PCR primers and vectors can be found in Additional file 1: Table S3 and Additional file 3: Table S2, respectively. PCR reactions were performed using the Phusion® polymerase (Finnzymes®) with HF-buffer and recommended conditions, unless stated otherwise. Cloned PCR products were sequenced. Restriction enzymes were obtained from Fermentas®, except for AlwNI, AvaI and HpaI which were obtained from New England Biolabs (NEB®).
A λ-phage cDNA library constructed from auxin-treated Arabidopsis roots  was used as a template for the amplification of the full length coding sequences of DAYSLEEPER, RHA1 and SNX1 YFP:SYP61 was obtained from Prof. P. Pimpl .
Binary vectors for promoter analysis using the gusA reporter gene
DAYSLEEPER promoter constructs were made in pCAMBIA1304 (CAMBIA foundation) to obtain promoter-reporter gene fusions. First, to separate the DAYSLEEPER promoter from promoter elements present on the pCAMBIA1304 vector, λ phage HindIII DNA marker (New England Biolabs®) was digested using KpnI and BamHI and the 5 kb fragment was cloned into the respective sites in the multiple cloning sites (MCS) of pCAMBIA1304, resulting in pCAMBIA1304λ. Using primer combination MK3 and MK4, 6.1 kb of the DAYSLEEPER upstream region was amplified from genomic DNA. This fragment was subsequently cloned into pJET1.2 (Fermentas®), giving rise to pJET1.2 6.1 kb pDAYSLEEPER. Using NcoI and EcoRI, the 35S promoter of the pCAMBIA1304λ vector was replaced by 3.6 kb of the DAYSLEEPER promoter, resulting in pSDM4328. The same was done using the enzymes NcoI and XbaI, giving rise to a pCAMBIA1304λ vector with 1 kb of DAYSLEEPER upstream sequence cloned in its MCS, resulting in pSDM4327. In both plasmids (pSDM4327 and pSDM4328) the mGFP5:gusA coding sequence is preceded by DAYSLEEPER upstream sequence, which in turn is spaced from elements present in the pCAMBIA backbone by a 5 kb stretch of λ DNA.
Binary vectors for DAYSLEEPER complementation and YFP fusions
DAYSLEEPER coding sequence was amplified using RedTAQ (Sigma Aldrich®) and primers PB1 and PB2 and cloned into pGEMtEASY (Promega®) to give rise to pGEMtEASY::At3g42170 (pSDM2099) and cloned into pAS2-1 (CLONTECH) to give rise to pSDM2304. In order to delete the central region of DAYSLEEPER, pSDM2304 was digested with Age1 and AlwNI (New England Bioscience; NEB®), blunted with T4 polymerase (Fermentas®) and ligated, giving rise to a shortened DAYSLEEPER coding sequence (Δ149-589) (pSDM4415).
To create a C-terminal deletion (Δ478-665), pSDM2099 was digested with the restriction enzyme AvaI and subsequently ligated, deleting the sequence between the 2 AvaI sites. The coding sequence was subsequently obtained with NcoI and SmaI and cloned into pAS2-1 (CLONTECH®), resulting in pSDM4416. To join the Δ149-589 and Δ478-665 shortened coding sequences with the native DAYSLEEPER promoter, the pAS2-1 vectors containing the C-terminal and central truncated DAYSLEEPER coding sequence were cut using NcoI and HpaI. The 3.6 kb fragment directly upstream of the ATG of the DAYSLEEPER coding sequence was inserted, after having been isolated using the same restriction enzymes. This 3.6 kb fragment was obtained from the DAYSLEEPER upstream fragment described in the paragraph “Binary vectors for promoter analysis using the gusA reporter gene”.
To create an N-terminal deletion of the DAYSLEEPER coding sequence (Δ1-142), PCR primers MK39.1 and MK40 were used to amplify bases coding for amino acid 142 until the stop codon, adding an NcoI restriction site at the 5′end of the fragment and an EcoRI site flanking the stop codon at the 3′end. The resulting PCR fragment was cut with NcoI and EcoRI (NEB®) and used for cloning into the pJET1.2 6.1 kb pDAYSLEEPER vector described in the paragraph “Binary vectors for promoter analysis using the gusA reporter gene”. This plasmid was cut with NcoI and SmaI (NEB®) and ligated with the digested PCR fragment, to give rise to a vector with DAYSLEEPER upstream sequence directly fused to the N-terminal (Δ1-142) truncated coding sequence of DAYSLEEPER.
Gateway cloning of binary vectors
Using Gateway-compatible primers the promoter::coding sequence fusions described above were amplified. This was performed using a slightly modified standard PCR protocol using Phusion polymerase and HF-buffer (Finnzymes®). The annealing temperature was set at 65°C for all reactions. The primers used to amplify the different fragments can be found in Additional file 3: Table S2. PCR reactions were performed on ~0.5 ng plasmid template per reaction, except for the amplification of the native DAYSLEEPER upstream region and coding sequence, which were amplified directly from genomic DNA. All PCR fragments were subsequently cloned into the vector pDONR207 (Invitrogen®), using BP Clonase II (Invitrogen®). The resulting pENTR clones were recombined with a binary destination vector. The three DAYSLEEPER versions, Δ1-142, Δ149-589 and Δ478-665 with DAYSLEEPER upstream sequence were recombined into pEARLEYGATE301 , using LR Clonase II (Invitrogen®), giving rise to pSDM4323 to 4325. The full length genomic sequence of the DAYSLEEPER locus was recombined into pGREEN179YFP:HA  following the same method, giving rise to pSDM4322.
Gateway cloning of protoplast vectors
The pENTR clones described above were cloned into a pART7-derived plasmid containing the appropriate Gateway cassette in frame with a fluorophore coding region [24, 25], using LR Clonase II (Invitrogen®), resulting in pSDM4337 and pSDM4341. For N-terminal fusions to fluorophores, HindIII fragments of the pSITEII 2C1 and 6C1 vectors  containing the expression cassettes were cloned into HindIII digested pSY vectors [18, 27]. This gave rise to the pSYSAT6 2xp35S TagRFP Gateway and pSYSAT6 2xp35S Cerulean Gateway vectors, respectively (pSDM4366 and pSDM4376).
Cloning of the DAYSLEEPER coding sequence into the pSY vectors
For the Bimolecular fluorescence-complementation assay in Arabidopsis protoplasts, the DAYSLEEPER coding sequence was isolated and restriction sites added (see: Additional file 3: Table S2) using PCR, cloned into pJET1.2 (Fermentas®) and sequenced. The DAYSLEEPER coding sequence was isolated from pJET1.2 using the appropriate restriction enzymes and subsequently cloned into the pSY728, 735, 736 and 738 vectors  (Additional file 1: Table S3 and Additional file 3: Table S2).
Seedlings were grown in vitro for 2 weeks, after being transferred to soil. Plants were grown with 12 hours of light at 21°C. Samples of Arabidopsis thaliana Col-0 were collected, flash-frozen in liquid nitrogen and stored at −80°C. The tissue was ground under liquid nitrogen in a TissueLyser II apparatus (Qiagen®). RNA was isolated with the RNeasy Mini Kit (Qiagen®) and 1 μg was treated with DNase I (Ambion®), according to the recommended protocol, with the addition of 0.5 μL RNasin (Promega®) per reaction. From each sample, 0.5 μg was used for subsequent random-primed cDNA synthesis, using an iScript cDNA kit (BioRad®). qRT-PCR was performed on 1 μl 20x diluted cDNA, using a standard PCR reaction mix for Phusion DNA polymerase (Finnzymes), with the addition of 1.25 μL 500x diluted SYBR Green (BioRad®) in DMSO. To measure DAYSLEEPER transcript levels, primer combination MK1/MK2 was used. Transcript levels were normalized against expression of the housekeeping gene β-6-TUBULIN (At5g12250). Primers were adopted from Czechowski et al. (Additional file 1: Table S3). Experiments were performed in triplicates on a Chromo4 Real-Time PCR Detection system (Biorad®). Data were processed using the Opticon Monitor 3.1 software (Biorad®) and the GeNorm normalization procedure .
Histochemical staining of gusA expressing plants
Seedlings of 10 days old were grown on solid ½ MS medium  and stained with bromo-4-chloro-3indolyl-Beta-D-glucuronic acid (X-GLUC). Organs (eg. flower buds, leaves, siliques) of mature plants grown on soil were cut off and stained with X-GLUC staining. Seedlings and various tissues were fixed in 90% acetone for 1 hour at −20°C, washed three times in 10 mM EDTA, 100 mM sodium phosphate (pH7.0), 2 mM K3Fe(CN)6 and subsequently stained for 2 h in 10 mM EDTA, 100 mM sodium phosphate (pH7.0), 1 mM K3Fe(CN)6, 1 mM K4Fe(CN)6 containing X-GLUC (Duchefa®). Tissue was post-fixed in ethanol-acetate (3:1), cleared in 70% ethanol and stored in 100 mM sodium phosphate (pH7.0).
Arabidopsis plant, protoplast transformation and microscopic analysis
Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used for floral dip transformation according to Clough and Bent, 1998 . Arabidopsis thaliana (Col-0) suspension cells were used to isolate and transform protoplasts . Protoplasts were observed after 16–18 hours of incubation at 25°C in the dark on a Zeiss Observer (Zeiss®) confocal microscope.
Observation of fluorescent constructs in Arabidopsis tissues and protoplasts
Seedlings were taken directly from ½ MS solid plates and observed on a Zeiss Imager confocal microscope (Zeiss®) prepared on a glass slide with cover slip . Older plants were dissected using a razor blade to allow observation of tissues using a glass slide and cover slip.
Fluorescent signals were visualized using a 63x oil objective on the Zeiss Imager and a 63x water objective with the Zeiss Observer confocal microscope. An Argon laser at 514 nm for excitation and a 530/600 nm band pass emission filter was used for GFP and YFP signals. FM4-64 was also excited with the 514 nm Argon laser and the emission was collected using a 530/600 nm band-pass filter. Cerulean was excited using a 458 nm laser and the emission was collected using a 475/525 nm band pass filter. TagRFP was visualized using a 543 nm laser and a 560/615 nm band pass filter. Images were processed using ImageJ  and Adobe Photoshop CS5 (Adobe®).