Design of synthetic microRNAs
The first step consisted of a BLASTN search  with the target cDNA to identify close homologues. Then, the alignment tool Clustal W  was used to help select sequence regions within candidate target cDNAs that show highest specificity for the chosen target. Candidate smiRNA sequences were selected on the basis that there should be no mismatches between the smiRNA and the mRNA target at the presumed cleavage site around nucleotides 10 and 11 of the smiRNA, and bulges and loops should be limited to one and two nucleotides, respectively, or even be avoided. The 5'end of the smiRNA should show near-perfect complementarity. As next steps, the program RNAhybrid http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/ was used to calculate minimal free energy (mfe) values for the candidate smiRNA:mRNA hybrid structures that should be -25 kcal/mol or below for optimal hybridization and to search for potential off-targets within the complete Arabidopsis transcriptome. Settings were: maximum internal loop size: 2 nt; maximum bulge size: 1 nt; minimal free energy cutoff: -25 kcal/mol; p-value cutoff: 0,001. Depending on the outcome of these tests the candidate smiRNA sequence was adjusted accordingly and the calculations were repeated (See additional file 1: In silico target predictions for smiRNA(CHS) using the program RNAhybrid).
Once the smiRNA sequence was chosen, the sequence of the corresponding smiRNA* was designed in a way that preserved the mfe value and the loop of the original MIR159A backbone within the smiRNA:smiRNA* hybrid structure. Arabidopsis MIR159A was amplified by PCR from genomic Arabidopsis thaliana Col-0 DNA and inserted into the XbaI and blunted SacI restriction sites of pUC19-3'GFP (Figure 1) [27, 41], thereby exchanging the GFP cDNA for pri-miR159a (Primers are given in additional file 6: Oligo nucleotide sequences). This MIR159A sequence, termed pri-miR159a in this work, is much longer than the miR159a precursor, however, it does not contain the transcription start site published by Xie et al. . RNAfold  was then used to check the secondary structure prediction of the resulting entire pri-smiRNA, and the smiRNA* sequence was adjusted and RNAfold analysis repeated if necessary. According to our in silico predictions, however, the pre-miR159a forms a very stable stem-loop structure within pri-miR159a that seems to tolerate different smiRNA:smiRNA* sequence variations without any effect on secondary structure (See additional file 2: Sequences and predicted secondary structures of pri-smiRNAs using the program RNAfold). Finally, primers were designed for overlap-extension PCR  to generate and clone the pri-smiRNA within the endogenous MIR159A primary transcript as backbone  using the "standard" procedure (Figure 1A, 1B). Alternatively, primers containing restriction sites (NheI - smiRNA* sequence and BsrGI - smiRNA sequence) were designed for the "ECV" procedure (Figure 1C; Primer sequences are given in additional file 6: Oligo nucleotide sequences). The ECV plasmid is available upon request. The expression cassettes, including the 35S promoter and the nopaline synthase terminator, flanked by the HindIII and EcoRI restriction sites were then excised and ligated into the HindIII and EcoRI sites of the binary vector pGPTV-BAR .
Plant material and growth conditions
Arabidopsis thaliana plants (ecotype Col-0) were cultivated on soil in a greenhouse at 24°C. They were first grown under short day conditions (8 h light/16 h darkness) for 2-3 weeks and then transferred to long day conditions (16 h light/8 h darkness) until seeds were harvested. Alternatively, surface-sterilized Arabidopsis thaliana seeds were sown on 0.8% agar plates containing 0.5 × Murashige-Skoog basal salts (Duchefa, Haarlem, Netherlands), with (4%) or without sucrose, kept at 4°C in the dark for 3 days for stratification, and then transferred to a light chamber with 16 h of light per day at 22°C until the plant material was harvested for further analysis.
Agrobacterium-mediated transformation of Arabidopsis thaliana
Agrobacterium tumefaciens (GV3101) was transformed with the binary vector pGPTV-BAR carrying the pri-smiRNAs under the control of the 35S cauliflower mosaic virus promoter. A small scale Agrobacteriumpre-culture was grown for two days at 28°C at 200 rpm in 5 ml YEP medium (1% DIFCO Bacto Tryptone (w/v), 1% DIFCO Bacto Yeast Extract (w/v), 0.5% NaCl (w/v)) containing appropriate antibiotics. This was used to inoculate a large scale culture of 500 ml YEP that was grown for two days at 28°C at 200 rpm. Sucrose (5%) and Silwet L-77 (0,02%, Lehle Seeds, USA) were added to the culture that was incubated for another 10 min at 28°C. Plants were dipped upside down in the bacterial solution for 2 min, drained, placed on the side, covered for 24 hours in Saran wrap, and then grown until seed harvest. Seeds of the T1 generation were harvested, T1 transgenic plants were selected by BASTA treatment, and the survivors were grown to harvest T2 seeds.
Small RNA Northern blots
Total RNA from Arabidopsis thaliana plants was isolated using TRI reagent (Molecular Research Center, Cincinnati, USA) and separated on a 17% denaturing polyacrylamide gel containing 7 M urea in TBE buffer (0.9 M Tris, 0.9 M boric acid, 0.02 M EDTA). The RNA was electro-blotted to Hybond N+ nylon membranes (GE Healthcare, UK) for 1 h at 400 mA using a trans-blot transfer cell (Bio-Rad, Hercules, CA, USA) and crosslinked by UV light (StrataLinker 1800, Stratagene, La Jolla, CA, USA). Pre-hybridization and hybridization of the blots with biotinylated single-stranded DNA probes (oligonucleotides that contained the reverse-complementary sequence of the smiRNA) were carried out in PerfectHyb Plus Hybridization Buffer (Sigma-Aldrich Co., St. Louis, MO, USA) at 42°C for 1 h and overnight, respectively. Blots were washed with decreasing concentrations of SSC/SDS (2 × SSC, 0.2% SDS; 1× SSC, 0.1% SDS; 0.5× SSC, 0.1% SDS) at 50°C. Detection was carried out with the Chemiluminescent Nucleic Acid Detection Module (Pierce, Rockford, IL, USA) according to the manufacturers' instructions, and membranes were exposed to BioMax XAR Films (Kodak, USA). Blots were stripped in between hybridizations by 10 min incubation with 1% SDS at 80°C. The second hybridization was with the U6 snRNA probe to detect U6 snRNA as loading control and a 21 nt-long RNA size marker.
Analysis of secondary metabolites in Arabidopsis thaliana
In order to analyze the content of flavonol derivatives in transgenic Arabidopsis thaliana plants, methanolic extracts were isolated from 7-days-old whole seedlings using 80% methanol and 10-15 zirconia beads of 1 mm diameter (Roth, Karlsruhe, Germany). Homogenized samples (Tissue Lyser, Qiagen, Retsch, Germany) were incubated for 10 min at 65°C and centrifuged for 10 min at 4°C at 14000 rpm in a standard table centrifuge. Supernatants were vacuum-dried in a SpeedVac (SPD111-V, Thermo Electron, Waltham, MA, USA) at 60°C. Dried pellets were dissolved in 1 μl of 80% methanol per mg fresh weight starting material. From each sample, 4 μl were used for high-performance thin layer chromatography  and spotted on 10 cm × 10 cm silica-60 HPTLC-plates (Merck, Darmstadt, Germany) as the stationary phase. The chromatography was carried out using ethyl acetate, formic acid, acetic acid and water (100:26:12:12) as the mobile phase in a closed glass tank. Separated compounds were stained by spraying a 1% (w/v) DPBA solution (Diphenyl boric acid-β-aminoethylester, Naturstoffreagenz A, Roth, Karlsruhe, Germany)  in methanol, followed by spraying 5% (w/v) methanolic polyethylene glycol 4000 solution. The stained HPTLC plates were examined under UV light (312 nm) and photographed.
For the visualization of flavonols in whole Arabidopsis seedlings, seeds were germinated and grown for 5 days on filter paper soaked with 3 ppm norflurazon in water (Sigma-Aldrich, Co., St. Louis, MO, USA) under long-day conditions. Bleached seedlings were stained to saturation for at least 1.5 h in a freshly prepared solution of 0.25% (w/v) DPBA in 0.00375% (v/v) Triton X-100. Fluorescence was visualized with a Leica DM5500 B epifluorescence microscope using Leica Filtercube A with an excitation wavelength of 340-380 nm and a 425-nm-long-pass splitter.
For the quantification of the accumulation of anthocyanins, transgenic plants were grown on 0.5 × MS medium enriched with 4% sucrose under long-day conditions to induce the formation of stress anthocyanins. Anthocyanins were extracted from whole 7 day-old seedlings by overnight incubation in acidic methanol (1% v/v HCl 37%, 99% v/v methanol p.a.) with gentle shaking. Samples were centrifuged for 1 min at 14000 rpm and room temperature in a standard table centrifuge and 0.5 ml of the supernatants were used to measure the absorption at 530 nm and 657 nm in triplicates. The relative quantity of anthocyanins per g fresh weight (FW) was calculated according to the equation Q(Ant) = (OD(530)-0,25 × OD(657))/FW [g-1].
Quantitative RT-PCR experiments
Primers for quantitative RT-PCR (qPCR) were designed to flank the predicted smiRNA binding site in the CHS (target) or At1g49390 (off-target) transcripts, respectively, and tested with BLASTN  against the Arabidopsis transcriptome to ensure specificity (Primer sequences are given in additional file 6: Oligo nucleotide sequences). Reverse transcription reactions were carried out in 20 μl volume using 4 μg DNaseI-treated total RNA (DNA-free kit, Ambion, Austin, TX, USA) and Superscript II reverse transcriptase (Invitrogen, Karlsruhe, Germany) according to the manufacturers' instructions. qPCR reactions were performed using the Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen, Karlsruhe, Germany) on a Rotor Gene 6000 Cycler (Corbett Research, Mortlake, NSW, Australia). Data was analyzed according to Pfaffl  and average results of two biological replicates, each with triple samples, are given.
Target Validation experiments
RNA ligase-mediated rapid amplification of 5'cDNA ends (RLM-5'RACE) was performed with RNA that was isolated with TRI reagent (Molecular Research Center, Cincinnati, USA) from Arabidopsis thaliana plants. An RNA primer was directly ligated to total RNA from whole seedlings using T4 RNA Ligase (New England Biolabs, Ipswich, England), and adaptor-ligated total RNA was used for reverse transcription with SuperScript II reverse transcriptase (Invitrogen, Karslruhe, Germany) using oligo dT primers. The cDNA was subjected to nested PCR using 5' adaptor-specific primers and 3' gene-specific primers. PCR products were gel-purified, cloned into TOPO TA vector (pCR2.1; Invitrogen, Karlsruhe, Germany) and then sequenced (Primer sequences are given in additional file 6: Oligo nucleotide sequences).