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In this work, the effect of ethylene on flower opening of cut …

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Materials and Methods
- Transcriptional regulation of ethylene receptor and CTR genes involved in ethylene-induced flower opening in cut rose (Rosa hybrida) cv. Samantha


Plant materials
Cut rose (Rosa hybrida) cv. Samantha, a cultivar whose opening is accelerated by ethylene treatment (Cai et al., 2002; Tan et al., 2006), was obtained from a commercial grower in Beijing. It was harvested at flower opening stage 2, and then placed immediately in tap water. Flower opening stages were described previously in Wang et al. (2004) and Ma et al. (2005): stage 0, unopened bud; stage 1, partially opened bud; stage 2, completely opened bud; stages 3 and 4, partially opened flower; stage 5, fully opened flower with anther appearance (yellow); stage 6, fully opened flower with anther appearance (black). Within 1 h of harvest, the flowers were delivered to the laboratory; after the stems were cut to a length of 25 cm under water, the flowers were placed in deionized water.

Ethylene and 1-MCP treatments
The effects of different ethylene (2–20 ppm) and 1-MCP (0.5–2 ppm) concentrations on rose flower opening were tested previously, and stable and repeatable results were obtained with 10 ppm ethylene and 2 ppm 1-MCP treatment. Therefore, for ethylene and 1-MCP treatments, flowers were sealed in chambers (64 l) with 10 ppm ethylene or 2 ppm 1-MCP at 25 °C for different times. For competitive experiments, flowers were treated by ethylene for 12 h or 24 h prior to 24 h 1-MCP treatment, or treated by 1-MCP for 12 h or 24 h prior to 24 h ethylene treatment. Control flowers were sealed with an air atmosphere. After treatments, flowers were placed in a vase with deionized water and under controlled conditions at 23–25 °C, 30–40% relative humidity, and a 12/12 h light/dark photoperiod at an illumination of ~40 µmol m–2 s–1. For each treatment, 10 flowers were randomly chosen for morphological observation.

Ethylene measurements
Petals of each individual flower were collected and placed in an airtight container (0.3 l). The containers were capped and incubated for 20 min at 25 °C. Then a head space gas sample of 1 ml was withdrawn, using a gas-tight hypodermic syringe, and injected into a gas chromatograph (GC 17A, Shimadzu, Kyoto, Japan) for ethylene concentration measurement. The gas chromatograph is equipped with a flame ionization detector and an activated alumina column. All measurements were performed with five replicates.

RNA extraction and northern blot analysis
Total RNA from petals was extracted using the hot borate method based on the method of Wan and Wilkins (1994) and with a modification. Briefly, petals were ground with liquid nitrogen and were homogenized with the preheated extraction buffer (200 mM sodium tetraborate decahydrate, 30 mM EGTA, 1% deoxycholic acid sodium salt, 10 mM DTT, 2% PVP 40, 1% NP-40) at a rate of 2–2.5 ml g–1 FW. After addition of Protease K (Merck), the extract was incubated at 42 °C for 1.5 h; then the extract was centrifuged at 12 000 g, 4 °C for 15 min. RNA was precipitated overnight with 2 M LiCl, washed by 2 M LiCl, dissolved in 1 M TRIS-Cl (pH 7.5). Then the RNA was precipitated with 2.5x volumes of 100% ethanol at –80 °C for 2 h.

Ten micrograms of total RNA was fractionated on 1.2% agarose gel containing 2.5% formaldehyde (v/v). Then the RNA was transferred onto a nylon membrane and fixed with a UV cross-linker (Spectroline, USA). DIG-labelled probes were generated by PCR using a PCR DIG probe synthesis kit (Boeringer Mannheim, Germany) with specific primers. The sequences of these primers have been described previously: Rh-ACS1, 2, 3 and Rh-ACO1 in Ma et al. (2005); RhETR1, RhETR3, RhETR5, RhCTR1, RhCTR2, RhEIN3-1, and RhEIN3-2 in Tan et al. (2006). The accession numbers of the genes studied are as follows: Rh-ACS1, AY061946; Rh-ACS2, AY803737; Rh-ACS3, AY803738; Rh-ACO1, AF441282; Rh-ETR1, AY953869; Rh-ETR3, AY953392; Rh-ETR5, AF441283; Rh-CTR1, AY032953; Rh-CTR2, AY029067; Rh-EIN3-1, AF443783; Rh-EIN3-2, AY919867.

Hybridization was carried out overnight at 46 °C for ACO, ethylene receptors, and EIN3; and at 43 °C for ACS and CTRs. The membranes were washed twice in 2x SSC at 37 °C and twice in 0.1x SSC for 30 min at 59 °C for ACO, ethylene receptors, and EIN3; and at 56 °C for ACS and CTR transcripts. The membranes were subjected to a chemiluminescent reaction with CDP-StarTM according to the manufacturer's protocol (DIG-Detection System, Boeringer Mannheim), and then exposed to Fuji Medical X-ray film.

In this work, one flower was regarded as an independent sample. Three flowers were taken as three replicates at each time point, and total RNA was extracted from the petals of each flower separately. The three RNA samples from each time point were then subjected to northern hybridization independently. Representative results are demonstrated here. All experiments were performed twice in 2003 and 2004.

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