CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL function synergistically in the circadian clock of Arabidopsis

Abstract

The circadian clock is an endogenous mechanism that coordinates biological processes with daily and seasonal changes in the environment. Heterodimerization of central clock components is an important way of controlling clock function in several different circadian systems. CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) are Myb-related proteins that function in or close to the central oscillator in Arabidopsis (Arabidopsis thaliana). Single mutants of cca1 and lhy have a phenotype of short-period rhythms. cca1 lhy double mutants show an even shorter period phenotype than the cca1 single mutant, suggesting that CCA1 and LHY are only partially functionally redundant. To determine whether CCA1 and LHY act in parallel or synergistically in the circadian clock, we examined their expression in both light-grown and etiolated seedlings. We have shown that LHY and CCA1 bind to the same region of the promoter of a Light-harvesting chlorophyll a/b protein (Lhcb, also known as CAB). CCA1 and LHY can form homodimers, and they also colocalize in the nucleus and heterodimerize in vitro and in vivo. In Arabidopsis, CCA1 and LHY physically interact in a manner independent of photoperiod. Moreover, results from gel filtration chromatography indicate that CCA1 and LHY are present in the same large complex in plants. Taken together, these results imply that CCA1 and LHY function synergistically in regulating circadian rhythms of Arabidopsis.

Figure 1.

The cca1-11 lhy-21 double mutant has a shorter period than the cca1-1 single mutant. A, Comparison of the CAB2∷LUC activity in wild type (Ws) and cca1-1. B, Comparison of the CAB2∷LUC activity in wild type (Ws) and cca1-11 lhy-21. CAB2∷LUC seedlings in wild-type (Ws), cca1-1, and cca1-11 lhy-21 backgrounds were grown in 12L:12D for 8 d and then transferred to LL. Mean bioluminescence traces ± SEM (n = 38–40) are shown. Each point was normalized to the average luminescence value for the entire run. Subjective day and subjective night are denoted by white and hatched bars, respectively. All of these experiments were done at least twice with similar results. WT, Wild type.

Figure 2.

CCA1 and LHY have similar expression patterns. A, Comparison of the expression of CCA1 and LHY in different tissues (RL, rosette leaf; CL, cauline leaf). Five-day-old (5d) seedlings were grown on plates, and the other tissues were harvested from 4-week-old soil-grown plants. All samples were collected at ZT-1. The RNA was subjected to qRT-PCR analysis to measure levels of CCA1 and LHY relative to RH8 (an internal control). Reactions were performed in triplicate. Error bars denote ± sd. B, CCA1 and LHY are expressed with similar kinetics in etiolated seedlings exposed to R. Time course of CCA1 and LHY expression in 5-d-old etiolated seedlings after exposure to 2 min of R. Gel blots of total RNA were hybridized with RNA probes of CCA1, LHY, and UBQ10 (control). All of these experiments were done at least twice with similar results.

Figure 3.

LHY binds to the same region of the Lhcb1*3 promoter as CCA1. A, EMSA analysis of CCA1 and LHY binding on the Lhcb1*3 promoter. The m1 probe contains the point mutations shown in C. WT, Wild type. B, Phenanthroline-copper footprinting of LHY on the wild-type A2 fragment of the Lhcb1*3 promoter. An EMSA gel with LHY and the wild-type A2 fragment was treated with phenanthroline-copper to cleave DNA, and the DNA from each band was recovered and separated on a sequencing gel. The lines between or next to the lanes highlight the regions protected by the proteins. F, Free probe. B1 and B2 refer to the DNA-protein complexes containing LHY used in the footprint analysis. C, Nucleotide sequence of the wild-type and m1 probes. WT and m1 denote the sequences of the wild-type A2 fragment of the Lhcb1*3 promoter and of the m1 mutated fragment, respectively. Dashes in the sequence of m1 represent nucleotides identical to those of the wild-type fragment. Solid and dashed lines above the sequence of the wild-type fragment represent the regions protected by LHY and CCA1, respectively. The region protected by CCA1 is from Wang et al. (1997). All of these experiments were done at least twice with similar results.

Figure 4.

CCA1 and LHY can physically interact in vitro, and the region of CCA1 downstream of the Myb domain is important for this interaction. A, CCA1 and LHY can homo- and heterodimerize in vitro. Glutathione-agarose beads containing approximately 1 μg of GST, GST-CCA1, or GST-LHY were mixed with 20 μL of ³⁵S-labeled CCA1, LHY, or LUC. Proteins interacting with GST, GST-CCA1, or GST-LHY were eluted and resolved in SDS-PAGE, followed by autoradiography. Input represents 10% of the ³⁵S-labeled protein used. B, The region between amino acids 136 and 316 of CCA1 is essential for interaction with LHY in vitro. The Myb domain of CCA1 is shown. The region of CCA1 filled with black and denoted with an asterisk indicates the 136- to 316-amino acid region that interacts with LHY. All of these experiments were done at least twice with similar results.

Figure 5.

CCA1 and LHY can form homodimers and heterodimers in vivo. A, CCA1 homodimerizes in planta. Immunoprecipitation (IP) by α-GFP antibody was performed on total protein extracts from N. benthamiana leaves transiently expressing YFP-CCA1 and MYC-CCA1. B, LHY homodimerize in planta. Immunoprecipitation by α-GFP antibody was performed on total protein extracts from N. benthamiana leaves transiently expressing YFP-LHY and HA-LHY. C, CCA1 and LHY colocalize in the nucleus. Left, CFP fluorescence localization of the transiently expressed LHY-CFP in N. benthamiana leaves; middle, YFP fluorescence localization of the transiently expressed YFP-CCA1 in N. benthamiana leaves; right, merged image of the CFP and YFP fluorescence in N. benthamiana leaves transiently expressing the two constructs. Bars = 10 μm. D, In planta heterodimerization of CCA1 and LHY requires the region between amino acids 136 and 316 of CCA1. Immunoprecipitation by α-LHY antibody was performed on total protein extracts from N. benthamiana leaves transiently expressing HA-LHY with MYC-CCA1 (lanes 1, 8, and 15), MYC-CCA1Δ86N (lanes 2, 9, and 16), MYC-CCA1Δ316N (lane 3, 10, and 17), MYC-CCA1Δ436N (lanes 4, 11, and 18), MYC-CCA1Δ86C (lanes 5, 12, and 19), MYC-CCA1Δ270C (lanes 6, 13, and 20), and MYC-CCA1Δ472C (lanes 7, 14, and 21). Input and supernatant after immunoprecipitation were run next to the immunoprecipitation product as a control. One hundred micrograms of total protein was analyzed as input. All of these experiments were done at least twice with similar results.

Figure 6.

CCA1 and LHY can interact in plants and their interaction is independent of photoperiod. Western-blot analysis of immunoprecipitation (IP) with α-LHY antibody and detection with antibody to LHY and CCA1. A, CCA1 coimmunoprecipitates with LHY in plant extract. Two-week-old seedlings grown in 12L:12D were harvested at ZT-1 and ZT-23. B, CCA1 coimmunoprecipitates with LHY throughout the diurnal cycle. Two-week-old seedlings grown in 12L:12D were harvested at different times as indicated. One hundred micrograms of total protein was analyzed as input. Light and dark periods are denoted by white and black bars, respectively. All of these experiments were done at least twice with similar results.

Figure 7.

CCA1 and LHY are present in the same large complex. A, CCA1 and LHY comigrate in gel filtration chromatography. Shown is western-blot analysis of fractions collected from gel filtration and detection with antibody to LHY and CCA1. The arrow indicates the band of CCA1 and the asterisks indicate cross-reactive bands. Total (T) represents 5% of the protein extract used for gel filtration. B, CCA1 coimmunoprecipitates with LHY in fractions 5, 6, and 7. Protein extracts were prepared from 2-week-old seedlings grown in 12L:12D and harvested at ZT-1. All of these experiments were done at least twice with similar results.