The out of phase 1 mutant defines a role for PHYB in circadian phase control in Arabidopsis

Abstract

Arabidopsis displays circadian rhythms in stomatal aperture, stomatal conductance, and CO(2) assimilation, each of which peaks around the middle of the day. The rhythmic opening and closing of stomata confers a rhythm in sensitivity and resistance, respectively, to the toxic gas sulfur dioxide. Using this physiological assay as a basis for a mutant screen, we isolated mutants with defects in circadian timing. Here, we characterize one mutant, out of phase 1 (oop1), with the circadian phenotype of altered phase. That is, the timing of the peak (acrophase) of multiple circadian rhythms (leaf movement, CO(2) assimilation, and LIGHT-HARVESTING CHLOROPHYLL a/b-BINDING PROTEIN transcription) is early with respect to wild type, although all circadian rhythms retain normal period length. This is the first such mutant to be characterized in Arabidopsis. oop1 also displays a strong photoperception defect in red light characteristic of phytochrome B (phyB) mutants. The oop1 mutation is a nonsense mutation of PHYB that results in a truncated protein of 904 amino acids. The defect in circadian phasing is seen in seedlings entrained by a light-dark cycle but not in seedlings entrained by a temperature cycle. Thus, PHYB contributes light information critical for proper determination of circadian phase.

Figure 1

Circadian rhythm in stomatal aperture in Arabidopsis. A, Epidermal peels were prepared from Col plants grown in a 14-h:10-h light-dark cycle. B, As in A, but plants were grown in a 14-h:10-h light-dark cycle and transferred into extended darkness. Hatched bars indicate subjective day. Each peel was scored by microscopy for the percentage of open stomates. Each point represents the mean of duplicate samples, each of at least 100 stomates.

Figure 2

The rhythm in stomatal aperture correlates with a rhythm in CO₂ fixation. A, Rates of CO₂ exchange of representative individual 5- to 6-week-old Col plants grown in a 16-h:8-h light-dark cycle and transferred into continuous light. Hatched bars indicate subjective night. For the first 3 d, plants were maintained under entraining conditions and, therefore, show respiration during the night. B, Rates of CO₂ exchange of several representative individual Col plants after transfer into continuous light. Hatched bars indicate subjective night.

Figure 3

Arabidopsis exhibits a rhythm in sensitivity and resistance to SO₂. Col plants were grown in a 14-h:10-h light-dark cycle, transferred into continuous light, and then treated with SO₂ for 30 min each time, at 2-h intervals. A, Plants gassed 4 h before subjective dawn (ZT20, where ZT refers to Zeitgeber Time, defined as the number of h after the onset of illumination). B, Plants gassed 2 h before subjective dawn (ZT22). C, Plants gassed at subjective dawn (ZT24). D, Diagrammatic representation of the screening strategy for putative clock mutants based on resistance to SO₂. Wild-type (WT) plants are resistant to the treatment only approximately 2 h before subjective dawn (ZT22 and ZT46) and, thus, do not develop lesions when treated with SO₂ at ZT46. A short-period mutant would already have advanced through the period of resistance and become sensitive because of its open stomata. A long-period mutant would not yet have reached the tolerance period (closed stomata) and would also be sensitive to SO₂. Phase mutants would display resistance to SO₂ with a WT period, but the timing of the window of resistance is shifted earlier (as shown) or later than that of Col plants. Plants were grown in a 12-h:12-h light-dark cycle and transferred into continuous light at T = 0; the gray area indicates subjective night.

Figure 4

The oop1 mutation causes an altered phase in circadian rhythms. A, Net CO₂ fixation levels in oop1 and Col. oop1 and Col plants were grown for 4 weeks in a 12-h:12-h light-dark cycle and transferred into continuous light. CO₂ assimilation levels were recorded for 6 d. B, Cotyledon movement in Col and oop1 seedlings. oop1 and Col seedlings were grown for 4 to 5 d in a 12-h:12-h light-dark cycle and transferred to 24-well cloning plates, one seedling per well, in continuous light. Cotyledon movement was recorded for 7 d. C, LHCB::LUC transcription. Col, oop1, and phyB-9 seedlings homozygous for the LHCB::LUC transgene were grown in a 12-h:12-h light-dark cycle and transferred into continuous light at T = 0. Luciferase activity was recorded from each group of seedlings after transfer into continuous light and temperature conditions and is presented as the average of multiple seedlings (see Table I) for four complete circadian cycles. Hatched bars indicate subjective night. Col, Blue squares; oop1, red circles; and phyB-9, black triangles.

Figure 5

The oop1 mutant is primarily impaired in red-light perception. A, oop1 was grown in continuous blue, far-red, or red light for 5 d before hypocotyl length was measured with NIH Image v1.62. The known photoreceptor mutants phyA-211, phyB-9, and cry1–304 were also used as controls for loss of PHYA-, PHYB-, or CRY1-mediated light perception, respectively. Hypocotyl length (mean ± sd) is given for each genotype and treatment. B, Hypocotyl phenotype of oop1 and phyB-9 in response to combinations of red and blue lights. Col, oop1, and phyB-9 seedlings were grown as described in A, under 25 μmol m⁻² s⁻¹ red light combined with 1, 5, or 25 μmol m⁻² s⁻¹ blue light. C, Enhancement of hypocotyl phenotype in oop1 and phyB-28. Col, oop1, phyB-9, and phyB-28 seedlings were grown as described in A, under a combination of 25 μmol m⁻² s⁻¹ red light and 15 μmol m⁻² s⁻¹ blue light. *, Hypocotyl length is significantly different (Student's two-tailed heteroscedastic t test, P < 0.001) from Col; °, hypocotyl length is significantly different (P < 0.001) from phyB-9.

Figure 6

oop1 is a new allele of PHYB. A, Map position of the oop1 locus. The number of recombinant chromosomes among a population of 41 plants homozygous for the oop1 mutation is indicated for each marker. mi398 and mi238 are RFLP markers at 29.27 and 39.02 cM, respectively. B, Mutations in the PHYB^oop1 gene. Sequencing results from Col and oop1 are shown. The positions of the mutations are highlighted by asterisks. C, dCAPS analysis of PHYB in oop1. Two dCAPS markers were developed, one specific to each mutation. The PCR products were amplified from Col and oop1 DNA and digested with StyI for the P872L mutation and BsrGI for the Q905X mutation. The restriction digests were run on a 10% (w/v) acrylamide gel and stained with ethidium bromide. Left, dCAPS results obtained for the P872L mutation. Right, Q905X mutation. M indicates the 100-bp Plus ladder (MBI Fermentas, Hanover, MD). D, Western-blot analysis of oop1 plants. Left, The membrane was probed with a PHYB-specific monoclonal antibody that recognizes an epitope in the carboxy terminus, which is not retained in oop1. This antibody fails to detect full-length PHYB protein in oop1. Right, The membrane was probed with a monoclonal antibody raised against a conserved epitope in the central region of the molecule, which recognizes all phytochromes; the oop1 sample shows a smaller protein species of the M_r predicted for a PHYB protein comprising the first 904 amino acids only. E, Maps of PHYB and PHYB^oop1. The functional domains of PHYB are indicated, as well as the position of the two mutations in oop1. The Q905X mutation introduces a premature stop codon at amino acid 904 and causes the loss of the His-kinase related domain and three of the four putative nuclear localization sequences.

Figure 7

Phase alteration of LHCB::LUC transcription in oop1 is not seen after entrainment by temperature cycles. Col and oop1 seedlings homozygous for the LHCB::LUC transgene were grown for 7 d under entraining conditions consisting of 12 h at 22°C followed by 12 h at 12°C. Luciferase activity was recorded from each group of seedlings after transfer into continuous light and temperature conditions and is presented as the average of multiple seedlings. Col, Blue squares; oop1, red circles; and phyB-9, black triangles. The hatched bars represent the subjective cold (12°C) period of the day.