Nakamichi et al. (2007); FKF1, CDF1, CO, FT; 16L:8D, 10L:14D; WT, prr7;5, prr9;7, cca1;lhy, cca1;lhy;prr7;5
| Property |
Value |
| BioDare ID |
13790777856363 |
| Author |
Nakamichi, N. |
| Institution |
Nagoya University |
| License |
CC_BY |
Description
Purpose
Photoperiodism allows organisms to measure daylength, or external photoperiod, and to anticipate coming seasons. Daylength measurement requires the integration of light signal and temporal information by the circadian clock. In the long-day plant Arabidopsis thaliana, CONSTANS (CO) plays a crucial role in integrating the circadian rhythm and environmental light signals into the photoperiodic flowering pathway. Nevertheless, the molecular mechanism by which the circadian clock modulates the cyclic expression profile of CO is poorly understood. Here, we first showed that the clock-associated genes PSEUDO-RESPONSE
REGULATOR (PRR) PRR9, PRR7 and PRR5 are involved in activation of CO expression during the daytime. Then, extensive genetic studies using CIRCADIAN
CLOCK-ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY) double mutants (cca1/lhy) and prr7/prr5 were conducted. The results suggested that PRR genes act coordinately in a manner parallel with and antagonistic to CCA/LHY, upstream of the canonical CO–FLOWERING LOCUS T (FT) photoperiodic flowering pathway. Finally, we provided evidence to propose a model, in which CCA1/LHY repress CO through GIGANTEA (GI), while PRR9, PRR7 and PRR5 activate CO predominantly by repressing CYCLING DOF FACTOR1 (CDF1) encoding a DNAbinding transcriptional repressor.
Comments
Sample preparation
Seedlings were grown on MS plates containing 2% sucrose under LD or SD. They were harvested to quantify the mRNA of genes involved in flowering regulation. Total RNA was purified by an RNeasy kit (Qiagen, Valencia, CA, USA). Northern blotting analysis was performed as described previously (Nakamichi et al., 2005a). To synthesize cDNA, 1 mg of each RNA was reverse transcribed with ReverTea Ace (TOYOBO, Osaka, Japan) and oligo(dT) primer. The synthesized cDNAs were amplified with iQ SYBR Green supermix (Bio-Rad Laboratories, Hercules, CA, USA) and a primer set using a MiniOpticon real-time PCR system (Bio-Rad Laboratories). The primer sets used in this study were, for CO, 5'-CTACAACGACAATGGTTCCATTAAC-3' and 5'-CAGGGTCAGGTTGTTGC-3'; for GI, 5'-GGGTAAATATGCTGCTGGAGA-3' and 5'-CAGTATGACACCAGCTCCATT-3'; for CDF1, 5'-TTTCCCGACGGTTTTAGAGG-3' and 5'-CCATGCTGTTGCATCTTGGA-3'; and for FKF1, 5'-GAAGTCTTCACTGGCTATCG-3' and 5'-GATCAACCAATGGGTGACG-3'. Primer sets for APX3 and FT were described by Hazen et al. (2005) and Endo et al. (2005), respectively. The following standard thermal cycling program was used for all PCRs: 95oC for 120 s, 40 cycles of 95oC for 10 s, and 60oC for 60 s. Data were analyzed using Opticon Monitor ver. 3.1 software (Bio-Rad Laboratories).
Measurement
Protocol
RT-PCR ()
Experimental conditions
Nakamichi2007_LD
Growth on MS plats 2% sucrose for 10.0 days (Nakamichi2007_LD).
Light
| Type |
Duration (days) |
Cycle (h) |
Start |
Duration |
Spectrum |
Source |
Intensity |
| diurnal light |
10 |
24 |
0:00 |
16:00 |
white |
LED |
90-100 |
Temperature
| Type |
Duration (days) |
Cycle (h) |
Base (°C) |
Warm (°C) |
Warm start |
Warm duration |
| constant temperature |
10 |
24 |
22 |
-- |
-- |
-- |
Nakamichi2007_SD
Growth on MS plates 2% sucrose for 10.0 days (Nakamichi2007_SD).
Light
| Type |
Duration (days) |
Cycle (h) |
Start |
Duration |
Spectrum |
Source |
Intensity |
| diurnal light |
10 |
24 |
0:00 |
10:00 |
white |
LED |
120-150 |
Temperature
| Type |
Duration (days) |
Cycle (h) |
Base (°C) |
Warm (°C) |
Warm start |
Warm duration |
| constant temperature |
10 |
24 |
22 |
-- |
-- |
-- |
Nakamichi2007_LD
Growth on MS plates 2% sucrose for 1.0 days (Nakamichi2007_LD).
Light
| Type |
Duration (days) |
Cycle (h) |
Start |
Duration |
Spectrum |
Source |
Intensity |
| diurnal light |
1 |
24 |
0:00 |
16:00 |
white |
LED |
90-100 |
Temperature
| Type |
Duration (days) |
Cycle (h) |
Base (°C) |
Warm (°C) |
Warm start |
Warm duration |
| constant temperature |
1 |
24 |
22 |
-- |
-- |
-- |
Nakamichi2007_SD
Growth on MS plates 2% sucrose for 1.0 days (Nakamichi2007_SD).
Light
| Type |
Duration (days) |
Cycle (h) |
Start |
Duration |
Spectrum |
Source |
Intensity |
| diurnal light |
1 |
24 |
0:00 |
10:00 |
white |
LED |
120-150 |
Temperature
| Type |
Duration (days) |
Cycle (h) |
Base (°C) |
Warm (°C) |
Warm start |
Warm duration |
| constant temperature |
1 |
24 |
22 |
-- |
-- |
-- |
Files
- nakamichi2007.xls
- STANDARD_RAWDATAFILE.txt
- STANDARD_DATAFILE.txt
- nakamichi2007.pdf