Circadian control of root elongation and C partitioning in Arabidopsis thalianapce_

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1 Plant, Cell and Environment (2011) 34, doi: /j x Circadian control of root elongation and C partitioning in Arabidopsis thalianapce_ NIMA YAZDANBAKHSH 1, RONAN SULPICE 1, ALEXANDER GRAF 2, MARK STITT 1 & JOACHIM FISAHN 1 1 Max Planck Institute of Molecular Plant Physiology, Potsdam/Golm, Am Mühlenberg 1, Germany and 2 Department of Metabolic Biology, John Innes Centre, Norwich Research Park, Norwich NR4/UH, UK ABSTRACT Plants grow in a light/dark cycle. We have investigated how growth is buffered against the resulting changes in the carbon supply. Growth of primary roots of Arabidopsis seedlings was monitored using time-resolved video imaging. The average daily rate of growth is increased in longer light periods or by addition of sugars. It responds slowly over days when the conditions are changed. The momentary rate of growth exhibits a robust diel oscillation with a minimum 8 9 h after dawn and a maximum towards the end of the night. Analyses with starch metabolism mutants show that starch turnover is required to maintain growth at night. A carbon shortfall leads to an inhibition of growth, which is not immediately reversed when carbon becomes available again. The diel oscillation persists in continuous light and is strongly modified in clock mutants. Central clock functions that depend on CCA1/LHY are required to set an appropriate rate of starch degradation and maintain a supply of carbon to support growth through to dawn, whereas ELF3 acts to decrease growth in the light period and promote growth in the night. Thus, while the overall growth rate depends on the carbon supply, the clock orchestrates diurnal carbon allocation and growth. Key-words: carbohydrates; circadian clock; root growth kinetics. Abbreviations: CCA/LHY, circadian clock associated late elongated hypocotyls; ELF3, early flowering 3; ELF4, early flowering 4; SEX, starch excess; PGM, phosphoglucomutase. INTRODUCTION Plants grow autotrophically using light, CO 2, nutrients and water that they acquire from the abiotic environment. As they need to maximize capture of these resources, plants are unavoidably exposed to changes in the environment. One of the most pervasive environmental changes is the daily alternation between light and dark (Geiger, Servaites & Fuchs 2000; Nozue & Maloof 2006; Smith & Stitt 2007). In the light, plants have a positive carbon (C) balance and can Correspondence: J. Fisahn. Fax: ; fisahn@ mpimp-golm.mpg.de use carbohydrates that are delivered by photosynthesis for growth. In the dark, growth depends on resources that have been stored in preceding light periods. In many species, starch is accumulated in the light and remobilized at night (Geiger & Servaites 1994; Geiger et al. 2000; Smith & Stitt 2007; Stitt, Usadel & Lunn 2010). Sugars and organic acids (Chia et al. 2000; Zell et al. 2010) also contribute to transient C storage. The rate of starch degradation in leaves at night is essentially linear, with about 95% of the starch being utilized by dawn (Fondy & Geiger 1985; Geiger & Servaites 1994; Matt et al. 1998; Smith et al. 2004; Gibon et al. 2004a; Graf et al. 2010). A correspondence between the time taken to degrade starch reserves and the length of the night is important to optimize growth in C-limiting conditions. Growth will be decreased if a significant fraction of the daily photosynthate remains as starch at the end of the night, rather than being invested in new leaf and root biomass (Rasse & Toquin 2006). Premature exhaustion of starch also carries a growth penalty. Mutant plants that are impaired in the synthesis or degradation of starch have strongly reduced biomass, except in continuous light or very long days (Caspar, Huber & Somerville 1985; Gibon et al. 2004a, 2009). This is partly due to inhibition of growth during the night (Wiese et al. 2007). In addition, in the first hours of the light period almost all the new photosynthate accumulates as sugars, indicating there is a delay until growth resumes (Gibon et al. 2004b). Plants adjust their starch turnover to changes in the amount of photosynthate that is fixed, or the length of the night. Thus, when plants are grown in lower light intensities, lower CO 2 concentrations or shorter light periods, they allocate more of their current photosynthate to starch in the light and degrade the starch more slowly at night (Stitt, Bulpin & Rees 1978; Chatterton & Silvius 1979, 1980, 1981; Mullen & Koller 1988; Lorenzen & Ewing 1992; Matt et al. 2001; Gibon et al. 2004a). As a result, a small amount of starch remains at the end of the night. Most remarkably, the rate of starch degradation in Arabidopsis adjusts immediately to a sudden and unexpected early or late onset of night. There is an immediate increase in the rate of starch degradation when the light period is suddenly lengthened and, as a consequence, the night is shortened (Lu, Gehan & Sharkey 2005) and there is an immediate decrease in the rate of starch breakdown after exposure to a premature 2011 Blackwell Publishing Ltd 877

2 878 N. Yazdanbakhsh et al. dusk and, as a consequence, a longer night (Lu et al. 2005; Graf et al. 2010). These observations imply that the rate of starch breakdown is gauged to the anticipated length of the night. Like other organisms, plants contain sophisticated biological clocks (Harmer, Panda & Kay 2001; Schaffer et al. 2001; Michael & McClung 2003; Schultz & Kay 2003; Webb 2003; de Montaigu, Tóth & Coupland 2010). Current clock models (Locke et al. 2006; Zeilinger et al. 2006; Nakamichi et al. 2010) contain a dawn loop, in which expression of the two Myb-related transcription factors LHY and CCA1 is modulated by PRR5, PRR7 and PRR9, and a dusk loop including the PRR family member TOC1, which is modulated by GI and LUX. LHY and CCA1 act to repress TOC1, and TOC1 acts to induce CCA1 and LHY via a process that is not fully understood but includes CCA1 HIKING EXPE- DITION (CHE), a member of the TCP family that acts as a transcriptional repressor of CCA1 (Pruneda-Paz et al. 2009). The multiple PRR genes of Arabidopsis uncouple events in the late night from light-driven responses in the day, increasing the flexibility of rhythmic regulation (Pokhilko et al. 2010). Based on an analysis of temperature responses, it was recently proposed that ELF3 is also part of the core circadian clock (Thines & Harmon 2010). Earlier studies attributed ELF3 a role in the regulation of light inputs into the clock (McWatters et al. 2000; Covington et al. 2001; Liu et al. 2001;Yu et al. 2008). It has also recently been proposed that ELF4 integrates the morning and evening loops of the clock (Kolmos et al. 2009). Most studies of the clock have been performed in leaves or hypocotyls. They emphasize the importance of interactions between light and the clock in light/dark cycles in entraining the clock (Nozue et al. 2007; de Montaigu et al. 2010). On the other hand, in a recent comparison of circadian rhythm in gene expression in shoots and roots, James et al. (2008) reported that the evening loop is absent in roots in continuous light, and that the root clock is resynchronized with the shoot clocks in light/dark cycles by a photosynthesis-related signal, which can be overridden by including sucrose in the medium in which the roots are growing. The clock plays a major role in the regulation of processes that are linked to day length, such as floral induction (Hayama & Coupland 2003; de Montaigu et al. 2010). It also regulates the expression of thousands of genes for metabolism and growth (Harmer et al. 2000; Schaffer et al. 2001). Transcripts for different sets of genes peak at different times in a free-running 24 h cycle (Harmer et al. 2000, 2001; Covington et al. 2008) leading to the proposal that circadian regulation anticipates diurnal changes. The importance of the clock for the regulation of growth in light/dark cycles is underlined by the finding (Dodd et al. 2005; Graf et al. 2010) that growth is decreased when the lengths of the circadian and diurnal cycle differ. Dodd et al. (2005) reported that the lower growth rates in mismatched conditions correlated with reduced chlorophyll and lower rates of photosynthesis. Graf et al. (2010) showed that starch was exhausted about 24 h after the last dawn, irrespective of the actual duration of the light/dark cycle. Thus, in a 14 h light/14 h dark cycle, starch was exhausted about 4 h before the actual dawn. Furthermore, the lhy/cca1 double mutant exhausted its starch about 20 h into the diel cycle (Graf et al. 2010), which corresponds to dawn as anticipated by the fast-running circadian clock in this mutant (Alabadi et al. 2002; Ding et al. 2007). Graf et al. (2010) concluded that the reduced growth of wild-type plants in 28 h days and lhy/cca1 in 24 h days is due to the inappropriately high rate of starch degradation, leading to a period of C starvation at the end of night. This would be analogous to the growth inhibition seen in starchless mutants, except that the recurring daily period of C starvation is due to an inappropriate regulation of diel starch turnover, rather than a block in the pathways of starch synthesis and degradation. Changes in the rate of starch mobilization, on their own, will not allow a plant to avoid periods of C starvation at the end of the night. It will also be necessary to decrease the rate of C utilization. This could, in principle, occur via coordinate regulation of starch breakdown and C utilization, or via a very sensitive regulation of the rate of C utilization in response to small changes in supply of sucrose and other metabolites that are synthesized from starch. Gibon et al. (2009) observed a strong positive correlation between the rate of starch degradation and the relative growth rate when Arabidopsis Col-0 was grown in a range of different photoperiods. However, this study only provided information about the average rate of growth. Highly time-resolved measurements of growth will be required to test whether inappropriate timing of starch breakdown leads to a timeof-day-dependent inhibition of growth, that is, at the end of the night. It is known that plant growth is highly rhythmic with respect to the time of day. In general, growth peaks at around dawn in leaves of dicot species (Schmundt et al. 1998; Walter & Schurr 2000, 2005; Wiese et al. 2007) and the middle of the day in monocot species (Watts 1974; Acevedo et al. 1979; Seneweera et al. 1995). In 12/12 light dark cycles, the Arabidopsis accessions Ler and Col-0 display a maximum soon after dawn, a subsequent decrease of growth during the day, and a minimum early in the dark period (Wiese et al. 2007). Hypocotyl growth in wild-type Arabidopsis growing in a light/dark cycle exhibits an even sharper maximum at the end of the night, followed by a decrease in the light (Nozue et al. 2007). Evidence for the involvement of the circadian clock in the control of hypocotyl growth in Arabidopsis seedlings was provided by the observation that the growth oscillations in continuous light are modified in lines with constitutive overexpression of CCA1 and in the elf3 mutant (Nozue et al. 2007). The following experiments investigate diel growth rhythms in wild-type Arabidopsis, in mutants in starch turnover, and in a set of clock mutants that have previously been shown to be affected in starch turnover or diel growth rhythms. We focus, for several reasons, on primary root growth in Arabidopsis seedlings. First, roots are completely dependent on the shoot for the provision of C. This allows

3 Circadian control of root growth 879 complicating effects of light on the local generation of energy or sugars to be excluded. It is also possible to complement changes in endogenous C provision by supplying sugars in the root medium. Second, local changes in water potential are likely to be less marked than in leaves. Third, root growth is essentially a unidirectional process and in seedlings occurs at one site, the primary root tip.this should make it easier to detect small and transient changes in the growth rate. Root extension growth can be measured by using digital calipers to determine root tip displacement, by marking root tip position on a transparent surface, or by capturing a series of time lapse records. Commercial software such as WINRHIZO (Arsenault et al. 1995), OPTIMAS analysis software (Media Cybernetics, or IMAGE J (Abramoff, Magelhaes & Ram 2004) has been introduced to assess root length. Recent studies have also quantified differences in root architecture (Armengaud et al. 2009). Using these approaches, it has been shown that there are rapid adaptations of growth in responses to modulation in light intensities and photoperiods (Aguirrezabal, Deleens & Tardieu 1994; Muller, Stosser & Tardieu 1998; Nagel, Schurr & Walter 2006). There are different growth zones at the root tip, which are differentially affected by different treatments (Walter, Feil & Schurr 2003; Walter & Schurr 2005). However, there is little information on how roots respond to diurnal stimuli or changes in the carbohydrate supply. We have developed a platform for high throughput analysis of Arabidopsis root growth kinetics (Yazdanbakhsh & Fisahn 2009, 2010). Seedlings are grown on agar plates in a custom-designed controlled climate chamber, in which the roots are illuminated by infrared diodes and are oriented and fixed in the focal plane of a charge-coupled device (CCD) camera that captures changes in the tip position of the primary root in time-lapse videos, and quantified to estimate the growth rate. We use this method to show that Arabidopsis wild-type seedlings exhibit robust oscillations in their root elongation rate in different photoperiods and in continuous light, and investigate the contribution of changes in the C supply and the clock to these diel rhythms. MATERIALS AND METHODS Plant material and growth conditions Measurements were performed on Arabidopsis thaliana wild-type Col-0 and Ws-2 as well as on mutants in starch metabolism (pgm, Caspar et al. 1985; sex1, Caspar et al. 1991) and in circadian clock mutants in ELF3 (Covington et al. 2001), ELF4 (Doyle et al. 2002) and CCA1/LHY.The elf3 and elf4 mutants were in Col-0 background (Yu et al. 2001; Kolmos & Davis 2007). The double mutant cca1/lhy originated from Ws-2 accessions (Mizoguchi et al. 2002). Seeds of all lines were surface-sterilized for 20 min with 10% sodium hypochlorite solution containing 0.1% surfactant (Triton X-100, Sigma-Aldrich, Munich, Germany), rinsed several times with sterile water and plated on the surface of solid nutrient agar (7.0% m/v) supplemented with half-strength Murashige Skoog medium (Murashige & Skoog 1962; M02 555, ph 5.6; Duchefa, Haarlem, Netherlands). After 4 d stratification Petri dishes were placed vertically in the phytotron (21 C constant day and night temperature, 100 mmol m -2 s -1 photon flux density, h light dark). At day 9, seedlings that had developed roots of >1 cm length were selected and distributed equally in a row 3 cm from the top on the surface of solid medium filled in mm rectangular Petri dishes, with seedlings per plate. After two further days in the phytotron, Petri dishes were used for measurement. Image acquisition and root elongation analysis Root growth kinetics was collected as described previously (Yazdanbakhsh & Fisahn 2009, 2010). In brief, a custom designed phytochamber housed the central measuring head of the plant root monitor (PlaRoM). The actinic photon flux density in the chamber at the surface of the leaves of the seedlings was 90 mmol m -2 s -1. Temperature was controlled by a cooling device providing 0.5 C accuracy. The PlaRoM imaging platform screens the surface of two Petri dishes and captures time lapse records of the seedlings growing on them (Yazdanbakhsh & Fisahn 2009). Image stacks were collected by a CCD camera (Panasonic Colour CCTV Camera, WV-CP210/G, Matsushita Communication Industrial Co. Ltd., Yokohama, Japan) mounted on the video port of the microscope. To monitor the seedlings regardless of actinic light requirements, an infra red light source (Infra- Red Illuminator CE-7710, Jenn Huey Enterprise Co., Ltd., Taipei, Taiwan) provided measuring light. Screening of the Petri dish and capturing of time lapse records was controlled by the PlaRoM imaging software application (Yazdanbakhsh & Fisahn 2010). The magnification of the microscopes was set such that the video stream covered a mm area of the surface of the Petri dish, and allowed images to be captured with a resolution of (5.96 mm 5.78 mm) pixel -1. The root extension profiling software application analysed the time lapse records and provided the growth velocity profiles and custom specified visualization of root extension profiles (Yazdanbakhsh & Fisahn 2010). Metabolite analysis Shoots and roots of seedlings were separated and immediately frozen in liquid nitrogen. Following homogenization and sub-aliquoting at -70 C, starch, sucrose, glucose, fructose and total amino acids were measured in roots and shoots as described in Cross et al. (2006). RESULTS Diel changes in root elongation in different photoperiods Root growth kinetics of wild-type Col-0 seedlings were investigated in 8/16, 12/12 and 16/8 light/dark cycles (Fig. 1).

4 880 N. Yazdanbakhsh et al. Figure 1. Diurnal changes in root extension growth rates in A. thaliana (Col-0) growing in different photoperiods. Plants were grown in a given photoperiod from germination onwards. Root extension rates of 11- to 15-day-old plants were monitored for three to five consecutive days. The result shows the mean absolute elongation rates for 71, 39 and 50 seedlings growing on 5, 5 and 3 separate plates in the 16 h/8 h, 12 h/12 h and 8 h/16 h light/dark photoperiod treatments, shown as blue squares, red dots and pink triangles, respectively. Black symbols denote the dark period. Error bars indicate the SE. Average elongation rates, as well as the timing and growth rate values of maxima and minima are listed in Supporting Information Table S1. The measurements were carried out for three to five consecutive days with 11- to 15-day-old plants that had been in that light/dark cycle from germination onwards. The average growth rate over a 24 h cycle was highest in a 16/8 ( mmh -1 ), intermediate in a 12/12 ( mmh -1 ) and lowest in an 8/16 ( mmh -1 ) light/dark cycle (Supporting Information Table S1). There were marked diurnal changes in all three light/dark cycles. The relative magnitude of the changes was smallest in the 16 (maximum and minimum rates of and mmh -1, n = 291), intermediate in 12 ( and mmh -1, n = 101) and largest in 8 h light periods ( and mmh -1, n = 239). At first glance, the diurnal growth profiles vary between the three light/dark cycles, but closer inspection indicates some common patterns. Firstly, after illumination, extension rates rose to a transient maximum after h. Secondly, after darkening, growth was depressed for 1 2 h. Thirdly, these rapid transient changes are superimposed on a more gradual oscillation, whose timing is largely independent of the length of the light period. Extension rates decrease to a minimum at 9 11 h after dawn, and gradually recover during the remaining h (Fig. 1). We next investigated whether the rhythms in root elongation persist in absence of a light dark cycle. To do this, wild-type Col-0 seedlings were entrained in light/dark cycles and then transferred to continuous darkness (Fig. 2) or continuous light (Fig. 3). Root elongation rates in continuous darkness To investigate the response after transfer to continuous darkness (Fig. 2), seedlings were grown in a 16/8 h light/ dark cycle to maximize the length of the period in which the responses could be compared. Seedlings were transferred to continuous darkness at the end of the night. One group of seedlings was grown without sucrose in the medium, and another was supplied with 1% sucrose to support growth in the long dark treatment (n = 16, n = 22, respectively). During the last light dark cycle, the pattern of root extension in seedlings grown without sucrose resembled that in 16 h photoperiod treatment of Fig. 1; root elongation showed a transient maximum early after light on, followed by a gradual decline for the next 7 8 h and a gradual recovery that started in the last part of the light phase and continued throughout the night, but was interrupted by a transient inhibition after darkening. Inclusion of 1% sucrose in the medium led to a ca. 50% increase in the growth rate, but the diurnal changes resembled those in the absence of sucrose. After transfer to continuous darkness, the transient increase seen after illumination was abolished. Root extension was maintained for 1 2 h into the subjective light period, and was then decreased (Fig. 2). In the absence of sucrose, elongation was rapidly inhibited, decreasing by >50% during the initial 10 h, and being completely inhibited by h. In the presence of exogenous sucrose, the rate of root extension stabilized and started to recover 8 9 h after the anticipated dawn (Fig. 2, arrow), that is, at the time at which growth would start to recover in a light/dark cycle, Figure 2. Root extension growth of A. thaliana (Col-0) seedlings in an extended night. Plants were grown in a 16 h photoperiod from germination onwards. Root extension rates of seedlings were monitored between 13 and 18 d. At time 0, when the seedlings were 15 d old, the night was extended for 4 d. Dark grey areas indicate subjective night; light grey areas indicate subjective day. Lower trace: Relaxation kinetics in the absence of exogenous sucrose (n = 8 seedlings). Upper trace: Relaxation kinetics in the presence of 1% extracellular sucrose (n = 11 seedlings). The results show the mean absolute elongation rates of each group. Error bars indicate the SE. The black arrow indicates the point of inflection of root growth in the subjective day.

5 Circadian control of root growth 881 and rose to a weak maximum in the middle of the subjective night. On the following days, growth continued to decline. This gradual decline may occur because 1% exogenous sucrose does not fully compensate for the lack of photosynthesis in prolonged darkness, or may also be due to additional signals that are derived from light and are needed to promote growth. Thus, an oscillation with a period of about 24 h appears to be superimposed on the gradual decline. Root elongation rates in free-running continuous light Figure 3. Oscillations in the rate of root extension growth of A. thaliana (Col-0) after transfer to continuous illumination. Subjective nights are marked by light grey shading; dark periods by dark grey. (a) Direct transfer from 12/12 h diurnal cycles to continuous light. Plants were grown in 12 h photoperiod from germination onwards. Root extension rates of 19-day-old seedlings were monitored during a 12/12 h light cycle followed by 5 d of continuous illumination (n = 9). (b, c) Recovery of root elongation growth in continuous light after 4 d of darkness. Prior to darkness plants were entrained to photoperiods of 16 h [(b), n = 10, 18 d] or 12 h [(c), n = 11, 15 d] light from germination onwards. They were then darkened for 4 d, before transfer to continuous light. The plots exhibit the mean absolute elongation rate values and the error bars indicate the SE. To investigate the response after transfer to free-running continuous light (Fig. 3) seedlings were grown without exogenous sucrose in a 12/12 light/dark cycle, and transferred to continuous light at the start of the light period (Fig. 3a). During the last light dark cycle, root growth showed a transient maximum in the first 1 2 h of the light period, followed by a decline until 9 10 h into the light period, and a gradual recovery during the night (as already seen in Figs 1 & 2). After transfer to continuous light the slow oscillations persisted, with a maximum at the subjective dawn, and a minimum towards the end of the subjective day (Fig. 3a). There was a trend to the minima being shifted forwards slightly. These oscillations were superimposed on a gradual increase in the rate of root extension, which rose after 3 d of continuous light to a value that was almost double that seen in a 12/12 light/dark cycle. The transients after illumination or darkening were abolished in a freerunning cycle. In a second set of experiments, plants growing in the absence of exogenous sucrose were entrained to photoperiods of 16/8 (long day) or 12/12, and then dark-adapted for 4 d. Growth completely ceased under these conditions.they were then re-illuminated in continuous light (Fig. 3b, c). Root growth recovered slowly, with almost no growth in the first 24 h, and a gradual rise over the next 5 d. Oscillations were superimposed on this gradual recovery, with a maximum at, or just after, the anticipated dawn, and a minimum before the anticipated dusk (Fig. 3b, c). The sharp transients after illumination or after transfer to darkness were again absent. The results in Figs 1 3 point to several conclusions. Firstly, in our growth conditions, the overall rate of root growth is limited by C. The average rate per 24 h cycle can be increased by providing exogenous sucrose, by extending the photoperiod, or by transfer to continuous light. Secondly, the use of C is tightly regulated to maintain reserves until the end of the 24 h diel cycle and to avoid acute limitation of growth by C. Across a wide range of photoperiod treatments, root extension rates increase through to the end of the night. Further, plants retain enough reserves to support a further 1 2 h growth when the night is extended. Thirdly, root extension growth shows strong diel changes, which can be divided into two components: (1) a rapid and transient stimulation at the start of the light and inhibition at the start of the night period, which depend on the occurrence of a light dark transition; and (2) slower oscillations

6 882 N. Yazdanbakhsh et al. whose timing is largely independent of the length of the light period. These slow oscillations are maintained in continued light and, to a certain extent, in continuous darkness. Further experiments were performed using mutants to investigate the importance of diurnal C allocation and (see further discussion) the circadian clock for the regulation of diurnal root extension within each 24 h cycle. Root elongation kinetics in a starchless and a SEX mutant Wild-type plants accumulate starch in the light and degrade it at night to provide C to support metabolism and growth in the dark. Two mutants were investigated in which the supply of C at night was disturbed in different ways. The pgm mutant lacks plastidic PGM and is unable to synthesize starch (Caspar et al. 1985). As a result, this mutant becomes acutely C-limited 3 4 h into the night (Gibon et al. 2004a; Usadel et al. 2008). The sex1 mutant lacks a glucan water dikinase (GWD), which is required to phosphorylate starch, and is impaired in starch degradation. This mutant contains large amounts of starch, which is only broken down slowly during the night (Caspar et al. 1991; Yu et al. 2001). The mutants and the corresponding Col-0 wild type were grown in a 16 h/8 h light/dark cycle with no added sucrose (open symbols), or in the presence of 1% sucrose (filled symbols; Fig. 4). Col-0 wild-type plants showed similar diel changes of growth to those seen in Figs 1 3, with a gradual decline in the later part of the light period, and a gradual recovery during the night. The transient peak at the start of the light period was less marked than in other experiments, possibly due to the lower temporal resolution of the growth measurements in this experiment. The diel response was strongly modified in pgm and sex1. Both mutants showed a strong inhibition of growth during the night and a gradual recovery of growth during the light period. The lag until extension growth was re-established after illumination was longer for pgm (3 4 h) than sex1 (within 1 h). Like wildtype plants, both mutants showed a slight minimum during the light period at 8 10 h, followed by a weak but sustained increase during the rest of the light period. Inclusion of 1% sucrose in the medium led to a stimulation of root extension in wild-type plants (see also Fig. 2), pgm and sex1. Although addition of sucrose did not alter the diel changes of root growth in wild-type plants (see also Fig. 2), it led to a marked change in the mutants. In particular, the inhibition of extension growth during the night was completely reversed in sex1, and almost completely reversed in pgm. These results show that the inhibition of root extension growth in the night in these mutants is due to a lack of sugars. This is presumably the consequence of the absence of starch (pgm) or the slower rate of starch breakdown (sex1). Together, these results show that starch turnover provides a reserve of C to fuel growth in the night and, more generally, that correct allocation of C is important to maintain root extension growth through the entire 24 h cycle. Figure 4. Diurnal root elongation rates in Col-0, pgm and sex1 in presence and absence of external sucrose. Twelve-day-old seedlings of Col-0, pgm and sex1 growing in 16 h photoperiod were transferred to two Petri dishes filled with solid agar medium, one of which contained additionally 1% sucrose. Three days later, root elongation was monitored for 5 d and the 24 h averaged root elongation pattern of each genotype/sucrose supply was calculated (mathematical equations in Yazdanbakhsh & Fisahn 2010). Col-0 is represented by red circles (18 individuals, n = 97, 24 individuals, n = 121). Empty symbols represent seedlings in absence of sucrose and filled symbols represent seedlings in the presence of 1% sucrose, pgm is shown by blue triangles (20 individuals, n = 102, 16 individuals, n = 84) and sex1 by green squares (6 individuals, n = 36, 4 individuals, n = 20, respectively). Plots exhibit the mean hourly absolute elongation rate values and error bars indicate the SE. They also show that a shortfall of C in the night leads to an impairment of root growth, including an acute inhibition of growth in the night and a delay before growth resumes in the following light period. This lag is already visible after a few hours of C depletion (Fig. 4). It extends to one or more days when plants are exposed to C starvation for more than 24 h (Fig. 3). Root elongation kinetics in circadian clock mutants The robust rhythms in root growth kinetics in light/dark cycles (Fig. 1) and free running conditions (Figs 2 & 3) indicate that the circadian clock is involved in control of root growth. We therefore analysed the growth kinetics of three Arabidopsis mutants that are known to be affected in circadian clock function: the double mutant cca1/lhy (circadian clock associated/late elongated hypocotyl; Mizoguchi et al. 2002), elf3 (Covington et al. 2001) and elf4 (Doyle et al. 2002) (Figs 5 & 6). The mutants and the corresponding wild-type lines were grown in a 12/12 light/dark cycle, with zero (open symbols) or 1% (closed symbols) sucrose in the medium. The cca1/lhy double mutant was in a Ws-2 background. Ws-2 wild-type plants (Fig. 5) exhibited similar diurnal root extension growth rhythms to Col-0 wild type. As in Col-0,

7 Circadian control of root growth 883 night. This resembles the pattern in cca1/lhy. In contrast to cca1/lhy, including 1% sucrose in the medium did not relieve the inhibition of root growth at the end of the night in elf3 and elf4. This indicates that the decline of root extension growth rates during the night in elf3 and elf4 is not primarily due to a lack of carbohydrate at the end of the night. Diurnal changes of starch and sugars in shoots and roots of wild-type Ws-2 and Col-0 and cca1/lhy and elf3 Figure 5. Diurnal growth rates of Arabidopsis thaliana Ws-2 wild type and circadian clock double mutant cca1/lhy in presence and absence of external sucrose. Seventeen-day-old seedlings growing in 12 h photoperiod from germination were transferred to two Petri dishes filled with solid agar medium, one of which contains additional 1% sucrose. Three days after transfer to new plates, root elongation was monitored for 4 d. The 24 h averaged root elongation pattern of each genotype/sucrose condition was calculated (based on Yazdanbakhsh & Fisahn 2010). Empty symbols represent seedlings in absence of sucrose and filled symbols represent seedlings in the presence of 1% sucrose, Ws-2 is represented by red circles (10 individuals, n = 40, 6 individuals, n = 24) and cca1/lhy by green squares (6 individuals, n = 24, 5 individuals, n = 20, respectively), empty symbols represent seedlings in absence of sucrose and filled symbols represent seedlings in the presence of 1% sucrose. Plots exhibit the mean hourly absolute elongation rate values together with the SE. Graf et al. (2010) reported that cca1/lhy mutants show a modified starch turnover phenotype, with starch being exhausted by ZT20-22 (i.e. 2 4 h before dawn), corresponding to the time at which dawn is anticipated in cca1/lhy in a light/dark cycle. Their experiments were carried out with 23-day-old plantlets growing on soil. We investigated whether the decrease in root growth rate towards the end of the night in cca1/lhy seedlings might also be due to a premature exhaustion of starch. We carried out similar measurements in elf3 because this mutant showed a similarly strong inhibition of root extension during the night that, in contrast to cca1/lhy, was not relieved by adding exogenous sucrose (see earlier discussion). The mutants and the these diurnal changes were maintained in the presence of 1% sucrose, although the relative amplitude was smaller. The cca1/lhy double mutant showed a strongly modified diurnal rhythm (Fig. 5). Root growth rates were similar to those of wild-type Ws-2 in the later part of the light period, but remained steady rather than rising in the first part of the night, and declined almost twofold in the second part of the night. After illumination in the morning, the rate of root elongation was initially lower than in wild-type Ws-2, but rose to a similar rate after 2 3 h in the light. As a result, growth rates in cca1/lhy were at a minimum at the end of the night, rather than towards the end of the day as in wild-type plants. The inhibition of growth in cca1/lhy in the second part of the night was largely relieved when 1% sucrose was included in the medium. This indicates that low C limits root growth of cca1/lhy at the end of the night. The elf3 and elf4 mutants are in a Col-0 background. The diurnal response for wild-type Col-0 (Fig. 6) resembled that seen in Figs 1 3. As already seen (Fig. 2), inclusion of 1% sucrose in the growth medium led to a higher overall growth rate (here twofold higher) but did not alter the diurnal rhythm in wild-type Col-0. elf3 and elf4 showed increased rates of root extension in the second part of the light period, and lower rates of growth in the second part of the dark period (Fig. 6). These changes were especially marked for elf3, which showed a twofold higher growth rate in the light period, and a twofold inhibition during the night. As a result, extension growth rates in elf3 were highest at the end of the light period, and lowest at the end of the Figure 6. Diurnal growth rates of Arabidopsis thaliana Col-0 and circadian clock mutants elf3 and elf4 in presence and absence of external sucrose. Seventeen-day-old seedlings growing in 12 h photoperiod from germination were transferred to two Petri dishes filled with solid agar medium, one of which contains 1% sucrose. Three days after transfer to new plates, root elongation was monitored for 4 d. The 24 h averaged root elongation pattern of each genotype/sucrose condition was calculated (based on Yazdanbakhsh & Fisahn 2010). Empty symbols represent seedlings in absence of sucrose and filled symbols represent seedlings in the presence of 1% sucrose, Col-0 is represented by red circles (eight individuals, n = 32, nine individuals, n = 36), elf3 by cyan triangles (seven individuals, n = 28, nine individuals, n = 36) and elf4 by blue squares (10 individuals, n = 40, 7 individuals, n = 28, respectively). Plots exhibit the mean hourly absolute elongation rate values together with the SE.

8 884 N. Yazdanbakhsh et al. Figure 7. Diurnal turnover of starch (a) and sucrose (b) in Col-0, elf3, Ws-2, and cca1/lhy. Plants were grown in 12 h photoperiod for 17 d before harvest. The results show the mean and standard deviation of four biological replicates of each genotype. Samples harvested during the dark period are denoted by a grey background. corresponding wild-type lines were grown in a 12 h/12 h light/dark cycle in the absence of exogenous sucrose. Four replicate samples were harvested at dawn, after 2 and 12 h light, and after 10 and 12 h darkness (i.e. 22 and 24 h after dawn). On the day of harvest, the night was also extended for a further 4 h, to allow a further set of plants to be harvested 28 h after the previous dawn. The plants were separated into shoot and roots, and then analyzed for carbohydrates, amino acids, nitrate and protein content (Fig. 7, see Supporting Information Table S2 for a summary of all the measurements). In wild-type Col-0 and Ws-2, starch increased during the light period and decreased at night, with about 17 and 20% of the initial starch content remaining at 22 h, 8.7 and 11.6% at 24 h, and almost none (4.7 and 7.0%) after an extension of the night for another 4 h (Fig. 7a). Leaf sugars rose slightly (Col-0) or about two fold (Ws-2) during the light period and decreased during the night. Leaf sugars decreased by a further two- to threefold when the night was extended by 4 h. Root sugars rose twofold in wild-type Col-0 and Ws-2 in the light, and decreased during the night, with a further twofold decrease when the night was extended for 4 h. Starch levels in the root were negligible (<0.25 and <0.1 mmol hexose equivalents/g FW in Ws-2 and Col-0, respectively, Supporting Information Table S2). The absolute levels of starch and sugars in these young wildtype seedlings are lower than in older plants (see, e.g. Gibon et al. 2004a; Bläsing et al. 2005; Gibon et al. 2009; Graf et al. 2010). Nevertheless, the diurnal changes of carbohydrates resemble those reported for older plants, with a large ca. 10-fold increase of starch in the day and almost complete re-mobilization of starch during the night, smaller (twofold or less) changes of sugars during the light/dark cycle, and complete exhaustion of starch and a decrease of sugars when the night is extended. These diurnal changes of sugars are consistent with the observation that root extension growth is maintained until the end of the night, but is inhibited when the night is extended by 3 4 h or more. The diurnal changes of carbohydrates were modified in a different manner in the two clock mutants. The cca1/lhy mutant accumulated marginally (although not significantly) higher levels of starch at the end of the day than Ws-2 wild-type (Fig. 7a). During the night, starch was degraded more quickly, with only 5.4 and 4.4% remaining after 22 and 24 h, respectively (see following for more data: Fig. 11). This resembles the levels in wild-type plants after a 4 h extension of the night. Leaf sugars in cca1/lhy resembled those in Ws-2 wild type at the end of the light period, but were strongly depleted at 22 h and 24 h when they resembled those seen in wild-type plants after a 4 h extension of the night (Fig. 7b). The depletion of sugars in the last hours of the night in cca1/lhy included a large decrease of sucrose and a smaller decrease of fructose, whereas glucose was unaltered. Root sugars in cca1/lhy were also depleted in the last hours of the night, falling to levels similar to those seen after a 4 h extension of the night in wild-type plants (Supporting Information Table S2). These results extend the finding that cca1/lhy exhausts its starch prematurely (Graf et al. 2010) to young seedlings. Indeed, while in Graf et al. (2010), the double mutant accumulated marginally less starch than Ws-2 in the light, in the present study cca1/lhy accumulated the same amount or marginally more starch than wild-type Ws-2. As a result, cca1/lhy shows not only changes in the timing of starch breakdown but also a higher absolute rate of starch breakdown than wild-type Ws-2. Further, we now demonstrate that the premature exhaustion of starch in cca1/lhy is accompanied by premature depletion of sugars in the shoot and in the root. This is consistent with the notion that an inappropriate timing of starch degradation is contributing to the decline in root growth at the end of the night in cca1/lhy. In contrast, elf3 exhibited higher levels of starch than the Col-0 wild type at all times of the diurnal cycle (Fig. 7a). The relative difference was especially large (>twofold) at the end of the night. Leaf sugar levels were increased in elf3 compared with Col-0 wild type at most times in the diurnal cycle, with an especially large increase at the end of the night (mainly due to glucose and fructose, see Supporting Information Table S2) and the beginning of the day (due to higher levels of sucrose, glucose and fructose, see Supporting Information Table S2).

9 Circadian control of root growth 885 We also investigated the diurnal changes of total amino acids in Ws-2 wildtype, cca1/lhy, Col-0 wild type and elf3 (Supporting Information Table S2). As previously seen (Bläsing et al. 2005; Gibon et al. 2006), total amino acids increased slightly in the light and decreased slighly in the night. Total amino acids were marginally lower in cca1/lhy than in Ws-2 wild type throughout the entire diurnal cycle. They were marginally higher in elf3 than Col-0 wild type at most times in the diurnal cycle. However, there was no evidence that either of these mutations led to a major depletion of amino acids. Root elongation kinetics of clock mutants in free running conditions To further investigate the effect of mutations in CCA1/ LHY, ELF3 and ELF4 on root elongation, we investigated the response after transfer from a 12/12 light dark regime to continuous illumination (Fig. 8). The diurnal growth kinetics of the wild-types Col-0, Ws-2 and the three clock mutants in the entraining light/dark cycle in Fig. 8 resembles that already shown in Figs 5 and 6; wild-type Col-0 and Ws-2 showed a rise in growth throughout the night, cca1/lhy rose to a peak in the middle of the night, followed by a decline to a minimum at the end of the night, elf4 showed a rise in the first part of the night and slight decline towards the end of the night, while elf 3 showed a maximum at the end of the light period and a decline throughout the entire night. Transfer of Col-0 (see also Fig. 3a) or Ws-2 wild type to continuous light results in a gradual doubling of the absolute extension rate, with superimposed strong oscillations that have a maximum at the subjective dawn and a minimum towards the end of the subjective day. The mutants showed contrasting responses in continuous light (Fig. 8). The cca1/ lhy double mutant displayed arrythmic root elongation kinetics during the first 48 h of continuous illumination. After 48 h continuous illumination cca1/lhy recovered a weak oscillatory growth phenotype, with a maximum at or just after the subjective dawn and a minimum towards the end of the subjective light period. However, individuals lost synchrony during this prolonged light treatment, as is revealed by the large error bars. Averaged growth rates in cca1/lhy were similar to wild type in the entraining light/dark treatment, but lower than wild-type plants in continuous light. In elf3, the depression of root extension during the subjective light period was abolished.the slow oscillations in continuous light were almost completely absent, and growth rates increased progressively for 36 h to a new and fairly stable plateau, which was about 60% higher than the average growth rate in wild-type Col-0 in continuous light. These results indicate that a clock-mediated restriction of growth in the light is abolished in elf3. Inelf4, the timing of the maximum moved forward slightly to the middle of the subjective night. The responses of root elongation in freerunning cycles have similarities to published changes in other circadian rhythm phenotypes in these three clock mutants (see Discussion). These results indicated that the clock regulates root growth in two different ways. One mechanism involves ELF3, is independent of starch turnover and restricts root extension in the subjective light period and promotes it in the subjective night. The other involves CCA1/LHY and impacts on root growth by regulating the turnover of starch and the supply of sugars for growth in the night. Figure 8. Root growth rates of Col-0, Ws-2, elf3, elf4 and cca1/lhy in free running conditions of continuous illumination. Seedlings were grown in 12 h photoperiod from germination. Root elongation of 18-day-old seedlings was monitored during the last cycle of a 12 h photoperiod and the following 5 d of continuous illumination. The plots show the 2 h averaged absolute elongation rate of each genotype (Col-0: red circles, n = 9, Ws-2: red downward triangles, n = 8, elf3: cyan triangles, n = 9, elf4: blue squares, n = 7, cca1/lhy: green diamonds, n = 5). Shaded areas represent subjective nights. The x-axis indicates time in continuous light. Error bars indicate the SE. Oscillations in root elongation kinetics of starch mutants in free running conditions To provide evidence that the slow oscillator is itself independent of starch turnover, we investigated root growth kinetics of pgm and sex1 in free running conditions in continuous illumination (Fig. 9). Prior to continuous light exposure, seedlings were forced to cease growth completely by treatment in continuous darkness for 4 d. As already seen, there was a delay of about 36 h until growth resumed, and growth rates continued to rise until at least 120 h after re-illumination. Once growth resumed, wildtype Col-0, pgm and sex1 exhibited synchronized oscillations in root elongation rate, with maximal extension growth 2 4 h after the anticipated dawn and minima 8 10 h later. Thus, continuous light induces wild-type-like oscillations of root extension rates in starchless and starch degradation mutants. Adjustment of root growth, starch turnover and sugar levels to a sudden early dusk Finally, we investigated the response to a sudden imposition of an early dusk. Graf et al. (2010) showed that

10 886 N. Yazdanbakhsh et al. Figure 9. Root growth recovery in Col-0, pgm and sex1 seedlings in continuous light after 4 d of continuous darkness. Col-0 (n = 9), pgm (n = 4) and sex1 (n = 5) seedlings were grown in a 12 h photoperiod from seed stage. Prior to growth measurement, seedlings were kept in continuous dark for 4 d, which resulted in complete cessation of root elongation. Subsequently, the 18-day-old seedlings were exposed to continuous illumination and root elongation was monitored for 128 h. Light grey bars denote subjective nights. Dark grey indicates the 4 d prolonged night. Each trace represents the 2 h mean absolute elongation rates together with the SE. resolve in the first 2 3 h after darkening, because darkening anyway leads to a transient decrease in the rate of root extension (see earlier discussion), and because the transition after 8 h illumination is close to the inflection point in the slow oscillation in extension rates. However, a clear additional inhibition of extension growth can be seen by 3 4 h after the premature dusk. Whereas the rate of root growth rises strongly during the night in a 12/12 cycle, it only increases slightly in the early dusk treatment. In the last 3 4 h of the night, growth declines in Col-0 but remains stable in Ws-2. Compared with the rate at the end of a normal night, the rate of root extension at the end of the first extended night is decreased >threefold in Col-0 and approximately twofold in Ws-2. sudden premature darkening of 23-day-old Arabidopsis plants leads to an immediate decrease in the rate of starch breakdown, compared with the rate in plants that were darkened at the end of the light period. As a result, starch reserves last until the end of the night. They argued that the clock acts as a timer, and that this information is used by an unknown mechanism to adjust the rate of starch breakdown to the anticipated length of the night. However, a decreased rate of starch breakdown, on its own, would not allow a plant to avoid a period of C starvation at the end of the night. This will also require a concomitant decrease in the rate of C consumption (see Introduction). Our experimental set-up allowed us to investigate the kinetics of root extension growth after subjecting plants to a sudden early dusk and directly test if the decrease in the rate of starch breakdown in the first night after a premature dusk is accompanied by an immediate decrease in the rate of root growth. Two separate experiments were performed: one with Col-0 wild type (Fig. 10a) and one with Ws-2 wild type and the cca1/lhy double mutant (Fig. 10b). In both experiments, plants were grown in a 12 h/12 h light/dark cycle for 13 d, and then subjected to an early night after 8 h in the light. These plants experienced a 16 h (instead of a 12 h) dark period, using reserves that had been built up in 8 h (rather than 12 h) of light. The early dusk was retained for the following 2 d. Root extension growth was monitored, starting a full day before the early dusk was imposed, and continuing for 2 d after the transition. Root extension growth kinetics of Col-0 and Ws-2 in a 12/12 light/dark cycle resembled those in previous experiments (Fig. 10a & b). In both wild types, an early unanticipated dusk led to an almost immediate decrease in the rate of root extension, compared with roots in a 12 h photoperiod (Fig. 10a & b). The precise kinetics are difficult to Figure 10. Adjustment of root growth rates to an unexpected pre-dusk. (a) Root elongation of Col-0 seedlings growing in a 12 h photoperiod was monitored for 1 d in a 12 h photoperiod followed by 3 d of 8 h illumination. Transition to 8 h cycles was performed on 15-day-old seedlings (n = 11). (b) Kinetics of root elongation in Ws-2 wild type and cca1/lhy double mutants during 1 day of a 12 h photoperiod followed by 3 d of 8 h illumination (n = 5, n = 6, respectively). Seedlings were grown in 12 h cycles for 16 d prior to the experiment. Dark grey shadings denote regular night time; light grey indicates darkness, which is applied earlier. The results exhibit the mean hourly averaged absolute elongation rates of each genotype. Error bars indicate the SE.

11 Circadian control of root growth 887 Figure 11. Diurnal turnover of starch (a) and sucrose (b) in wild type (Ws-2) and cca1/lhy double mutants in a 12 h light cycle and a 4 h advanced transition to dark. Pre-dusk is indicated by a dark grey bar; the light grey bar denotes the regular 12 h night. The result shows the average of four biological replicates and standard deviation. Samples taken in a 12 h light/dark cycle are marked in green while the premature night samples are in red. (a) Starch content in shoots of Ws-2 (empty squares) or cca1/lhy (filled squares) in a regular 12 h cycle (green) and the first night of transition to 8 h photoperiod (red). (b) Diurnal changes of sucrose levels in shoots of Ws-2 (empty squares) and cca1/lhy (filled squares) seedlings growing in a regular 12 h cycle (green) and the first night of transition to an 8 h photoperiod (red). On the day after the premature dusk, the normal diurnal pattern was re-established, with a peak 2 3 h into the light period, a decline later in the light, and a gradual recovery to a maximum at the end of the night. This pattern was retained on the second day after the transfer. The absolute rates are about twofold lower than in 12/12 light dark cycles; this resembles the growth responses in plants that were grown from germination in a 12/12 or a 8/16 light/dark cycle (Fig. 1; Supporting Information Table S1). The cca1/lhy doublemutant showed a different response to a premature dusk (Fig. 10b). The transient inhibition after darkening is followed by an increase in the rate of growth, similar to that seen in a normal night. Growth is then strongly inhibited in the last hours of the night. Further, cca1/lhy does not adjust to the long night, but instead continues to show a strong inhibition of growth in the last part of the night for at least the next two nights. Ws-2 wild-type and cca1/lhy plants were harvested to measure shoot carbohydrate levels during a 12/12 light dark cycle, and on the first night after the premature dusk (Fig. 11, see Supporting Information Table S3 for the original data). Starch was degraded in a near-linear manner in wild-type Ws-2 (Fig. 11a, see also Fig. 7a). Premature darkening led to a halving of the rate of starch degradation, with the result that starch reserves lasted through the lengthened night (Fig. 11a). There was also an immediate decrease in the levels of sucrose (Fig. 11b) and glucose (Supporting Information Table S3). In cca1/lhy, starch accumulated slightly more rapidly in the light and was degraded more rapidly at night in a 12/12 light/dark cycle (Fig. 11a, see also Fig. 7a). When cca1/lhy was exposed to an early night, the rate of starch degradation remained similar to that in a 12/12 cycle, and was >twofold higher than in wild-type Ws-2 after a premature dusk. As a result, after a premature dusk, cca1/lhy depleted its starch several hours before the end of the night. Furthermore, sucrose levels in cca1/lhy were higher than in wild-type Ws-2 in the first part of the night, and lower in the last part of the night (Fig. 11b). These results show that in wild-type Arabidopsis the rate of starch degradation adjusts immediately to an early dusk, resulting in a lower rate of starch degradation and conservation of starch reserves until the end of the night (see also Graf et al. 2010). Crucially, the decrease in the rate of starch degradation is accompanied by a rapid decrease in the rate of root extension growth. Further, cca1/lhy is unable to adjust the rates of starch degradation or root growth to this sudden challenge. DISCUSSION Arabidopsis exhibits a strong diel rhythm for root extension growth Arabidopsis exhibits a diel rhythm in primary root extension growth in a range of different photoperiods, and in the absence and presence of external sucrose. This extends previous reports of diel rhythms in Arabidopsis root elongation (Yazdanbakhsh & Fisahn 2009, 2010). There appear to be at least two components: (1) a gradual oscillation in which growth decreases in the first part of the 24 h cycle and then recovers; and (2) transient changes after illumination and darkening. These components were observed repeatedly in independent experiments. The gradual inhibition of root elongation during the major part of the light period, and gradual recovery of extension growth during the remainder of the 24 h diel period was seen in a range of photoperiods (Figs 1, 2, 3a, 4 6, 8 & 10). This gradual oscillation still occurs in the presence of exogenous sugar (Figs 2 & 4 6) and is retained in free running conditions (Figs 2, 3b c, 8 & 9), indicating that it is not due to changes in the C supply but is, rather, driven by the clock (see below for further discussion). The sharp transient stimulation of growth 1 2 h after illumination and the transient inhibition of growth 1 2 h after darkening were also seen in all photoperiods (Figs 1, 2,