The 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 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 expression of the Populus LATE ELONATED HYPOCOTYL (LHY) 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 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 (hybrid- asp). 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 post- translationella 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 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 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 ...... 10 Photoperiodism in plants...... 11 Light perception...... 12 Clock genetics ...... 14 Clock model organisms ...... 15 ...... 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 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 in various species...... 35 EBI 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

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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 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))

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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.).

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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).

Advance +4

+2 Dead zone shift 0

Phase ‐2 ‐4 Delay 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.

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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.

Period amplitude

2x

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.

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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 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 (Espinosa- Ruiz 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 re- activated 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 gene- of-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 over- expression 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 , 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.

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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 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 cell- autonomous, 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.

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The molecular clock in Arabidopsis In Arabidopsis, the first clock gene to be characterized was the evening- expressed 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).

Figure 4: The genes and proteins CCA1/LHY 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 CCA1/LHY TOC1 levels and thus a decrease in CCA1 and LHY. This allows TOC1 transcription to increase TOC1 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.

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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/PAS- domain, 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). 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), LIGHT- REGULATED WD1/ -2 (LWD1-2), LUX ARRHYTHMO (LUX), SENSITIVE TO FREEZING6 (SFR6), TEJ, TIME FOR COFFEE (TIC),

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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 1 P K 1 L F K F L T Z I I G G ” 3 p R o 5 R o l Y P R Y

R g P n i n e v E ” P 1 1 C C O O T T 3 F L E X 4 F E E ” L H H p E C C o o l

X e r o C ” Y Y H H L L 1 1 A A C C C C ” p o o l

g n i n r o M ” 9 9 R R R R P P 7 R 7 R R P R P

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.

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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 differe n t 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).

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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 chilling- sensitive 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.

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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.

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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 free- running 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.

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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.

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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 wild- type, 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).

1.5

1

0.5 Ws-2

Normalized Luminescence Normalized 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.

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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 low- confidence 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 (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 back- crossing. 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-X1- like 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 back- crossing 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-1-

33 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 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 C- termini (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 co- immunoprecipitation 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 ztl- 21. 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.

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4 EBI ebi1 EBI ZTL ebi-1 ZTL ZTL Empty 3

2

1 Normalized expression Normalized 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.

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ZT6 (day) ZT18 (night) Figure 10: Speculative model for EBIs mode of action. EBI represses expression of CCA1 during the day, EBI while located in the nucleus. During ZTL ebi1 ZTL ebi1 ZTL ZTL the night ZTL binds EBI, which allows CCA1 expression. Ebi1 seems to allow transcription of TOC1 both during the day and during the night, CCA1 Cytosol CCA1 Cytosol TOC1 TOC1 possibly causing the short period phenotype of ebi-1. Genes are written in italics and proteins are described EBI Nucleus Nucleus 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 s- 1synthetic 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.

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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% down- regulation 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 down- regulation 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 down- stream 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 up- regulated (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 cold- treated 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 down- regulation 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 clock- controlled 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, aPttPHYA- 1 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:

• 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.

46

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 NF- X1 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 clock- thinking 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 gadget- guide 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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