Cryptochromes are required for phytochrome signaling to the circadian clock but not for rhythmicity

Abstract

The circadian clock is entrained to the daily cycle of day and night by light signals at dawn and dusk. Plants make use of both the phytochrome (phy) and cryptochrome (cry) families of photoreceptors in gathering information about the light environment for setting the clock. We demonstrate that the phytochromes phyA, phyB, phyD, and phyE act as photoreceptors in red light input to the clock and that phyA and the cryptochromes cry1 and cry2 act as photoreceptors in blue light input. phyA and phyB act additively in red light input to the clock, whereas cry1 and cry2 act redundantly in blue light input. In addition to the action of cry1 as a photoreceptor that mediates blue light input into the clock, we demonstrate a requirement of cry1 for phyA signaling to the clock in both red and blue light. Importantly, Arabidopsis cry1 cry2 double mutants still show robust rhythmicity, indicating that cryptochromes do not form a part of the central circadian oscillator in plants as they do in mammals.

Figure 1.

Effect of Light Intensity on Period Length of the Circadian Rhythm of CAB2::LUC Bioluminescence in Wild-Type and Phytochrome-Deficient Seedlings. Seedlings were germinated and grown in 12-hr-white-light/12-hr-dark cycles for 6 days and then transferred to constant red or blue light at the fluence rates indicated for 5 days. Values shown are means (±se) for wild type (closed circles) and mutants (open circles). (A) Wild type and phyA mutant in red light. (B) Wild type and phyB mutant in red light. (C) Wild type and phyA phyB double mutant in red light. (D) Mean period length for wild-type, phyA phyB, phyA phyB phyE, and phyA phyB phyD mutant seedlings in red light of 50 μmol m⁻² sec⁻¹ (solid bars) and 150 μmol m⁻² sec⁻¹ (open bars). (E) Wild type and phyA mutant in blue light. (F) Wild type and phyB mutant in blue light. (G) Wild type and phyA phyB double mutant in blue light. Asterisk, P < 0.01 (Student's two-tail heteroscedastic t test).

Figure 2.

Effect of Blue Light Intensity on Period Length of the Circadian Rhythm of CAB2::LUC Bioluminescence in Wild-Type and Cryptochrome-Deficient Seedlings. Seedlings were germinated and grown in 12-hr-white-light/12-hr-dark cycles for 6 days and then transferred to constant blue or red light at the fluence rates indicated for 5 days. Values shown are means (±se) for wild type (closed circles) and mutants (open circles). (A) Wild type and cry1 mutant in blue light. (B) Wild type and cry2 mutant in blue light. (C) Wild type and cry1 cry2 double mutant in blue light. (D) Circadian rhythm of CAB2::LUC bioluminescence in wild-type and cry1 cry2 double mutant seedlings in blue light at 53 μmol m⁻² sec⁻¹. (E) Wild type and cry1 mutant in red light. (F) Wild type and cry2 mutant in red light. (G) Wild type and cry1 cry2 double mutant in red light. Asterisk, P < 0.01 (Student's two-tail heteroscedastic t test).

Figure 3.

Effect of Red Light Intensity in Hypocotyl Length in Wild-Type and Photoreceptor-Deficient Seedlings. One-day-old dark-grown wild-type and mutant seedlings were either maintained in darkness (solid bars) or transferred to low-fluence-rate red light (0.3 μmol m⁻² sec⁻¹; striped bars) or high-fluence-rate red light (30 μmol m⁻² sec⁻¹; open bars) for 3 days, after which hypocotyl lengths were measured. Mean (±se) hypocotyl lengths are normalized to dark-grown wild-type seedlings.

Figure 4.

Effect of White Light Intensity on Period Length of the Circadian Rhythm of CAB2::LUC Bioluminescence in Wild-Type and Phytochrome- and Cryptochrome-Deficient Seedlings. Seedlings were germinated and grown in 12-hr-white-light/12-hr-dark cycles for 6 days and then transferred to constant red light at the fluence rates indicated for 5 days. Values shown are means (±se) for the wild type (closed circles) and mutants (open circles). (A) Wild type and phyA mutant. (B) Wild type and cry1 mutant. (C) Wild type and phyA cry1 double mutant. Asterisk, P < 0.01 (Student's two-tail heteroscedastic t test).

Figure 5.

Circadian Rhythm of CCR2 Expression in Wild-Type, phyA phyB, and cry1 cry2 Seedlings. Seedlings were germinated and grown in 12-hr-white-light/12-hr-dark cycles for 6 days and then transferred to constant darkness. Tissue was harvested after 72 hr and then at 3-hr intervals thereafter for an additional 33 hr. Total RNA was extracted, and CCR2 RNA was quantified as described in Methods. Wild-type, closed circles; phyA phyB, open triangles; cry1 cry2, open squares.

Figure 6.

Elements Involved in Light Input to the Circadian Clock in Arabidopsis. Both red light and blue light act in light input to the clock. The phytochromes phyB, phyD and phyE perceive high-fluence-rate red light signals, whereas the cryptochromes cry1 and cry2 perceive high-fluence-rate blue light signals. Low fluence rates of both red and blue light are perceived by phyA, with cry1 acting in a light-independent manner as a signal transduction component necessary for phyA action.