The Circadian Clock
in Annuals and Perennials
Coordination of Growth with
Environmental Rhythms
Mikael Johansson
Akademisk avhandling
som med vederbörligt tillstånd av Rektorsämbetet vid Umeå universitet för
avläggande av filosofie doktorsexamen i Växters cell- och molekylärbiologi
framläggs till offentligt försvar i KB3B1, KBC-huset, Umeå universitet,
fredagen den 1 Oktober kl. 10:00. Avhandlingen kommer att försvaras på
engelska.
Fakultetsopponent:
Dr. Paul Devlin,
Royal Holloway University of London,
United Kingdom
Umeå Plant Science Centre
Department of Plant Physiology,
Umeå University, Umeå, Sweden
2010
The Circadian Clock in Annuals and Perennials:
Coordination of Growth with Environmental Rhythms
Mikael Johansson
Umeå Plant Science Centre, Department of Plant Physiology, Umeå University
ISBN: 978-91- 7459-062-3
Abstract
Since the first signs of life on planet earth, organisms have had to adapt to
the daily changes between light and dark, and high and low temperatures.
This has led to the evolution of an endogenous time keeper, known as the
circadian clock. This biological timing system helps the organism to
synchronize developmental and metabolic events to the most favorable
time of the day. Such a mechanism is of considerable value to plants, since
they in contrast to animals cannot change location when the environment
becomes unfavorable. Thus is the ability to predict coming events of central
importance in a plants life. This thesis is a study of the molecular machinery
behind the clockwork in the small weed plant Arabidopsis thaliana as well as
its close relative perennial; the woody species Populus. We have
characterized a novel component of the circadian clock, EARLY BIRD
(EBI). EBI is involved in transcriptional and translational regulation, via
interaction with the known post-translational clock regulator ZEITLUPE
(ZTL). In Populus, we describe the role of the circadian clock and its
components with respect to entry and exit of dormancy and show that gene
expression of the Populus LATE ELONATED HYPOCOTYL (LHY)
genes are crucial importance for freezing tolerance and thereby survival at
high latitudes. Furthermore, the input to the Populus clock is mediated via
the phytochrome A (phyA) photoreceptor.
Keywords: Arabidopsis, Populus, Circadian Clock
The Circadian Clock
in Annuals and Perennials
Coordination of Growth with
Environmental Rhythms
Mikael Johansson
Umeå Plant Science Centre
Department of Plant Physiology,
Umeå University, Umeå, Sweden
2010
© Mikael Johansson, 2010
Detta verk skyddas enligt lagen om upphovsrätt (URL 1960:729)
ISBN: 978-91- 7459-062-3
Cover: Arabidopsis seedlings expressing CAB:LUC over 24 hours
Electronic version available at http://umu.diva-portal.org/
Printed by: Arkitektkopia, Umeå, Sweden 2010
ii
Till min kära mor
iii
Abstract
Since the first signs of life on planet earth, organisms have had to adapt to
the daily changes between light and dark, and high and low temperatures.
This has led to the evolution of an endogenous time keeper, known as the
circadian clock. This biological timing system helps the organism to
synchronize developmental and metabolic events to the most favorable
time of the day. Such a mechanism is of considerable value to plants, since
they in contrast to animals cannot change location when the environment
becomes unfavorable. Thus is the ability to predict coming events of central
importance in a plants life. This thesis is a study of the molecular machinery
behind the clockwork in the small weed plant Arabidopsis thaliana as well as
its close relative perennial; the woody species Populus. We have
characterized a novel component of the circadian clock, EARLY BIRD
(EBI). EBI is involved in transcriptional and translational regulation, via
interaction with the known post-translational clock regulator ZEITLUPE
(ZTL). In Populus, we describe the role of the circadian clock and its
components with respect to entry and exit of dormancy and show that gene
expression of the Populus LATE ELONATED HYPOCOTYL (LHY)
genes are crucial importance for freezing tolerance and thereby survival at
high latitudes. Furthermore, the input to the Populus clock is mediated via
the phytochrome A (phyA) photoreceptor.
Keywords: Arabidopsis, Populus, Circadian Clock
iv
Sammanfattning
Liv på jorden har alltid behövt anpassa sig till de dagliga växlingarna mellan
främst ljus och mörker. Detta har lett till evolutionen av en intern, biologisk
klocka, känd som den circadianska klockan, efter latinets ”circa diem”, som
betyder ”ungefär en dag”. Denna inre klocka hjälper organismer att styra
biologiska processer till den tid på dygnet som är mest gynnsam för deras
utveckling och överlevnad. Denna mekanism är av stort värde för växter,
eftersom de inte kan söka skydd på mera lämpliga platser om de blir utsatta
för olika former av stress. Det gör att förmågan att förutse kommande
händelser är av yttersta vikt för växter. Denna avhandling är en studie av det
molekylära nätverk som styr denna biologiska klocka i den lilla örtplantan
Arabidopsis thaliana (backtrav), och den besläktade träd-arten Populus (hybridasp). Vi har karaktäriserat en ny komponent i den circadianska klockan i
Arabidopsis, EARLY BIRD (EBI). EBI är involverad i transkriptionell och
translationell reglering av klockan, via interaktion med den kända posttranslationella klock-regulatorn ZEITLUPE (ZTL). I Populus har vi beskrivit
den interna klockan och dess roll i processer som invintring, vinterdvala och
återstart av tillväxt. LATE ELONATED HYPOCOTYL (LHY) generna i
Populus är avgörande för förvärv av köld-tolerans och således överlevnad på
högre latituder. Dessutom har vi visat att signaler till den circadianska
klockan i Populus är medierade via fotoreceptorn phytochrome A (phyA).
v
Abbreviations
All abbreviations are explained when they first appear in the text.
vi
List of Papers
This thesis is based on the following publications, which will be referred to
in the text by their Roman numerals (I-V).
I
Mikael Johansson*, Harriet G. McWatters*, László Bakó, Naoki
Takata, Péter Gyula, Anthony Hall, David E. Somers, Andrew J.
Millar and Maria E. Eriksson. Partners in time: EARLY BIRD
reveals novel regulatory function of ZEITLUPE in the
Arabidopsis clock Manuscript
II
Kevin Ashelford*, Maria. E. Eriksson*, Christopher M Allen, Linda
D’Amore, Mikael Johansson, Peter Gould , Susanne Kay, Andrew
J. Millar, Neil Hall and Anthony Hall. Full genome re-sequencing
reveals a novel circadian clock mutation in Arabidopsis
Manuscript
III
Mikael Johansson, Iwanka Kozarewa and Maria E. Eriksson
ZEITLUPE promoter activity is modulated in an EARLY
BIRD dependent manner in response to abscisic acid
Manuscript
IV
Cristian Ibáñez*, Iwanka Kozarewa*, Mikael Johansson*, Erling
Ögren, Antje Rohde, and Maria E. Eriksson. Circadian Clock
Components Regulate Entry and Affect Exit of Seasonal
Dormancy as Well as Winter Hardiness in Populus Trees
Plant Physiology (2010) 153:1823–1833
V
Iwanka Kozarewa*, Cristian Ibáñez*, Mikael Johansson, Erling
Ögren, David Mozley, Eva Nylander, Makiko Chono, Thomas
Moritz and Maria E. Eriksson. Alteration of PHYA expression
change circadian rhythms and timing of bud set in Populus.
Plant Mol Biol (2010) 73:143–156
*
These authors contributed equally to respective manuscript
Paper IV is published with the kind permission of the American Society of Plant Biologists.
Paper V is published with the kind permission of Springer/Kluwer Academic Publishers.
vii
Clock linguistics
Annual and perennial plants – Annual plants complete their life cycle
over one season, while perennial plants lives for three or more seasons.
Bud burst – Breaking of buds, growth resumption following, for
example, winter dormancy
Bud set/formation – The process following cessation of elongation
growth when the apical meristem is enclosed by bud scales
Circadian, Circannual – A circadian rhythm repeats itself daily, a
circannual rhythm repeats yearly.
Critical day length – The length of photoperiod/darkness required to
trigger a photoperiodic response, for instance dormancy or flowering.
Diurnal, nocturnal – Diurnal organisms are active during the day,
nocturnal during the night.
Dormancy – The inability to initiate growth from meristems under
favourable conditions. A state during the winter season in woody species
such as Populus, when no visible growth occurs.
Entrainment – The process that sets the pace of a circadian rhythm with
an external cue.
Free-running – A rhythm that is not affected by any zeitgebers and thus
runs freely.
Growth cessation – Inhibition of internode elongation or cambial growth
Input, output – Input is a signal that goes to the clock and then,
depending on (for example) time of day, will result in an output, e.g. a
physiological response.
Long/short day plants – Plants that require a short/longer period of
darkness to flower
Masked rhythms – rhythms that are hidden by other non-circadian
events that happen to have the same pattern.
Oscillator – The core clock genes keeping track of time are often
collectively referred to as a biological oscillator
Period, Phase, Amplitude – Nomenclature to describe a rhythm; see
‘Expression of Clock Genes’.
Rhythm - The repetitive pattern of for example gene expression.
Zeitgeber – All signals that have the ability to entrain the endogenous
clock.
viii
Table of Contents
Abstract…………………………………………………………………iv
Sammanfattning………………………………………………………...v
Abbreviations…………………………………………………………...vi
List of Papers…………………………………………………………..vii
Clock Linguistics……………………………………………………..viii
Preface .................................................................................. 1
Introduction ......................................................................... 3
History of clock research .......................................................... 3
Plant clock history ......................................................................................... 4
What is a circadian rhythm? ......................................................................... 6
Gating.............................................................................................................. 7
Entrainment ................................................................................................... 7
Zeitgebers ....................................................................................................... 8
Expression of clock genes............................................................................ 9
Photoperiodism ...........................................................................................10
Photoperiodism in plants ..............................................................................11
Light perception...........................................................................................12
Clock genetics ..............................................................................................14
Clock model organisms ..............................................................................15
Cyanobacteria..............................................................................................15
Neurospora..................................................................................................16
Drosophila ..................................................................................................16
Mammals (including humans)......................................................................16
Plant clocks...................................................................................................17
The molecular clock in Arabidopsis............................................................18
The Circadian clock at the whole plant level...........................................23
Populus and other plant species ..................................................................24
Temperature limits.......................................................................................24
Conclusions ..................................................................................................26
Aims of thesis .......................................................................... 26
Methodology and Experimental Techniques .................... 27
Experimental conditions to observe circadian rhythms ........................27
Time series measurements .........................................................................28
Physiology.....................................................................................................28
Leaf movement.............................................................................................28
Hypocotyl length ..........................................................................................29
ix
Flowering.....................................................................................................29
Luciferase constructs: imaging of gene transcription.............................29
Results and Discussion ...................................................... 31
The search for EARLY BIRD................................................. 31
EBI characterization - Core clock phenotype .........................................31
The mapping of ebi-1 (Paper II) ...............................................................33
Sequencing...................................................................................................33
Biological confirmation .................................................................................34
Phylogeny.....................................................................................................35
NFX1-like proteins in various species..........................................................35
EBI protein actions (Paper I) ....................................................................36
EBI opposes ZTL’s effect on the clock.........................................................37
Control of stress responses by ZTL and EBI (Paper III) .....................39
Effects of abscisic acid on EBI and ZTL .....................................................39
The circadian clock in the perennial Populus ........................ 41
The Clock and seasonal effects (Paper IV)..............................................41
Growth cessation..........................................................................................41
Cold acclimation ..........................................................................................42
Input signals into the Populus clock (Paper V).........................................44
Photoperiodic effects......................................................................................44
Light and the clock ......................................................................................45
Final conclusions ............................................................... 47
Further perspectives................................................................ 48
Acknowledgements ............................................................ 49
References .......................................................................... 50
x
Preface
As it often is, it was somewhat of a coincidence that I ended up working
with the circadian clock, in plants. However, I have no regrets; the
mechanistic thinking of the circadian clock is intriguing, although it can at
times be somewhat frustrating. How do you find the starting point of a
loop? Understandably, this can be rather abstract for people not within the
specific field (and for me). Circadian clocks are also very interdisciplinary,
which is a nice feature. You can often have better understanding with
people working with fish than other plant scientists.
The idea of an internal clock can at first seem like a redundant feature, since
there is no single process that is solely controlled by the clock. On the other
hand, life on earth has been subjected to daily light/dark changes for
millions of years. That evolution would not have been influenced by that
would be even stranger to me. This thesis is an attempt to expand the
knowledge of circadian clocks in plants, specifically Arabidopsis thaliana, and
define its role in Populus trees. I have learned a lot during this process;
hopefully the reader(s) of this thesis will learn something too.
Finally, I can’t believe that more than five years can pass so quickly, maybe
there is something wrong with my clock…
Mikael Johansson, 2010
1
Tempus Rerum Imperator
2
Introduction
History of clock research
The earth is rotating while travelling around the sun. This leads to daily
changes between light and dark and therefore in temperature. There are also
varying lengths of day in different locations due to the sun’s varying
position relative to the earth’s axis. This dynamic environment has affected
evolution since the beginnings of life.
Presumably, organisms with a sense of time-of-day, and thus an ability to
predict environmental changes, had an adaptive advantage over those
organisms that simply responded to the changes. The mechanism that
provides such predictions of daily transitions is called the biological clock,
or more scientifically, the circadian clock after the latin, “circa diem”, which
means around or about a day. This “clock” can sustain a rhythm of about
24 h without any cues indicating time of day, closely matching the length of
a day. Additionally, it can keep track of seasonal changes by measuring day
length, which is more crucial at high latitudes, but still is of importance near
the equator.
Leaf movement was the first observed clock phenotype, as noted by the
French astronomer Jean Jacques d’Ortous de Marian, who observed the
continuous movement of leaves on plants placed in constant darkness in
1729 (de Marian, 1729). However, at that time this phenomenon was not
connected to any innate rhythms (Dunlap et al., 2004).
Carl Linnaeus also observed time-dependent behaviour in plants. In 1751,
he published a garden plan for a flower clock, showing the time by using
plant species that opened and closed their flowers at different times of the
day (Linnaeus, 1751). This also caught the attention of Charles Darwin, who
published “The Power of Movement in Plants” in 1881.
Much later, in 1928, the German plant physiologist Erwin Bünning started
studying circadian rhythms and suggested that these rhythms have an
adaptive value (Bünning, 1964). His work is considered to mark the start of
plant photoperiod research.
However, the foundation of modern chronobiology is usually attributed
to the two scientists Colin Pittendrigh and Jürgen Aschoff, who started
studies in this field during the 1950’s. Much of the nomenclature that is still
used in the field of clock research was developed by them, using a variety of
models, ranging from flies, to finches, to humans.
(An excellent and more extensive background to the circadian research field can be
found in Protein Reviews Vol. 12: Circadian Clocks.(Albrecht, 2010))
3
Plant clock history
In plants, research at the molecular level accelerated when Steve Kay and
Andrew Millar introduced the firefly LUCIFERASE (LUC) reporter gene,
fused with the photosynthesis gene CHLOROPHYLL A/B BINDING
PROTEIN 2 (CAB2) (Millar and Kay, 1991; Millar et al., 1992) into the
model plant Arabidopsis thaliana. CAB2 expression is regulated both by the
circadian clock and by phytochrome (phy) photoreceptors (Millar and Kay,
1991; Anderson et al., 1994). This gave the researchers a convenient tool for
performing mutant screens in a non-invasive manner, with the focus on
finding altered clock phenotypes. This advance also allowed extensive
experimental characterisation of clock performance in the identified clock
mutants.
Photosynthesis is fundamental for plant growth and many plant circadian
rhythms are connected to photosynthesis (Millar, 1999). The probable role
of the clock in the light harvesting process is to anticipate the light period,
allowing the plant to prepare for photosynthesis. Thus energy uptake over
the day is maximized by having the photosynthetic machinery running from
first light.
A particular feature of the circadian clock is its ubiquitous nature, both
within and between organisms. It has similar functions and works in similar
ways in most aspects of development and metabolism in different tissues
and different organisms. The significance of an endogenous timekeeper and
the importance of it having a period matching the light/dark cycle of the
organism’s environment have been reported for several organisms. For
instance, cyanobacteria that have endogenous rhythms that match their
environment out-grow their competitors with non-matched periods
(Ouyang et al., 1998). This phenomenon of matching inner- and external
rhythms is termed ‘resonance’ and is also important for plants, since it has
been shown that plants with an internal clock period matching the
surrounding environment grow much better and accumulate more starch
than plants with a mis-matched inner clock period (Dodd et al., 2005). This
illustrates that synchronization between internal and external periods is
important across kingdoms and can also be applied to growth processes.
Thus, the circadian clock is important in directing metabolic and
developmental events to the most favourable time of the day, thereby
maximizing the organism’s growth and chance of survival (Blasing et al.,
2005; Dodd et al., 2005; Graf et al.).
4
What is a circadian rhythm?
There are certain rules or properties of a rhythm that need to be observed,
in order for it to be considered as circadian, differentiating it from rhythms
of other period lengths, or those that are directly driven by cycling
conditions, such as light-on and light-off. A circadian rhythm persists in the
absence of all time cues, i.e. in constant conditions, with a periodicity of
about 24 h. Also, it has to be entrainable, meaning that it can re-set itself in
response to transitions between light and dark, high and low temperatures
or metabolic signals. Furthermore, it needs to be buffered against
environmental ‘noise’, such as stochastic variation in light and temperature,
thus only responding to changes in the environment that need to be in
synchrony with the surrounding day-night cycle. As part of this mechanism,
the circadian clock is most responsive to light around dusk and dawn, while
it is most insensitive around the middle of the day/night. This characteristic
is referred to as ‘gating’ and is more extensively described below. To be able
to deal with random temperature shifts, the clock is temperature
compensated over certain, species-dependent, temperature intervals. This
means that the period of the rhythm stays the same, even if the temperature
fluctuates within this interval. All these characteristics make the clock
sufficiently stable to function as a timekeeper, but flexible enough to adjust
itself, and processes influenced by the clock, to the surrounding
environment, on both a daily and a seasonal basis.
Conceptually, the circadian clock can be divided into three parts, as
illustrated below: Input pathways that transmit signals, such as light and
temperature, from the environment to the Central oscillator, which functions
as a negative feedback loop determining what is done with the signals
(Figure 1). This results in the signals being directed to an Output pathway,
which produces overt rhythms (Millar and Kay, 1997; Somers et al., 1998;
Devlin, 2002). This is a simplification, as recent research indicates that the
clock consists of a number of interconnected transcription/translation
feedback loops, several components of which may be acted upon by light
and temperature. This is described in more detail, later in this thesis.
5
Input
Oscillator
Output
Gating
Figure 1: Function of a circadian oscillator.
Gating
The clock regulates its own responsiveness to light, for example CAB2
expression responses to light vary over the circadian cycle (Millar and Kay,
1996; Covington et al., 2001; Allen et al., 2006). This means that the clock is
sensitive to changes in light at certain times of its cycle, while at other times
the light signals are blocked, or the gate for light signals is closed. In this
way it is possible to distinguish between gated and acute responses to light;
the former only occur at certain key intervals of the circadian cycle, while
the latter are immediate responses that are not dependent on timing.
Entrainment
The process by which the biological clock re-sets itself following
partucilar environmental signals is called entrainment. The most obvious cues
for entrainment are the daily changes between light and dark, although
shifts between low and high temperatures are also important and sufficient
to entrain clock-controlled rhythms.
Another factor influencing the period of a circadian rhythm is light
intensity. Jürgen Aschoff found that there is an inverse linear relationship
between irradiance and period length. Diurnal organisms kept at a high light
intensity will exhibit shortened free-running periods compared to with
counterparts kept under lower light intensities, while the opposite is true for
nocturnal organisms (Aschoff, 1960). This behaviour of the clock is also
known as parametric entrainment (outlined below) and this particular
phenomenon is referred to as ‘Aschoff’s rule’. Aschoff based this theory on
results of activity experiments with various species, for instance mice for
6
nocturnal and finches for diurnal behaviour. Although this generalization
might have been taxonomically biased by the fact that most diurnal animals
studied were birds and the nocturnal animals were mammals (Albrecht,
2010), plants follow this rule in a diurnal manner (which is not surprising,
considering the properties of, and their dependence on, photosynthesis).
Two theories have been postulated to explain how entrainment occurs.
The continuous or parametric entrainment theory suggested by Aschoff,
summarized in Dunlap et al. (2004), assumes that light affects the clock
throughout the whole circadian cycle. One line of evidence for this model is
the effect of light intensity on the period (Aschoff’s rule). In contrast,
according to the discrete theory, first proposed by Pittendrigh, the clock is
reset by the light/dark shifts at dawn and dusk instantly, correcting any
mismatch with the environment (Daan and Pittendrigh, 1976). This model
has been more successful in predicting entrainment, for example in
Drosophila and rodents. Both theories are considered to be relevant (Daan,
2000) and Aschoff also speculated that some kind of combination of these
two mechanisms was responsible for entrainment (Aschoff, 1963).
Zeitgebers
Any environmental input that has the ability to reset the clock is called a
Zeitgeber (time giver). The most obvious Zeitgebers are changes between
light and dark or light pulses, but daily temperature changes can also act as
Zeitgebers in plants (Jones, 2009). To illustrate how the clock responds to
stimuli in the discrete entraining model, one can construct a Phase
Response Curve (PRC) (Johnson, 1990). A PRC is a map of how the clock
responds to light signals over one circadian cycle. It is constructed to show
how the phase of the clock is changed depending on when it is given a pulse
of a stimulus (usually a light pulse) (Figure 2).
Phase shift
Advance
Delay
+4
+2
Dead zone
0
‐2
‐4
Subjective day
Subjective night
Circadian time (h)
Adapted from Dunlap et al (ed) 2004
Figure 2: A phase response curve, describing the shift that will
occur in phase if a Zeitgeber is applied over the circadian cycle.
7
According to theory, the clock has a point of singularity, which is the point
where the clock starts again. If light pulses are given before this point, the
phase will be delayed, while if light pulses are given after this point, the
phase will be advanced. This point usually occurs in the middle of the
subjective night in a 12 h light/12 h dark cycle (Daan and Pittendrigh, 1976;
Salome and McClung, 2005). There is also a “dead” zone in which light
signals have no effect on the phase, in the middle of the subjective day. By
constructing such a PRC, it is thus possible to predict when light should be
applied to achieve stable entrainment, providing information about the
behaviour of the clocks in the examined organism.
Expression of clock genes
The cycling expression of many genes involved in the regulation of the
clock can be described as an oscillating rhythm (Figure 3). One cycle is then
referred to as the period of this rhythm.
2x amplitude
Period
Phase
Time (h)
Figure 3: A circadian oscillation
The phase of this rhythm is the velocity of the increase of the gene
expression, or the angle between peak and trough. The amplitude is half the
height difference between the peak and trough. The appearance of and
differences in oscillating patterns between different components or outputs
of the clock will guide conclusions about their role in the clock circuitry.
8
Photoperiodism
The variable difference between day/night lengths over the seasons of the
year is a cue for developmental processes in many organisms. The length of
the light period of a day is called the photoperiod and its effects on this on
organisms is called photoperiodism.
There are two possible ways to visualize how measurement of time could
occur. It could work as an hour-glass mechanism that simply is reset, or
“turned over” after a certain period of time. The other option is that it
works as a clock, continuously measuring the time in an oscillating manner.
In a stable environment, an hourglass timer would be sufficient to predict
an event that will occur after a certain period, for example dusk 12 h after
dawn. However, since the photoperiod changes over the year in many
locations on the planet, this would make an hourglass timer only correct
twice a year, at the equinoxes. An oscillating clock is more flexible; the peak
of the oscillation can be set to occur at a certain time, for example dawn,
and then the oscillations could be further adjusted depending on the
prevailing photoperiod.
Edward Bünning was the first to suggest that an endogenous diurnal
rhythm controls reproductive responses, based on its relation to day lengths
(Bünning, 1936). He divided the day into a photophil and a scotophil phase,
comparable to the part of the day that is expected to be light (subjective
day) and dark (subjective night). He then predicted that the response of a
plant would depend on how the length of the received period of light.
According to this scheme, short day responses would appear when the
rhythm was restricted to the photophil phase, and long day responses when
the rhythm extends into the scotophil phase.
Colin Pittendrigh later conceived two similar models, the external and
internal coincidence models (Pittendrigh and Minis, 1964). According to the
external coincidence model, the endogenous rhythm will produce a
response when it is in phase with external light stimuli. The internal
coincidence model was based on the theory that an organism has several
independent oscillators, connected to either dawn or dusk. When two such
oscillators are synchronized at a certain time of the season, they will
produce a response. Although the internal coincidence model is very
difficult to test, since one would have to first identify and then be able to
couple and uncouple two separate oscillators, it has received renewed
interest since dual- (or more) oscillator models have been proposed
(Goldman, 2001). The external coincidence model can be applied to
flowering responses in plants, where long-day plants start flowering when
their CONSTANS (CO) levels coincide with the light period of the day
(Roden et al., 2002; Imaizumi and Kay, 2006). CO will then induce
9
expression of the flowering regulator FLOWERING TIME (FT), which
leads to flowering (Putterill et al., 1995; Samach et al., 2000; Suarez-Lopez et
al., 2001; Corbesier et al., 2007; Turck et al., 2008).
Photoperiodism in plants
Seasonal changes in the environment over the year are important signals
for plants. At high latitudes, as in northern Sweden, obvious changes are the
pronounced variations in day length between the seasons (in Umeå, ~21 h
of daylight in June compared to ~5 h in December). The photoperiodic
responses to these changes in light conditions control, inter alia, flowering
periods in plants. Arabidopsis is a facultative long-day plant, which means
that its flowering process is accelerated as the days become longer than the
nights, during the spring. Other plant species, such as tobacco, will only
flower under short days. The circadian clock has an important role in the
perception of these changes (Jarillo and Pineiro, 2006). To perceive these
temporal changes correctly, the clock has to run accurately and not be
sensitive to random, short-term environmental fluctuations.
The connection between the clock and flowering is mainly controlled
through the genes GIGANTEA (GI) and FLAVIN-BINDING, KELCH
REPEAT, F-BOX 1 (FKF1) who acts on the transcription factors CYCLIN
DOF FACTOR (CDF1-2). The CDF genes are transcriptional repressors of
CO. GI stabilizes FKF1 specifically under long days, leading to degradation
of transcriptional repressors of CO, enabling CO transcription (Sawa et al.,
2007; Fornara et al., 2009). However, there are genetic redundancies in
these families, complicating these interactions (de Montaigu et al., 2010).
CO, in turn, regulates FLOWERING TIME (FT) in photoperiod-dependent
manner, and FT triggers flowering (Suarez-Lopez et al., 2001; Imaizumi et
al., 2003; Imaizumi et al., 2005; de Montaigu et al., 2010). FT, together with
its relative TWIN SISTER OF FT (TSF), is expressed in vascular tissues,
and FT protein is then (probably together with TSF) translocated through
the sieve elements to the meristem, where it signals reprogramming of
transcription and thus triggers flower development (Kobayashi and Weigel,
2007; Turck et al., 2008).
In perennials, such as hybrid aspen (Populus tremula x P. tremuloides), the
ability to suspend and resume growth in response to seasonal rhythms is an
important life strategy. These processes are called growth cessation and
dormancy; cessation, or discontinuation, of growth occurs first, and then
the trees enter dormancy, which can be defined as the inability to initiate
growth even under favourable conditions (Rohde and Bhalerao, 2007).
During dormancy, a period of chilling is required for the tree to be able to
10
break buds and continue growth in spring. This is true for elongation
growth (Myking and Heide, 1995), as well as cambial growth (EspinosaRuiz et al., 2004). Growth cessation is provoked by various environmental
cues, for example photoperiod, cold and drought. Through these
photoperiodic cues, it is plausible that the clock has a role in these
processes.
Trees have developed a critical daylength (CDL), around which they enter
the process of growth cessation. The CDL varies along latitudinal clines
(Böhlenius et al., 2006). The phytochrome A (phyA) photoreceptor is
important for this process, since over-expression of oat phyA leads to
photoperiodic insensitivity, while reduction of its endogenous expression
increase sensitivity (Olsen et al., 1997; Kozarewa et al., 2010).
The plant hormone gibberellin (GA) also plays a role in the growth
cessation process; it is down-regulated in response to short days via activity
of GA 20-oxidase, and over-expression of GA 20-ox postpones the process
(Moritz, 1995; Eriksson et al., 2000; Eriksson and Moritz, 2002).
Bud burst is the process in spring in which growth and development is reactivated and the trees resume growth. The timing of bud burst is
dependent on environmental factors such as winter chilling and temperature
(Jimenez et al.; Linkosalo et al., 2006; Fan et al.), but photoperiod can also
be of importance (Linkosalo and Lechowicz, 2006; Li et al., 2009) .
Light perception
Light is one of the most important factors affecting plants. The
mechanisms whereby plants collect and utilize light as energy and for
signalling have been intensively investigated. To perceive the surrounding
light, plants use different photoreceptor families for different wavelengths,
although there is some overlap in the wavelengths they perceive (Millar,
2003; Chen et al., 2004; Demarsy and Fankhauser, 2009). The
photoreceptors can be found in every cell of the plant, in contrast to, for
example mammals that only have photoreceptors in the eye. The light
perception pathway has been studied most extensively in Arabidopsis.
Light has three functions in the photoperiod response mechanism: It
entrains the clock, promotes the blue light-dependent interaction between
FKF1 and GI, and it regulates CO stability, as outlined by (Jackson, 2009).
The phytochromes (phyA-E) perceive red and far-red light, and function
as molecular switches (Franklin and Quail, 2010). In their inactive form,
they perceive red light, which activates them. If they then are exposed to
far-red light, they will be inactivated again. Under natural light conditions,
there is equilibrium between the active and inactive forms of phytochromes,
11
depending on the red/far-red ratio. PhyA has some unique characters
distinguishing it from the other phytochromes. It can rapidly convert
between its active and inactive form and thus function as a highly sensitive
light antenna. This is important when etiolated (dark-grown) seedlings reach
light and need to switch to de-etiolation growth quickly. Another process
the red:far-red ratio controls is shade avoidance, an adaptive response of
plants that increases the chances of plants growing out of the shade of other
plants or objects.
Phytochromes interact with a subfamily of transcription factors known as
PHYTOCHROME INTERACTION FACTORS (PIFs). These PIFs have
a number of regulatory functions in photomorphogenesis (Castillon et al.,
2007). PIF4 and PIF5 have, for example, been shown to be of importance
under diurnal conditions, being repressed during the day via clock-mediated
repression, but promoting growth in the dark (Nozue et al., 2007).
PhyA, B, D and E have been shown to affect the clock, probably by
affecting the perception of photon irradiance (Somers et al., 1998; Devlin
and Kay, 2000). Increases in irradiance lead to shortening of circadian
output rhythms of leaf movement and gene expression periods in
Arabidopsis and Populus (Millar et al., 1995; Somers et al., 1998; Kozarewa et
al., 2010). Phytochromes are also important for the resetting of the clock
(Yanovsky et al., 2000; Yanovsky et al., 2001).
The chryptochromes (cry1-2) are blue light receptors. They are
phosphorylated by the action of blue light and thus become biologically
active (Banerjee and Batschauer, 2005).
Cryptochromes affect, via the E3 ubiquitin ligase COP1, inputs to both
the clock and flowering pathways (Yu et al., 2008). The PHY and PRY gene
families are in turn targets of circadian regulation at the transcriptional level,
although circadian regulation at the protein level is of low amplitude (Millar,
2004).
Phototrophins (phot1-2) also perceive blue light and are important for
light-dependent processes, such as phototropism, light-induced opening of
stomata and chloroplast movement (Christie, 2007). These proteins have
LOV domains, a feature they share with the ZEITLUPE (ZTL)/LOV
KELCH PROTEIN 2 (LKP2)/FKF1 family of blue light receptors
(Demarsy and Fankhauser, 2009). This last family is of great importance to
the circadian clock, as well as for photomorphogenesis. These proteins
contain, in addition to the LOV-domain, an F-box domain and a number of
Kelch repeats, indicating a role in light-dependent protein degradation. ZTL
is also known to be involved in the degradation of TOC1 (Más et al., 2003;
Harmon et al., 2008) and PRR5 (Kiba et al., 2007), and to interact in a
stabilizing manner with GI (Kim et al., 2007). Recently, LKP2 and FKF1
12
have been shown to have similar roles in the regulation of the same proteins
(Baudry et al., 2010).
In Populus, there is one PHYA gene and two PHYB genes, PHYB1 and
PHYB2 (Howe et al., 1998). PHYA participates in photoperiodic control of
dormancy and bud set (Olsen et al., 1997; Kozarewa et al. 2010), while
PHYB2 is co-localized with a QTL for bud set (Frewen et al., 2000). Two
SNPs in the PHYB2 gene reportedly explain some of the variation in timing
of bud set in a collection of European aspens (Ingvarsson et al., 2008).
Knowledge about chryptochromes and the function of downstream targets
as PIFs is still very limited in woody species (Olsen, 2010).
Clock genetics
Most efforts to elucidate the mechanisms of biological clocks in various
species have been carried out on mutants with phenotypes suggesting a
malfunctioning clock. Mutants obtained from classic forward and reverse
genetic approaches led the way to the discovery of genes coding for
proteins with a clock function. In forward genetics, selection is based upon
a phenotype. In clock research, an altered phase of clock controlled gene
expression, e.g. an early- or late phase or arrhythmia or short period in gene
expression or leaf movements, will often be selected for. In reverse genetics,
the starting point is to choose a gene of possible interest and then
investigate the (clock) phenotypes that arise from manipulation of the geneof-interest. Via such mutational analyses, it is possible to study complex
systems, altering a single component while keeping all other parts constant
(Dunlap, 1993). However, the resulting phenotype reflects the interaction of
many factors. The goal of these types of genetic studies is to provide insight
into the rhythmic biochemistry of the clock (Dunlap, 1993).
Finding a mutant with a relevant phenotype is usually the easy part.
Defining the function of the mutated gene within the clock system is more
challenging. Depending on the location of the induced mutation within a
gene, the resulting phenotype might differ considerably, e.g. toc1-1 mutants
have a light-dependent phenotype, but not toc1-2 mutants (Millar et al.,
1995; Somers et al., 1998; Strayer et al., 2000; Más et al., 2003). The most
straightforward type of mutant is a knock-out, in which the mutation or
insertion is usually situated in an exon and completely disrupts
transcription/translation. However, loss-of-function mutants may also be
obtained, in which the mutated gene may be transcribed, but is unable to
perform its wild-type function. For example, a translated protein may not
interact with its substrate due to a conformational change in the protein or
lack of phosphorylation. A third type is a gain-of-function mutant, in which
13
the mutated gene/protein gains a new function, for instance overexpression mutants may constantly promote expression of another protein.
It is difficult to predict the types of mutation carried by clock mutant
from their clock phenotypes. A knock-out never disrupts the rhythm, but
usually the phase and/or period becomes shorter or longer. To disrupt the
clock completely, several genes have to be knocked-out or they need to
over-express the right component (Wang and Tobin, 1998; Ding et al.,
2007).
An additional factor to consider is the timing; disruption of an interaction
between two proteins at a certain time of day may lead to an out-of-phase
clock, while protein levels remain largely unchanged. Considering all these
variables, it can be challenging to firmly establish the type of mutation and
its effects, while trying to define a new piece of the clock machinery.
Clock model organisms
By focusing research on certain organisms as models for other, similar
species, the molecular science community has made many major
discoveries. The typical model organism is easy to work with and has
relatively simple genetics, but can still be used to describe mechanisms in
more complex species. The use of such model organisms has been essential
for elucidating the molecular biology of the circadian clocks in nature. A
brief summary of model systems (other than plants) that are of great
importance for molecular clock research follows.
Cyanobacteria
Even small bacteria, with a very short life cycle, have been shown to have
an advantage in fitness from a circadian clock running in phase with the
environment (Ouyang et al., 1998; Johnson et al., 2008). The first rhythm to
be characterized in Cyanobacteria was nitrogen fixation, regulated to occur
during the night, so as not to interfere with photosynthesis during the day.
Further experiments revealed that almost all promoters in the cyanobacterial
genome are regulated by the circadian clock (Liu et al., 1995). Underlying
these rhythms, there are three main proteins, KaiA, KaiB and KaiC, which
form a negative feedback loop. These three proteins have even been shown
to be able to form a cycling feedback loop in vitro (Nakajima et al., 2005).
The relative simplicity of this system makes it ideal for studying the basic
molecular mechanisms behind the circadian clockwork.
14
Neurospora
The simple eukaryote Neurospora crassa has been used in circadian research
for over 50 years (Merrow et al., 2006; Heintzen et al., 2007). The most
common way to visualize clock rhythms in Neurospora is the ‘Race tube
assay’, where a glass tube is filled with a growth medium and inoculated
with the fungus, which will subsequently produce zones of mycelia followed
by zones of denser asexual spores in a circadian manner.
The genes considered to be at the core of the Neurspora clock include
FRQ, FRH, WC-1, and WC-2, of which WC-1 and WC-2 works as positive
elements in a feedback loop, while FRQ and FRH are negative elements.
FRQ expression is also temperature regulated and different forms of the
FRQ protein are produced, depending on the temperature that regulates
splicing of the transcript (Diernfellner et al., 2005). Studies of the underlying
mechanisms of temperature compensation in Neurospora may give hints
about how this works in other species.
Drosophila
Drosophila melanogaster, the fruit fly, is an organism that has become an
important model for circadian clockwork research in animals. Examples of
circadian outputs are adult emergence (eclosion) and locomotor activity
(Hardin, 2005). Three genes are central to current understanding of clock
mechanisms: the transcription factor Clock (Clk) and the two transcriptional
repressors, Period (Per) and Timeless (Tim). The Drosophila clock is
composed of two feedback loops, one involving the Per and Tim genes and
the other one the Clk gene. In addition to these, a number of other genes
are involved in the fine tuning of the clock. Oscillators have been identified
in a number of tissues, often operating autonomously (Hardin, 2005).
Studies in Drosophila have also revealed a large variation in clock genes over
latitudinal clines (Kyriacou et al., 2008). This is a feature that it shares with
genes important for growth cessation in Populus trees (Hall et al., 2007).
Mammals (including humans)
In mammals, the master circadian clock is located in the suprachiasmatic
nuclei (SCN), part of the hypothalamus in the brain (Reppert and Weaver,
2001). It perceives light signals from the environment via the retina, thus
providing entrainment signals for the clock. In the SCN, all clocks are cellautonomous, while there are several “slave” clocks in various tissues (for
example, the liver) that are entrained by the SCN clock mechanism. In
mammals, the molecular clock also has the form of a feedback loop,
15
involving three Period-genes and two Cryptochrome genes. Their rhythmic
expression is driven by two transcription factors, Clock and Bmal1.
Mammals are also the only known organisms with a specific
photoperiodic hormone, called melatonin (Pandi-Perumal et al., 2006).
Although melatonin has been found in many other species, including plants,
knowledge of its photoperiodic effects is so far limited to mammals.
Melatonin is secreted at night and promotes sleepiness.
In humans, knowledge about the biological clock is important for many
reasons. Circadian rhythms are believed to be involved in the regulation of
many metabolic networks and disturbed expression of clock genes may lead
to decreased metabolic health, and diseases such as diabetes and obesity
(Marcheva et al., 2009). Other studies indicate that disruption of normal
circadian rhythms may lead to increased risk of cancer, for example when
shift working (Davis and Mirick, 2006).
The fact that the molecular clock is based on similar mechanisms in
different organisms, with little homology between the genetic components
may indicate that although the circadian circuitry has evolved separately,
such feedback loops are of significant advantage in the perception of the
environment in a cycling world. The complexity of these regulatory
networks also indicates that the ability to adjust to the local environment is
important for most organisms, since each added component gives the clock
system further ability to adjust its regulation.
Plant clocks
One major difference between plants and most other organisms is that the
vast majority of terrestrial plants do not move. They have to cope with
whatever environment they establish in, without the possibility of moving
to a more favourable location. This makes the ability to predict fluctuations
in the environment even more important for these organisms. Another
significant difference between animals and plants is that each plant cell
contains its own autonomous clock that can be entrained separately (Thain
et al., 2000; Young and Kay, 2001). The role of the circadian clock has been
most extensively studied in the small annual weed plant Arabidopsis,
common name Thale Cress. This plant is the subject of most basic plant
molecular research, due to its compact, fully sequenced genome and
relatively fast life cycle (seed to seed in about six to eight weeks), among
other favourable characteristics.
16
The molecular clock in Arabidopsis
In Arabidopsis, the first clock gene to be characterized was the eveningexpressed TIMING OF CAB EXPRESSION1 (TOC1) (Somers et al., 1998),
which was found in a mutant screen using Arabidopsis seedlings expressing a
construct of the CAB2 reporter fused to the LUCIFERASE (LUC)
reporter gene (Millar et al., 1995). CIRCADIAN CLOCK ASSOCIATED1
(CCA1) (Wang et al., 1997) and LATE ELONGATED HYPOCOTYL
(LHY) (Schaffer et al., 1998) are two single MYB domain transcription
factor genes that have a peak expression of both transcripts and proteins
around dawn (Kim et al., 2003).
These three genes form what can be considered the central feedback loop
of the clock (Figure 4), in which TOC1 protein promotes expression of the
CCA1 and LHY genes during the night, while CCA1 and LHY proteins in
turn repress expression of the TOC1 gene during the day, by binding
directly to its promoter (Alabadi et al., 2001; Alabadi et al., 2002; Perales
and Mas, 2007). When TOC1 levels decrease, promotion of CCA1 and
LHY transcription decreases, allowing TOC1 levels to be increased and
again promote CCA1 and LHY transcription (Lu et al., 2009). All this
happens in a cycle over approximately 24 h (Figure 4).
CCA1/LHY
CCA1/LHY
TOC1
TOC1
Figure 4: The genes and proteins
making up the circadian core
oscillators.
TOC1
promotes
expression of CCA1 and LHY,
which
in
turn
represses
transcription of TOC1. This leads
to a decrease in TOC1 protein
levels and thus a decrease in
CCA1 and LHY. This allows
TOC1 transcription to increase
again and TOC1 protein can once
more promote CCA1 and LHY.
All this happens over a ~24h
cycle.
This model illustrates the basic feedback mechanism of the clock, but it is
too simple to represent the full functionality of the clock. Several other
proteins are involved in the regulation of the clock, forming several
interconnected loops.
17
To understand how this intricate system is configured, mathematical
modelling based on experimental and theoretical data has been helpful
(Locke et al., 2005; Locke et al., 2006; Zeilinger et al., 2006). In the most
current, but incomplete, theoretical model of the clock, at least three
interlocked feedback loops make up the clock, and two unknown factors X
and Y are predicted to be significant players. These factors most likely
comprise the action of several unknown genes. These loops are then further
co-ordinated by additional interactor(s), e.g. EARLY FLOWERING 4
(ELF4) (Doyle et al., 2002; McWatters et al., 2007; Kolmos et al., 2009).
TOC1 is part of a gene family called the PSEUDO-RESPONSE
REGULATORS. It includes five genes: TOC1 (PRR1) and PRR3, PRR5,
PRR7 and PRR9 (Eriksson et al., 2003; Matsushika et al., 2005). All of these
genes have a role in the clock, although their single mutant phenotypes are
subtle (Matsushika et al., 2000; Nakamichi et al., 2003; Matsushika et al.,
2005; Nakamichi et al., 2005). They all oscillate with different peak times
during the circadian cycle (Matsushika et al., 2002; Nakamichi et al., 2003;
Farré and Kay, 2007; Ito et al., 2007; Fujiwara et al., 2008).
PRR7 and 9 form a second feedback loop with CCA1 and LHY(Farré et
al., 2005), acting in the morning, when they are promoted by CCA1 and
LHY. In turn, they repress expression of CCA1 and LHY transcript; PRR7
and PRR9 proteins have been recently shown, together with PRR5 protein,
to bind to the promoters of CCA1 and LHY in a repressive manner
(Nakamichi et al., 2010).
PRR5 has been shown to interact with, and be degraded by, ZTL through
interaction between the Light-Oxygen-Voltage (LOV) domain of ZTL and
the PR domain of PRR5 (Kiba et al., 2007). PRR5 is also genetically linked
to GIGANTEA (GI), and plays a role in regulating photoperiodic flowering
time, by acting on the flowering regulator CDF1 (Ito et al., 2008). PRR5
also interacts with TOC1 and promotes its phosphorylation, as well as
regulating the nuclear import of TOC1 (Wang et al., 2010).
ZTL, is an F-box-containing protein that is part of a Skp/Cullin/F-box
(SCF) E3 ubiquitin ligase complex (Somers et al., 2000; Han et al., 2004;
Kevei et al., 2006). The protein structure also contains a LOV/PASdomain, allowing it to sense blue light, and a number of kelch repeats. It
interacts with both TOC1 and PRR5 via its LOV domain, which leads to
their degradation via the proteasome pathway (Más et al., 2003; Kiba et al.,
2007). ZTL interacts with GI via the LOV domain which stabilizes ZTL
protein in a cyclic manner (Kim et al., 2007). GI additionally interacts with
the ZTL homologue FKF1 depending on the action of blue light (Baudry et
al., 2010). Although ZTL mRNA levels seem to be constitutive, ZTL
18
protein levels oscillate (Kim et al., 2007). All the protein-protein interactions
ZTL is involved with show the importance of post-translational regulation
of clock components by ZTL and other components (Jones, 2009; Somers
and Fujiwara, 2009).
PRR3 stabilizes TOC1 protein by inhibiting ZTL-dependent degradation
of TOC1 (Para et al., 2007).
The evening-expressed gene GI is involved in both regulation of the clock
and flowering (Park et al., 1999; Mizoguchi et al., 2005). It has also been
suggested to comprise at least part of the predicted factor Y in the clock
model (Locke et al., 2006).
EARLY FLOWERING 3 (ELF3) has been shown to act as a gatekeeper
of input signals to the circadian clock (McWatters et al., 2000; Covington et
al., 2001). elf3 mutants are responsive to light signals during the night, when
the gate for such signals is closed. In addition, ELF3 has been shown to
affect GI protein stability, together with COP1 (Yu et al., 2008), as well as
being important for the clock’s temperature responses (Thines and
Harmon, 2010). The gating of light into the clock is also controlled in a
more specific manner; FAR-RED ELONGATED HYPOCOTYL3 (FHY3)
has been shown to gate red light signals to the clock during the day (Allen et
al., 2006).
ELF4 is important for entrainment and rhythm sustainability under
constant conditions (McWatters et al., 2007), probably by repressing both
the morning and evening loops (Kolmos et al., 2009).
To put all these pieces together, mathematical modelling suggests that the
clock consists of at least three feedback loops (Figure 5): a core loop
consisting of CCA and LHY and TOC1, in which TOC1 acts via a factor X
on CCA1 and LHY, a morning loop consisting of CCA1 and LHY and
PRR5/9, and an evening loop with TOC1 and the unknown factor Y, at
least partly consisting of GI.
Recently, CCA1 HIKING EXPEDITION (CHE) has been shown to
bind to the CCA1 promoter, repressing its activity, possibly by interfering
with TOC1 activity (Pruneda-Paz et al., 2009) and it might be a part of the
predicted factor X.
A number of other proteins are involved in regulation of the clock that
have minor or not yet fully understood roles. To describe them all in this
thesis, in addition to the already numerous mentioned components, would
most likely only add confusion so they are only mentioned by name, as
follows:
FIONA1 (FIO1), LIGHT INSENSITIVE PERIOD1 (LIP1), LIGHTREGULATED WD1/ -2 (LWD1-2), LUX ARRHYTHMO (LUX),
SENSITIVE TO FREEZING6 (SFR6), TEJ, TIME FOR COFFEE (TIC),
19
XAP5 CIRCADIAN TIMEKEEPER (XCT), (Panda et al., 2002; Hall et al.,
2003; Hazen et al., 2005; Allen et al., 2006; Kevei et al., 2007; Kim et al.,
2008; Knight et al., 2008; Martin-Tryon and Harmer, 2008; Wu et al., 2008;
Pruneda-Paz et al., 2009). The fact that roles of these proteins have not yet
been fully characterized shows that our understanding of the clock
mechanism in Arabidopsis, and plants in general, is incomplete, and it is
highly probable that further proteins involved in these complex processes
will be found.
20
LKP1
CCA1
ELF4
CCA1
LHY
LHY
X
CHE
CHE
X
ELF3
TOC1
TOC1
P
Y
PRR5
PRR3
GI
ZTL
FKF1
GI
Y
”Evening loop”
”Core loop”
PRR9
PRR7
PRR7
PRR9
”Morning loop”
Figure 5: Current view of the circadian molecular network in plants, with three
interconnected feedback loops. Transcription is described by dashed arrows. Genes are
written in italics and proteins are described as coloured ovals. Promotion is described
with arrows and repression with bars.
21
The Circadian clock at the whole plant level
Clocks in different plant tissues have been shown to run with different
phases (Hall et al., 2002), or to have a slightly different make-up (James et
al., 2008). A slightly different composition of the clock has also been
suggested following temperature entrainment (Michael et al., 2003).
Additionally, clocks in different tissues can be entrained autonomously and
are tissue-specific (Thain et al., 2000; Thain et al., 2002). The clock in root
cells has disengaged from the morning-phased loop with the central and
evening-phased loops (James et al., 2008). Root clocks can also be entrained
by signals from the shoot clock.
The clock also has a temperature-compensating mechanism, in which
CCA1 and LHY levels increase at higher temperatures, while they decrease
and TOC1 levels increase at lower temperatures (Gould et al., 2006). GI
may be important for maintenance of the 24 h period at extreme
temperatures. Different internal oscillators also respond differently to
various responses; CAB2 expression entrains preferentially to light-dark
cycles over temperature cycles, while the opposite is true for CATALASE
3 (CAT3) (Michael et al., 2003). A dampening of clock rhythms, in the form
of decreased amplitudes also occur under cold conditions (Bieniawska et al.,
2008). Together, this suggests that there are yet unknown layers of
regulation of the clock on whole plant level. These layers might be tied to
the clock’s role in co-ordinating hormone biosynthesis and response
(Covington and Harmer, 2007; Legnaioli et al., 2009; Rawat et al., 2009),
since plant hormones are able to shift the periodicity of circadian rhythms
(Hanano et al., 2006). An additional level of regulation has been suggested,
through oscillations of the cytosolic signalling molecule, cyclic adenosine
diphosphate ribose (cADPR) (Dodd et al., 2007). cADPR drives cycling
Ca2+ release in the cytosol and has an effect on the core transcriptional
feedback loop.
A role for the circadian clock in stress responses has also been suggested.
The central oscillator genes CCA1 and LHY have been implicated in stress
responses, with the cca1lhy double mutant showing reduced stress resistance
(Kant et al., 2008). Also, transcriptional studies indicate that the clock has a
role under cold stress (Bieniawska et al., 2008; Nakamichi et al., 2009) and
GI has been implicated in cold stress responses (Cao et al., 2005).
Transcriptional regulation of clock genes has also been shown to depend on
rhythmic changes in chromatin structure, probably as a way to synchronize
development with external surroundings (Perales et al., 2006; Stratmann and
Mas, 2008).
22
Populus and other plant species
Whilst the short life cycle of Arabidopsis has many advantages, the species
is not a suitable model for research on every aspect of the biological clock,
or for every type of plant. Notably, in trees and other species with a longer
lifespan, other aspects and lengths of cycles need to be considered, for
example dormancy in winter and bud burst in spring (Luttge and Hertel,
2009).
Populus is the model of choice for tree research. It is relatively close
taxonomically to Arabidopsis and thus very suitable for homology searches; a
gene in Arabidopsis most likely has at least one homologous cousin in Populus
(Jansson and Douglas, 2007). The CCA1, LHY and PRR genes are highly
conserved among plant species (Izawa and Kwang, 2007; Okada et al., 2009;
Takata et al., 2010). Populus contains two LHY genes (Populus tremula x
tremuloides LHY1 and PttLHY2), but lacks a CCA1 homologue (Takata et
al., 2008). A TOC1 homologue is present, while the other PRR genes are
somewhat differentiated. There is no PRR3 homologue, while PRR7 and
PRR9 are duplicated, suggesting the clock system has a slightly different
setup in Populus (Takata et al., 2010). The central clock components oscillate
in a similar way as in Arabidopsis, with the PttLHYs peaking around dawn
and PttTOC1 about 8 h later, and with a periodicity of about 24 h (Ibanez et
al.). Other clock-related genes, such as PttGI, PttFKF1 and PttCO2, also
oscillate as expected from Arabidopsis (Ibanez et al., 2010; Kozarewa et al.,
2010).
As reviewed by (Song et al., 2010), although differences in the setup of the
circadian system exist, it works in a similar way in all examined plant
species. As an example of a difference, in Physcomitrella the clock seems to
consist of a single loop (Okada et al., 2009; Holm et al., 2010).
Other plant species in which the circadian clock and photoperiodism have
received attention include Oryza sativa (rice) (Lee and An, 2007), Brassica
(cabbage) (Salathia et al., 2007), barley (Kikuchi and Handa, 2009), Capsella
burs-pastoris (Slotte et al., 2007), Solanum (Yanovsky et al., 2000), Lemna
Gibba (Miwa et al., 2006; Serikawa et al., 2008), Ginkgo (Christensen and
Silverthorne, 2001) and Crassulacean acid metabolism plants such as
Kalanchoe (Dodd et al., 2002).
Temperature limits
Plants respond to changes in temperature in various ways, sometimes
quickly and sometimes slowly, for example rapidly when breaking buds or
slowly during vernalization (Penfield, 2008). Knowledge about how plants
perceive temperature is limited, although many known plant temperature
23
responses have common signalling components, indicating the existence of
a plant temperature signalling network that (inter alia) receives and acts on
temperature cues during dormancy (Penfield, 2008). The circadian clock is
temperature-compensated, meaning that the cycling period is kept the same
over certain temperature intervals, but can also be entrained using
temperature cycles (Salome and McClung, 2005).
In Castanea sativa (chestnut), the circadian clock has been shown to be
disrupted during cold and winter dormancy. The CsTOC1 and CsLHY
homologues, as well as CsPRR5, CsPRR7 and CsPRR9, cycle as expected at
22ºC, but become constitutively expressed at 4ºC or colder temperatures
(Ramos et al., 2005; Ibanez et al., 2008). This indicates that the circadian
clock is disrupted at colder temperatures. The oscillations resume when
leaves are returned to 22ºC. Similar results have been found in Populus
(Ibanez et al., 2010).
Njus et al. (1977) found that the circadian rhythms of bioluminescence in
the algae Gonyaulax polyedra disappeared when the temperature of its
environment was decreased from 20 to 12 ºC, but re-appeared, with a phase
correlated to the time of transition when the temperature was returned to
20 ºC. Loss of periodicity can also be induced in a similar manner by bright
light, and resumed by darkening the organism’s environment. Thus, synergy
between light and temperature signals seems to exist in Gonyaula.
Cold temperature pulses can phase shift circadian rhythms in cotton
seedlings (Rikin, 1991). Such pulses are more effective during the chillingsensitive phase, the light period. The effects of extreme temperatures on the
clock have also been studied by examining the rhythms of carbon dioxide
fixation in Bretfeldiana fedtschenkoi (Anderson and Wilkins, 1989). These
authors found that rhythms of CO2 fixation were active at temperatures
between 10ºC and 30ºC, disrupted at 2ºC or 40ºC, and restarted if the
temperature was changed from either extreme to 15ºC.
Thus, in plants the circadian clock seems to be sensitive to temperature
shifts, but is only active over certain, species-dependent, temperature
intervals.
24
Conclusions
The circadian clock has been shown to be important in a wide range of
processes affecting the life of plants. By coordinating numerous processes,
the clock helps to optimize the plant’s ability to survive in specific
environments. Although we are beginning to understand the involvement of
the endogenous clock in development, growth and adaptation in plants and
other organisms, the picture is far from complete. The aim of the studies
this thesis is based upon was to solve a little more of the puzzle that is clock
science
Aims of thesis
Except for the obvious goals of scientific training and bringing some new
knowledge to the world of plant science, there were also some more
concrete goals of the projects this thesis is based upon; The focus of this
PhD project has been to study and expand knowledge of the circadian clock
in the annual plant species Arabidopsis and the perennial woody species
Populus. More specifically, work has concentrated on characterizing the
novel clock component EARLY BIRD (EBI), by studying a mutant allele,
early bird-1 (ebi-1), a short period mutant, and examining its link to stress
responses. In Populus, inputs to the circadian clock, and its role in the
processes of growth cessation, bud set, bud burst and cold tolerance, have
been studied.
25
Methodology and Experimental Techniques
Many common molecular biology-techniques have been used in this work,
including examining gene expression with Real-time RT-PCR, protein levels
with Western blotting and protein interaction with Co-immunoprecipitation
(Co-IP). Gene constructs have also been made using classical cloning
techniques. Nevertheless, there are certain methods that are rather specific
for work with the circadian clock. These are briefly described below, and
more details are given in the methods sections in the attached articles and
manuscripts.
Experimental conditions to observe circadian rhythms
Since the circadian clock resets itself via changes in light and temperature,
it is necessary to remove all such cues to observe any differences in clock
regulation. Only if a rhythm persists under constant conditions, can it be
considered as circadian (Figure 6). The procedure applied to measure
differences in the clock is usually as follows. First, the plants are grown
under cycling conditions to entrain the clock under a regime of
environmental cues, such as light/dark cycles, high/low temperature cycles
or variations in hormones or other signalling compound levels, chosen in
accordance with the purpose of the study. After a certain number of
circadian cycles, the plants are then kept under constant conditions with
respect to the environmental cue. In most cases, this means constant light,
to keep the photosynthesis running and avoids starving the plants, but it can
also mean constant darkness or temperature. Any difference in the freerunning period or phase between plants of interest and controls is then
most likely due to changes in clock behaviour.
Entrainment
Free-run
24 h
Figure 6: Common setup of a circadian experiment. A period of entrainment is followed
by constant conditions, when the free-running period is observed.
26
It is important to avoid masking when examining circadian rhythms. This
is a phenomenon that occurs when the endogenous rhythm is overruled by
external factors and thus cannot be detected. To avoid this, as many
conditions as possible should be kept constant, after a period of
entrainment, in order to visualize the true circadian rhythms.
Time series measurements
Since an inherent aspect of clock research is observations of changes over
circadian time, the sampling methods applied differ from those used in
many other branches of research, notably of course changes are often
examined in time series, usually over at least 24 hours, to detect changes in
the rhythm of expression of clock-related proteins. Depending of the
resolution required, samples are usually taken over periods ranging from
every 20 minutes to every six hours. Ideally, this is done automatically, but it
is often necessary to go to the lab in the middle of the night and collect the
samples. Further, there will usually be numerous samples to process and
measure, before any real results can be harvested. The time series of
acquired data are then analyzed in various ways, mostly examining changes
in transcript or protein levels of genes/proteins of interest. Various
statistical methods, such as Student’s t-test or ANOVA, followed by
multiple comparison tests such as Student-Newman-Keul’s can then be
used to evaluate results and confirm differences.
Physiology
Leaf movement
Variations in clock behaviour will manifest in changed outward rhythms
under clock control. Several such rhythms can be studied at the
physiological level, among them leaf movement (Swarup et al., 1999).
The movement of leaves over a day was the first output of the
endogenous clock studied in plants (de Marian, 1729) and the response is
also found in Arabidopsis. Today, we use digital cameras that capture
photographs of plants under constant conditions every 20 minutes,
collecting a time series of images. By tracing the movement of the leaves
with computer software (Metamorph, Molecular Devices, USA), we can
then translate this into a rhythm and measure the phase, period and
amplitude of the response.
27
Hypocotyl length
To see if a clock phenotype is somehow related to the input of light (a
strong clock Zeitgeber), one can measure the length of the hypocotyls.
After radicle emergence, the hypocotyl is the elongating part of germinating
seed that lifts the growing tip upwards towards the light. The length of the
hypocotyl is inversely proportional to the amount of light perceived by the
plant. A long hypocotyl indicates that the plant has perceived little light. By
growing plants under light of different wavelengths, one can determine
which of the plant photoreceptors are involved in a particular phenotype
(Fankhauser and Casal, 2004).
Flowering
A physiological development process that is often, but not always,
affected by the clock is the time of flowering. The faster the clock runs,
often the earlier the flowering response. This also applies under entrained
conditions. Since the number of days after planting to flowering can differ
considerably between individual plants, it is common practice to also use a
developmental scale of measurement. In the studies reported here, the
numbers of rosette and shoot leaves after Arabidopsis plants started shooting
were used for this purpose, from the time their shoots were ca. 1 cm tall.
Luciferase constructs: imaging of gene transcription
One of the most important discoveries in clock research was the utility of
the firefly protein luciferase (LUC) as a reporter of gene expression (Millar
et al., 1992). A construct containing the promoter of the gene-of-interest
fused with the luciferase gene will emit photons whenever expressed, as
long as a substrate called luciferin is available. By using very light-sensitive
cameras, this allows the monitoring of gene expression of clock-related
genes over time in living plants. The camera arrangement used in the
Eriksson laboratory is shown in Figure 7. The procedure most commonly
used during the studies this thesis is based upon was to entrain the plants
for 6-7 days in cycling conditions, then monitor the expression of
promoter:LUC constructs for approximately 144 h (6 days) under constant
light using a camera. After that, the pictures were collected and processed as
a time series, using the MetaMorph software package (Molecular Devices,
USA). This allows the intensity of light from each individual seedling at
each time point (in each photo) to be logged and compared over time to see
how gene expression patterns differ between mutant and control plants. To
confirm the results statistically, BRASS (Biological Rhythm Analysis
28
Software System, available at www.amillar.org) software was used, in which
gene expression cycling periods are calculated by Fast-Fourier transform
nonlinear-least square analysis (FFT-NLLS) (Plautz et al., 1997; Locke et al.,
2005). Using a unit called Relative Amplitude Error (RAE), each rhythm
could be scored and its reliability could be determined, thus the stability of
the entrainment could be assessed. A high RAE (>~0.6) indicates that the
rhythm is not very reliable. If an entire dataset consists of high RAE
rhythms, the clock is clearly disrupted in the genotype under investigation.
RAE scores also help to exclude outliers, i.e. systems that for some reason
do not oscillate properly.
Camera control
LUC pic
CCD camera
FFT‐NLLS (BRASS)
LED light source
3
2
1
0
Luciferin (substrate)
00
24
48
72
96
~7 days old seedling
Photons
pCAB2
LUC
Promoter:LUC insertion
Figure 7: Setup for monitoring Luciferase expression in the Eriksson lab. A highly
sensitive CCD camera is used to capture photons emitted via a promoter:LUC construct
in transgenic plants. This is logged in a computer and then processed to show the
oscillating pattern of expression.
29
Results and Discussion
In this section, the results achieved in the projects this thesis is based on
are presented and discussed. First, the work performed in Arabidopsis is
discussed (paper I-III), followed by the Populus work (paper IV and V).
Further details on each experiment can be found in respective paper.
The search for EARLY BIRD
Efforts to elucidate the character and function of EBI and identify the
mutation causing the ebi-1 phenotype were parallel and intertwined
processes, leading to two separate manuscripts. The first (I), describes
studies that were parts of the main PhD-project; characterization of the
novel clock component EBI and its role in regulation of the clock
mechanism. The second paper (II) describes the mapping and sequencing
effort required to find the gene harbouring the ebi-1 mutation that is
responsible for the ebi-1 phenotype.
The search for the unknown clock regulator EARLY BIRD (EBI) began,
as such searches often do, with a screen for novel components of the clock.
The ebi-1 mutant was discovered in a screen for Arabidopsis mutants with
altered CAB2-expression phase, in an EMS-mutagenized population of the
Ws-2 accession. The population carried a promoter::reporter fusion
between CAB2 promoter and the firefly LUC gene. Thus it was possible to
measure the out-put as ‘hands of the clock’ of the mutant seedlings, using a
TopCount photon counter (Perkin Elmer, USA). After back-crossing to
remove unwanted mutations, the period of ebi-1 was tested under various
light conditions, showing that it was a short-period mutant, although with a
relatively subtle 1-1.5 h shortening of the period compared with its wildtype, i.e. Ws-2 carrying the CAB:LUC transgene that was used for
mutagenisation (Figure 1, paper II).
EBI characterization - Core clock phenotype
In the search for novel clock regulators, when characterizing a mutant it is
important to determine whether the mutational effect is located in the clock
or is an effect of altered input. To test whether the mutant phenotype was
related to an input pathway to the clock or the clock itself, the length of
hypocotyls of mutants and controls grown under light of various different
intensities and wavelengths was measured. No significant difference
30
between mutants and Ws-2 was found in this respect. (Figure S2, paper I).
Temperature entrainment also did not rescue the mutant phenotype from
CAB:LUC expression (Table II, paper I). Thus, light and temperature
pathways were not apparently affected by the mutation.
The next step was to examine any effects on known clock regulators and
thus attempt to elucidate how the novel component is connected to them.
Transcript levels of seedlings entrained in 12 h:12 h light:dark (LD 12:12)
conditions for six days, followed by three days of constant light before the
start of sampling, showed shifted expression patterns of the core genes
CCA1, LHY, TOC1 and GI (Figure 2, paper I). Hence, the ebi-1 mutation
affects the core of the clock. Levels of EBI transcript were weakly rhythmic
under constant light as well as under entrained (12 h:12 h) conditions, with
slightly elevated levels in the ebi-1 mutant. A gating experiment under red
light (15 µmol m2 s-1) showed an increased response to light 4 h before
dawn, indicating that the gating of light to the clock is impaired in the
mutant (Figure 1C, paper I). This earlier sensitivity to light could explain the
short period phenotype, making the mutant anticipate dawn prematurely.
The effect of the mutation on core clock components was further
confirmed using a TOC1:LUC construct, showing short period phenotype
and a severely damped expression in constant light (Figure 8, unpublished).
Normalized Luminescence
1.5
1
0.5
Ws-2
ebi-1
0
00
48
96
144
Time in LL (h)
Figure 8: Illuminescence as reported by a TOC1:LUC construct in
Ws-2 and ebi-1 under constant synthetic white light after entrainment.
A cca1-11;lhy-21;ebi-1 triple mutant showed extremely short period (16 h)
of CAB:LUC expression, although with retained rhythmicity (Figure S3,
paper I). Thus, ebi-1 acts to extend the period of the clock via an additional
pathway to the CCA1 and LHY-TOC1 cycle.
31
The ebi-1 mutant is early flowering, a trait shared with other clock
mutants. It flowers early under both short (SD) and long day (LD)
conditions, although with a larger number of leaves under SD, indicating
that ebi-1 mutants retain the ability to distinguish between long and short
photoperiods (Table III, paper I). The cca1-11;lhy-21;ebi-1 triple mutant
flowered early, but not earlier than cca1-11;lhy-21, so ebi-1 did not have a
similar additive effect as that observed on CAB2 expression.
The characterization thus far could be done without knowing the exact
location of the mutation causing the phenotype. For further studies, at the
protein level, the locus needed to be known. In order to pin-point the
mutation we initially used a gene mapping approach using F2/F3
populations from a cross between the ebi-1 mutant in Ws-2 background and
the Columbia-0 (Col-0) accession. Mapping was followed by sequencing of
the Ws-2 parent and back-crossed ebi-1 mutant, which provided detailed
information on the nature and location of the point mutation, a project that
resulted in a separate manuscript (Paper II).
The mapping of ebi-1 (Paper II)
The initial approach for finding the mutation carried by the ebi-1 mutant
was map-based cloning. The mutant plant was out-crossed with Col-0 and
the F2 generation was coarsely mapped using molecular markers and
followed up in F3 to confirm the phenotype. This proved to be difficult,
due partly to the relatively subtle clock phenotype and partly to the higher
plasticity of clock rhythms in the Col-0 ecotype compared with Ws. Because
of this, the ebi-1 and Ws-2 genomes were sequenced using an ABI SOLiD
(version 2) sequencing machine in parallel to the mapping effort. Thus,
candidate polymorphisms for the mutant were expected to be found.
Sequencing
Since EMS does not typically yield insertion or deletion mutations, the
focus was on finding single nucleotide polymorphisms (SNPs). The SNP
causing the mutation was presumed to be homozygous, thus selection
criteria were optimized to find such SNPs. The results from a“Corona Lite”
SNP-discovery pipeline (Life Technologies) outputs were scored, by
studying coverage and SNP score. By ignoring loci below a threshold
coverage depth low confidence SNPs were excluded.
Also the SOLiD SNP score obtained was used to filter out lowconfidence SNPs. A high score indicates high confidence in an SNP, taking
into account the location of the SNP within each read. In this manner,
SNPs located among the more error-prone bases towards the distal end of
32
the reads score lower than those located in the proximal end (Figure 2,
paper II). Only SNPs with 5 x coverage in both ebi-1 and Ws-2
backgrounds, and a SOLiD score of 0.7 or more were finally considered.
One hundred and nine SNPs were found to be unique for ebi-1, mostly
located on the north arm of Chromosome (chr) V, and to a lesser extent,
chromosome I. (Figure 3, paper II). The mapping had located the mutation
to the northern part of chr V, thus the high number of SNPs there were
due to other mutations “hitch-hiking” on the ebi-1 mutation during backcrossing. Of the 76 SNPs found on chr V, 32 were non-synonymous,
meaning that they would lead to changes in the amino acid composition of
translated proteins. Since most clock-related genes are themselves
rhythmically expressed, the Diurnal database (diurnal.cgrb.oregonstate.edu,
(Mockler et al., 2007; Michael et al., 2008) was used to evaluate these 32
SNPs. Two experimental setups were considered, constant light after LD
12:12 entrainment and constant dark after temperature entrainment. Genes
were considered rhythmic if they had a correlation with an expression
pattern model consistent with circadian regulation. This revealed that only
one SNP-containing gene, PRR7, was rhythmic under all tested conditions,
while one other gene, ARABIDOPSIS THALIANA NF-X-LIKE 2
(AtNFXL-2), showed high correlation in constant dark.
Biological confirmation
The obvious candidate for the mutation was then PRR7, since it is a
known clock-related gene, although the prr7-3 mutant allele results in a
longer cycle period, contrary to the effect of ebi-1. The SNP was confirmed
with Sanger sequencing and a dCAPS marker. This point mutation causes
an R to H amino acid substitution, although not in a functional domain.
The other candidate, AtNFXL-2, was also confirmed using Sanger
sequencing and a dCAPS marker. The AtNFXL-2 shares homology with the
mammalian zinc finger transcription factor NF-X1. There are two NF-X1like genes in Arabidopsis, AtNFXL-1 and AtNFXL-2 (Lisso et al., 2006).
Neither of these has been suggested to have a role in the circadian clock.
The SNP in ebi-1 causes a V to I amino acid substitution in a Zinc finger
motif.
Based on the location of the SNP, AtNFXL-2 was now the stronger
candidate. To test this possibility, four lines were generated using backcrossing and dCAPS markers: ebi-1-clean-1 and ebi-1-clean-2, containing only
the AtNFXL-2 SNP; and prr7-clean-1 and prr7-clean-2, containing only the
PRR7 SNP. CAB2 rhythms of these lines were then examined under red
light after entrainment. The results showed that the ebi-1-clean-1 and ebi-133
clean-2 lines have a short-period phenotype, comparable to that of ebi-1.
Prr1-clean-1 and prr7-clean-2 showed almost wild type (WT) behaviour,
indicating that these SNPs make very little or no contribution to the ebi-1
phenotype (Figure 5A, paper II). Furthermore, fine-mapping narrowed
down the mapping interval to between the two molecular markers nga158
and CIW18, consequently excluding PRR7.
Finally, a T-DNA line containing an insertion in the promoter region of
EBI was found to phenotypically mimick ebi-1 (ebi-2). A homozygous line
was transformed with a CAB2:LUC construct and its circadian period was
examined. Similar to ebi-1, ebi-2 had a short period phenotype in constant
light and an early peak in the dark (Figure 5B-C, paper I). Thus, my
colleagues and I concluded that the AtNFXL-2 gene is the novel clock
regulator EBI.
Phylogeny
AtNFXL-2 (EBI) has previously reported involvement in the regulation
of salt and osmotic stress responses (Lisso et al., 2006). Atnfxl2-1 and
AtNFXL-2 antisense plants show increased stress tolerance, similar to
overexpressors of EBI’s sister gene AtNFXL-1, suggesting a possible
antagonistic role. AtNFXL-2 also has at least three different splice variants.
In Arabidopsis, the most well characterized gene in the family is AtNFXL-1,
which is involved in various stress and defence responses (Lisso et al., 2006;
Asano et al., 2008; Larkindale and Vierling, 2008).
NFX1-like proteins in various species
The human NF-X1 homologue of AtNFXL-1 and AtNFXL-2 was the
first member of this type of proteins to be characterized (Song et al., 1994).
It was described as a repressor of class II MHC (major histocompability
complex) gene expression and shown to interact with X-box promoter
elements. Two splice variants have also been identified, NF-X1-123 and
NF-X1-91, with some functional differences due to differences in their Ctermini (Gewin et al., 2004). mice, the mNFX1 protein shows particular
similarity to the human NF-X1-123 protein, and represses MHC class II
gene expression, suggesting the proteins have conserved functions in mice
and humans (Arlotta et al., 2002). In yeast, the NF-X1 protein FAP1 has
been identified as a repressor of rapamycin toxicity and reportedly interacts
with a peptidyl-propyl cis-trans isomerase, FKBP12 (Kunz et al., 2000). In
Drosophila, the shuttle craft (STC) homologue of NF-X1 has been shown to be
important for embryo development (Stroumbakis et al., 1996). In plants,
34
two NF-X1-like genes have been found in Arabidopsis, rice and in Populus,
where there are two duplicates of each gene, amongst other species, while
only one gene homologue has been found in Physcomitrella and Clamydomonas
(Figure S1, paper I).
All NF-X1-like proteins, including the plant proteins, have a characteristic
N-terminal zinc finger motif, called a RING finger domain, and a Cys-rich
region with several repeated zinc-finger motifs (Müssig et al., 2010). RING
finger proteins can be found in both the cytoplasm and the nucleus. Many
are ubiquitin ligases (E3s) that together with ubiquitin conjugating enzymes
(E2s) ubiquitinate target proteins. However, this has not been shown for
plants, although it is plausible (Müssig et al., 2010).
The Cys-rich region has been shown to be necessary for binding to
specific promoter elements in human NF-X1 (Song et al., 1994; Gewin et
al., 2004). There are also specific differences between the plant proteins and
those of other organisms; some domains are conserved in NF-X1-like
genes, PAM2 and R3H motifs, but not in plants. This could reflect
functional differences. NF-X1-like proteins are likely to control the
expression of specific genes and may regulate their own location and that of
other proteins, by regulating their own levels and the activity of other
transcription factors, or by histone modifications (Müssig et al., 2010).
The SNP causing the ebi-1 phenotype is located in a zinc-binding domain,
thus it may affect, for example, the structure or binding of the protein, but
it is currently not known. The fact that also the ebi-2 allele causes an early
phase/short period phenotype (ebi-2) suggests that EBI may be of general
importance for clock rhythms in Arabidopsis.
EBI protein actions (Paper I)
Knowing the EBI locus allowed the creation of constructs to probe the
role of EBI at the translational level. Much of the regulation in the clock
occurs post-translationally, via the interactions of different proteins. These
interactions add an extra level of regulation, probably allowing processes to
be fine-tuned. Therefore, interactions between EBI with the known clock
proteins TOC1, ZTL and GI were tested, by co-expressing the proteins
using His- or Myc-tagged constructs in protoplast cell cultures. A coimmunoprecipitation assay revealed that EBI interacts strongly with ZTL,
weakly (possibly indirectly) with TOC1 and not at all with GI (Figure 4,
paper I). Since ZTL is known to degrade TOC1, a test was performed to
see if ZTL also degraded EBI. CHX was added to the cell cultures to block
protein synthesis and then samples were taken over a time series. TOC1 was
degraded within 30 min together with ZTL, as expected. However, EBI was
35
not degraded in a ZTL-dependent manner. It was degraded later, possibly
through other degradation pathways (Figure 6, paper I).
To further examine the EBI-ZTL interaction, a number of ZTL,
constructs with mutants in their Kelch repeat, LOV and F-box domains,
were tested for their ability to interact with EBI. The results showed that
both the LOV and F-box domains of ZTL are required for binding EBI
(Figure 5, paper I).
To see whether EBI affected ZTL transcription or translation, the ebi-1
mutant was examined in a time series using a ZTL antibody, and ZTL
transcript levels were measured. Earlier accumulation of ZTL transcript was
observed in the ebi-1 mutant, but no apparent differences were detected in
ZTL protein levels (Figure 5, paper I). Thus, there seems to be no feedback
between EBI and ZTL transcription, highlighting the importance of the
protein-protein interaction.
EBI opposes ZTL’s effect on the clock
To test the internal relationship between EBI and ZTL, mutants carrying
ebi-1 and the ZTL mutant allele ztl-21 were crossed. Ztl-21 carries a
mutation in the LOV domain of the protein, making it unable to bind to
EBI. The ztl-21 mutant has a long period, while ebi-1 mutant has a short
period phenotype. The ebi-1;ztl-21 double mutant has a long period
phenotype, compared with the wild type (Ws-2), but shorter than that of ztl21. EBI has an opposing effect to ZTL on a common target, since it has a
shortening effect on periodicity in a ztl-21 background (Table I, paper I).
To examine if EBI had an effect on other clock components at the
transcriptional level, Myc-epitope tagged EBI or ebi1 mutant protein was
co-expressed in protoplasts with ZTL, then transcript levels of the morning
clock gene CCA1 and the evening clock gene TOC1 were measured. At
Zeitgeber time 6 (ZT6), six hours after lights on in a LD 12:12 cycle,
repression of CCA1 transcript levels by EBI/ebi1 was observed. If
EBI/ebi1 was co-expressed with ZTL, however, the repression was
removed, indicating that these two proteins together act as promoters of
CCA1 expression (Figure 7, paper I). At ZT18, in the middle of the dark
period, EBI/ebi1 upregulate CCA1 transcription, while combined
EBI/ebi1 and ZTL expression leads to even higher upregulation, indicating
that these two proteins together may to promote CCA1 expression at this
time (Figure 9, unpublished). This shows that EBI regulates CCA1
transcription during the night. It does not, however, explain the ebi-1
phenotype.
36
Normalized expression
4
EBI
EBI ZTL
ZTL
ebi1
ebi-1 ZTL
Empty
3
2
1
0
CCA1
CCA1
TOC1
TOC1
ZT6
ZT18
ZT6
ZT18
Figure 9: Expression of CCA1 and TOC1 at two different time points
in a protoplast assay. EBI and ebi1 were co-expressed together with ZTL
to probe transcriptional effects on CCA1 and TOC1
TOC1 was significantly upregulated if ebi1 and ZTL was co-expressed, at
both ZT6 and (to a lesser extent) ZT18. This earlier accumulation of
transcript could be a plausible explanation of the ebi-1 short period
phenotype, since an earlier rise in TOC1 levels act to advance the clock in
ebi-1. In the ebi-1 mutant, the gate is not able to fully close, leading to early
accumulation of TOC1 transcript when mutant ebi1 binds to ZTL. This
would mean that also CCA1 and LHY are promoted prematurely, affecting
period length.
Since ZTL resides in the cytosol (Kim et al., 2007) and EBI has a putative
nuclear localization domain (Lisso et al., 2006), the interaction between
ZTL and EBI could regulate EBI’s transcriptional role. When EBI and ZTL
are bound together during the day, no effect on CCA1 transcription can be
seen. However, during the night, EBI-ZTL interaction leads to induction of
CCA1, possibly by ZTL binding to EBI and preventing it from repressing
CCA1 (Figure 10). The fact that CCA1 transcript is induced when
EBI/ebi1 is expressed without ZTL at ZT18 could be due to additional
factors affecting the process while the gate is “open”. Additionally, ZTL
protein levels cycle with a peak around ZT12, which could regulate EBI’s
window of activity, by binding to the EBI protein at certain times during
the circadian cycle.
37
ZT6 (day)
ZTL
EBI
ebi1
ZTL
CCA1
ZTL
Cytosol
TOC1
EBI
ZT18 (night)
Nucleus
ebi1
ZTL
CCA1
Cytosol
TOC1
Nucleus
Figure 10: Speculative model for
EBIs mode of action. EBI represses
expression of CCA1 during the day,
while located in the nucleus. During
the night ZTL binds EBI, which
allows CCA1 expression. Ebi1 seems
to allow transcription of TOC1 both
during the day and during the night,
possibly causing the short period
phenotype of ebi-1. Genes are written
in italics and proteins are described
as coloured ovals. Promotion is
described with arrows and repression
with bars
Control of stress responses by ZTL and EBI (Paper III)
Effects of abscisic acid on EBI and ZTL
Since the EBI allele AtNFXL-2 had been previously described as
important for stress responses, the effects of the ebi-1 mutation on stress
responses were explored. First, ebi-1 mutants were tested to confirm that
they respond to stress in a similar way to earlier reports. No difference in
resistance to salt stress could however be detected, thus it is possible that
the effect of the ebi-1 allele is clock specific (Figure I, paper III).
While abscisic acid (ABA) has been implicated as important for several
clock components, ebi-1 as well as ztl-21 and the ebi-1;ztl-21 double mutant
were entrained for 7 days after which the seedlings were transferred to
plates containing 20 µM ABA or 0.01% DMSO and their free-running
period was observed. Also, a ZTL:LUC construct was used to examine ZTL
transcript levels with and without ABA.
ABA had a shortening effect on period of CAB:LUC in all tested
genotypes, and caused a dampening in amplitude (Figure 2/Table I, paper
III). This is in contrast to earlier reports (Hanano et al., 2006), but could
possibly be explained by the difference in light intensities used. We used
100 µmol m-2 s-1 for entrainment and free-ran the plants in 20 µmol m-2 s1
synthetic white light while Hanano et al used 10 µmol m-2 s-1 for
entrainment and free-ran in 2 µ m-2 s-1. However, it seems like ABA has an
effect on period, although light dependent.
The ZTL:LUC constructs showed weak rhythmicity under constant light,
and interestingly ABA stabilized these oscillations (Figure 3, paper III). ZTL
transcript has earlier been reporter as constitutive (Somers et al., 2000; Kim
et al., 2007). This might be due to differences in conditions between the
38
experimental set-up or the deployment of different accessions. However,
previous studies were performed by measuring RNA levels by northern
blotting or real-time PCR, in contrast to our ZTL:LUC construct that gives
a higher resolution (hourly) and provides a more dynamic read-out because
we are looking at the de novo synthesis of LUC driven by the ZTL
promoter.
The periodicity of ZTL transcript was more pronounced in ebi-1
background, something that was even clearer after addition of ABA (Figure
3, paper III). Thus is seems that ZTL transcript oscillates under certain
conditions, and that EBI mediates these oscillations. Additionally, no
differences in period could be found between ebi-1 and Ws-2 in ZTL
oscillations suggesting that they may be part of or controlled by two
different oscillator set-ups.
As a means to address stress signalling, investigation of the expression of
the stress response gene RD29A together with ZTL and EBI under an
entraining LD 12:12 cycle using ebi-1, ztl-21 and Ws-2 genotypes was
performed. No difference in RD29A could be seen between ebi-1 and Ws-2
at 6 h resolution, but the peak of RD29A expression was delayed in ztl-21,
in accordance with its 2 h long period phenotype as reported by Kevei et al.
(2006)(Figure 4, paper III). ZTL transcript was weakly rhythmic, with an
earlier peak and somewhat lower expression levels in ztl-21. EBI levels were
unchanged. The late peak of RD29A in ztl-21 confirms that it is under
significant clock control as reported by Dodd et al (2006).
To summarize, ZTL transcript is oscillating in Arabidopsis. These
oscillations seem conditional, and perhaps related to stress signalling, since
addition of ABA, which is a plant growth regulator much associated with
plant’s stress adaption, acts in a stabilizing manner. The lack of a difference
in period length between the mutant ebi-1 and Ws-2 control plants could
mean that the ZTL oscillations are controlled by a separate oscillator. EBI is
involved in this regulation, since ZTL oscillations in ebi-1 background are
more rhythmic than in Ws-2 controls. This is an additional feature of EBI,
plausibly mediating ABA signals to ZTL and acting as a link between these
two regulatory pathways.
39
The circadian clock in the perennial Populus
The Clock and seasonal effects (Paper IV)
Although it is very practical to work with, Arabidopsis lacks many
characteristics of other plant species, due to its very short life span.
Therefore, the role of the circadian clock in the Populus model system is
being studied. This enables more to be learned about the clock in perennials
and the importance of the clock in responses to seasonal variations, which
is of particular importance at high latitudes, especially the Scandinavian
region, considering the valuable forest industry and the dramatic shifts in
light and temperature over the year.
In Populus, there are two LHY genes that are homologous with the
Arabidopsis LHY gene, PttLHY1 and PttLHY2 (Takata et al., 2008).
Transcripts of both were detected in WT plants grown under 18 h day/6 h
night, long-day (LD) conditions. Levels peaked 1 h after dawn (ZT1), with
PttLHY2 showing five times higher expression levels than PttLHY1 (Figure
S2, paper IV).
To study their role in Populus, transcripts of both genes were targeted with
an RNA interference (RNAi) construct, which led to 20-50% downregulation of their expression levels. Assayed separately, PttLHY1 showed
~35-60% expression, while PttLHY2 was expressed at 60-80% of control
levels. There is also a single TOC1 homologue in Populus, which has a peak
expression at ZT9 under long-day conditions (Figure 1, paper IV). PttTOC1
was also targeted with an RNAi construct, leading to similar level of downregulation as for the lhy lines.
Growth cessation
A major seasonal event for trees at these latitudes is the cessation of
growth and the beginning of dormancy, in preparation for the coming
winter. To examine whether these genes have any effect on growth
cessation in Populus, selected lines were grown under a photoperiod slightly
shorter than the critical day length (CDL) of the wild-type plants (15.5 h,
(Olsen et al., 1997). After six weeks, most of the wild type plants had set
buds, while less than 30% of the lhy and toc1 lines showed any signs of
growth cessation (Figure 2, paper IV). A shift of an additional hour under
CDL prompted all lines to set buds, indicating that the CDL for lhy and toc1
lines is about 1 h shorter than for wild type. Shifts from LD to SD showed
that lhy tended to enter growth cessation slower, but only one lhy line
differed significantly from wild type. Thus, RNAi lines were concluded to
40
have no overall response deficiency, but are altered in daylight sensing near
the CDL.
To study the effects of the RNAi constructs on the circadian clock, two
representative RNAi lines putatively representing the repressive and
promoting regulators of the core Populus circadian clock respectively, lhy-10
and toc1-5. They were transformed with the heterologous clock reporter
construct AtCCR2:LUC, allowing the monitoring of the expression of
CCR2. Wild-type lines cycled robustly with a peak around ZT14 (Figure 3,
paper IV). Transformed lhy-10 lines showed peaks ~4 h earlier than wild ,
while toc1-5 peaked ~1 h earlier than wild type under 18 h light/6 h dark
cycles. When released into constant light lhy-10 showed weak periodicity,
while toc1-5 had a higher amplitude than wild type lines. When transferred
to constant dark, the RNAi lines showed similar behaviour, with lhy-10 and
toc1-5 showing arrhythmia at subjective dawn and dusk respectively (Figure
3B, paper IV). Period estimates showed a shortening of period of about 2.5
h (lhy-10) and 1.5 h (toc1-5) compared with wild-type Controls. These results
are comparable with observations in Arabidopsis and other species (Song et
al., 2010). These studies thus confirmed a role within the Populus oscillator
for these gene products.
Gene expression studies of circadian genes and genes involved in
photoperiodic responses were performed on lhy-lines, since they showed
clear periodic and phase defects using AtCCR2:LUC reporter gene
expression. Gene expression was analyzed after six days of SD entrainment.
Showing that the combined expression of PttLHY1 and PttLHY2 (PttLHY)
peaked at dawn in WT, while it was advanced ~4 h in lhy-lines, consistent
with the results detected from CCR2:LUC expression. (Figure 4, paper IV).
Populus homologues of GI and FKF1 were examined, showing advanced
phase, as well. This was also true for PttCO2expression, these gene’s
advances in peak times relative the 12 h light/ 12 h dark cycle together
could explain the change in CDL sensing and delay in growth cessation in
lhy-lines.
Cold acclimation
Processes associated with growth cessation and bud set may affect downstream processes, such as winter cold hardiness and dormancy release. To
see whether decrease in PttLHY1 and PttLHY2 expression had any impact
on freezing tolerance, wild type and the lhy representative lhy-10 were
subjected to SD to induce growth cessation, followed by 6 weeks of cold
hardening at 6oC. Electrolyte leakage and tissue discoloration were assayed,
and the results showed that wild type resisted freezing without injury, while
lhy-10 consistently showed freezing injury (Figure 5, paper IV). By contrast,
41
toc1-5 showed greater freezing tolerance than wild type, in a separate
experiment (Figure S4, paper IV). To examine if this had anything to do
with an abnormal clock response to cold, plants were entrained in 18 h
light: 6 h dark cycles and then shifted to 24 h darkness and 4oC from lights
off at ZT18. Subsequent examination of their transcript levels showed that
levels of PttLHY1, PttLHY2, PttTOC1 and PttPRR5 were high and constant
in wild-type plants, but lhy-10 plants were unable to up-regulate PttLHY1
and PttLHY2, and responses of their PttTOC1 and PttPRR5 levels were
weaker. (Figure 6, paper IV). Additionally, PttC-REPEAT/DRE BINDING
FACTOR 1 (PttCBF1), a cold-stress-response gene, was expressed in wild
type with a peak around ZT28, while there was no expression of PttCBF1 in
lhy-10 plants. Notably, in toc1-5, which had increased freezing tolerance in
the freeze test, PttLHY1, PttLHY2 and PttCBF1 expression was upregulated (Figure S5, paper IV), showing that the level of expression of
PttLHY1 and PttLHY2 are instrumental of cold-response of PttCBF1 as
well as for building freezing resistance.
Furthermore bud burst was examined in buds that had set under CDL
conditions and were cold-treated for 18 weeks. Transcription levels of
PttLHY, PttTOC1 and PttPRR5 were monitored throughout the bud burst
process, due to the observed cold responses of PttLHY and PttPRR5.
Examination of gene levels at ZT4 and ZT12 showed that PttLHY
expression was decreased in lhy-10, compared with wild type as expected for
these down-regulated line (Figure 7, paper IV). PttTOC1 levels were slightly
lower and PttPRR5 levels were higher. Since high PttPRR5 levels in coldtreated wild-type plants was observed (Figure 6, paper IV), the increase in
PttPRR5 levels in lhy-10 could indicate an impaired response to warming
temperatures and delayed bud burst. Thus, expression of PttLHY is also
needed for proper clock gene expression during bud burst and for the speed
of growth resumption.
Therefore, we have showed that the clock in Populus is involved in the
control of growth cessation and is important for cold tolerance. It also has
an effect on bud burst and growth resumption. Taken together, this shows
the importance of a functioning clock for proper timing of growth in
response to season and was importantly also shown to be crucial for
freezing tolerance. All crucial for a perennial life style and survival of winter
temperatures at high latitudes.
42
Input signals into the Populus clock (Paper V)
The most important seasonal signal for trees is the changing duration of
light over the year. The photoreceptor phyA perceives light and mediates
signals to downstream systems, thus controlling light-dependent responses.
One of the systems affected by light is the circadian clock. Therefore, the
role of phyA in regulation of the circadian clock and tree dormancy was
examined. Tests with decreasing photoperiod showed that under CDL,
strong down-regulation of PttPHYA gene causes earlier timing of growth
cessation and bud set (Table I, paper V). Under short days, moderate downregulation was sufficient to prompt earlier growth cessation. When SD
conditions were extended with 4.5 h of FR-enriched light, all genotypes
responded with fast elongation and reduced leaf number. 3h end-of-day
(EOD) treatment led to significantly earlier growth cessation in aPttPHYA
lines, while wildtype lines sustained growth longer.
Photoperiodic effects
To examine the dependence of the coincidence between endogenous
rhythms and external light conditions, short light/dark day cycles were used
to see if they were capable of induced growth cessation in the transgenic
Populus over-expressing oatPHYA (oatPHYAox) that show daylength
independent growth and fail to set bud under any so far tested photoperiod
(Olsen et al., 1997). Plants were entrained to a LD 12:12 cycle, then exposed
to a 6 h light:6 h dark (LD 6:6) cycle. After 63 days, wild-type and
aPttPHYA plants were starting to show signs of growth cessation (Figure 2,
paper V). After 80 days, wild type had completed bud set. Within 112 days,
oatPHYAox had also stopped growth and 80% of oatPHYAox plants had set
bud. They were still significantly smaller with fewer leaves than wild-type
plants in agreement with earlier experiments (Olsen et al., 1997; Møhlmann
et al., 2005). The dramatic difference between endogenous rhythms and
external light/dark cycles prompted growth cessation, even in the
oatPHYAox line that was previously unable to cease growth. Since proper
growth cessation is required for the acquisition of freezing tolerance, the
cold hardiness of oatPHYAox plants that had stopped growing was
examined. Thus, oatPHYAox plants together with aPttPHYA and wild-type
plants were tested for capability to withstand freezing. Interestingly,
oatPHYAox plants grown in LD 6:6 cycles had marginally lower tolerance
compared to wild-type, while oatPHYAox plants grown under LD 12:12
were not cold hardened at all. In contrast, wild-type and anti-sense lines
were equally competent. Thus, proper alignment of inner and external
phases/cycles was able to rescue normal growth cessation/bud set
properties as well as freezing tolerance in oatPHYAox plants. This means
43
that determination of daylenght in Populus depends on a properly
functioning circadian oscillator.
Light and the clock
The circadian clock’s probable role in daylength detection, by timing the
regulation of downstream components, prompted the study of such
components in wildtype, aPttPHYA-1 and oatPHYAox lines. Two clockcontrolled promoter:reporter constructs were used to follow the innate
period of rhythms in the plants AtCCA1:LUC and AtCCR2:LUC.
Luminescence was followed under LD and constant darkness, following SD
entrainment. Under LD, the peaks of CCA1 and CCR2 expression appeared
at ZT2 and ZT13, respectively, similar to findings of previous studies in
Arabidopsis (Figure 4, paper V). Under constant darkness, the internal mean
periodicity of their expression was 23.7 h and 23.5 h, respectively. PttLHY
levels were similar in wildtype and aPttPHYA-1 plants, although with a
slightly deeper trough in the anti-sense plants (Figure 5, paper V). PttTOC1
was out of phase with PttLHY, as reported earlier. The double peak in wild
type might have been due to incomplete entrainment. PttFKF1 expression
peaked at ZT8, with lower levels in anti-sense plants. PttCO2 peaked at ZT0
and ZT8 in wildtype, but only at ZT0 in anti-sense plants, suggesting that
PttCO2 was down-regulated in the anti-sense plants after only two SD
cycles. This suggests that PttPHYA is necessary to support its expression, as
well as PttTOC1 and PttFKF1.
Since the circadian clock probably controls the phase of expression of the
photoperiodic genes PttFKF1 and PttCO2, this function of the circadian
clock was examined. Circadian period can be measured by following leaf
movements under constant light. We measured this in wildtype, aPttPHYA1 and oatPHYAox. The mean period of leaf movement was significantly
shorter in oatPHYAox compared with wildtype and longer in aPttPHYA-1
(Figure 6, paper V). To confirm the lengthening of the period in aPttPHYA
plants, PttLHY expression was assayed under constant light. Peaks were
delayed in both tested lines (Figure 7, paper V). PttFKF1 expression was
also changed under these conditions. Using the AtCCR2:LUC construct,
period length was also measured in constant darkness, and the results
showed that the anti-sense lines again had a longer periodicity than wild
type (Table II, paper V).
To also examine effects of FR light on selected clock and photoperiodic
genes, aPttPHYA and wild-type plants were adapted to constant dark for
four days and then subjected to a 1 h FR light pulse. Expression of
PttLHY1 increased in anti-sense plants, suggesting that FR light represses
this gene (Figure 8, paper V). In contrast, PttFKF1 and PttFT expression
44
was reduced in anti-sense plants, suggesting a promoting effect of FR on
these genes. These results fit with the hypothesis that de-repression of
PttLHY1 in response to reduced phyA leads to a longer circadian period.
This shows the importance of far-red light signalling on the circadian clock
and its response to seasonal changes.
45
Final conclusions
The studies this thesis is based upon have extended knowledge of the
circadian clock and its role in several physiological plant processes. By
studying the relatively well-characterized annual species Arabidopsis, a new
component connected to the circadian circuitry, EBI, has been found.
Formerly, this had only been identified as a stress response regulator (Paper
I-III). It has been shown that this component is important for maintaining
the correct phase and period of the circadian oscillator and that it interacts
at the protein level with ZTL, a known regulator of the stability of central
clock components. The short period phenotype in the cca1-11;lhy-21;ebi-1
triple mutant points towards the period lengthening effect of EBI being
additive to that of CCA1 and LHY. However, studies of the effect of
epitope tagged EBI and mutant ebi1 in a protoplast assay indicate that EBI
is involved in transcriptional regulation of CCA1, as well as TOC1. The
mutation predominantly seems to affects the expression of TOC1, which in
agreement with the genetic data likely is the primary cause for the mutant
phenotype of ebi-1.
The effects of down-regulation of clock genes in the perennial Populus
(Paper IV) were also studied and the importance of the clock for growth
cessation and bud set was demonstrated, in addition to its role in the
acquisition of cold tolerance. The clock control of such photoperiodic
responses is mediated through the phytochrome photoreceptors, in
particular Populus phyA (Paper V).
Thus, we can conclude:
46
•
EBI is a novel component of the circadian clock in Arabidopsis,
which is involved in both transcriptional and translational
regulation.
•
EBI has dual roles, since it is implicated in stress responses and
could act as a link between the clock and stress.
•
The circadian clock is responsible for regulating seasonal growth
patterns and acquiring tolerance to freezing in Populus.
•
Light perceived and mediated via phyA modulates the behaviour of
the circadian clock and its regulation of growth in Populus.
Further perspectives
Several things still need to be examined before the role of EBI is fully
understood. For instance, studying the regulation of EBI protein and its
localization would indicate where EBI is expressed and when. It would also
indicate if it moves to the nucleus and if that too is a timed process. The
transcriptional regulation of EBI itself is not yet known, studies of such
features might provide further insight to clock regulation. The
phylogenetically conserved protein structure of EBI and the role of the NFX1 protein in humans suggest that ubiquitination may be involved in
regulation of the protein. Thus, it would be logical to examine whether EBI
and its related proteins are ubiquitinated. However, since the role of EBI in
the circadian clock and its role in stress responses have not been examined
in other plant species, and there are likely to be major differences between
species in the roles and regulation of these proteins, species-specific
approaches and studies are required.
These results indicate that EBI together with ZTL could be involved in
responses to stress and hormones (ABA). This of course raises the question
of whether the stress function of EBI is a consequence of its role in the
clock or a distinct role. Since CCA1 and TOC1 have been independently
implicated in stress responses, this can first be addressed by following the
expression of CCA1:LUC and TOC1:LUC and other stress-related genes
under such conditions.
Since Populus is already being studied as a model system, it would be a
natural step to examine the role of EBI homologues in the perennial plant
clock, to further elucidating the role of EBI in the circadian oscillator.
Expanding knowledge of the roles of EBI and its homologues in seasonal
regulation of growth and winter hardiness should follow.
47
Acknowledgements
There are a number of people I would like to acknowledge, and surely I will
forget someone. My regards to those people as well. ☺
My supervisor Maria, always committed and interested in my work. Thanks
for letting me develop freely and allowing me to sneak away to conferences
as far away as I could find them.
Harriet McWatters, thanks for hosting me in Oxford and providing clockthinking on the highest level, superbly putting my results into the proper
context.
Laszlo Bako, the protein interactions wizard of UPSC, crucial to our
success and always ready to help.
Current and former members of the Eriksson group:
Naoki, the working ant from Japan (I’m certain the lucky token you gave
me has boosted me while writing), the always super-friendly Cristian, now
spreading knowledge around him at home in Chile and the whirlwind
Iwanka..
Peter, we have followed the same road for quite some time now, I can’t
imagine a better study/drinking/skiing/diving/conferencing/traveling
buddy. Good luck on the other side!
All PhD students at UPSC; the inimitable Bas, Hanna med koll and all the
others. (And also the unforgettable mr (dr?) Soolanayakanahally that made a
legendary guest appearance).
Everyone else working at UPSC, for creating a really nice atmosphere for
science, especially all of you that take care of everything we scientists are
too lazy and distracted to do (watering plants, doing dishes, etc, etc).
Jag vill även tacka gänget som förgyllde min studietid här i Ume; Shandy,
mannen med världens näst bästa musiksmak, den levande superdatorn
Gabbe, globetrottern Henke och hela världens lillebror Omme.
48
Det gamla goda korridors-gänget kräver erkännande, främst Dr David G,
med svart bälte i alfapet och Jeopardy-meriter och alltid lika glada och snälla
Helena. Har varma (men kanske lite suddiga...) minnen av stenhårda
korridors-fotbollsmatcher och Madonna på repeat.
I 08-land vill jag tacka Nykvarns-gänget: Håkan, min f.d. ständiga guide på
Stockholms okända smågator, numera baserad i världens ände (a.k.a.
Nynäshamn). Tack för en titt på min kappa från någon som inte är helt
insnöad i det akademiska. Stig-finnaren och F1-fantasten Daniel och vår
småländske London-emigrant Rickard. Hoppas på fler (skid)resor i
framtiden.
Även Tälje/Enhörna-gänget är värt ett omnämnande: uppslagsverket
Tobbe, fixaren Norrman, spjut-Wallin, herr Selegård med flera.
Min familj; min far som verkar vilja förstå vad jag håller på med på riktigt
och alltid uppmuntrat mig till att göra det jag vill. Min bror och gadgetguide Robert (och hans Tina), och resten av släkten, som iallafall låtsas(?)
att det jag gör är intressant 5 minuter vid varje julbord. ☺
Till sist,
Franziska, immer so liebevoll. Mot nya äventyr!
49
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