Transformation of Arabidopsis and plant growth
The Spcdc25 construct, BTX::Spcdc25, comprising the Spcdc25 ORF driven by an attenuated 35S CaMV promoter as described in , was used to transform Arabidopsis thaliana cv. Columbia plants by the floral dip method . Expression of the transgene was checked in several independent transformant lines by RT-PCR using primers specific for the transgene P7 5'-TTAGGTCCCCTTCTCCGATG and P101 5'- TCAATGAGTCCTCCCTTCACG). Two lines were selected for their expression profile (BTX::Spcdc25 line 9 and line 10). Initial experiments showed that the phenotype of the two lines was very similar and hence further experiments were performed only with line 9. From here on, the genotypes used in this study are referred to as WT (wild type) or Spcdc25 (BTX::Spcdc25 expressing) plants.
For root measurements, seeds were surface sterilised and sown aseptically 1.5 cm apart onto Murashige and Skoog (MS) medium  agar in 90 mm diameter Petri-dishes. Seedlings were stratified at 6°C for 24 h and grown vertically at 21°C with 16 h light (fluence rate = 300 μm m-2 s-1) 8 h darkness in a Sanyo-Gallenkamp Arabidopsis chamber. To score lateral root primordia, seedlings were fixed in 3 to 1 absolute ethanol to glacial acetic acid and Feulgen stained . Primary root length, numbers of lateral roots and lateral root primordia were recorded using a stereo dissecting microscope (Nikon Z100). Additionally, hypocotyls were dissected from 24 day old seedlings and the number of adventitious roots were scored.
Analysis of root apical meristem phenotypes
Ten day old seedlings grown as above were used for the RAM analyses. Roots were fixed and mounted on slides in 8:3:1 chloral hydrate:distilled water: glycerol taking care to apply a coverslip gently . Cell length, width and number were measured in three tissues of the RAM: epidermis, cortex and stele using a ZeissAxiophot set for D.I.C. interfaced to Image analysis software (PixiLINK (C) Capture S.E.). Measurements were taken along longitudinal cell files of epidermis, cortex and mid-stele until the parameter spanned a cell that suddenly increased its cell length/width substantially compared with the previous one. For all genotypes, and for all tissues, this was between a 1.40- to 1.95-fold increase. This was the transition point beyond which those cells began to elongate substantially and is taken to be the basipetal border of the promeristem for each tissue. The cellular measurements were undertaken at either x20 or x40. Images of whole root tips were captured using a low power, x10 objective.
For confocal images, roots were prepared as described in  and visualised using a Leica TCS SP2 (Spectral Confocal and Multiphoton System) confocal scanning laser microscope interfaced to ImageJ software (ImageJ64).
RNA extraction and real time RT-PCR
RNA extractions from roots of Spcdc25 and WT seedlings, grown as described above, were performed using 2 ml TRI reagent (SIGMA-ALDRICH, Dorset, UK) according to the manufacturer's protocol, following grinding to a powder in liquid nitrogen using a mortar and pestle. RNA was treated with RQ1 Dnase (PROMEGA, Southampton, UK) to remove residual genomic DNA. cDNA was synthesised as described in .
Seedling root cDNA (30 ng) was amplified in qRT-PCR reactions in a 20 μl total volume containing: 10 μl 2× PowerSYBR Green PCR Master Mix (Applied Biosystems); 400 nM of each primer; 30 ng cDNA. Reactions were performed in triplicate with the following cycles: 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min. To test primer specificity, melting curve analysis (from 60°C to 95°C with an increasing heat rate of 0.5°C s-1) was performed after amplifications.
Relative quantification of gene expression data was carried out with the 2-DDCT or comparative CT method . Expression levels were normalized with the CT values obtained for the housekeeping gene UBQ10 (At4G05320F, 5'- CACACTCCACTTGGTCTTGCGT-3'; At4G05320R, 5'- TGGTCTTTCCGGTGAGAGTCTTCA-3'; . The primers used for qRT-PCR of target genes ERF/AP2 (At5g61590), ACC oxidase (At1g12010), EBF1 (At2g25490) and AHK3 (At1g27320) are listed in Additional file 6.
Messenger RNA was amplified from purified total RNA using the MessageAmp aRNA kit (AMBION, Huntingdon, Cambs, UK) according to the manufacturer's protocol from 5 μg extracted total RNA. The CyScribe Post-Labelling Kit (AMERSHAM BIOSCIENCES, Little Chalfont, Bucks. UK) was used to label 1 μg aRNA, with either Cy3 or Cy5 according to the manufacturers' instructions, using 1 μg of aRNA. Each labelled RNA pair was freeze-dried and resuspended in 50 μl of hybridisation buffer containing 25% formamide, 5 × SSC, 0.1% SDS, 0.5 mg/ml poly(dA), 0.5 mg/ml yeast tRNA. CATMA v 3 arrays comprising 24 000 gene probes (; http://www.catma.org) were hybridised with the labelled RNA which was heated to 95°C for 5 min and applied to the microarray slide surface. A second microarray slide was lowered over the labelled RNA and the slides were hybridised back-to-back overnight in a humid chamber at 42°C. Four slides were used for hybridisations including a dye swap for each probe pair. Following hybridisation, the slides were washed and scanned using an Affymetrix 428 array scanner with the supplied software (AFFYMETRIX, Santa Clara, CA, USA) at 532 nm (Cy3) and 633 nm (Cy5). Scanned images were quantified using Imagene version 5 software (BIODISCOVERY, El Segundo, CA, USA). Spot quality labelling (flags) was defined for empty spots with a signal strength threshold of 1, and for shape regularity with a threshold of 0.4. The median signal intensity across each spot and the median background intensity were calculated in both channels, and these data were exported into GeneSpring version 6 (AGILENT, Santa Clara, CA, USA). Background intensity was subtracted from spot intensity for both channels giving the background-corrected spot intensity.
Data analysis was carried out using the GeneSpring microarray data analysis package. Ratios between mutant and wild type expression were calculated and levels for the four replicates were averaged. Genes showing an average ratio above 2 or below 0.5 were selected and, following a Benjamini and Hochberg false discovery rate test (BHFDR), genes with P value > 0.05 were excluded.
Hypocotyl and ethylene measurements
WT and Spcdc25 seeds were surface sterilised and stratified as above and seedlings grown in either continuous dark or 16 h L/8 h D at 21°C. Hypocotyl length was recorded at 3, 4 and 7 days following germination. Effects of exogenous ethylene were measured by sealing plates with Nescofilm and injecting 0, 10 or 100 ppm ethylene through a predrilled, sealed hole in the lid of the Petri dish. Hypocotyl measurements were then made as above. To measure endogenous ethylene, 0.015 g seeds of WT or Spcdc25 were sown onto the surface of 5 ml 1 × MS in 10 ml glass head space vials (Chromacol, Thermo Scientific, Loughborough, UK), seeds were stratified at 4°C for 24 h then grown at 21°C for 10 days in the dark. Headspace concentration of ethylene was measured on a Clarus 500, modified model 2101 analyzer (PerkinElmer, MA, USA), retention time was confirmed using pure ethylene and quantification was calibrated using a standard gas mixture (Scott Specialty Gases, mixture 54).
Cytokinin and IAA measurements
Frozen samples were ground in liquid nitrogen and transferred to Bieleski  solvent (10 ml/g fresh weight). Deuterated standards (100 pmol [13 C6-phenyl]- IAA (Cambridge Isotope Laboratory Inc., Andover, MA, USA) and 10 pmol each for cytokinins ([2H5]DHZ, [2H5][9R]DHZ, [2H5](9 G)DHZ, [2H6]iP, [2H6][9R]iP, [2H6](9 G)iP, [2H5](7 G)DHZ, [2H5](OG)DHZ, [2H5](OG)[9R]DHZ, [2H6](7 G)iP), OlchemIm Inc. Olomouc, Czech Rep) were added to the extracts and incubated overnight at -20°C. Cytokinins were purified by solid phase extraction combining DEAE-Sephadex (HG Healthcare, Uppsala Sweden) and RP-C18 (500 mg, Varian, Middelburg, The Netherlands) cartridges. Purified extracts were loaded onto immunoaffinity columns containing monoclonal antibodies against isoprenoid cytokinins (OlchemIm). Cytokinins were analysed by UPLC-TQD (ACQUITY TQD mass spectrometer (Waters, Milford, MA, USA) using an ES + interphase (based on [54, 55]). Chromatograms were analysed using Masslynx and Quanlynx v4.1 software (Waters, Milford, MA, USA) and cytokinin concentrations were calculated according to the principles of isotope dilution.