysis of RISE and SHINE mRNA expression. Peak RISE and SHINE transcript
levels occur just prior dawn and dwindle throughout the day. The 18S r RNA
transcripts demonstrate a constant expression level throughout the day. The bar above
reflects the light/dark cycles to which the plants were exposed for this experiment.
The black boxes correspond to periods of darkness.


SHINE we


18S











0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72


Hours


LHY


CCAl


'Ir ~ I~


18S




Figure 2-5. Analysis of LHY and CCA1 mRNA expression. Peak transcript levels of the
transcription factors occur just prior dawn and become greatly reduced by evening.
The 18S rRNA transcripts demonstrate a constant expression level throughout the
day. The bar above reflects the light/dark cycles to which the plants were exposed for
this experiment. The black portions represent the evening hours.

























Fiur 2-6 Exrsso oLH anCA1iAr bidpi ise H n C tasrpswr

lctdibohyugan maur Arbdpi s flrltsu.Teloaiaino h
tancipts~ wa deemne yi st yridztos LHYtrans1cript weeprsnti
A) th lrlmrse fm n h lrlpimri f)o h ougtsu n nB
the ~ ~ ~ gyoeiu (g) ovle (0,ates()adple ran p)o h auefoa
tisue.CCA trnscipt wee dtecedinC ) one iseseiial ntef n

fpadi )teodrtsu ntesm lcto sosre ihL Y ,0 n
pg E heses poe onrl hwsnosgnl
























Figur 2-.RS n HN rncit r xrse nbot on n atr lrltsseo
the ~ ~ ~ 9 California~- pop ln.Telclzto ftetasrpswsdtr ine b i
siuhyrdiaios RS tas ritweepeetiA)hefoamrseaicegn



(f),prmaur eaes(1 adinth ailay ud (x)o te ouge isuewhlei
B)7 theI olde tise hihexrsion was deetdi h aplc)vls()nhr





shw no igal










































Figure 2-8. Proposed mechanism of the central oscillator of Eschscholzia californica. RISE and
SHINE serve as negative regulators of a gene 'X' (designated by a blunted arrow), a
TOC1 homolog. That gene in turn should induce expression of RISE and SHINE
(pointed arrows). Candidate genes for component 'X' have yet to be identified.









CHAPTER 3
CONCLUSION

Elucidating the mechanisms that lie beneath the circadian oscillator has become the

primary focus of chronobiologists. A wealth of knowledge stands to be gained since nearly all

processes important for species survival involve rhythmicity of one or more elements. Research

on the crucial components that are required to maintain rhythmicity could provide insight into

possible therapies and treatments for diseases that target the clock system. In addition, by

analyzing the mechanisms and proteins of the central oscillator and comparing them among

different species, the evolutionary history of the clock can be examined.

In this study, it was proposed that a common clock ancestor exists among higher plant

species. In Eschscholzia californica, two homologs of Arabidopsis Myb transcription factors

were identified as potential components of the biological clock. Other probable homologs to

components of the Arabidopsis central oscillator LHY and CCAl have been identified in other

species including Cucumis sativus (cucumber), Asparagus ofjicinalis, Liriodendron tulipifera

(tuliptree), Salruma henryi (standing ginger), Acorus amnericanus (the American Sweet Flag) and

Nuphar advena (water lily). Further investigation of these species should provide relevant

information on the clock mechanism and its conservation. Genetic screens for clock mutants aids

in the assignment of potential components to roles within the circadian clockwork. Altered

expression, either increased or greatly reduced, of these constituents should result in aberrant

clock phenotypes. Although it is proposed that the RISE and SHINE genes of Eschscholzia

californica play a role in the central oscillator of the plant' s clock, further studies similar to those

mentioned above are required for verification.










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BIOGRAPHICAL SKETCH

Meredith Lynn Sullivan was born on January 13, 1979 in Tuscaloosa, Alabama. The

younger of two children, she graduated from Central High School in 1997. Following graduation,

Meredith enrolled at Shelton State Community College where she was a member of the women' s

varsity soccer team. She subsequently enrolled at the University of Alabama (UA) where she

earned her B.S. in Biology in 2002.

Upon receiving her B.S. degree, Meredith enrolled in graduate school at the University of

Florida (UF) in 2003. As a graduate student in the Department of Botany, she pursued molecular

biology- based studies to elucidate the components and mechanisms involved in flower

regulation. This information allowed her to analyze the evolutionary significance of circadian

clocks in development.

Upon completion of her M. S. degree, Meredith will pursue a career in medical research.

She plans to utilize the knowledge she has obtained throughout her education to aid in the

identification of therapeutic drugs for certain illnesses.





PAGE 1

1 A COMPARATIVE EXPRESSIONAL ANALYS IS OF A FAMILY OF CCA-LIKE MYB TRANSCRIPTION FACTORS IN TWO HIGHER PLANT SPECIES by MEREDITH L. SULLIVAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

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2 2007 Meredith L. Sullivan

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3 To my parents with love and respect

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4 ACKNOWLEDGMENTS Most of all, I thank my family for their endle ss love and support. They are the pillars of my strength. I also thank my advisor, Dr. David Oppenheimer for affording me the opportunity to attend the University of Florida. I also express gratitude to Dr. Bernard Hauser for his support of this work. A special thanks to Zhengui Zheng for his time and assistance with this project. Also I thank Xiaoguo Zhang and Stacey Jeffries for the invaluable advice they offered and the support they provided me throughout my time at UF. Fina lly I extend special appr eciation to the love of my life, Brad for his continued encouragement.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF FIGURES................................................................................................................ .........6 ABSTRACT....................................................................................................................... ..............7 CHAPTER 1 LITERATURE REVIEW.........................................................................................................9 Biological Rhythms............................................................................................................. .....9 The Circadian Clock............................................................................................................ ...10 ClockControlled Genes........................................................................................................13 The Arabidopsis Central Oscillator........................................................................................18 Evolution of Core Clock Components....................................................................................21 2 TWO CCA-LIKE MYB TRANSCRIPT ION FACTORS ARE PRESENT IN CALIFORNIA POPPY...........................................................................................................27 Summary........................................................................................................................ .........27 Introduction................................................................................................................... ..........28 Materials and Methods.......................................................................................................... .28 Plant Growth Conditions.................................................................................................28 In situ Hybridizations......................................................................................................30 Results........................................................................................................................ .............32 RISE and SHINE Encode CCA1-lik e Myb Transcription Factors.................................32 RISE and SHINE Expression is Under Circadian Control..............................................32 Discussion..................................................................................................................... ..........34 Feedback Loop Mechanism as the Ba sis of the Circadian Oscillator.............................34 The RISE and SHINE Genes Encode MYB Transcrip tion Factors That Are Similar to LHY and CCA1.......................................................................................................34 The Biological Importance of Circadian Clock Genes....................................................38 3 CONCLUSION..................................................................................................................... ..49 LIST OF REFERENCES............................................................................................................. ..50 BIOGRAPHICAL SKETCH.........................................................................................................57

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6 LIST OF FIGURES Figure page 1-1 The three basic components of the circadian clock...........................................................25 1-2 Positive and negative factors act upon the circadian clock................................................25 1-3 Comparisons of three conser ved regions of LHY and CCA1............................................26 1-4 The Arabidopsis circadian oscillator.................................................................................26 2-1 Alignment of the LHY/CCA1 genes in Arabidopsis thaliana and Eschscholzia californica ..........................................................................................................................41 2-2 A phylogenetic analysis of CCA-like family of genes......................................................42 2-3 Alignment of the RISE (eca_4_183384) and SHINE (eca_4_184056) EST sequences using the BLASTN program from the FGP da tabase (Albert et al, 2005; Carlson et al., 2006)..................................................................................................................... .......43 2-4 Analysis of RISE and SHINE mRNA expression..............................................................44 2-5 Analysis of LHY and CCA1 mRNA expression.................................................................45 2-6 Expression of LHY and CCA1 in Arabidopsis tissue.........................................................46 2-7 RISE and SHINE transcripts are expressed in bot h young and mature floral tissue of the California poppy plant.................................................................................................47 2-8 Proposed mechanism of th e central oscillator of Eschscholzia californica .......................48

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7 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science A COMPARATIVE EXPRESSIONAL ANALYS IS OF A FAMILY OF CCA-LIKE MYB TRANSCRIPTION FACTORS IN TWO HIGHER PLANT SPECIES By Meredith L. Sullivan August 2007 C hair: David Oppenheimer M ajor: Botany Circadian clocks are ubiquitous among most liv ing species. Since life on Earth originated i n the presence of light/dark cycles, organisms ha d to evolve mechanisms to cope with such e nvironmental fluctuations. An accurate timekeeping apparatus affords an organism with t emporal organization of crucial molecular and cellular processes. The components that c onstitute the central oscillator of the clock vary greatly among plants and animals but the basic a rchitecture appears similar. This resemblance serves as a foundati on on which evolutionaryb ased investigations on the conservation of su ch machinery can be conducted. In the plant A rabidopsis thaliana two genes involved in circadia n regulation were identified and c haracterized as members of a family of Myb transcription factors ch aracterized by only one M yb repeat sequence. These genes, CCA1 and LHY are necessary to maintain rhythmicity in the p lant and have been shown to have a role in floral induction. Using th e Floral Genome Project ( FGP) database of known flowering genes, two EST homologs of CCA1 and LHY were i dentified in Eschscholzia californica eca_4_183384 and eca_4_184056 (E1 and E2), based on s equence similarity. These orthologs demonstrate transcript oscillations over a 24hr period with p eak levels of expression o ccurring just prior to dawn. In situ analyses revealed similar patterns o f expression in young and older floral tissue of both plant species. In this paper I present

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8 molecular evidence that the transcriptiontransl ationbased feedback loop mechanism of the circadian oscillator is conserve d in these higher plant species a nd suggest that this mechanism can also be observe di n other higher plants.

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9 CHAPTER 1 LITERATURE REVIEW Biological Rhythms The daily rotation of the earth leads to periodic fluctuations in environmental conditions. Because life on Earth originated in the presence of a cyclical environment, many organisms have evolved timing mechanisms to organize important events. In order to adapt to the changes presented by the environment, organisms must m odulate their behaviors with the daily cycles of light and temperature variation, and establish an effective met hod for tracking time. Since the environment imposes a period of approximately 24 hours, organisms with rhythmic behavior that matches these oscillations have high er fitness than those that do not. The first noted rhythmic behavior occurred in the fourth century BC when the sleep movements of the tamarind tree were not ed by Androsthenes, a Greek philosopher (Chandrashekaran, 1998). In the mid-1700s, a Fren ch astronomer named JeanJacques dOrtous de Mairan recorded the da ily leaf movements of the Mimosa pudica plant and demonstrated that the rhythms persisted for several days when the plant was subjected to complete darkness (Golden and Strayer, 2001; Sweeney, 1987). This was the first eviden ce that the rhythmic behavior must be endogenous and that the r hythmicity, once established, continues in the absence of environmental cues. Erwin B unnings work during the 1930s, in which he demonstrated a rhythm in the eclosion behavior of the fruit fly Drosophila further supported the idea of an endogenous timekeeping mechanism. Since the rhythms occurred with a 24 hour period, the term circadian was coined from the Latin words circa and dias meaning about a day (Chandrashekaran, 1998; Halberg, 1959). Circadian rhythms are variations in physiologi cal and behavioral activities that occur over a period of about 24 hours (Hardin, 2000). In the context of a biological process, the time

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10 interval between two successive events is de scribed as a biological rhythm (Kumar, 2002). Biological rhythms are found in almost all livi ng organisms. They have been described extensively in mammals, insect s, fungi, plants and bacteria (Dunlap et al., 1999; McClung, 2001; Ouyang et al., 1998). These rhythms occur over a large range of time scales: from millisecond oscillations to seasonal changes. The ubiquity of circadian rhythmicity across a broad taxonomic spectrum suggests that adaptive fitness is enhanced by the synchronization of certain events with the diurnal cycle imposed by the environment (McClung 2000). In a number of different species, a plethora of activities are regulated by circadian rhythms. Cyanobacteria demonstrate daily oscillations in nitrogenase activ ity, photosynthesis and metabolic activities (Kondo and Is hiura, 1999). In vertebrates, a number of behavioral and life processes, ranging from the molecular level to the cellular and systemic levels, are driven by circadian rhythms including eati ng, sleeping, seasonal migration a nd cell proliferation (Gillette and Sejnowski, 2005). Similarly, a variety of ev ents such as leaf m ovement, stomatal opening and regulation of flowering time and fragran ce emission are tightly regulated by the plant timekeeper. In other organisms, such as Neurospora and Drosophila, development, cell signaling and stress responses w ith self-sustaining rhythms can be regulated by the circadian clock. The role that diurnal rhythms play in a number of different activ ities suggest that an endogenous timekeeping system provides an adaptiv e advantage, enabling the anticipation of environmental change and the coordination of crucial events to occur at specific phase relationships with the en vironment (Ms, 2005). The Circadian Clock Circadian or biological rhythms, although they parallel the environmental cycles of light and dark, are generated within an organism by a complex timekeeping system (Hardin, 2000). Many organisms have evolved an endogenous chronom eter, the circadian clock, to temporally

PAGE 11

11 coordinate important processes with the daily variations in the e nvironment. Circadian rhythm of gene expression has been shown to function as the underlying mechanis m of the clock (Wang and Tobin, 1998). Two important attributes of the circad ian clock, entrainment and temperature compensation, ensure synchrony between important rhythmic activities an d the surroundings of an organism. The first, entrainment, is the manne r in which the clocks are set to local time by environmental cues such as the light / dark cycles. The second, temperature compensation, describes the ability of the cloc k to run at the same rate inde pendent of temperature changes (Hardin 2000). The notion that biological rhythms are created with in an organism stimulated interest in the mechanism that maintains these oscillatory patte rns. In theory, numer ous regulatory schemes could achieve such fluctuations. The most co mmon method involves a regul atory circuit with a positive and negative product (Fig. 1-2). The negative element feeds back to slow down the rate of the process itself and creates a delay in the execution of the feedback. A positive element is required to activate the clock and prevent it from winding down (Dunlap et al., 1999). The generally conserved clock mechan ism consists of an autoregula tory feedback loop in which positive factors act on genes encoding negative factor s that in turn feedback to inhibit their own expression (Strayer et al ., 2000; Dunlap, 1999). A circadian system often consists of one or more interconnected feedback loops. The circadian system is divided into three main components: input pathways, a central oscillator and output pathways (Fig. 1-1). Input pathways, also known as entrainment pathways, transmit environmental signals to the central ti mekeeping apparatus. In plants, this signal transduction pathway adjusts the clock in respons e to external cues most frequently through the

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12 action of cryptochromes and phytochromes. The entr ainment of circadian cl ocks to the light/dark cycles is usually mediated by light-induced change s in the level of a component of the oscillatory feedback loop. The ability to re-entrain the cl ock ensures synchrony with the environment and allows the anticipation of dawn and dusk (Devlin, 2002). The core timekeeping component of the circad ian clock is the central oscillator (Hardin, 2000). It serves as the systems pacemaker and is responsible for generating circadian rhythms. In order for the oscillator to function in th e absence of environmental cues it must be synchronized with the external time via the i nput pathways. Environmen tal transitions between dawn and dusk help to adjust th e endogenous period created by the oscillator to precisely match the 24hour period found in nature (Ms, 2005). Completing the circadian clock model are th e output pathways which provide a link between the central oscillator and the rhythmic physiological responses they control. These pathways are activated at specific times of the circadian cycle and the outputs are in phase with the oscillation of light cycles (Dodd et al., 2005). In both an imals and plants, there are a substantial number of physiologi cal and metabolic processes that are regulated by the circadian outputs. These events include such varied respons es such as olfactory responses in Drosophila, leaf movements and hypocotyl growth in plants and enzyme activity. The rhythmic behaviors that are generated in response to day length are known as photope riodic responses (Schultz and Kay, 2003). The discovery of components of the circadian oscillator has enabled scientists to concentrate on the mechanisms that interconnect the three components to form an effective timekeeping system. Recent studies of several model organisms including Drosophila Neurospora and mice have revealed a common molecular mechanism at the center of the

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13 circadian oscillator (Barak et al., 2000). Cloc k proteins, encoded by clock genes, serve as negative elements that repress their own expres sion by blocking their tran scriptional activators, or positive elements (Fig. 1-2). A decrease in cl ock transcripts and protei ns results in the derepression of the transcriptional activat ors and thus reinitiates the cycle. ClockControlled Genes The identification of genes which function at the center of the clock machinery has rapidly increased over the last decades. These clock-c ontrolled genes, or ccgs, encode pieces of the central oscillator. Their products produce and maintain the oscillat ions that drive other circadian rhythms. A substantial number of clock-associated genes whose e xpression relies on the rhythms generated by the oscillator have been identifi ed in the genomes of many organisms. Recent microarray analyses of the model plant Arabidop sis indicated that up to 6% of the genes are rhythmically expressed (Harmer et al., 2000; Schaffer et al., 2001). Components of the biochemical feedback l oop whose rhythmic activity is required for oscillator function are referred to as state variables. A number of criteria have been defined to identify and characterize state variables of th e circadian system (Ar onson et al., 1994; Kay and Millar, 1995). First, the component itself must demonstrate circad ian oscillations in its activity and expression. The second criterion requires th e component to control its own levels by feedback inhibition of its s ynthesis. Additionally, clamping of the amount of the putative oscillator component at any le vel from null to high stops the clock and thus rhythmicity. The final criterion states that i nduced transient perturbations in the abundance of the component should cause a phase shift in the clock output. Thes e criteria are applied in the investigations of putative oscillatory components to assign positive and negative roles to the elements. Knowledge of interconnected loops is obtained from the id entification through classical genetics of genes that are within one of the core loops of the oscillator (Roenneberg and Merrow, 1998).

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14 The molecular basis of circadian clocks is best understood in Neurospora and Drosophila The genetics of rhythms originat ed in these two organisms and mu ch of what scientists have learned about how mammalian clocks operate clos ely parallels the behavior of one or both of these species. In each of these model systems, ge netic screens for rhythm mutants that affect the period length of the clock or abol ish its activities were found (Hardin and Siwicki, 1995; Dunlap, 1996). The first clock mutations to be discovered were the period ( per ) mutant from Drosophila and the frequency (. frq ) mutant from Neurospora (Dunlap, 1993; Konopka and Benzer, 1971; Feldman and Hoyle, 1973). The PER gene product, as well as its transcript, oscillates with a circadian rhythm and the PER protein was require d for a feedback regulation of its own gene products (Williams and Sehgal, 2001). Oscillati ons of PER transcripts and the protein they encode persisted in continuous dark conditions suggesting that the gene must be under the control of the circadian clock. The phases of the per messenger RNA (mRNA) and protein rhythms were noticed to be quite distinct; PER protein level peaked in abundance approximately six hours after the peak in PER mRNA levels. This difference accounts for the delay or lag time in oscillations required by the central timekeeper In addition, mutant per fruit flies exhibited altered rhythmic behavior, which suggests a role for this gene in the oscillator. The tim mutant was identified in a screen for recessive mutants that affected the eclosi on behavior of the fly. Similar to the per mutant, the tim mutants exhibited arrhythmic behaviors, periods that were shortened or lengthened, and in some mutant al leles, PER expression was dampened (Allada et al., 2001). PER contains a protein interaction domain known as a PAS domain, which enables the protein to interact with the second Drosophila circadian gene identified, timeless ( tim ). PER and

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15 TIM form a heterodimer that serves as part of a feedback loop to inhibit per and tim transcription. In return, the PER-TIM heterodimer activates a gene called Drosophila Clock (dClk), a transcription factor th at is also rhythmically expr essed (Scully and Kay, 2000). dCLK interacts with another transc ription factor, CYCLE (CYC) a nd together the two form a heterodimer complex that is required for the activation of PER and TIM transcription. The dCLK-CYC complex binds to a specific sequence in PER and TIM promoters known as the Ebox motif, a consensus hexanucleotide sequence found in basic helix-loop-helix (bHLH) transcriptional factors (Fairman et al ., 1993). Binding of the complex to the E-box motif allows transcription of PER and TIM and results in a increase in PER-TIM heterodimers in the cytoplasm. When expression is high, usually in the evening, these complexes move into the nucleus where PER binds to the dCLK-CYC co mplexes. This releases the dCLK and CYC proteins from the promoters and results in the shutting off of PER and TIM transcription. As the levels of PER and TIM decline, the dCLK-CYC dimers are rel eased and thus the dCLK-CYCdependent repression of dClk expression is lifted. As the levels of dCLK begin to rise, usually in the morning, an increased number of dCLK-CYC complexes are formed and per and tim transcription is re-activat ed (Glossop et al., 1999). Since the products of the per gene and the dClk gene inhibit their own s ynthesis it appears that the Drosophila oscillator consists of two interconnected negative feedback loops: a per tim loop and a dClk loop (Glossop et al., 1999). In this model PER and TIM serve as the negative elements while dCLK and CYC serve as the positive elements (Dunlap et al., 1999). The second timekeeping component identified through forward genetic screens for clock mutants was the FREQUENCY ( FRQ ) gene in the fungus Neurospora crassa Mutations of this gene resulted in arrhythmic expressions, altere d periodicity and defici encies in temperature

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16 compensation (Aronson et al., 1994). The FRQ RNA and protein levels cycle with a circadian rhythm and the protein negatively regulates its own transcript resu lting in a feedback loop similar to observed in Drosophila (Dunlap, 1996). The expression of FRQ is activated by two PASdomain-containing transcripti on factors, WHITE COLLAR 1 (WC-1) and WHITE COLLAR-2 (WC-2) that form the white collar complex (WCC), which bi nd to circadian photoreceptor connecting light signals and the oscillator (He et al., 2002). Afte r the accumulation of FRQ, the proteins begin to dimerize, enter the nucleus a nd interact with the WCC diminishing its activity and dampening FRQ expression (Froelich et al., 2003). FRQ also promotes the synthesis of WC1 increasing the level of WCC. This results in a mass of WCC that is held inactive by FRQ until the protein is phosphorylated and targeted for ubiquitination, accounting fo r the delay that is required by the circadian oscillator. Therefore Neurospora has a transcripti onal/translational negative feedback loop at the core of its oscill ator with WC-1 and WC-2 acting as the positive elements and FRQ as the negative (Dunlap et al., 1999). Recent evidence suggests that this FRQbased oscillator might work in cooperation with other oscillators within the organism as well (Correa et al., 2003). Over recent decades, numerous advances in understanding the mechanisms underlying the biological oscillator in mammals have been made. The first cloned mammalian clock component identified by forward genetics was the CLOCK ( CLK ) gene of Mus musculus the mouse (Antoch et al., 1997). Like transcription fact or proteins that are central to the clock in other organisms, the mouse Clock gene contains 1) a PAS domain, 2) its le vels of mRNA and pr oteins oscillate, and 3) in clk mutant mice, the cyclic expression the mPeriod ( mPer ) homolog is reduced. These data suggest that CLOCK, as a member of the oscillat or, controls the transcri ption of circadian genes (Gekakis et al., 1998). It was shown that CLOC K binds to another tran scription factor, BMAL1

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17 (Brain and Muscle Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT)-Like1) and this complex activates the transcription of the PER and CRYPTOCHROME ( CRY ) genes. The mPER and CRY proteins form heterodimers and hom odimers and upon translocation to the nucleus, they inhibit the activity of the CLOCKBMAL1 complex, which in turn suppresses PER and CRY transcription (Panda et al., 2002). Once th e mPER and CRY proteins are phosphorylated, they are targeted for degrada tion and transcriptional repres sion is relieved. Although other oscillators might be present in the mouse, central to its core oscillator is the negative feedback loop in which the negative elements, the PER homologs repress the activation of the positive elements CLOCK and BMAL1 (Dunlap, 1999). Circadian rhythms, once only thought to be a feature of eukaryotic organisms, have recently been identified in some prokaryotes. The cyanobacteria Synechococcus elongatus serves as the model system for molecular investigatio ns of this group. Approximately one hundred clock mutants identified from an ethylmethansu lfonate (EMS) mutagenesis screen that were characterized by arrhythmia, atypical periods an d some mutants that could be rescued by the introduction of wild-type DNA from a Synechococcus genomic library (Lorne et al., 2000). One cluster of DNA fragments that c ould be rescued represented the kai cluster of genes: kaiA kaiB and kaiC Transcribed from two different promoters, PkaiA and PkaiBC a monocistronic kaiA mRNA and a dicistronic kaiBC mRNA are produced and both transcripts cycle in abundance. Overexpression studies revealed that KaiC represses the activation of the PkaiBC promoter while KaiA enhances PKaiBC transcription (Ishiura et al., 1998) Since KaiC represses its own transcription it functions as the ne gative element of the negative f eedback loop at the core of the Synechococcus elongatus oscillator while KaiA, which helps drive expression from PkaiBC functions as the positive element. A role for KaiB has yet to be determined.

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18 Although circadian rhythms were first obs erved in photosynthetic organisms, the molecular mechanisms underlying the circadian os cillator in plants ha ve been difficult to elucidate. Much of what we know about circadia n rhythms has come from the studies of animal systems. Over the last decade there has been substantial effort put into identifying and understanding the roles of oscilla tor genes in the model plant Arabidopsis and in a few other plant species. Plant researchers are working to determine if a mechanism similar to those observed in other organisms is conser ved among members of the plant family. The Arabidopsis Central Oscillator The molecular basis of circadian rhythms has been thoroughly studied in Drosophila, Neurospora, mice and cyanobacteria model sy stems. The common denominator among these organisms is a biological clock based on a central oscillator that uses transcriptional feedback loops to generate a circadian oscillator with a 24 hour period that regul ates circadian outputs (Dunlap, 1999). The oscillator re sponds to environmental signals through input pathways, which entrain the clock, and controls output pathwa ys that generate a r hythm in phenotype or biochemical pathway Mizoguchi et al., 2002). Al though some of the first recorded circadian rhythms were identified in plants, the mol ecular mechanisms underlying these rhythms have remained unclear until the last decade. Advances in the identification and characterization of plant circadian components have been made primarily through genetic studies of Arabidopsis thaliana The first Arabidopsis clock mutant was identifi ed by fusing a luciferase marker gene to the CAB2 ( chloropyhll a/b binding ) promoter to investigate clockregulated gene expression in different populations. The result wa s the identification of a mutant, timing of CAB ( toc ) that altered the period of the clock (Millar et al., 1995). The TOC1 protein was shown to contain a pseudo-response regulator motif, similar to those in two-co mponent signal transduction

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19 pathways, at its amino terminus and a CONSTA NS (CO)-like motif at its carboxyl terminus (Strayer et al., 2000). Interestingl y the CO family represents a gr oup of plant transcription factors that are involved in flowering response (Putte rill et al., 1995). Since the TOC1 protein shows similarity to the CO family, TOC1 might play a role in flower ing as well. The levels of TOC 1 mRNA cycled in light-dark conditions and peak levels of transcript were observed late in the day while minimal levels were observe d at dawn. In addition, since the toc1-1 mutant was characterized by a circadian os cillator with a shortened pe riod which demonstrated that TOC1 products reduce their own expression (Strayer et al., 2000). Based on the observed data, TOC1 appears to be a component of the central circadian oscillat or of Arabidopsis. A second potential Arabidopsis clock compone nt was identified as a result of the identification of a day length-insens itive flowering mutant. The mutant late elongated hypocotyl ( lhy ) caused an elongated hypocotyl and reduced chlo rophyll, as well as an altered flowering phenotype (Schaffer, 1997). lhy mutants were also arrhythmic for leaf movements and for the expression of several othe r clock-regulated genes. Rhythmic expression of LHY was observed with levels of the transcript peaking at daw n.The sequence of the LHY protein was used to screen the GenBank database us ing the TBLASTN program to iden tify any potential homologs in the plant. The Arabidopsis DNA-binding protein CIRCADI AN CLOCK ASSOCIATED-1 (CCA1) was most closely related to LHY (Schaffer et al., 1998) CCA1 was first identified as a factor that binds to the promoter of the Chlorophyll a/b-binding li ght-harvesting complex ( LHCB ) gene in Arabidopsis and functions in the phytochrome signaling pathway to induce the transcription of LHCB (Wang et al., 1997). Later studies revealed that cca1 mutants display a shorter period of circadian rhyt hms. Overexpression of this pr otein disrupted rhythmicity in several clock outputs including hypocotyl elongation, leaf m ovements and circadian gene

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20 expression (Green and Tobi n, 1999; Wang and Tobin, 1998). CCA1 transcripts also oscillate with peak levels of expressi on early in the morning and in constant conditions the rhythms persist suggesting this gene is under circadian control (Wang and Tobin, 1998). In lhy cca1 double mutants circadian rhythms were observed with an abnormal phase and oscillations of transcripts were dampened. Early expression of LHY and CCA1 (morning genes) and some evening genes were also observed in the double mu tants suggesting that these two genes function as components of a negative feedba ck loop (Schaffer et al., 1998). LHY and CCA1 genes function redundantly and are required for the maintenance of circadian rhythms in Arabidopsis (Alabad et al., 2002). Both ge nes are closely related MYB-like transcription factors but are unique in that they only possess a single MYB repeat sequence whereas other myb transcription factors usually contain two to three of the motifs. LHY and CCA1 are also related outside of the MYB domain sharing other regi ons that exhibit at least 80% identity (Fig. 1-3). Overall, the two genes are 46% identical to one another (Schaffer et al., 1998). This sequence analysis suggests that LHY and CCA1 encode related DNA-binding proteins with a single MYB repeat that function as transcription factors. This notion is supported by evidence that shows LHY and CCA1 bind speci fically to a sequence known as the evening element (EE) in the promoter of many genes w hose expression peaks nears dusk (Alabad et al., 2001; Harmer et al., 2000). In Arabidopsis the model for the plant clock compone nts is based on the regulation of the three plant genes described above, CCA1 LHY and TOC1 These components appear to operate in a transcriptional/translationalbased negative feedback loop similar to that observed in other studied systems. Light activation of LHY and CCA1 expression results in tr anscript levels that peak at dawn followed by a peak in proteins appr oximately two hours later. Both of the proteins

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21 bind to the EE motif located in the promoter of the TOC1 gene, a positive element, and repress TOC1 expression during the day (Alabad et al., 2001). A drop in TOC1 protein results in the reduction of LHY and CCA1 transcript and protein levels. Th e low levels of expression of the two genes results in the derepression of TOC1 transcription. In return, levels of TOC1 protein peak during the late evening resulting in the activation of LHY and CCA1 transcription just prior to dawn (Carr and Kim, 2002). This crossregulation between LHY, CCA1 and TOC1 is proposed to function as the central oscillator of the Arabidopsis clockwork where LHY and CCA1 function as the negative elem ents and TOC1 serves as the positive element (Fig. 1-4). This central clockwork regulates numerous genes in Arabidopsis responsible for photosynthesis, nitrogen assimilation, biosynthesis of photo-prot ective pigments, lipid modification, hypocotyl elongation and flowering (Harmer et al., 2000; Schaffer et al., 2001; Ms, 2005). Evolution of Core Clock Components The circadian clock has been we ll characterized in organisms from cyanobacteria to fungi, mice and plants. In these organisms, the central oscillator measures time with a molecular feedback loop or loops that cycle with a 24hr period (Dunlap, 1999). The central timekeeper generates rhythms by controlling transcription of numerous cl ock genes. Regulation of the feedback loop is based on negative elements, which repress their own expression and positive elements that stimulate transcription. The negativ e feedback along with a delay is sufficient to produce oscillations. Although the basic architectu re of the circadian oscillator appears conserved among different species, the mechanisms at the core of the feedback loop differ. Biological clocks have either evolved multiple ti mes to perform similar tasks thus they are an example of convergent evolution. The use of positive and negative elements to regulate transcriptional and translational activity in a feedback loop is common among the well-studied circadian systems (Fig. 1-6). The

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22 positive elements serve as the transcriptional activ ators in the loop and they have been found in Synecohcoccus (kaiA), Neurospora (WC-1 and WC-2), Drosophila (CLK and CYCLE), mammals (CLOCK and BMAL1) and Arabidopsis (TOC1). Similarly, negative elements also compose a portion of the feedback mechanism by inhibiting the action of the positive elements and these include kaiC, FRQ, PER and TIM, PER and CRY and CCA1 and LHY in cyanobacteria fungi fruit flies mice and plants, respectively (Dunlap et al., 1999). Yet despite their similarities, the time at wh ich these elements are expressed, late in the evening or early morning, differs among the organisms. Transcription factor proteins serve important roles in the ci rcadian oscillatory system. The type of transcriptional inducer varies among model systems. In mammals and Drosophila the activators are basic helix loop helix (bHLH) protei ns which contain a spec ific region which binds to DNA (Gekakis et al., 1998; Da rlington et al., 1998). The Neurospora positive elements are similar to the Drosophila complex but they contain an additi onal zinc finger binding domain thus they are categorized as zinc finger factors (L oros and Dunlap, 2001). In the plant circadian oscillator, the activation of tran scription is induced by MYB-like tr anscription factors (Carr and Kim, 2002). The oscillatory mechanism of cy anobacteria is still under investigation. Another difference between the circadian mach inery of different organisms is the number and location of oscillators. Mammals have a master circadian pacemaker that is localized to the suprachiasmatic nucleus (SCN) lo cated within the hypothalamus of the brain. The SCN entrains multiple clocks that are located in the periphe ry of the organism (Yamazaki et al., 2000). In contrast, plants contain at leas t one oscillator in each cell and these oscillators function autonomously and independently of any central pacemaker (Thain et al., 2000; Barak et al.,

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23 2000). This organization makes it is possible to set different r hythms of gene expression to different phases in varying pa rts of a single plant or organ. The general organization of the circadian appa ratus suggests a selec tive advantage in the rhythmic control of physiological and behavioral processes. The ubiquity of the system implies that the endogenous circadian pr ograms enhance fitness. Eviden ce from cyanobacteria suggests that an organism with a circadia n rhythm close to that of its external environment is favored under competition as a result of soft selecti on (Futuyama, 1998). Circad ian clocks are also important since they provide a timing mechanism re quired for the response of organisms to daily and seasonal changes in light. Te mporal organization of processe s such as those involving photolabile enzymes in plants is crucial for the optimization of important endogenous events. The close connection between the clock and light sign aling pathways allow an organism to predict environmental changes even in their absence. Thus, the circadian clock provides an adaptive advantage by enabling the anticipation of th e external transitions and the temporal synchronization of physiological events with specific phases of th e environment (Johnson, 2001). Since a number of discrepanc ies exist between th e circadian components of different systems, the next phase of chronobiology concentr ates on elucidating the molecular mechanisms that underlie the oscillator in ot her species. The identific ation of similar elem ents allows insight into the evolutionary lineage of the clock apparatus as well as the resulting rhythmic outputs while the differences between systems provide relevant information on species-specific adaptations. From this data, it is possible to cr eate evolutionary relati onships between specific clock-controlled genes across numerous taxa. One such project, the Floral Genome Project (FGP) examines gene families in a number of di fferent plant species that play a role in the

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24 evolution of flowering (Albert et al., 2005). One identified family of genes involved in floral initiation was shown to be similar to the LHY and CCA1 transcription factors of the Arabidopsis central oscillator. Genetic and molecular analyses have proven va luable tools in the elucidating the central oscillatory mechanism of circadian clocks. In this study, two EST homologs to LHY and CCA1, the MYB-like family of tr anscription factor s in Arabidopsis, are identified in Eschscholzia californica the California poppy plant. Expressional an alyses suggest that these genes are rhythmic components of the circadian oscillator that participate in the initiation of flowering. This suggests that the feedback loop mechanism of the plant circad ian oscillator is conserved in these two species.

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25 Figure 1-1. The three basic components of the circadian clock. Numer ous input and output pathways function within the system. Figure 1-2. Positive and negative factors act upo n the circadian clock. Negative elements block the activation of the positive elements (bl unted arrow) which in turn promotes the expression of the negative element (pointed arrow).

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26 Figure 1-3. Comparisons of th ree conserved regions of LHY and CCA1. The number at the end of each row corresponds to the last amino acid shown within the original protein. Conserved amino acids are highlighted. Figure 1-4. The Arabidopsis circadian osci llator. Light activat es expression of LHY and CCA1 inducing their transcription near da wn. Their gene product binds to the TOC1 promoter and inhibits its expression during the early day. As LHY and CCA1 expression dwindles, the repression of TOC1 is lifted and TOC1 protein accumulates in the evening. TOC1 then induces the expression of LHY and CCA1 starting the cycle over again.

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27 CHAPTER 2 TWO CCA-LIKE MYB TRANSCRIPTION FA CTORS ARE PRESENT IN CALIFORNIA POPPY Summary Biological clocks play an important role in the lives of many organisms. This machinery allows species to coordinate important activities or behaviors in relation to their environment. The clock generates rhythms over which these events operate with a period of approximately twentyfour hours, thus the term circadian rhyt hms. The clock is primarily composed of three main components: input pathways, a centr al oscillator and output pathways. An analysis was conducted to determine if two circadian clock genes found in the California poppy plant, RISE and SHINE, are rela ted to two transcription factors involved in maintaining circadian rhythms in Arabidopsis, LHY and CCA1. In Arabidopsis, these two genes function in a feedback loop of the central circadian oscillator and are crucial for maintaining rhythms within the organism. A comparison of the genomic sequences revealed that there was an acceptable degree of homology between the two seque nces. In addition, an expressional analysis revealed that the levels of messenger RNA (mRN A) of the genes oscilla ted over a twentyfour hour period which suggests circad ian control. The location of expression was also similar between the two plant species. In young tissue, the transcripts were localized to the meristematic regions as well as the premature leaves. In older tissue, expression was highest in the reproductive organs and pollen grains. Since the circadian clock plays a role in promoting flowering and the release of polle n, it is no surprise the transc ripts were localized to these various regions. Due to these similarities this paper proposes that homologs of LHY and CCA1 exist in the California poppy plant and function as critical components of the negative feedback loop at the center of the circadian oscillator. Additional potential homologs of LHY and CCA1 have been

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28 identified in other species based on sequence similarity. This discove ry suggests that the circadian machinery is conserved among higher pl ants. In addition, temporal organization of important events seems to confer a se lective advantage for the organisms. Introduction Biological clocks are important in maintain ing the rhythmicity of crucial events in different species. In Arabidopsis thaliana two genes, LHY and CCA1 play important roles in generating and maintaini ng the rhythms within the organism (Schaffer, 1997; Schaffer et al., 1998; Wang et al., 1997). They have been identifie d as components of the central oscillator of the clock, one of its three core components. LHY and CCA1 function in a feedback loop, along with the TOC1 gene, in which they negatively regulate their own expression (Alabad et al., 2001; Carr and Kim, 2002). The paus es that occur as a result of this feedback loop are efficient for generating the observed oscillations. These osci llations, in turn, are conveyed as changes in the organisms physiological or behavi oral changes via th e output pathway. Two potential homologs of the LHY and CCA1 genes were identified in Eschscholzia californica on the FGP database (Albert et al., 2005). These two EST sequences could potentially serve as components of a central oscillator in Californ ia poppy. Designated as RISE and SHINE these genes might be the functional equivalents of LHY and CCA1 This paper reveals that these genes show an acceptable degree of sequence homology and share similar expressional patterns suggesting they are co mponents of the central oscillator in Eschscholzia californica Materials and Methods Plant Growth Conditions Arabidopsis thaliana and Eschscholzia californica plants were grown under ideal temperate conditions in the University of Florida Department of Botany greenhouse in

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29 Gainesville, Florida between March and July 2004 and June through September of 2005. California poppy was grown on sterile soil under normal light conditions with a period that matched the exogenous environment. Arabidopsis wa s sowed on autoclaved soil at an irradiance of 100 mol m s as recommended by Kranz a nd Kirchheim (1987), and the day length was set so that it matched the period of the environment. Tissue for in situ hybridizations was harvested mid-morning and included floral meristems and fl oral buds. The collected material was placed in 4% paraformaldehyde to prepare for fixation. Fo r RT-PCR analysis, plant tissue consisting of small leaves, floral meristems and buds was coll ected from each species every four hours for three days. Each sample was placed in liqui d nitrogen and stored in a -80C freezer. Sequence Analysis and DNA Isolation The CCA1 and LHY genomic sequences, identif ied as genes At2g46830 and At1g01060, were identified in the Floral Genome Project (FGP ) database (Albert et al, 2005; Carlson et al., 2006) and recognized as a distinct family of transc ription genes associated with the circadian clock. The sequences were used to search for homo logs in other plant spec ies associated with the FGP database using the sites BLAST program a nd a number of candidate genes were identified. In Eschscholzia californica (California poppy) two ESTs or e xpressed sequence tags, which are small fragments of genes that have been cloned, demonstrated a notab le level of similarity to the Arabidopsis genes. The FGP identification numbers for these two sequences are eca_4_183384 and eca_4_184056 which I will refer to as RISE a nd SHINE respectively. Alignments of the cDNA and protein sequences of CCA1, LHY, RI SE and SHINE were constructed using the GenomeNet database program CL USTALW (Thompson et al., 1994) Clones of the RISE and SHINE sequences we re received from Penn State University, a participant in the FGP grant. Their preparation has been described previously (Carlson et al.,

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30 2006). Luria-Bertani (LB) media was prepared and 2.5 mL cultures were prepared with ampicillin at a concentration of 50g/mL. The cultures were grown for 16 hrs in a shaking incubator at 37C. It was noted that the cultures grew slowly due to their low turn-over rate. The alkaline lysis mini-prep protoc ol (Morelle, 1989) was used to purify plasmid DNA from 1 mL of culture. Arabidopsis genetic material wa s obtained from wild type plants as previously has been described (Edwards et al., 1991). In situ Hybridizations Except for the modifications noted below, pr eviously described methods were used for in situ hybridization (Jackson, 1991; Drews et al., 1991). To generate templates for probe synthesis, DNA from plasmids containing the RISE and SHINE EST sequences as well as genomic DNA isolated from Arabidopsis was PCR amplifie d. The T7 RNA polymerase promoter sequence (TAATACGAGTCACTATAGGG) was pl aced in front of each reverse primer which allowed direct synthesis of digoxigenin-labeled antise nse probes from PCR products. The sense control probes were designed with the T7 promoter sequence in front of th e forward primers. In addition, probes were designed within the exons of the ge nomic sequences of each species in order to hybridize to corresponding messenger RNAs (mRNAs) in situ Since Myb transcription factors contain a similar conserved motif in their amino terminus or 5 region, it was important to design primers in the carboxyl or 3 region that would be unique to each sequence. The following primers were used to amplify RISE templates for probe synthesis: TCTCTTTCGCCTCT ACCGAACA and TAATACGACTCACTATAGGGAAGCACTCTTCAGGGAACCTCA. The primers used to amplify SHINE DNA were ACCACCACCAACTGCAACTCCTAT and TAATACGACTCACTATAGGGTGTACGGCGATTA CTGAAGGGT. Amplification of LHY

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31 DNA used the following primers: CAGTTCCAACTCCAGCAATGAC and TAATACGACTCACTATAGGGCTGAAACGCTATA CGACCCTCT. The primers for CCA1 (TCTGGTTATTAAGACTCGGAAGCCAT and TAATACGACTCACTATAGGGTTCATTGGCCATCTCAGG ATGC) were used to amplify its PCR product. RNA probes were synthesized usin g the Dig-RNA labeling kit (Roche Applied Science, Indianapolis, IN). The cRNA products, 263bp for RISE, 266bp for SHINE, 476bp for LHY and 361bp for CCA1, were synthesized and a dded to the hybridization buffer a so the final concentration was 500 ng mL. Slides were hybrid ized at 45C overnight and washed at 50C. For signal detection, a few grains of tetramisol e hyrochloride (Sigma, St. Louis, MO) was added to the Western Blue substrate (Promega, Madiso n, WI). Slides were evaluated using a Zeiss Axiostar Plus Microscope (Carl Zeiss, In c, Thornwood, NY) and images were photographed with an Axiocam MRc5 camera (C arl Zeiss, Inc., Thornwood, NY). Quantitative RT-PCR For each plant species, fresh tissue including leaves, floral buds and meristematic tissue was collected every four hours for three days and placed immediately in liquid nitrogen and stored at -80C. Total RNA was isolated from the tissues using the RNeasy plant RNA isolation kit (Qiagen, Valencia, CA). RNA concentration was measured using Ribogreen dye (Molecular Probes, Eugene, OR) and a TBS-380 Mini Fluoro meter (Turner BioSystems, Sunnyvale, CA). The RNA templates were transcribed using the Re verse Transcriptase product protocol (Roche Applied Science, Indianapolis, IN). PCR was us ed in order to determine the differences in transcript expression using the same primers described previously for probe construction. Differences in the 18S ribosomal RNA (rRNA) positive control transcripts were determined by using the following primers: TTGTGTTGGCTTCGGGATCGGAGTAAT and

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32 TGCACCACCACCCATAGA ATCAAGAA (Cho and Cosgrove 2000). PCR products were separated by size on agarose gels stained with ethidium bromide and visualized under a UV light. Images of the gels were captured using a ChemImager 4400 (Alpha Innotech Corp., San Leandro, CA) and the relative sizes of bands we re determined by comparison to a standard 1kb plus DNA ladder (Invitrogen, Carlsbad, CA). Results RISE and SHINE Encode CCA1-like Myb Transcription Factors Sequence similarity among genes of different species can provide re levant information about the evolution of particular gene familie s and the conservation of important mechanisms. RISE and SHINE share significant sequence identity with the CCA1 and LHY genes of Arabidopsis. In particular, RISE was shown to be comparable to LHY with over 40% identity in a region at the C-terminus. SHINE displayed sim ilarity to the CCA1 sequence with 30% identity in the C-terminal region (Fig. 21). These regions located at the carboxyl or 5 end of the genes and ESTs corresponds to a DNA-binding domain that is found in plant Myb transcription factors. This motif is highly conserved among the Myb gene family and provides evidence that RISE and SHINE are indeed part of the family (Fig. 2-2). Additionally, RISE and SHINE share 40% identity in the region inves tigated which demonstrates th e redundancy between the two components (Fig. 2-3). RISE and SHINE Expression is Under Circadian Control Circadian clock genes (ccgs) show a rhythmic pattern of transcript and protein expression. In Eschscholzia californica this is no exception. The RISE and SHINE transcripts oscillate over a 24 hr period. Both RISE and SHINE oscillat e in a pattern analogous to CCA1 and LHY in Arabidopsis (Fig. 2-4). The tr anscripts abundance varies during the day. Peak levels of transcription occur just prior to dawn and decr ease throughout the day. By evening, the levels of

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33 RISE and SHINE transcripts are greatly reduced but begin to ri se in the early hours of the morning. This evidence supports th e notion that RISE and SHINE are activated by a light signal similar to the mechanisms of CCA1 and LHY (Fi g. 2-5). The transcripts also oscillate with a period of approximately 24 hrs, wh ich matches the external envir onment of the organism, further supporting their role in the circadian clock. Similar Expression Patterns in a CCA1like Family of Myb Transcription Factors In situ hybridizations are ideal for determining th e location of transcri pt expression within an organism. This method was used to analy ze the expression pattern of CCA1 and LHY in Arabidopsis thaliana and RISE and SHINE in Eschscholzia californica For both species, two stages of development were analyzed: a younger stage characterized by premature inflorescence meristems and a later stage which is exemplified by floral buds. In the young Arabidopsis tissue, a strong LHY si gnal is detected in the meristematic region and in the stamen and carpel primordia (Fig 2-6A ). In older tissue, LHY is expressed in the gynoecium, ovules, and anthers and to a lesser degree in pollen grains (Fig. 2-6B). The expression of the LHY homolog, CCA1, is similar to its counterpart. High transcript levels are detected in the young developing fl oral meristem and include the premature reproductive organs (Fig. 2-6C). Expression of CCA1 in older tissue is limited to the reproductive tissues (Fig. 2-6D). The sense probe does not have a signal (Fig. 2-6E). The expression patterns of the CCAlike Myb transcription factors in Eschscholzia californica is similar to the patterns observed in Ar abidopsis. No signal coul d be detected on the sense probe control (Fig. 3-7E). High levels of RISE transcri pt were detected in the young developing tissue in the meristema tic region, premature leaves and axillary buds (Fig. 2-7A). In older tissue, expression is highest in the carpel, ovules, anthers and pollen grains but is still

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34 detected in the developing petals (Fig. 2-7B ). Like its Arabidopsis counterpart, SHINE transcripts are detected in both the young and ol der stages of the poppy plant. Transcripts are detected in the sepal primordia, cauline leaves and the floral meristem (Fig. 2-7C). SHINE expression in older tissue is c onfined to the reproductive orga ns and petals (Fig. 2-7D). However, it should be noted that a lower expr ession level can be detected for both genes throughout the specimen (Fig. 2-7A-D), showing th at RISE and SHINE transcripts are located within a variety of tissue types.. Discussion Feedback Loop Mechanism as the Ba sis of the Circadian Oscillator An autoregulatory feedback loop involving both positive and negative elements is central to the circadian oscillator. Cir cadian systems are often composed of one or more interconnected loops. Knowledge of these interloc ked loops results from the identif ication of genes that function within the core loop of the oscillator. In Arabidopsis thaliana three genes with required roles in maintaining rhythmicity have been identified: LHY, CCA1 and TOC1 (Schaffer, 1997; Wang et al., 1997; Millar et al., 1995). The two Myb tran scription factors LHY and CCA1 serve as the negative elements of the core loop and function to block the ac tivation of the positive element TOC1. The positive regulator, TOC1 activates e xpression of LHY and CCA1. In this study, we have identified two potential homologs to LHY and CCA1 in Eschscholzia californica These genes are hypothesized to serve similar roles in the core oscillator of the poppy plant, thus providing a conserved mechanism for main taining rhythmicity in higher plants. The RISE and SHINE Genes Encode MYB Transcriptio n Factors That Are Similar to LHY and CCA1 Sequence analyses revealed that two Califor nia poppy EST sequences located in the FGP database (Albert et al, 2005; Carlson et al., 2006) share sequence identity with known

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35 components of the central circadian oscillator from Arabidopsis (Fig. 2-1). The Myb domain, which functions as a DNA-binding domain, shares the most sequence similarity with RISE and SHINE. Research shows that LHY and CCA1 bind to an evening element (EE) located within the promoter of TOC1, an evening gene (Alaba d et al., 2001; Harmer et al., 2000). Because of the similarity between the components of these tw o circadian systems, I wa nted to determine if RISE and SHINE function in a similar manner to CCA1 and LHY in the Arabidopsis central oscillator. A prerequisite for a protein to function as a ne gative element in the circadian clock is that its expression and activity must oscillate in s ynchrony with the environmental oscillations. In addition, this component regulates its own tran scription by negative feedback which creates a delay in the rhythmic cycle. In the Arabidopsis model plant, LHY and CCA transcripts were shown to oscillate over a 24 hr period with peak levels accumulating just prior to dawn (Mizoguchi et al., 2002). Simila r results for this species we re obtained (Fig. 2-5). The RISE and SHINE transcripts displayed a similar pattern of expression with minimal levels of mRNA detected in the evening (Fig.2-4). The accumulation of transcripts just prior to dawn shows that LHY and CCA1, as well as RISE and SHINE, are regulated by a light signal and are entrained to anticipate dawn. The genes at the center of the Arabidopsis cir cadian oscillator serve as either positive or negative factors to influence th e rate of transcription. The ac tivation and inhibition of ccgs occurs at particular points with in the circadian cycle and when coupled, form a loop in which the components serve crucial roles in generating and maintaining rhythmicity within an organism. The LHY and CCA1 gene products in Arabidopsis function in a manner that is antagonistic to TOC1. In Eschscholzia californica putative homologs for LHY and CCA1 have been identified

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36 but other components of the os cillator remain unknown. Based on the previously described similarities between the two systems, it is reasonable to hypothesize that a TOC1 like gene also functions in the poppy oscillator (Fig. 2-8 ). The spatial expression pattern s of the Myb transcription factors in Arabidopsis and California poppy provide relative information about th eir functional similaritie s. In both species, the young tissue contained a high level of expressi on in the meristem and sepal primordia (Fig. 2-6A, C; Fig. 2-7A, C). The older tissues were ch aracterized by high levels of transcript in the reproductive organs and petals (F ig. 2-6B, D; Fig. 2-7B, D). The similarity in the expression pattern suggests that th ese genes might be true orthologs stemming from a common ancestor. In addition, the location of expressi on provides relevant information on the processes regulated in that particular region. The high level of transcript expression in pollen grains in the older tissue of Arabidopsis and poppy could control the timed release of pollen, a mechanism that evolved for maximizing reproductive succe ss (Subba et al., 1998). The fact that the circadian clock regulates expression of floral path way genes that in turn activate floral meristem identity genes (Vijayraghavan et al., 2005) seems logical to ex plain the high level of expression of the Myb transcription factors in the meristematic regions of the young tissue. Together these sequence comparison and mRNA expression data suggest that RISE and SHINE encode Myb transcription factors that coul d function as the negative elements in the oscillator of the California poppy plant similar to the manner of LHY and CCA1 in Arabidopsis. An Evolutionary Conserved Clock Mechanism in Higher Plants Although the above data suggests similaritie s exist among the circadian systems of Arabidopsis thaliana and Eschscholzia californica little is known about the elements and mechanisms underlying the clocks of other highe r plants. It is possible that the molecular

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37 components that form the clock machinery are uni que to higher plants. In this case it is important to determine whether other plant species have homologs for each of the Arabidopsis clock components and whether they share simila r functions. This paper demonstrates that homologs for two Arabidopsis clock genes exis t in the California poppy plant and that they appear to be expressed in a similar manner to their counterparts. In other plant species, components of the central oscillator remain unknown however, recent evidence has identified several clock-associated genes th at are involved in the input pathway to the clock. Studies of Pisum sativum peas, have revealed circad ian clock gene homologs of TOC1 CCA and LHY referred to as TOC1 and MYB1 (a CCA1 / LHY homolog) respectively (H echt et al., 2007). Two additional Arabidopsis orthologs, EARLY FLOWERING4 ( ELF4 ) and LATE BLOOMER1 ( LATRISE ), were characterized in pea plants and their diurnal rhythm expression conformed closely to those associated with their counterparts, ELF4 and GIGANTEA ( GI) In Arabidopsis, ELF4 promotes clock entrainment and is requir ed for sustained rhythms in the absence of environmental cues (McWatters et al., 2007). The GI gene regulates flower ing in long day (LD) conditions in a clock-controlled pathway, where it acts as an intermedia te between the central oscillator and the FLOWERING LOCUS T ( FLT ) gene (Mizoguchi et al., 2005). Investigations in the clock components in other species continue, including Oryza sativa (rice), Medicago trunculata ( a legume) and Lycopersicon esculentum (tomato). In addition, sequence analysis of the Myb family of transcription factors using the FGP database (A lbert et al, 2005; Carlson et al., 2006) revealed one CCA1/LHY homolog in Cucumis sativus (cucmber), Asparagus officinalis Liriodendron tulipifera (tuliptree) and Saruma henryi (standing ginger) an d two homologs in Acorus americanus (the American Sweet Flag) and Nuphar advena (water lily). The presence of similar sequences across a wide variety of species suggests that the oscillator mechanism that

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38 involves CCA1 and LHY in Arabidops is is conserved in higher pl ants. The conservation of this mechanism and its components implies that such organization is beneficial for the organism. The ubiquity of the feedback loop mechanism of plant circadian oscillato rs suggests that an adaptive advantage results from the spatial and temporal organization of important rhythmic activities. A recent experiment compared the perf ormance of wild type Arabidopsis plants with lines having mutations that alter period length in a range of en vironmental period lengths that were either matched or mismatched to the endogenous clock. The re sults showed that a photosynthetic advantage was conferred by matc hing the endogenous clock period with the light/dark period (Dodd et al., 2005). Incorrect matching of the peri ods resulted in reduced leaf chlorophyll, reduced assimilation, reduced growth and increased mortality (Dodd et al., 2005). Optimization of physiological parameters by the circadian clock probably has been selected during plant evolution. Similar results have been described in the cyanobacteria Synechococcus as well (Ouyang et al., 1998) The Biological Importance of Circadian Clock Genes Although this paper addresses the circadian oscillator and its key mechanisms in higher plants, the importance of the circadian machiner y also resonates through out the animal kingdom. In addition, elucidating the compone nts underlying the feedback loops of the oscillator in either plants or animals provides relative information on the general architectur e of the mechanisms. Both plants and animals use circadian clocks to temporally organize important processes involving reproduction and developmen t which are crucial in the evolution of every species. In humans, many behaviors are regulat ed by the circadian clock including the sleep/wake cycle, feeding patterns, hormone production and cell re generation (Edgar et al ., 1993; Stokkan et al., 2001; Czeisler and Klerman, 1999; Shibata, 2004).

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39 A number of human illnesses are attributed to a dysrhythmia in a behavioral or physiological process. Abnormal circadian rhythms ha ve been associated with affective disorders like A number of human illnesses are attributed to a dysrhythmia in a behavioral or physiological process. and the existing therapy drugs used to treat these disorders such as lithium act upon the circadian cycle (Hallonquist et al., 1986). Insomnia and sleep problems also result from abnormal circadian rhythmicity and usually are characterized by an endoge nous clock that runs faster or slower than the norm (Zisapel, 2001). Individuals that suffer from attention-deficit hyperactivity disorder (ADHD) are often plague d by sleep disturbances which result from a dysrhythmic clock (Owens, 2005). In women who suffer from menopa use, hot flashes disrupt the clocks rhythm resulting in a clock that is mi sentrained. This abnormal entrainment results in sudden awakenings during the sl eep cycle (Freedman et al., 1995). Recently a role for the circadian clock has been identified in cancer studie s. Research suggests that at least eight central clock genes coordinate many ba sic functions, including cell pro liferation, tumor growth and apoptosis in circadian time. This work indicates that circadia n clock genes and their products potentially represent novel ta rgets for the control of cancer growth (Wood et al., 2006). Elucidating the mechanisms that lie beneat h the circadian oscillator has become the primary focus of chronobiologists. A wealth of know ledge stands to be gained since nearly all processes crucial for species su rvival involve rhythmicity of one or more elements. The availability of technologies to analyze global gene expression s hould become a powerful tool in clock research. This advancement should aid in the identification of new genes affected by the timekeeping apparatus and help char acterize the interactions of t hose clock proteins that have been previously identified. Howeve r, the question of how the gene s involved in the clocks are regulated is just star ting to be addressed.

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40 In this study, two homologs of the Arabi dopsis Myb transcription factors CCA1 and LHY were identified in Eschscholzia californica Sequence analyses suggest that these genes are true orthologs and are similar in their temporal a nd spatial expression. This information provides evidence that there is a conserved transcriptionaltransl ational feedback loop at the center of the circadian oscillator in higher plants. Based on this congruence, other circadian clock genes involved in the maintenance of rhythms in Ca lifornia poppy should resemble those described in Arabidopsis (Fig. 2-8).

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41 CCA1 TYP--------------------MHIPVLVPLGSSITSSLSHPPSEPDSHPHTVAGDYQS LHY NHPSGMVSQDFMFHPMREETHGHANLQATTASATTTASHQAFPACHSQDDYRSFLQISST RISE -----------------------------------------------------------SHINE TTEQN------------------SHTSRSSVHQTLPNFPPPFAPLHNPETYRSFANMSST CCA1 FPNHIMSTLLQTPALYTAATFASSFWPPDSSG------------GSPVPGNSPPNLAAMA LHY FSNLIMSTLLQNPAAHAAATFAASVWPYASVGNSGD--------SSTPMSSSPPSITAIA RISE ------------------ALFRREVLP---------------------QSFSPP-----SHINE FPCFLMSALLQNPAAHMAATLAASLWPGSNGETSLDSSSMPLGGFPLGQASPTPNLAAIA : .. ..* CCA1 AATVAAASAWWAANGLLPLCAPLSSGGFTSHPPSTFGPSCDVEYTKASTLQHGSVQSREQ LHY AATVAAATAWWASHGLLPVCAPAPITCVPFSTVAVPTPAMTEMDTVENTQPFEKQNTALQ RISE -----------------------------------------------------------SHINE ATVAAASAWWAAHGMMPLCPP-------------------------------------CCA1 EHSEASKARSSLDSEDVEN-------KSKPVCHEQPSATPESDAKGSDGAGDRKQVDRSS LHY DQNLASKSPASSSDDSDETGVTKLNADSKTNDDKIEEVVVTAAVHDSNTAQKKNLVDRSS RISE ---------------------------------------------------------SHINE -----------------------------------------------------------CCA1 CGSNTPSSSDDVEADASERQEDGTNGEVKETNEDTNKPQTSESNARRSRISSN------I LHY CGSNTPSGSD-AETDALDKMEKDKE-DVKET--DENQPDVIELNNRKIKMRDNNSNNNAT RISE -----------PDLKKALFREPQNSIMVTEQIQDEKDENMLQLN--------------LM SHINE -------------LHPSFSYPPPPPPTATPMDINQAPPVN-------------------N .. : CCA1 TDPWKSVSDEGRIAFQALFSREVLPQSFTYREEHREEEQQQQEQRYPMALDLNFTAQLTP LHY TDSWKEVSEEGRIAFQALFARERLPQSFSPPQVAENVNRKQSDTSMPLAP--NFKSQDSC RISE SNSWGEVNP-----------------NPPPPSDNNNVEKDSFLS---------------SHINE NEKQDNIPE-------------------DPPWEVQQLDPEQSEATKPPNPS--------.: .: .: : .. CCA1 VDDQEEKRNTGFLGIGLDASKLMSRGRTGFKPYKRCSMEAKESRILNNNPIIHVEQKDPK LHY AADQE-----GVVMIGVGTCKSLKTRQTGFKPYKRCSMEVKESQVGNIN--NQSDEKVCK RISE -----------IETVGLGSGKFKAR-RTGFKPYKRCSVEAKESRMSNGN----CEEQGPK SHINE ---------PKSPSLSSSDSADSGGARSDYIKPISTANEDNPSVIAVHD-----SNKSKA :. ::.: : : : : .:: CCA1 RMRLETQAST LHY RLRLEGEAST RISE RIRLEGEPSA SHINE RKKX-----* : Figure 2-1. Alignment of the LHY/CCA1 genes in Arabidopsis thaliana and Eschscholzia californica. The nucleotide sequences of LHY an d CCA1 were used to search the FGP database (Albert et al., 2005; Carls on et al., 2006) using BLASTN in order to form an alignment with the SHINE and RI SE ESTs. The residues shaded in gray represent the bases that are conserved among all four genes. Those highlighted in yellow represent the nucleotides that ar e conserved between LHY and RISE. The residues in blue illustrate the c onserved bases among CCA1 and SHINE.

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42 Figure 2-2. A phylogenetic analys is of CCA-like family of gene s. This tree illustrates the relationships among the CCA-like family of Myb transcri ption factors in Arabidopsis thaliana (CCA, LHY), Eschscholzia californica (SHINE, RISE) and Nuphar advena (NAD), a basal angiosperm. Here, the NAD gene was identified in the FGP EST database and serves as the outgroup for th is analysis. Multiple sequence alignment and tree construction were produced usi ng the MAFFT program (Katoh et al., 2002; Katoh et al., 2005).

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43 1 60 RISE --------------------GCTCTCTTCAGGAGAGAA-GTTCTG---CCACAAAGTTTT SHINE GCAGAAACTACTGACGGGGAAAATTGTTCAGAAGTCATTACCCTGATTCGAGAAACTTCT ***** ** *** *** ** 61 120 RISE T----CCCCACCACCTGATCTGAAAAAAGCACTCTTCAGGGAACCTCAAAAC---AGTAT SHINE TGTGTTTCTATCCCTTCTGCAAAGAAAAGTTTGATTTCAACATCTGCTGTACTCAAAAGT * * ***** ** ** 121 160 RISE TATGGTAACAGAACAGATTCAAGA----TGAGAAAGATGAAAATATGTTGCAATTAAACC SHINE TCCTGCACCTTTATGGAGTTTGTGCCATTGAGAAAGGAACCAACTCAAAATAGTAAACAG * ** ******** ** ** 161 180 RISE TTATGAGTAATTCATGGGGAGAAGT-AAATCCTAATCCCCCTCCTCCGTCCGACAACAAT SHINE GTCGAAGTTGATGATTCACAGAAATTGGACAAAAATGACCCCAGATTTTCTGATACCGAG *** ** **** *** *** ** ** 181 240 RISE AACGTAGAGAAGGATAGTTTTTTGTCTAT-AGAAACTGTAGGGCTTGGATCAGGAAAGTT SHINE AATATAAGCTTGGGTGAAGCTCTGAAGTTGAAAAACAGCAAGATTAACTCTAATGAGAAA ** ** ** ** **** * 241 300 RISE CAAGGCCCGTCGAACAGGCTTTAAACCGTATAAGAGATGTTCGGTAGAGGCGAAAGAGASHINE CTAGTCCTGGAGAACAATACCGAAA----AACAAAAGCAACCAGAAAAACCGAGTAACTT ** ** ***** *** * *** 301 360 RISE GCAGAATGAGTAA-CGGAAACTGTGAGGAGCAAGGTCCGAAGAGGATACGTTTAGAAGGT SHINE ACATACTGAGGAAGTACAAGGTAACCAGAGCTACCCACGGCATGTTCCTGTCCATATTGT ** **** ** ** **** ** ** ** 361 420 RISE GAACCTTCAGCTTGA--------------------------------------------SHINE GGATATGAGCTCTGGCCCAGTTGATATTTGCCCTTCATCCAAAATGTACCATTCAGGAGC ** Figure 2-3. Alignment of the RISE (eca _4_183384) and SHINE (eca_4_184056) EST sequences using the BLASTN program from the FGP da tabase (Albert et al, 2005; Carlson et al., 2006). Conserved residues are shaded in gray.

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44 Hours 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 RISE SHINE 18S Figure 2-4. Analysis of RISE and SHINE mRNA expression. Peak RISE and SHINE transcript levels occur just prior dawn and dwi ndle throughout the day. The 18S r RNA transcripts demonstrate a constant expre ssion level throughout the day. The bar above reflects the light/dark cycles to which the plants were e xposed for this experiment. The black boxes correspond to periods of darkness.

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45 LHY CCA1 18S Hours 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 Figure 2-5. Analysis of LHY and CCA1 mRNA expression. Peak transcript levels of the transcription factors occur just prior dawn and become greatly reduced by evening. The 18S rRNA transcripts demonstrate a c onstant expression level throughout the day. The bar above reflects the light/dark cy cles to which the plants were exposed for this experiment. The black porti ons represent the evening hours.

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46 Figure 2-6. Expression of LHY and CCA1 in Arabidopsis tissue. LHY and CCA1 transcripts were located in both young and mature Arabidopsis floral tissue. The localization of the transcripts was determined by in situ hybridizations. LHY transcripts were present in A) the floral meristem (fm) and the floral primordia (fp) of the young tissue and in B) the gynoecium (g), ovules (o), anthers (a) and pollen grains (pg) of the mature floral tissue. CCA1 transcripts were detected in C) you nger tissue specifically in the fm and fp and in D) the older tissue in the same location as observed with LHY: g, o, a and pg. E) The sense probe control shows no signal. B C E fp fm fp fm o o fm f p pg pg g g a a A D f p

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47 Figure 2-7. RISE and SHINE transcripts are expressed in bot h young and mature floral tissue of the California poppy plant. The localization of the transcripts was determined by in situ hybridizations. RISE transcripts were present in A) the floral meristematic region (fm), premature leaves (l) a nd in the axillary buds (ax) of the younger tissue while in B) the older tissue, high expres sion was detected in the carpel(c), ovules (o), anthers (a) and pollen grains (pg) while lower expres sion was observed in th e floral tube (ft) and developing petals (pe). SHINE transcripts were detected in C) younger tissue in a pattern identical to RISE: fm, l and ax. SHINE transcripts in D) older tissue was restricted to the c, o, a and pg as well as the ft and pe. E) The sense probe control shows no signal. A B C D E fm fm l l ax ax c c o o a a pg pg ft ft ft pe p e pe ft o c pe p e pg a

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48 Figure 2-8. Proposed mechanism of the central oscillator of Eschscholzia californica. RISE and SHINE serve as negative regul ators of a gene X (desi gnated by a blunted arrow), a TOC1 homolog. That gene in turn sh ould induce expression of RISE and SHINE (pointed arrows). Candidate genes for co mponent X have yet to be identified.

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49 CHAPTER 3 CONCLUSION Elucidating the mechanisms that lie beneat h the circadian oscillator has become the primary focus of chronobiologists. A wealth of know ledge stands to be gained since nearly all processes important for species su rvival involve rhythmicity of one or more elements. Research on the crucial components that are required to ma intain rhythmicity could provide insight into possible therapies and treatments for diseases that target the clock system. In addition, by analyzing the mechanisms and proteins of th e central oscillator a nd comparing them among different species, the evolutionary hi story of the clock can be examined. In this study, it was proposed that a co mmon clock ancestor exists among higher plant species. In Eschscholzia californica, two homologs of Arabidopsis Myb transcription factors were identified as potential co mponents of the biological clock. Other probable homologs to components of the Arabidopsis cen tral oscillator LHY and CCA1 have been identified in other species including Cucumis sativus (cucumber), Asparagus officinalis, Liriodendron tulipifera (tuliptree), Saruma henryi (standing ginger), Acorus americanus (the American Sweet Flag) and Nuphar advena (water lily). Further investigation of these species should provide relevant information on the clock mechanism and its conser vation. Genetic screens for clock mutants aids in the assignment of potential components to roles within the circadian clockwork. Altered expression, either increased or greatly reduced, of these constituents should result in aberrant clock phenotypes. Although it is proposed that the RISE and SHINE genes of Eschscholzia californica play a role in the central oscillator of the plants clock, further st udies similar to those mentioned above are required for verification.

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57 BIOGRAPHICAL SKETCH Meredith Lynn Sullivan was born on January 13, 1979 in Tuscaloosa, Alabama. The younger of two children, she graduated from Ce ntral High School in 1997. Following graduation, Meredith enrolled at Shelton State Community College where she was a member of the womens varsity soccer team. She subsequently enrolled at the University of Alabama (UA) where she earned her B.S. in Biology in 2002. Upon receiving her B.S. degree, Meredith enrolled in graduate school at the University of Florida (UF) in 2003. As a gra duate student in the Department of Botany, she pursued molecular biologybased studies to eluc idate the components and mech anisms involved in flower regulation. This information allo wed her to analyze the evoluti onary significan ce of circadian clocks in development. Upon completion of her M.S. degree, Meredith will pursue a career in medical research. She plans to utilize th e knowledge she has obtained througho ut her education to aid in the identification of therapeutic drugs for certain illnesses.