Ito et al. (2008); GI, FKF1, CDF1, CO, FT; 16L:8D, 10L:14D; WT, toc1, prr5, toc1;prr5
| Property |
Value |
| BioDare ID |
13790751359110 |
| Author |
Ito, S. |
| Institution |
Nagoya University |
| License |
CC_BY |
Description
Purpose
In Arabidopsis thaliana, many circadian clock-associated genes have been identified. Among them, the eveningexpressed TOC1 (TIMING OF CAB EXPRESSION 1)
gene plays a role by forming a transcriptional feedback core loop together with the morning-expressed CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) gene and its homologous LHY (LATE ELONGATED HYPOCOTYL) gene. TOC1 encodes a member of the PSEUDORESPONSE REGULATOR (PRR) family, including PRR9, PRR7, PRR5, PRR3,and PRR1/TOC1. The PRR genes other than TOC1 (or PRR1) also appear to be crucial for certain circadian-associated events. To clarify missing genetic linkages amongst these PRR genes, here we constructed a toc1 prr5 double knockdown mutant. In freerunning circadian rhythms, the resulting toc1-2 prr5-11 mutant plants showed an extremely short period and reduced amplitude phenotype, which was more severe than that of the toc1-2 single mutant plant, suggesting a non-linear genetic interaction between TOC1 and PRR5. Surprisingly, the hallmark early flowering phenotype of toc1-2 in the shortday conditions had been converted to a markedly late flowering phenotype in the long-day conditions, when combined with the prr5-11 allele, which itself showed a subtle flowering phenotype. This unexpected genetic result (i.e. phenotypic sign conversion) suggested that the TOC1 and PRR5 genes are coordinately implicated in a non-linear and closed genetic circuitry. In the toc1-2 prr5-11 double mutant, the diurnal expression profile of CDF1 (CYCLING DOF FACTOR 1) was markedly de-repressed in the evening in the long-day conditions. These and other results of this study led us to propose the novel view that TOC1 might play bipartite roles in the control of flowering time within a closed circuitry; the one is a GI (GIGANTEA)-dependent negative role through CCA1/LHY, and the other is a CDF1-dependent positive role through cooperating closely with PRR5.
Comments
Sample preparation
For real-time PCR, seedlings were grown on MS plates containing 1% sucrose under the conditions of LoD (16 h light/8 h dark) or ShD (10 h light/14 h dark) for 10 or 7 d. They were then harvested (3 h intervals) to quantify mRNA of genes involved in flowering regulation. Total RNA was purified by an RNeasy plant kit (Qiagen, Valencia, CA, USA). To synthesize cDNA, 1 mg of each RNA was reverse transcribed with Rever Tra 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 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 version 3.1 software (Bio-Rad Laboratories). The oligonucleotide primers used in this study have all been described previously (Niwa et al. 2007).
Measurement
Protocol
RT-PCR ()
Experimental conditions
Ito2008_LD
Growth on MS 1% sucrose for 10.0 days (Ito2008_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 |
-- |
-- |
-- |
Ito2008_SD
Growth on MS 1% sucrose for 7.0 days (Ito2008_SD).
Light
| Type |
Duration (days) |
Cycle (h) |
Start |
Duration |
Spectrum |
Source |
Intensity |
| diurnal light |
7 |
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 |
7 |
24 |
22 |
-- |
-- |
-- |
Ito2008_LD
Growth on MS 1% sucrose for 1.0 days (Ito2008_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 |
-- |
-- |
-- |
Ito2008_SD
Growth on MS 1% sucrose for 1.0 days (Ito2008_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
- ito2008.xls
- STANDARD_RAWDATAFILE.txt
- STANDARD_DATAFILE.txt
- ito2008.pdf