The Circadian Clock That Controls Gene Expression in Arabidopsis Is Tissue Specific

Expression of CHS:LUC in Roots and Leaves

Fusions of firefly LUC to the promoters of the white mustard (Sinapis alba) chalcone synthase (mCHS) and Arabidopsis chalcone synthase (CHS) genes were transformed into Arabidopsis plants of the Landsberg erecta(Ler) and C24 strains, respectively. Figure1B shows that luminescence driven by theCHS promoter was evident in the leaves, hypocotyl, and roots of 12-d-old seedlings. Particularly high expression occurred in the shoot apical region and in young lateral roots. Younger, 5-d-old seedlings showed no CHS expression in the hypocotyl (data not shown), consistent with previous reports (Kaiser and Batschauer, 1995; Kaiser et al., 1995). The mCHS promoter had an identical pattern of expression (data not shown). In contrast,CAB:LUC activity was largely confined to green tissues, with a gradient down the hypocotyl and no detectable activity in the roots (Fig. 1D).

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Fig. 1.

Tissue-specific expression of the CHSpromoter. Reflected-light (A and C) and luminescence (B and D) images of CHS:LUC (A and B) andCAB:LUC (C and D) seedlings after 12 d of growth in LD (12, 12) of 150 μmol m⁻²s⁻¹. Luminescence video images were processed in false color (see “Materials and Methods”): White and red shades represent the highest photon counts, and darker blues, the lowest. A reflected-light image of the plant is shown to the left of each false color image. E through H, Leaf sections were prepared from plants grown in LD (12, 12) of 150 μmol m⁻²s⁻¹ for 7 d and then transferred to 250 μmol m⁻² s⁻¹ for 5 d. E, Microtome section of a CHS:β-glucuronidase (GUS) leaf, stained for GUS activity and counter-stained with ruthenium red. F through H, Thick hand section of a CHS:LUCleaf: bright field (F), luminescence (G), and overlaid (H) images. E and F, Scale bars represent 60 μm. E and H, Arrowheads indicate areas where CHS expression is very weak or absent from the mesophyll.

The spatial pattern of CHS promoter activity was examined in greater detail in leaf sections from CHS:LUCplants and plants carrying a CHS-GUS fusion transgene. TheCHS:LUC signal was highest in the upper epidermis, but both reporters showed patchy expression also in the palisade mesophyll layer (Fig. 1, E and G). The epidermal expression ofCHS:LUC was about 2-fold higher than the mesophyll expression (in Fig. 1G, for example, average counts per pixel per minute were 0.36 for epidermis and 0.19 for upper mesophyll). A minor contribution from rhythmic CHSexpression in the mesophyll would not be distinguished in our assays. Luminescence from the mesophyll is appreciably reduced by passage through the epidermis (Wood et al., 2001), indicating that theCHS:LUC rhythm preferentially reflects epidermal luminescence. CHS-GUS expression was also evident in all cell layers of root cross sections (data not shown).

The CHS Expression Rhythm Is Distinct from theCAB Expression Rhythm

The strong CHS:LUC activity in the root might make this a useful marker for circadian rhythms specifically in this organ, where root pressure and ion fluxes are the only previously reported, rhythmic markers (Vaadia, 1960; Parsons and Kramer, 1974;Gorr et al., 1995; Henzler et al., 1999). The circadian rhythm ofCHS:LUC activity was tested under several fluence rates of constant light, because CHS mRNA levels increase in response to higher fluence rates (Feinbaum and Ausubel, 1988; Peter et al., 1991; Fuglevand et al., 1996). Transgenic seedlings were grown for 12 d on vertical agar plates under white light of 60, 150, or 250 μmol m⁻² s⁻¹ fluence rate. Seedlings grown and assayed in white light of 60 μmol m⁻² s⁻¹ showed low expression of CHS:LUC andmCHS:LUC (data not shown). Luminescence rhythms were measured after the plants were transferred to constant light of either the same fluence rates (Fig. 2, A and C), or of an increased fluence rate (from 150 to 250 μmol m⁻² s⁻¹; Fig. 2B).CHS:LUC activity showed circadian rhythmicity in aerial organs and roots under all conditions. The peak of activity on the 1st d in constant light occurred before predicted dawn under all conditions (at zeitgeber time [ZT] 20–22; ZT is defined as the number of hours since lights-on), 6 or 7 h before the peak ofCAB expression. Transgenic plants carrying themCHS:LUC fusion in the Ler background showed very similar luminescence rhythms (Fig. 2D).

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Fig. 2.

Rhythmic luminescence in roots and aerial organs of Arabidopsis seedlings expressing CHS:LUC. CHS:Luc (A, B, C, and E) ormCHS:LUC (D) expression was assayed by video imaging (see “Materials and Methods”) at the times indicated. A, B, D, and E, Seedlings were grown on vertical agar plates, in 7 d of LD (12, 12) at 150 μmol m⁻²s⁻¹. Seedlings in A and D were transferred (at 0 h) to continuous light of 150 μmol m⁻²s⁻¹; seedlings in B were transferred to continuous light of 250 μmol m⁻²s⁻¹; and seedlings in E were transferred first to 250 μmol m⁻² s⁻¹ for 12 h and then to constant darkness. Seedlings in C were both grown and tested at 250 μmol m⁻²s⁻¹. The data are representative of at least five independent experiments. White box on time axis, Light interval; black box, dark interval. Lv, Luminescence from aerial organs; Rt, luminescence from roots.

The lighting conditions affected the amplitude of rhythmicCHS expression. The high-amplitude rhythm in the 1st d of constant light was followed by a reduction (damping) in the amplitude of the CHS:LUC circadian rhythm within two to three cycles. mCHS:LUC was less affected (Fig.2D), possibly because of a difference between the C24 and Ler genetic backgrounds. CAB:LUCactivity continued to cycle with high amplitude under constant light of all fluence rates. In plants transferred from lower to higher fluence rates of light (Fig. 2B), CAB:LUC luminescence levels were reduced compared with CHS:LUC. When 12-d-old plants expressing CHS:LUC were transferred to continuous darkness, transcription from theCHS promoter was greatly attenuated (Fig. 2E). A single, high-amplitude peak at the expected phase was followed by rapid damping of CHS:LUC activity, with a complete loss of rhythmic amplitude by 72h.

The CAB and CHS rhythms had different free-running periods under constant light. The lag (phase angle) between the rhythms, therefore, changed progressively during our experiments, most obviously when Ler plants were imaged on the 3rd to 7th d under constant light (Fig. 2D). The altered period was clear: The first peak of mCHS expression shown (at 68 h) occurred 10 h before the CAB peak (at 78 h), but by 147 h, the mCHS peak occurred with or slightly after that for CAB. Period estimates were derived from luminescence rhythms measured in the aerial tissues, where both CHS:LUC and CAB:LUCare expressed (Table I). The period of the CHS rhythm (25.4 h) was significantly longer than the period of the CAB rhythm (23.7 h). A similar period difference was observed between CHS and CABexpression rhythms in the Ler background (as in Fig. 2D). In contrast, the period of rhythmic CHS expression in the root was not significantly different from that in the leaf, in either genetic background (Fig. 2; Table II; data not shown). These results suggested that the circadian system that controlled CHS in aerial organs was distinct from that controlling CAB.

de-etiolated 1 (det1) and timing of CAB expression 1 (toc1) Mutations Shorten the Period of Both CHS and CAB Rhythms

The det1 mutant has a severe short-period phenotype forCAB expression (Millar et al., 1995b), along with other phenotypes principally related to light signaling (Chory and Peto, 1990; Pepper et al., 1994). toc1 is a short-period mutant that affects only clock-regulated processes (Millar et al., 1995a; Kreps and Simon, 1997; Somers et al., 1998b;Dowson-Day and Millar, 1999); TOC1 encodes one of the best candidates for a plant circadian oscillator component (Strayer et al., 2000; Alabadi et al., 2001). The CHS:LUCconstruct was crossed into these mutant backgrounds to test whether these genes are involved in the circadian systems that control both theCAB and CHS markers. det1 andtoc1 mutant seedlings expressedCHS:LUC rhythmically in both leaves and roots under constant light (Fig. 3A; data not shown). Light-grown det1 seedlings express theCHS promoter in all leaf cell layers, and expressCAB genes inappropriately in roots and in aerial tissue (Chory and Peto, 1990). CAB:LUC activity indet1 roots was barely detectable and was too low for us to measure circadian rhythms (data not shown). The det1mutation prevents the damping of rhythmic CAB expression in darkness (Fig. 3B; Millar et al., 1995b). It had little effect on CHS expression, which damped out in the dark similarly in the mutant (Fig. 3B) and wild type (Fig. 2E). Consistent with previous data, the toc1 mutation did not affect the mean expression level or damping of CAB and CHS expression rhythms (data not shown; Millar et al., 1995a).

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Fig. 3.

Circadian rhythms of CHS:LUCactivity in det1. Expression was assayed as in Figure 2. Seedlings were grown in LD (8, 16) at 20 μmol m⁻² s⁻¹ to confirm thedet1 phenotype. Seedlings were transferred to LD (12, 12) at 150 μmol m⁻² s⁻¹, 2 d before the start of the experiment and to constant light of 250 μmol m⁻² s⁻¹ at 0 h (A) or to 250 μmol m⁻²s⁻¹ at 0 h and constant darkness at 12 h (B). Luminescence data were analyzed from whole seedlings, without separate analysis of roots and aerial tissues. The data are representative of at least four independent experiments. White box on time axis, Light interval; black box, dark interval.

Quantitative comparisons showed that the period of CHSexpression was longer than the period of CAB expression in both mutant backgrounds (Tables I and II), reinforcing the conclusion that separate clocks control these two promoters. However, both rhythmic markers were very similarly affected by the mutations (TablesI and II), with much shorter periods in mutant progeny than in wild-type controls. This result indicates that TOC1 andDET1 function similarly in the circadian clocks that regulate CAB and CHS.

Regulation of CHS

The expression of CHS exhibited a very similar circadian rhythm in all tissues (Fig. 2). The phase of the circadian rhythm of CHS was earlier than CAB, with a peak occurring in the late subjective night (ZT 20–22). Chalcone synthase enzyme activity and mRNA abundance have previously been reported to exhibit diurnal and circadian regulation (Peter et al., 1991; Deikman and Hammer, 1995; Harmer et al., 2000; Schaffer et al., 2001). The reported phase of peak enzyme activity lags slightly behind the peaks of mRNA abundance and of transcriptional activity, suggesting that the circadian system principally regulates CHS expression at the transcriptional level. It may be advantageous for the plant to accumulate photoprotective pigments in advance of the daily photoperiod (Harmer et al., 2000); the timing of CHS transcription before dawn is consistent with this notion.

Increasing the fluence rate of white light from 150 to 250 μmol m⁻² s⁻¹ had little effect on the phase or period of CHS or CAB expression (Fig. 2; data not shown). However, mean expression levels were differentially affected by the lighting conditions. Plants entrained at 150 μmol m⁻² s⁻¹ but assayed at 250 μmol m⁻²s⁻¹ showed reduced levels ofCAB:LUC activity compared withCHS:LUC activity (Fig. 2B), perhaps reflecting a requirement for increased photoprotection and reduced light-harvesting capacity. High-fluence rate light was required to maintain high expression levels of CHS transcription in wild-type plants:CHS:LUC activity was very low in plants assayed at 60 μmol m⁻² s⁻¹(data not shown) or in darkness (Fig. 2E). The det1 mutation increases CAB expression levels in dark-adapted plants but had little effect on the level of CHS expression (Fig. 3B;Chory and Peto, 1990; Millar et al., 1995b). This presumably reflects a differential involvement of DET1 in the phototransduction pathways that regulate these promoters (Mustilli et al., 1999; Jenkins et al., 2001).

Differences in the Circadian Regulation of CHS andCAB

The rhythm of CHS expression was very similar in roots and in aerial organs under constant light, indicating that the clocks controlling CHS in these organs do not differ significantly. The period of CHS expression in aerial tissues was approximately 1.5 h longer than the period of CABexpression. This difference was maintained in two wild-type accessions and in the det1 and toc1 mutants (Tables I andII). Distinct circadian clocks, therefore, control the rhythms ofCAB and CHS expression, although both are nuclear genes that could in principle respond to the same regulator. The phase of a single rhythm (free calcium concentration) has recently been shown to vary among tissues of transgenic tobacco (Nicotiana tabacum; Wood et al., 2001). The authors point out that a phase difference could result from tissue-specific clocks or from tissue-specific responses to a single, common clock. Where period differences are observed, the latter interpretation can be ruled out (Sai and Johnson, 1999).

It is not surprising that the period difference is small. Studies on cyanobacteria indicate that a clock with a period that matches the environmental light-dark cycle provides a competitive advantage (Ouyang et al., 1998). Balancing selection is, therefore, likely to maintain periods in a narrow range around 24 h. Consistent with this notion, we have previously shown that the similar periods of several Arabidopsis accessions are the result of balancing long- and short-period alleles at multiple loci (Swarup et al., 1999).

Period differences under constant conditions are relatively easy to measure. Under light-dark cycles, however, clocks with different periods will entrain to different phases (for example, Ouyang et al., 1998; Somers et al., 1998b). A clock with a longer period (such as that controlling CHS) will be set to a later phase, all else being equal. The longer period moves the peak of CHSexpression away from midnight and toward dawn. If CAB was controlled by the same, longer-period clock, then the peak ofCAB expression would also be later in the day, all else being equal. The delay between the peak of CHS expression and the peak of CAB expression would thus be greater than we observe. Independent clocks allow the temporal sequence of metabolic processes to be fine-tuned: A smaller delay between the peaks ofCHS and CAB expression might be one example of this. Such a flexible timing system has potential selective advantages (Roenneberg and Mittag, 1996), although these remain to be demonstrated experimentally.

Differential Regulation of Circadian Period

The circadian clocks that control CAB andCHS might differ fundamentally in their oscillator mechanism, or they might alternatively be separate copies of a common biochemical mechanism with only minor modifications leading to the period difference (Millar, 1998). det1 and toc1mutations are thought to affect the circadian rhythm of CABexpression via the input pathway and the oscillator, respectively (Millar et al., 1995a, 1995b; Strayer et al., 2000; Alabadi et al., 2001). Each mutation shortens the circadian period ofCHS in aerial organs in parallel with CAB (TablesI and II). This result shows that both circadian clocks share at least the TOC1 and DET1 functions, so the clocks are not radically different. A parsimonious explanation is that theCHS and CAB promoters are controlled by separate copies of the same clock mechanism (Fig.4). The characteristics of circadian timing also vary among rodent organs, outside the brain (Yamazaki et al., 2000). Differences in input pathways might be particularly important in that case (Damiola et al., 2000; Stokkan et al., 2001). The same canonical clock genes seem to be involved in diverse anatomical locations (Ripperger et al., 2000).

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Fig. 4.

Autonomy and specialization of the circadian clock. A simplified model of the circadian clock is shown in the mesophyll and in the epidermis, with the output markers and clock-related genes tested in this work. The clock is depicted as containing the same components in each case; TOC1 is thought to function in the oscillator, and DET1 is thought to affect the light input pathway from the photoreceptors. Rhythmic output from the oscillator is shown regulating transcription factors at theCAB or CHS promoter. Two hypothetical tissue-specific factors are shown: modification of the light input pathway and an unknown tissue-specific factor (X). One or both modify the function of an oscillator component, resulting in a longer period in the epidermis.

CHS is expressed principally in the epidermal cell layer of wild-type aerial organs, whereas CAB is expressed in the mesophyll layers. It is most likely that their different circadian periods reflect tissue-specific modifications of the clock in epidermal and mesophyll cells, respectively. In support of this conclusion, we have recently shown that the PHYTOCHROME B gene, which is expressed in the epidermis, also has a longer period than CAB (A. Hall, L. Kozma-Bognar, F. Nagy, and A.J. Millar, unpublished data). These data imply that epidermal circadian clocks are not tightly coupled to clocks in the mesophyll of the same leaf. We have previously demonstrated that several autonomous clocks exist within the mesophyll of a single leaf (Thain et al., 2000). Taken together, our results suggest that circadian control is local to one or a few cells, not widely coupled within or between tissues, at least for gene expression rhythms in the leaf.

The molecular cause of the observed period difference is unclear. Any circadian input pathway might contribute (Fig. 4). Photoreceptor genes are differentially expressed in the epidermal and mesophyll cell layers (Somers and Quail, 1995), and photoreceptors are known to alter circadian period in a dose-dependent manner (Somers et al., 1998b; Devlin and Kay, 2000). The light input pathways might thus control circadian period in a tissue-specific fashion. Observed distinctions between circadian rhythms suggest that further specialization of the circadian clock might be present in the cells that sense or respond to photoperiodic signals (for example, Salisbury and Denney, 1971; Fowler et al., 1999) or control rhythmic leaf movement (Hennessey and Field, 1992; Fowler et al., 1999; Park et al., 1999). Rhythmic reporters that peak at various phases in restricted spatial patterns are required to determine how much the circadian clock is modulated for specialized timing functions. Recent microarray experiments suggest many candidate promoters (Harmer et al., 2000;Schaffer et al., 2001), which can now be tested in detail usingLUC fusions.