Multiple bHLH proteins form heterodimers to mediate CRY2-dependent regulation of flowering-time in Arabidopsis

Abstract

Arabidopsis thaliana cryptochrome 2 (CRY2) mediates light control of flowering time. CIB1 (CRY2-interacting bHLH 1) specifically interacts with CRY2 in response to blue light to activate the transcription of FT (Flowering Locus T). In vitro, CIB1 binds to the canonical E-box (CACGTG, also referred to as G-box) with much higher affinity than its interaction with non-canonical E-box (CANNTG) DNA sequences. However, in vivo, CIB1 binds to the chromatin region of the FT promoter, which only contains the non-canonical E-box sequences. Here, we show that CRY2 also interacts with at least CIB5, in response to blue light, but not in darkness or in response to other wavelengths of light. Our genetic analysis demonstrates that CIB1, CIB2, CIB4, and CIB5 act redundantly to activate the transcription of FT and that they are positive regulators of CRY2 mediated flowering. More importantly, CIB1 and other CIBs proteins form heterodimers, and some of the heterodimers have a higher binding affinity than the CIB homodimers to the non-canonical E-box in the in vitro DNA-binding assays. This result explains why in vitro CIB1 and other CIBs bind to the canonical E-box (G-box) with a higher affinity, whereas they are all associated with the non-canonical E-boxes at the FT promoter in vivo. Consistent with the hypothesis that different CIB proteins play similar roles in the CRY2-midiated blue light signaling, the expression of CIB proteins is regulated specifically by blue light. Our study demonstrates that CIBs function redundantly in regulating CRY2-dependent flowering, and that different CIBs form heterodimers to interact with the non-canonical E-box DNA in vivo.

Figure 1. Multiple bHLH promote flower initiation in long day condition.

(A–H) Flowering phenotype of different transgenic lines in long day. Three independent overexpress lines expressing 35S::Myc-CIB2 (A–B), 35S::Myc-CIB3 (C–D), 35S::Myc-CIB4 (E–F), 35S::Myc-CIB5 (G–H) and the WT control were grown in LD (16-h light/8-h dark) for 23 days when the pictures were taken. The quantitative flowering times measured as days to flower and the number of rosette leaves at the day floral buds became visible, and the standard deviations (n≥20) are shown. (I–J) Three independent overexpress lines expressing 35S::Myc-CIB4 (I) or 35S::Myc-CIB5 (J) in cry1cry2 background and the cry1cry2 control were grown in LD (16-h light/8-h dark). The quantitative flowering times measured as days to flower and the number of rosette leaves at the day floral buds became visible, and the standard deviations (n≥20) are shown.

Figure 2. CIB proteins promote flowering redundantly by activating FT mRNA expression.

(A–D) Flowering phenotype in long day. Three independent 35S::VP16-Myc-CIB1 (A–B) transgenic lines and the WT control were grown in long day for 23 days when the picture was taken. 35S::Myc-CIB1-EAR (C–D) plants and the WT control were grown in long day for 33 days when the picture was taken. The quantitative flowering times measured as days to flower and the number of rosette leaves at the day floral buds became visible, and the standard deviations (n≥20) are shown. (E–F) The cib15 double and the cib125 triple mutant showed a mild but statistically significant delay of flowering under a photoperiodic inductive condition. Plants were grown in short-day photoperiod (9 hL/15 hD) for 20 days, transferred to long-day photoperiod (16 hL/8 hD) for 4 days, and moved back to short-day to continue grow until flowering. Days from sawing to flowering and number of rosette leaves at the time of flowering are shown with the standard deviations (n>20). (G) A comparison of the FT mRNA expression in the cib1cib2cib5 triple mutant and the wild type. Plants were grown in short-day photoperiod (9 hL/15 hD) for 20 days and transferred to long-day photoperiod (16 hL/8 hD) for 4 days, samples were collected every 3 hr for 24 hr in the fourth day of long day at the time indicated for the qPCR analysis. (H–I) Quantitative PCR results showing mRNA expression of FT in the wild type (WT), transgenic lines expressing the 35S::Myc-CIB2, 35S::Myc-CIB4, 35S::Myc-CIB5 or 35S::Myc-CIB1-EAR, 35S::Myc-CIB4-EAR, 35S::Myc-CIB5-EAR transgene in the wild-type background grown in long-day (16 hL/8 hD) for 6 days then moved to continue white light for one day. Samples were collected every 3 hr for 24 hr in the continuous white light. Each experiment was performed at least three times with similar results.

Figure 3. Blue light-dependent CRY2-CIB5 interaction in plant cells.

(A) Fluorescent microscopy images showing that CIB3, CIB4 and CIB5 (Green) all co-localize with CRY2 (Red) in the nucleus. (B) Bimolecular fluorescence complementation assays of the in vivo protein interaction. Leaf epidermal cells of N. benthamiana were cotransformated with cCFP–CRY2 and nYFP, or nYFP-CIB1, or nYFP-CIB2, or nYFP-CIB4, or nYFP-CIB5. BF, Bright Field; Merge, overlay of the YFP and bright field images. (C) The co-immunoprecipitation assay showing the blue light dependent CRY2-CIB5 interaction in planta. Co-IP assays of samples prepared from 12-day-old 35S::MycCIB5 seedlings grown in continuous red light, pre-treated in MG132, then exposed to white light (W), or red light (R), or blue light (B, 20 µmol m−2 s−1, 20 min). Total proteins (Input) or IP product of anti-CRY2 antibody (CRY2-IP) or preimmune serum (Preim) were probed, in immunoblots, by the anti-CRY2 antibody (CRY2), stripped and reprobed by the anti-Myc (MycCIB1) antibody.

Figure 4. ChIP-qPCR showing interaction of CIB4 and CIB5 with chromatin regions of the FT gene.

(A) GUS staining of seedlings expressing CIB2::GUS, CIB4::GUS, CIB5::GUS transgene. (B) A diagram depicting the putative promoter (arrow), 5′ UTR (grey line), exons (black boxes), introns (dashed boxes), 3′ UTR (grey line) of the FT gene. Black solid lines depict the DNA regions that were amplified by ChIP-PCR using the indicated primer sets. (C) Representative results of the ChIP-qPCR assays. Chromatin fragments (∼500 bp) were prepared from 7-day-old transgenic seedlings expressing 35S::Myc-CIB4 or 35S::Myc-CIB5, immunoprecipitated by the anti-Myc antibody, and the precipitated DNA were qPCR-analysised using the primer pairs indicated. The IP/input ratios are shown with the standard deviations (n≥3). (D) Structure of the FT promoter–driven dual-Luc reporter gene. 35S promoter (black arrow), FT promoter (−2000 bp–0 bp) (white arrow head), REN luciferase (REN), firefly luciferase (LUC), and T-DNA (LB and RB) are indicated. (E) Relative reporter activity (LUC/REN) in planta with different effectors (CIB1/2/4/5) expression. Control: transiently expressed reporter only, CIB1: transiently expressed reporter and CIB1, CIB2: reporter and CIB2, CIB4: reporter and CIB4, CIB5: reporter and CIB5. Tobacco leaves were transfected with the reporter and the effector (CIB1 or CIB2 or CIB4 or CIB5); kept in white light for 3 days. The relative LUC activities normalized to the REN activity are shown (LUC/REN, n = 3).

Figure 5. CIB1 interacts with CIBs.

(A) Fluorescent microscopy images showing that CIB2, CIB4 and CIB5 (Green) all co-localize with CIB1 (Red) in the nucleus. (B) Bimolecular fluorescence complementation assays of the in vivo protein interaction. Leaf epidermal cells of N. benthamiana were cotransformated with cCFP–CIB1 and nYFP , or nYFP-CIB1, or nYFP-CIB2, or nYFP-CIB4, or nYFP-CIB5. BF, Bright Field; Merge, overlay of the YFP and bright field images.

Figure 6. CIB heterodimers bind to the non-canonical E-box sequence of the FT promoter.

(A–C) Competitive electrophoretic mobility shift assay (EMSA) showing binding of CIB2 (A), CIB4 (B), and CIB5 (C) to the G-box DNA (canonical E-box) in vitro. Relative amounts of the un-labeled competitive oligonucleotide containing the G-box sequence used in the reactions are indicated on the top. (D–E) An EMSA experiment showing association of the CIB heterodimers, but not monomers, with the non-canonical E-box DNA of the FT promoter (region c in Figure 4B). The indicated CIB proteins were expressed and purified from E. coli, and incubated with the labeled oligonucleotide containing the E-box DNA of the FT promoter (D) or the same sequence except that the E box was replaced with AAAAAA sequence (E) (F–H) Transient assays show CIBs (CIB1/2/4/5) activation of the FTpro::LUC reporter gene. (F) Control: transiently expressed reporter only, CIB1: reporter and CIB1, CIB2: reporter and CIB2, CIB1 CIB2: reporter, CIB1 and CIB2 together. (G) CIB4: reporter and CIB4, CIB1 CIB4: reporter, CIB1 and CIB4. (H) CIB5: reporter and CIB5, CIB1CIB5: reporter, CIB1 and CIB5. Tobacco leaves were transfected with the reporter and the effectors; kept in white light for 3 days. The relative LUC activities normalized to the REN activity are shown (LUC/REN, n = 3). Error bars indicate SD of three biological repeats.

Figure 7. Immunoblots and luciferase assays showing light regulation of CIBs protein expression.

(A) Transgenic plants expressing the 35S::Myc-CIB4 and 35S::Myc-CIB5 transgenes were grown in long day for 3 weeks, treated with blue light (Blue) for 16 hr, and transferred to dark (Dark), red light (Red, 20 µmol m−2 s−1), or far red light (FR, 5 µmol m−2 s−1) for the indicated time (Left). Alternatively, the 3-week-old plants were first treated with red light for 16 hr (Red), and transferred to blue light (Blue, 35 µmol m−2 s−1) or kept in red light (Red, 20 µmol m−2 s−1) for the indicated time. (B) Immunoblot showing the inhibition of CIB4 and CIB5 degradation in darkness by the proteasome inhibitor MG132. Plants expressing the 35S::Myc-CIB4 or 35S::Myc-CIB5 transgenes were grown in continuous white light (CW) for 3 weeks, leaves were excised and incubated with MG132 (50 µmol/L) or mock solution (0.1% DMSO) in darkness for the indicated time. (C–D) A luciferase assay showing decreased levels of LUC-CIB1, LUC-CIB2, LUC-CIB4, and LUC-CIB5 fusion proteins in the absence of blue light. Transgenic Arabidopsis seedlings expressing the indicated LUC-fusion CIB proteins were grown in continuous blue light for 7 days, and transferred to dark (C) or red light (D) for the indicated time. The luciferase activity was measured by a CCD camera (C) or by a luminometer (D). For (C)), the bioluminescence/20 seedlings were measured by a CCD camera and shown after background subtraction. For (D), the relative levels of LUC activity (REU) was calculated by the formula [LUC^Red/mg^Red]/[LUC^Blue/mg^Blue]. LUC^Blue and LUC^Red: luciferase activity of dark- or blue light-treated samples, mg^Red and mg^Blue: total proteins (mg) of dark- or blue light-treated samples.