| Property | Value |
|---|---|
| BioDare ID | 13790787578160 |
| Author | Kim, W.Y. |
| Institution | Ohio State University |
| License | CC_BY |
The circadian clock regulates many aspects of plant development, including hypocotyl elongation and photoperiodic induction of flowering. ZEITLUPE (ZTL) is a clock-related F-box protein, and altered ZTL expression causes fluence rate-dependent circadian period effects, and altered hypocotyl elongation and flowering time. EARLY FLOWERING 3 (ELF3) is a novel protein of unknown biochemical function. elf3 mutations cause light-dependent circadian dysfunction, elongated hypocotyls, and early flowering. Although both genes affect similar processes, their relationship is unclear. Here we show that the effects of ZTL and ELF3 on circadian clock function and early photomorphogenesis are additive. The long period of ztl mutations and ELF3 overexpressors are more severe than either alone. Dark-release experiments showing additivity in phase advances suggest that the arrthymicity caused by ZTL overexpression and that of the elf3-1 mutation arise through independent pathways. A similar additive effect on hypocotyl elongation in red and blue light is also observed. In contrast, ELF3 and ZTL overexpressors act similarly to control flowering time in long days through the CONSTANS/FLOWERING LOCUS T (CO/FT) pathway. ZTL overexpression does not delay flowering through changes in GIGANTEA or FLAVIN-BINDING, KELCH REPEAT, F-BOX levels, but through a ZTL-mediated reduction in CO expression. In contrast, ELF3 negatively regulates CO, FT, and GIGANTEA transcript levels, as the expression of all three genes is increased in elf3-1. The elf3-1 co-1 double mutant flowers much earlier in long days than co-1, although FT message levels remain very low. These results show that elf3-1 can derepress late flowering through a CO-independent mechanism. ELF3 may act at more than one juncture, possibly posttranscriptionally.
Seedlings were grown for 7 d (70 mmol m-2 s-1 white fluorescent light) in 16-h-light/8-h-dark cycles and harvested on day 8 at the appropriate times. Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s instruction. Except in Figure 6B, transcripts of Actin2, CO, FT, FKF1, and GI were quantified by RT-PCR, followed by DNA gel-blot analysis as described previously (Somers et al., 2004). The primer sequences and annealing temperatures used to amplify each gene are as follows: CO, 48oC, 5'-ACGCCATCAGCGAGTTCC-3' and 5'-AAATGTATGCGTTATGGTTAATGG-3'; FT, 55oC, 5'-ACAACTGGAACAACCTTTGGCAATG-3' and 5'-ACTACTATAGGCATCATCACCGTTCGTTACTCG-3'; GI, 52oC, 5'-CTGTCTTTCTCCGTTGTTTCACTGT-3' and 5'-TCATTCCGTTCTTCTCTGTTGTTGG-3'; and FKF1, 55oC, 5'-GTCGTAACTGTCGATTCCTACA-3' and 5'-ATCTCCAGTGTTCCAGTTATCT-3'. A portion of At3g18780 (ACTIN2; ACT2) was amplified using oligonucleotides 5'-AAAACCACTTACAGAGTTCGTTCG-3' and 5'-GTTGAACGGAAGGGATTGAGAGT-3' with the annealing temperature of 55oC and used as an internal control to normalize the amount of cDNA. The exponential range of amplification was empirically determined for each gene, and 18 cycles were used for Actin2; 22 cycles were used for GI, FKF1, and FT; and 23 cycles for CO. For Figure 6B, transcripts of Actin2 and FT were measured by quantitative RT-PCR, essentially as previously described (Mockler et al., 2004). cDNAs were prepared from DNase-treated (Turbo DNAfree,Ambion) RNA samples using the Omniscript RT kit (Qiagen). Oligonucleotide primers were designed using PRIMER EXPRESS V2.0 software (Applied Biosystems) and were as follows: FT, 5'-CCTTTGGCAATGAGATTGTGTG-3' and 5'-TTCCTGCAGTGGGACTTGG-3'; FD, 5'-TCCCGCGCTAGGAAACAG-3' and 5'-CCTGCAAGTGAGCAACTTCAAG-3'; and ACT2, 5'-ACCTTTAACTCTCCCGCTATGTATGT-3' and 5'-GGCACAGTGTGAGACACACCAT-3'. Expression level was calculated based on standard curves constructed for each primer set and normalized to ACT2 in arbitrary units.
RT-PCR ()
Growth on MS 3% sucrose 0.8% agar for 7.0 days (Kim2005).
| Type | Duration (days) | Cycle (h) | Start | Duration | Spectrum | Source | Intensity |
|---|---|---|---|---|---|---|---|
| diurnal light | 7 | 24 | 0:00 | 16:00 | white | LED | 70 |
| Type | Duration (days) | Cycle (h) | Base (°C) | Warm (°C) | Warm start | Warm duration |
|---|---|---|---|---|---|---|
| constant temperature | 7 | 24 | 22 | -- | -- | -- |
Growth on MS 3% sucrose 0.8% agar for 1.0 days (Kim2005).
| Type | Duration (days) | Cycle (h) | Start | Duration | Spectrum | Source | Intensity |
|---|---|---|---|---|---|---|---|
| diurnal light | 1 | 24 | 0:00 | 16:00 | white | LED | 70 |
| Type | Duration (days) | Cycle (h) | Base (°C) | Warm (°C) | Warm start | Warm duration |
|---|---|---|---|---|---|---|
| constant temperature | 1 | 24 | 22 | -- | -- | -- |