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First published online July 9, 2008; 10.1104/pp.108.123901 Plant Physiology 148:293-303 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
SENSITIVE TO FREEZING6 Integrates Cellular and Environmental Inputs to the Plant Circadian Clock1,[W],[OA]School of Biological and Biomedical Sciences, Durham University, Durham DH1 3LE, United Kingdom (H.K.); Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh EH9 3JH, United Kingdom (A.J.W.T); and Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom (H.G.M.)
The sensitive to freezing6 (sfr6) mutant of Arabidopsis (Arabidopsis thaliana) is late flowering in long days due to reduced expression of components in the photoperiodic flowering pathway in long-day photoperiods. Microarray analysis of gene expression showed that a circadian clock-associated motif, the evening element, was overrepresented in promoters of genes down-regulated in sfr6 plants. Analysis of leaf movement rhythms found sfr6 plants showed a sucrose (Suc)-dependent long period phenotype; unlike wild-type Arabidopsis, the clock in sfr6 plants did not have a shorter rhythm in the presence of Suc. Other developmental responses to Suc were unaltered in sfr6 plants, suggesting insensitivity to Suc is restricted to the clock. We investigated the effect of sfr6 and Suc upon clock gene expression over 24 h. The sfr6 mutation resulted in reduced expression of the clock components CIRCADIAN CLOCK ASSOCIATED1, GIGANTEA, and TIMING OF CAB1. These changes occurred independently of Suc supplementation. Wild-type plants showed small increases in clock gene expression in the presence of Suc; this response to Suc was reduced in sfr6 plants. This study shows that large changes in level and timing of clock gene expression may have little effect upon clock outputs. Moreover, although Suc influences the period and accuracy of the Arabidopsis clock, it results in relatively minor changes in clock gene expression.
Plants experience many fluctuations in their environment, but the daily change between dark and light conditions is both predictable and extremely significant in terms of the direct effect it has on photosynthesis. Green plants need to tune their metabolism and biochemistry to take account of times when photosynthesis is or is not possible. Many of the changes that occur throughout the diurnal cycle are controlled at the transcript level, such that 30% to 50% of genes in the model plant Arabidopsis (Arabidopsis thaliana) are rhythmically expressed in light:dark conditions (Blasing et al., 2005  ). A core of about 6% of genes continues to be rhythmic under constant light conditions (Harmer et al., 2000), indicating that these genes are under the direct control of the circadian clock. The clock controls the timing of key areas of plant metabolism, including the synthesis and degradation of starch and other carbohydrates (Harmer et al., 2000), allowing plants to optimize their metabolism to suit daily changes in the environment. The Arabidopsis clock is made up of a series of interlocked transcription-translation feedback loops (Locke et al., 2005, 2006; Zeilinger et al., 2006) centered on the highly homologous MYB transcription factors CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY; Schaffer et al., 1998; Wang and Tobin, 1998; Mizoguchi et al., 2002). These proteins, together with their transcripts, have peak expression around dawn. They drive the expression of other clock components such as TIMING OF CAB1 (TOC1), whose transcript is maximally expressed in the evening (Strayer et al., 2000; Alabadi et al., 2001), and its homologues, the PSEUDORESPONSE REGULATOR family, which are expressed in sequential waves throughout the day (Matsushika et al., 2000; Makino et al., 2002). CCA1 and LHY repress TOC1 transcription (Alabadi et al., 2001; Perales and Mas, 2007), whereas the level of TOC1 is regulated by ZEITLUPE (ZTL; Somers et al., 2000). ZTL targets TOC1 protein for degradation via the proteasome, a process that is dark dependent (Mas et al., 2003b).
The clock receives inputs from the external environment from several types of photoreceptors, including phytochromes and cryptochromes (for review, see Millar, 2003
The clock has a large influence on flowering time via the photoperiodic flowering pathway (Imaizumi and Kay, 2006
While much is known about the plant clock's responses to light and temperature (Gould et al., 2006
During the course of routine plant growth, we observed that the Arabidopsis sensitive to freezing6 (sfr6) mutant (Knight et al., 1999
sfr6 Is Late Flowering We observed that sfr6 plants flowered significantly later than wild type in long days (LD 16:8), having on average (under our conditions) 24 rosette leaves at the time of flowering compared with only 15 in wild-type plants (P < 0.0001, one-tailed t test; Fig. 1A ). A significant difference in the age of plants at the time of flowering was also recorded (Supplemental Fig. S1; data shown in Fig. 1A and Supplemental Fig. S1 represent 22 plants per genotype).
To examine the cause of late flowering in sfr6, we measured expression of genes known to be involved in the control of flowering in Arabidopsis (Fig. 1, B and C). When we examined CO expression in seedlings in LD 16:8 photoperiods, we found that expression of CO was reduced during the late day and night in sfr6 (Fig. 1B). Moreover, expression of FT was severely reduced in sfr6 seedlings in long days (Fig. 1C). As CO is involved in photoperiodic time measurement and FT levels determine the timing of the floral transition, reduction in expression of these genes explains the late flowering of sfr6 plants.
We also examined expression of genes that act upstream of CO in the photoperiodic pathway regulating flowering time. The gi mutant resembles sfr6, as it is also late flowering in long days (Fowler et al., 1999
Clock-Controlled Genes Are Down-Regulated in sfr6
Having observed an effect on the pathway leading to control of flowering time, we sought to discover whether the circadian clock also was affected in sfr6. Microarray analysis was used to compare global gene expression in wild-type and sfr6 seedlings grown in LD 16:8 photoperiods during the light period. When we analyzed data from a full genome chip, we found 209 genes were least 2x down-regulated in sfr6 (Supplemental Table S1). Analysis of 500 bp of upstream sequence from these genes by the Regulatory Sequence Analysis Tools (van Helden, 2003
The overrepresentation of the EE among genes down-regulated in sfr6 coupled with the knowledge that many flowering time mutants also have defects in their circadian clocks led us to investigate whether sfr6 plants had altered circadian function. We tested this possibility by examining leaf movement rhythms in sfr6 and wild-type seedlings. As the leaf movement rhythm is controlled by the circadian clock, determination of its frequency in constant light provides a measure of the endogenous, free-running period of the plant. Leaf movement experiments are commonly performed using plants grown with supplemental Suc, but, as our flowering time and microarray experiments had been conducted on plants grown without Suc, we wanted to observe clock behavior in plants growing under similar conditions. We therefore measured leaf movement rhythms in wild-type and sfr6 plants grown without supplementary Suc. There was no difference in period length between wild-type and sfr6 seedlings under these conditions (ecotype Columbia [Col0] period = 25.12 h versus sfr6 period = 25.15 h; one-tailed t test, t = –0.06, not significant [ns]; Fig. 3A ; breakdown of both experiments is in Table I ).
To allow comparison with the majority of previously published leaf movement data, we repeated the leaf movement recordings using plants grown on media supplemented with 3% (w/v) Suc. Analysis of rhythms showed that under these conditions, sfr6 plants had a significantly longer period than wild type (Col0 period = 24.44 h versus sfr6 period = 25.24 h; one-tailed t test, t = –4.28, P < 0.001; Fig. 3D; breakdown of all experiments is in Table I). This shows that the sfr6 mutation caused a conditional circadian phenotype that resulted in a leaf movement rhythm longer than wild type but only in the presence of Suc. It appears that this difference occurs because the free-running period of the wild-type Arabidopsis clock is sensitive to Suc, but sfr6 is unaffected by the presence of Suc (Fig. 3, A and C); analysis of the leaf movement data found wild-type plants had shorter clock periods when they were grown with Suc (mean period for all Col0 seedlings on 1x Murashige and Skoog [MS] 25.1 h versus mean period on 1x MS with 3% Suc 24.4 h; t = –2.43, P < 0.01, one-tailed t test), but sfr6 plants did not (mean period for all sfr6 seedlings on 1x MS 25.1 h versus mean period on 1x MS with 3% Suc 25.2 h; t = 0.24, ns, one-tailed t test). We noticed that each genotype returned a very broad range of period values when grown without Suc (Fig. 3, A and B), but values were much more tightly clustered around the mean when plants were grown with Suc (Fig. 3, C and D). As both genotypes showed similar behavior in this respect, this is unlikely to result from the sfr6 mutation. The light intensity, photoperiod, and temperature were all equivalent in both sets of experiments; hence, this result may indicate that Suc acts to facilitate entrainment to the light:dark cycle.
To determine the robustness of leaf movement rhythms, we determined the relative amplitude error (RAE) for each genotype grown with and without Suc (Johnson and Frasier, 1985
We did not observe an abnormal appearance of seedlings grown with supplementary Suc (Fig. 4, A–D ). However, this does not rule out the possibility that the effect of Suc on clock function was due to osmotic stress imposed by Suc supplementation. Therefore, as a chemical control for the higher osmotic strength of MS medium containing Suc, we compared growth of wild-type and sfr6 seedlings on plates containing 1x MS medium supplemented by 3% Suc or mannitol at isoosmolar concentrations (concentrations of equivalent osmotic strength). Both genotypes grown on mannitol were stunted and appeared yellow when compared with plants grown on Suc (Fig. 4, E and F; Supplemental Fig. S2).
This could have indicated either that mannitol exerted a negative effect on Arabidopsis seedlings that could not be attributed to its osmotic strength alone or that Suc does not in fact impose the level of osmotic stress on Arabidopsis that is caused by an isoosmolar concentration of mannitol. To distinguish between these possibilities and to test whether this was an effect specific to mannitol, plants were grown on 1x MS medium containing 3-O-methyl Glc (3-OMG); 3-OMG is a Glc analog that is not recognized by plants as a metabolizable sugar (Cortes et al., 2003 ). Like Suc, 3-OMG can be taken up by plant cells. Plants grown on a concentration of 3-OMG equivalent to the osmotic strength of 3% Suc were also stunted, similar to those grown on mannitol (Supplemental Fig. S2). Finally, we calculated the osmolarity of MS medium and grew plants on 2.9x MS medium, which has an osmolarity equivalent to that of 1x MS plus 3% Suc. Again, we observed stunted plants, though less severely stunted than those grown on 3-OMG or mannitol (Supplemental Fig. S2). These observations led to the conclusion that the Suc in our circadian experiments was not likely to impose a major osmotic stress. Suc-grown plants appeared healthy and unlike plants grown on two alternative sugars at equivalent osmolarity or on a medium with a similar osmolarity due to an increased salt concentration. Thus, we concluded that there was no appropriate chemical osmotic control for Suc in such experiments; therefore, MS medium minus Suc was the most appropriate control treatment in these and subsequent experiments. Plants grown on these three alternative media were all developmentally and physiologically quite different to those grown on Suc or non-Suc medium and therefore would not have been appropriate control plants for our experiments. The lack of osmotic stress imposed by Suc is likely to be due to the very fact it is metabolized and therefore does not remain in the media at the concentration initially added to the plates.
We sought to determine if the lack of effect of Suc on the clock was due to insensitivity to Suc of sfr6 plants. Suc is known to delay germination in wild-type plants, and at high concentrations, Glc has been shown to block it, but these effects are reduced or absent in sugar-insensitive mutants such as hexokinase mutants (Jang et al., 1997
A delay in germination in response to Suc is observed in wild-type seeds (Dekkers et al., 2004
We measured hypocotyl elongation as a further test of sensitivity to Suc, which has been shown to inhibit hypocotyl growth (Jang et al., 1997
SFR6 and Suc Both Modulate Circadian Gene Expression To investigate how the sfr6 mutation was interacting with Suc to cause the effects on the clock, we used real-time quantitative PCR to measure level and timing of transcript accumulation for the clock components CCA1, TOC1, and GI. Expression of these genes was measured in seedlings of both genotypes grown with or without supplementary Suc (Fig. 5 ). This experiment was designed to replicate the free-running conditions of the leaf movement experiments. For this reason, seedlings were entrained in LD 12:12 photoperiods and then allowed to free-run in LL for 24 h before sampling began. Samples were collected at 3-h intervals during the second day in LL (subjective day, 24–36 h; subjective night, 36–48 h) to avoid after-effects of entrainment to the light:dark cycle.
CCA1 We examined expression of the central clock component CCA1 in wild-type and sfr6 plants to observe the effects of the mutation and Suc supplementation on expression of a morning clock component. In both genotypes, CCA1 expression was rhythmic with peak levels occurring, as expected, in the morning shortly after subjective dawn; however, the amplitude of the CCA1 rhythm was reduced in sfr6 seedlings. The expression maximum was much lower than in wild-type regardless of whether Suc supplementation was provided (Fig. 5, A and B). Thus, the sfr6 mutation leads to a reduction in peak CCA1 expression. Our results indicate Suc caused small but significant changes in CCA1 expression in wild type. In samples collected at and before subjective dawn, CCA1 expression in wild-type plants was higher when plants were grown with Suc (compare expression in Col0 at 24 and 48 h; Fig. 5, A and B). Expression of CCA1 in sfr6 seedlings did not change in the presence of Suc; CCA1 expression in sfr6 plants was not increased by Suc at dawn or any other time. Hence, SFR6 is needed for the Suc enhancement of the increase in CCA1 levels seen at dawn. Examination of CCA1 expression in sfr6 in LD 16:8 photoperiods showed that it remained rhythmic, but, as in free-running conditions, the amplitude of the rhythm was reduced relative to that in wild-type plants (Supplemental Fig. S3a).
We measured expression of TOC1, a clock component expressed during the evening and early night, to determine if the evening arm of the clock was affected by the sfr6 mutation. As CCA1 negatively regulates TOC1, changes in CCA1 expression should be reflected in TOC1 profiles. The main peak of TOC1 expression in wild-type seedlings occurred in the late subjective day at time 33 h (Fig. 5, C and D). Both the peak of TOC1 expression and the amplitude of the TOC1 rhythm were reduced in sfr6 seedlings; in addition, the time of peak expression was delayed in sfr6 relative to wild type (Fig. 5, C and D). Consistent with this result, TOC1 expression in sfr6 was reduced in amplitude and rhythmicity in long-day photoperiods (Supplemental Fig. S3b). Suc supplementation increased the amplitude of rhythmicity of TOC1 expression in free-running wild-type plants (Fig. 5, C and D). It also produced an overall increase in TOC1 expression; total TOC1 expression across all time points in Col0 grown with Suc was 133% of that found in plants grown on nonsupplemented media. In contrast, although Suc strengthened the amplitude of TOC1 rhythm in sfr6, it barely affected the level of expression in sfr6; TOC1 expression of Suc-grown sfr6 plants was 103% of the expression seen in plants grown without Suc (Fig. 5, C and D). Therefore, the increase in TOC1 expression in response to Suc supplementation may require SFR6. Expression of both CCA1 and TOC1 was also low relative to wild type in sfr6 plants maintained in long-day photoperiods (Supplemental Fig. S3), showing that the reduced expression of clock components is an integral part of the sfr6 phenotype rather than being caused by a rapid damping of rhythmicity in the mutant following release from an entraining photoperiod.
It has been demonstrated previously that TOC1 is activated by GI (TOC1 expression is reduced in the gi;cca1;lhy triple mutant relative to the cca1;lhy mutant; Locke et al., 2006
Unlike its effects on CCA1 and TOC1 transcript levels, the direction of effect of Suc on GI expression varied with genotype; the height of the GI peak in LL was increased when Suc was present in the growth media in wild type but reduced in sfr6 (compare expression in Fig. 5, E and F). This implies that GI is directly sensitive to Suc and this response requires SFR6. In contrast to this finding, a recent study has suggested that GI expression does not respond to Suc (Usadel et al., 2008
SFR6 Regulates the Photoperiodic Pathway and Clock Gene Expression We have shown that, like other late flowering mutants, sfr6 shows reduced expression of genes active in the photoperiodic pathway. The reduction and or delay in expression of GI, FKF1, CO, ZTL, and FT support the conclusion that sfr6 plants have reduced sensitivity to daylength, because SFR6 is required for activation of the photoperiodic flowering pathway. A notable aspect of the sfr6 phenotype is the simultaneous reduction in expression of morning and evening clock components. Taken together, our results revealed an interesting contradiction. Analysis of clock component expression showed that, consistent with its effects upon the photoperiodic pathway, sfr6 caused an overall decrease in level of clock gene expression in free-running plants, regardless of Suc supplementation. However, sfr6 exhibited a clock phenotype differing from wild type only when plants were grown with Suc. Therefore, the large changes in clock gene expression apparent in sfr6 were not in themselves sufficient to alter clock behavior; when grown without Suc, sfr6 and wild-type plants had equivalent free-running periods of leaf movement despite major differences in clock gene expression between genotypes. This is an important result, as the sfr6 mutation appears to separate expression of clock components from clock function.
Clock gene expression was less responsive to Suc in sfr6 than in wild type; however, this reduced response appears to be restricted to clock pathways only. sfr6 showed normal wild-type behavior on Suc in terms of inhibition of hypocotyl elongation, germination timing, and success, suggesting that sfr6 is not globally insensitive to Suc but that Suc-responsive pathways leading to clock function specifically are regulated by SFR6. The changes in clock gene expression caused by Suc in wild type may be responsible for the shortened period observed; however, given that the larger differences in sfr6 and wild-type clock transcript levels measured cause no change in clock function, alternative explanations remain a possibility. Although Suc does cause moderate changes in clock gene transcript levels, it is likely that it exerts its major influence on circadian function downstream (or independently) of clock gene transcript regulation.
This study reveals the sensitivity of the Arabidopsis circadian clock to Suc. The significance of Suc in regulating the period of the circadian clock has not been recognized until now, with experiments routinely performed on plants grown on media containing 3% Suc (Harmer et al., 2000
As the three transcripts CCA1, GI, and TOC1 peak at different times relative to the environmental cycle, the similar effects of the sfr6 mutation upon them suggest that SFR6 acts upstream of the clock. In sfr6, transcript levels of the evening genes GI and TOC1 are reduced and their peaks delayed, suggesting that SFR6 acts at night. Our results imply that the effects of SFR6 and Suc may not be mediated solely via the CCA1-TOC1-GI feedback loops, and that, as might be expected, additional factors contribute to integrate the clock with other signals.
According to the current model of the Arabidopsis clock (Locke et al., 2005
sfr6 was originally isolated on the basis of its inability to cold-acclimate to tolerate freezing temperatures (Warren et al., 1996
More recently, it has become apparent that there is a relationship between flowering time and the pathways leading to cold acclimation (Kim et al., 2004
As already noted, the late flowering and low CO and FT aspects of the sfr6 phenotype resemble plants with altered GI and ZTL expression (Fowler et al., 1999 Our study shows that in addition to its role in cold gene expression and acclimation, SFR6 plays an important part in regulation of the circadian clock and in the control of flowering time. We have also demonstrated a role for sugars in clock regulation and we suggest that SFR6 may serve to integrate information such as temperature and sugar status to elicit a suitable response. Cloning and analysis of the SFR6 gene may give insights into the mechanism by which this is achieved.
Plant Materials, Growth Conditions, and Chemicals All experiments were carried out using the homozygous sfr6 ethylmethanesulfonate mutant of the Arabidopsis (Arabidopsis thaliana) Col0 accession; Col0 plants were used as a wild-type control in all experiments. Except for flowering experiments, seedlings were grown on plates containing 1x MS salts with 0.8% to 1% agar and, where indicated, supplemented with 3% (w/v) Suc or isoosmolar concentrations of mannitol (96 mM) or 3-OMG (87 mM). In all cases, media pH was corrected to pH 5.8. Unless otherwise stated, seeds were surface-sterilized before being stratified in the dark at 4°C for 48 h prior to transfer to the growth chamber. Plants were grown in Sanyo MLR-350 growth chambers at a constant temperature (20°C). The light level during photoperiods or constant light was 50 µmol m–2 s–1. All chemicals were purchased from Sigma except for the agar used in the germination and hypocotyl experiments purchased from Duchefa Biochemie (m1002.0500). To determine the effects of mannitol, 3-OMG, or 2.9x MS on growth, plants were grown for 14 d on 1x MS-agar plates with appropriate supplementation for 14 d in LD 12:12 at 20°C.
The microarray experiment was performed on plants grown at a constant temperature of 20°C in an LD 16:8 photoperiod and has been described elsewhere (Garnet Affymetrix array http://affy.arabidopsis.info/narrays/experimentpage.pl?experimentid=194). A total of 500 bp of promoter sequence was analyzed for each gene that showed at least 2-fold higher expression in wild type than in sfr6. Only genes giving a "present" call in both sfr6 and wild type were included in this analysis. Promoter sequences were analyzed using the regulatory sequence analysis tools pattern matching function (van Helden, 2003
Flowering time experiments were conducted in growth chambers in LD 16:8 photoperiods and 20°C constant temperature. Two-week-old seedlings were transferred to sterilized soil following surface sterilization of seeds, stratification, and germination of seedlings on 1x MS plates without Suc. Plants were checked daily until the floral meristem was clearly visible. At this point, plants were dissected to count the total number of rosette leaves.
Col0 and sfr6 seedlings were grown on MS-agar plates, as described above, with and without Suc, in LD 12:12 photoperiods for 12 d postgermination (clock gene expression experiments). At dawn on day 13, seedlings were transferred to LL. Samples of 20 to 30 seedlings were collected every 3 h and snap-frozen in liquid nitrogen; the first sample was collected at subjective dawn on day 14, after 24 h in LL. Similarly, for analysis of flowering time gene expression, seedlings were grown on plates as described above in LD 16:8 photoperiods for 13 d and sampled every 2 h on day 14 in a free-running cycle. RNA was extracted (RNeasy kit, Qiagen [74904] with additional DNAse digestion) from each sample and cDNA synthesized (Taq-Man, Applied Biosystems [N808-0234] reverse transcriptase kit) for each time point. Real-time reverse transcription (RT)-PCR was carried out in triplicate with SYBR Green PCR Master mix (Applied Biosystems [4309155]) using an Applied Biosystems Prism-7300. Levels of specific mRNA and βTUBULIN4 (βTUB4) controls were calculated by the standard curve method (Applied Biosystems user bulletin 2); the relative expression (arbitrary units) of each gene of interest was obtained by dividing by contemporaneous βTUB4 expression. No-RT and no-template controls were included as negative controls for each set of reactions. Two or three independent biological repeats gave similar results. Gene-specific primers were as follows: CCA1, forward 5'-TCTGTGTCTGACGAG GGTCGAATT-3', reverse 5'-ACTTTGCGGCAATACCTCTCTGG-3'; CO, forward 5'-TGGCTCCTCAGGGACTCACTACAA-3', reverse 5'-TTGACTCCGGCACAACACCAGT-3'; FKF1, forward 5'-TCTTGGTCGTAACTGTCGATTCC-3', reverse 5'-GACGCCTTTGAGCTCGAGG-3'; FT, forward 5'-CCATTGGTTGGTGACTGATATCC-3', reverse 5'-CTCATTGCCAAAGGTTGTTCC-3'; GI, forward 5'-CATTGCTGAGTTGGTCCGG-3', reverse 5'-CCAGCACATCGTCTAAAAGTCG-3'; TOC1, forward 5'-ATCTTCGCAGAATCCCTGTGATA-3', reverse 5'-GCACCTAGCTTCAAGCACTTTACA-3'; βTUB4, forward 5'-TTTCCGTACCCTCAAGCTCG-3', reverse 5'-TGAGATGGTTAAGATCACCAAAGG-3'; and ZTL, forward 5'-CCGTCTTCGAAATGGTTACAGG-3', reverse 5'-CTCTACATTGCAAGAAGCGGC-3'.
Seeds were surface-sterilized and stratified for 72 h at 4°C. Seedlings were grown on plates containing 1x MS salts with 2% agar with or without 3% Suc in LD 12:12 photoperiods at 22°C and were 11 d old at the commencement of imaging. Plants were assayed in constant light at 22°C, starting at subjective dawn 24 h after the discontinuation of the photoperiod; light level during photoperiods or constant light was 50 µmol m–2 s–1. Circadian rhythms of leaf movement were measured by video imaging under constant light and analyzed using the BRASS interface (Brown, 2004
Seeds were sown on square petri plates (120 cm) containing 1x MS with or without Suc and 1% agar, stratified as described above, and placed vertically in the growth chamber for 7 d in darkness or in constant light (50–60 µmol m–2 s–1). All plates were subjected to a 2-h light treatment before the commencement of this period to allow seedlings in DD to germinate. After this period, plants were photographed and hypocotyl lengths were measured using ImageJ software (http://rsb.info.nih.gov/ij/). Germination rates were measured on horizontally grown MS agar plates as in all other experiments, with or without Suc, and in DD after an initial 2-h light period to allow germination. Germination was scored by checking for the emergence of a radical every 12 h.
The following materials are available in the online version of this article.
We are grateful to Andrew Millar and the IMPS (University of Edinburgh) for access to the growth imaging system, and to Jane Langdale, Marc Knight, and Andrew Smith for helpful discussions and comments. We thank Paul Sidney (Durham University) for excellent photographic assistance. We are most grateful to Bethan Taylor, who conducted the preliminary experiments that led to the initiation of this study. Received June 3, 2008; accepted June 30, 2008; published July 9, 2008.
1 This work was supported by Queen's College, Oxford (H.G.M.), and by the Biotechnology and Biological Sciences Research Council (grant no. 43/P18613). H.G.M. is a Royal Society University Research Fellow.  The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) are: Harriet G. McWatters (harriet.mcwatters{at}plants.ox.ac.uk) and Heather Knight (p.h.knight{at}durham.ac.uk).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.123901 * Corresponding author; e-mail harriet.mcwatters{at}plants.ox.ac.uk.
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