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Dodd et al. (Reports, 14 December 2007, p. 1789) reported that the Arabidopsis circadian clock incorporates the signaling molecule cyclic adenosine diphosphate ribose (cADPR). In contrast, we found that there is no rhythm of cADPR levels nor are there any significant effects on the rhythm by cADPR overexpression, thus raising questions about the conclusions of Dodd et al.
Circadian (daily) rhythms regulate many biological processes to enhance fitness (1). In plant and animal cells, there is a daily oscillation in the level of cytosolic free calcium (Ca2+) that is likely to rhythmically modulate the activities of myriad Ca2+-regulated cellular processes (2–4). A question of central importance is what factor or factors couple the rhythm of Ca2+ to the core circadian pacemaker.
From their experiments on the plant Arabidopsis thaliana, Dodd et al. (5) contend that this Ca2+-regulating factor is the intracellular level of cyclic adenosine diphosphate ribose (cADPR). Cyclic ADPR would seem to be an excellent potential candidate for a regulator of Ca2+ rhythms, because it is known to promote release of Ca2+ from internal stores in plants and animals (6, 7). Moreover, cADPR mediates hormone responses in Arabidopsis (8, 9). Dodd et al. (5) reported that cADPR levels undergo significant circadian oscillations in Arabidopsis and, further, that manipulations of cADPR levels have significant effects on circadian rhythms. Our attempts to confirm a circadian rhythm of cADPR levels or to detect an impact of changes in cADPR levels upon the circadian system in Arabidopsis were unsuccessful. First, Dodd and colleagues reported a circadian oscillation of cADPR with higher levels in the subjective daytime (5). We repeated these measurements using the same assay as that used by Dodd and colleagues, which had been previously developed and applied to both plant and animal tissue (fig. S1) (10). The first discrepancy we noted was that Dodd and colleagues reported basal cADPR levels in wild-type Arabidopsis seedlings to range between 0.1 and 0.5 pmol/μg protein (100 to 500 pmol/mg protein), which is ~1000 times as high as the levels we measured (Fig. 1 and fig. S2) and 100 to 500 times as high as those measured by an independent group (9). More important, we found no evidence for a circadian oscillation in cADPR levels. We performed two separate 48-hour time course experiments in constant light (LL) using the same Arabidopsis accession line as that of Dodd et al. (Col-0) and a third 48-hour experiment using a different Arabidopsis accession (Ws) (Fig. 1, A and B and fig. S2) (11). Cosinor analyses (12) on the data depicted in Fig. 1A revealed that less than 3% of the variance in cADPR levels could be attributed to a 24-hour oscillation and is therefore insignificant (R2=0.029) (11). Moreover, when all three separate experiments were analyzed in a linear mixed model design (11), the 24-hour component to the data was not significant (fig. S3). We conclude that there is no consistent or reproducible circadian oscillation of cADPR levels.
A major experimental support to the argument of Dodd et al. of the importance of cADPR in the plant circadian system was that 10 to 50 mM nicotinamide suppressed the amplitude of their cADPR rhythm and that 50 mM nicotinamide lengthened the period of the leaf movement and CAB2 promoter activity rhythms (5). However, extracellular nicotinamide treatment has been reported to have many cellular effects, including affecting pyridine nucleotide levels and redox status, inhibiting key enzymes in multiple pathways (e.g., PARP, Sir2, and ADP-ribosyl transferases), increasing plant growth rate, altering plant hormone levels (IAA, GA3, cytokinins, and ABA) or intracellular ion levels (Na+, K+, Ca2+, Mg2+, Cl−, and others), stimulating plant cell apoptosis, and reducing insulin secretion by β cells (11). In particular, the high concentrations used by Dodd et al. (10 to 50 mM) will be expected to have many nonspecific effects. Therefore, we argue that a more specific modulator of cADPR levels is needed before an effect of cADPR on the Arabidopsis circadian system can be inferred.
Dodd et al. (5) sought to provide a more specific manipulation of cADPR levels by constitutively expressing Aplysia ADP ribosyl cyclase. This treatment would be expected to clamp cADPR levels at a high level, thereby swamping rhythmic expression of processes under putative circadian control by cADPR. Nevertheless, Dodd et al. reported only a modest effect on rhythms by cADPR elevation; there was no significant effect on circadian period, but the authors reported that there was a significant increase in the variability of the leaf movement rhythm as assessed by the relative amplitude error (RAE) (5). We repeated the constitutive expression of Aplysia ADP ribosyl cyclase using the estradiol-inducible ADP ribosyl cyclase system (pER8ADPRc) previously developed for Arabidopsis (9) with the aequorin reporter of cytosolic Ca2+ (2) that should be the proximal responder to changes in cADPR. We found that after treatment with 17-β-estradiol, cADPR levels begin to rise substantially between 6 and 24 hours after treatment and that these high levels are maintained for at least 96 hours in both of two pER8ADPRc transgenic lines (Fig. 1D and fig. S4). However, these elevated cADPR levels had no significant effect on the circadian Ca2+ rhythms. Regardless of estradiol administration in the morning (CT 4) versus afternoon (CT 13) on the first day in LL, there was no correlation between the period of the Ca2+ oscillation and the level of cADPR elevation among controls and the two transgenic lines (Fig. 1 and figs. S4, S5, and S6A). Nor did cyclase overexpression consistently phase-shift the Ca2+ rhythm at CT4 or CT13 (fig. S6B). In contrast to the findings of Dodd et al., cADPR elevation did not reduce the robustness of the circadian Ca2+ rhythm; the RAE was not reproducibly different between the controls and cyclase overexpression for treatment at CT4 or CT13. In fact, the rhythms for cADPR elevation appear to be more robust (lower RAE) than for the controls (fig. S6A). Finally, not only does cADPR elevation have no significant effect upon the Ca2+ rhythm, it also does not appear to affect basal Ca2+ levels themselves, as can be seen by the absence of an effect on the Ca2+ rhythm's baseline after overexpression of cyclase (Fig. 1C and fig. S5).
Our data indicate that there are no circadian rhythms of cADPR in Arabidopsis seedlings. Moreover, overexpression of cADPR does not alter circadian rhythms of Ca2+ (neither period, amplitude, nor phase), or indeed basal cytosolic Ca2+ levels. We therefore conclude that the Arabidopsis circadian clock does not incorporate a cADPR-based feedback loop and that the basis for the plant Ca2+ rhythm must be sought elsewhere.
We thank N. Chua and J.-P. Sanchez for providing the Arabidopsis lines transformed with 35Saequorin and pER8ADPRc. We thank S. Servick for technical assistance. This research was supported by grants from NIH and NSF (NIMH MH043836 to C.H.J.; NIGMS GM061568 to R.G., Q. Hao, and H. C. Lee; and NSF IOB-0517111 to C. R. McClung).
Supporting Online Material www.sciencemag.org/cgi/content/full/326/5950/230-b/DC1 Materials and Methods Figs. S1 to S6 References