Comment on "The Arabidopsis circadian clock incorporates a cADPR-based feedback loop"

Abstract

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.

Fig. 1

[cADPR] fluctuation shows no circadian rhythm, and circadian [Ca²⁺]_cyt oscillates independently of the elevated cADPR level. (A) cADPR concentration in constant light in Col-0 wild type. For cADPR analysis, aerial tissues were collected 24 hours after the entrained seedlings were released to constant light (LL). Each point represents data from three to five assays of an extract taken at the indicated time point, plotted as mean ± SEM. (B) Mean ± SEM level of cADPR from subjective day and night separately. By Student's t test, there was no statistically significant difference between the mean day versus night levels in either the second day (P = 0.453) or the third day (P = 0.567) of LL. (C) Mean traces of circadian [Ca²⁺]_cyt oscillation as monitored by aequorin luminescence (2) in 8-day-old Arabidopsis seedlings treated with 30 μM 17-β-estradiol at CT 13 on the first day in LL. Filled circles, empty vector control; open triangles and gray squares, overexpression of Aplysia ADPR cyclase, lines 1 and 4 respectively. Mean for 8 to 11 samples per point (the same data are shown in fig. S5 with error bars). (D) cADPR levels in transgenic lines expressing Aplysia ADPR cyclase. Sampling was done after 0, 6, 24, and 48 hours induction with 30 μM 17-β-estradiol. Open bars, empty vector control; expression of Aplysia ADPR cyclase, lines 1 (striped bars) and 4 (cross-hatched bars). The extent of the cADPR increase was 4.6-fold in line 1 and 7.8-fold in line 4 at 48 hours of treatment. Mean values of five independent induction treatments are plotted with SEM bars.