Rneasy plant kit

Plants were grown from wild-collected seed (locations shown in supplementary table S1, Supplementary Material online). Seeds were germinated on damp filter paper and seedlings were transferred to a soil/vermiculite mix in a growth room set at 19–21 °C with a 16-h photoperiod. To maximize the number of transcripts present, apical tissues were harvested from each plant (inflorescence, stem, and first apical leaf) when the first inflorescence opened, and frozen in liquid nitrogen. Tissue samples were ground while frozen and RNA was extracted with a Qiagen RNeasy plant kit (Qiagen, Crawley, UK) according to manufacturer’s instructions. The extraction procedure included an optional treatment with DNase (Qiagen). 3 µg of RNA per specimen was sent to the Wellcome Trust Centre for Human Genomics, Oxford (WTCHG) for sequencing. Paired-end libraries were prepared individually, barcoded, and then combined prior to sequencing. Libraries were sequenced in a single run using the Illumina Hiseq 2000 sequencing platform to produce 100 base-pair (bp) paired-end reads.

According to the RNA extraction protocols, total RNA of different samples was extracted using a Qiagen RNeasy plant kit according to the manufacturer’s instructions, followed by DNase I treatment, phenol/chloroform extraction and precipitation with ethanol plus sodium acetate. The concentration and purity quotient of RNA were determined by the measurement of 260 nm absorbance and 260/280 nm absorbance, respectively. The integrity of total RNA was determined by 2% agarose gel electrophoresis and ethidium bromide staining. The resulting RNA preparation was incubated at 70°C for 10 min and snap-cooled on wet ice for 5 min and then used as template for reverse transcription- polymerase chain reaction (RT-PCR). cDNA synthesis was carried out according to manufacturer’s instructions. The synthesized cDNA was kept at -80°C until further use.

Microarray analysis was performed on complete arabidopsis transcriptome containing 24,576 GSTs corresponding to 22,089 genes from arabidopsis [30 (link)]. (Hilson et al., 2004). Three independent biological replicates were performed. For each biological replicate we pooled stems from 4 to 6 plants to generate each sample. We collected the first 2 cm at the base of a 20 cm-long floral stems. The plants were grown in a greenhouse under long-day conditions. Total RNA was extracted with the RNeasy Plant Kit (Qiagen) according to the manufacturer’s instructions. For each comparison, one technical replicate with fluorochrome reversal was performed for each biological replicate (i.e., four hybridizations per comparison). We labeled cRNAs with Cy3-dUTP or Cy5-dUTP (Perkin-Elmer-NEN Life Science Products) and performed hybridization and scanning of the slides as previously described [31 (link)]. (Lurin et al., 2004).

To compare the arr1/10/12 triple mutant with the wild type, three-days-old seedlings were collected without any treatment. For cytokinin treatment, 10 µM 6-BA or an equal volume of ethanol was applied to each sample. RNA was isolated using RNeasy plant kit (Qiagen, cat #74904) and libraries were prepared by NeoPre Library Prep System (Illumina). Alignments were done by Tophat 2(v2.0.8, using TAIR10, Bowtie 2, and default parameters)^{79 (link)} and differential expression was called by CuffDiff (Cufflinks v2.1.1, using TAIR10 and default parameters)^{80 (link)}. The significantly differentially expressed genes used 1.6-fold change and q-value <= 0.05 as cutoff.

To analyze the genomic DNA for integration of the J1-1 gene, pepper genomic DNA was isolated using a DNeasy Plant Maxi Kit (Qiagen, Hilden, Germany) as described by the manufacturer. For Southern blot analysis, 15 µg of each DNA sample was digested with EcoRI and separated on a 1.0% (w/v) agarose gel. The digested DNA was then transferred to a nylon membrane and hybridized with HPT or J1-1 gene probes that was labeled with [α³²P] dCTP using the Rediprime II Random Prime Labeling System (Amersham Biosciences, UK). After hybridization, the membranes were exposed at −80°C on Kodak XAR-5 film (Kodak, Rochester, NY) using an intensifying screen.
For Northern blot analysis, total RNA was extracted from the pepper fruits using a RNeasy Plant Kit (Qiagen, Hilden, Germany). 10 µg of the total RNA was separated on 1.2% denaturing agarose gels and blotted onto a Hybond N⁺ membrane (GE Healthcare, Buchinghamshire, UK). The blots were then hybridized with [α³²P] dCTP-labeled respective probes that were amplified by PCR. The primers used for probes were shown in Table S1.

Samples were prepared as described above. Total RNA was extracted from the roots of dmas-kd1 or WT plants using an RNeasy Plant Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. Using ReverTra Ace qPCR RT Master Mix with genomic DNA Remover (Toyobo, Tokyo, Japan), contaminated genomic DNA was removed from the total RNA, and first-strand cDNA was synthesized. For RT analysis, the primers for dmas-kd1 and dmas-kd2 were forward 5′-GAGGAGGAGAGGCAGAGGAT-3′ and reverse 5′-TCAACACGATCGTCAAGAGC-3′. The primers used for the qPCR of OsNAS1–3, OsNAAT1, OsYSL15, OsYSL2, OsIRT1, and OsIRO2 were as described previously by Kobayashi et al. (2009) (link). The primers used for the qPCR of TOM1 and OsActin1 were as previously described by Nozoye et al. (2011) (link). The fold-change between two samples was calculated according to the comparative CT method (Schmittgen and Livak, 2008 (link)) and expressed as copies/OsActin 1.

RNA for quantitative PCR analysis was obtained from 30-day-old plants. The full rosette was harvested by grinding in liquid nitrogen, and RNA was obtained using the Qiagen RNeasy Plant kit from approximately 100 mg tissue (fresh weight). One μg total RNA was used in 20 μL reverse transcriptase reactions (Roche) using manufacturer’s instructions and a poly dT₁₈V anchored primer at 48°C. cDNA synthesis reactions were halted after one hour by heating at 70°C and then diluted 1:10 (v/v) with pure water. One μL was used as template in a PCR that also included 10 μL 2X SYBR Green mix (Roche), 0.6 μL each forward and reverse primers (10 μM), and pure water for a final volume of 20 μL. Primers DXR-F (5’- A G T A G C G G A T G C G T T G A A G C) and DXR-R (5’- G C G G A T G A A T G A C A A T C T C T A T A T C G) were used in these experiments. cDNA loading in individual reactions was normalized to the levels of APT1 and RP2ls genes, whose sequence and stability under these conditions were previously reported [36 (link)]. Six individual plants were used for each transgenic or wild type line and each biological replicate was analyzed in three technical replicates for each gene of interest or normalizer. Relative fold calculations were performed using the efficiency corrected model [37 (link)].