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Functional characterization of tomato Sl-IAA3 and Sl-hls genes. Role ...

Functional characterization of tomato Sl-IAA3 and Sl-hls genes. Role ...

Chapitre II:

Chapitre II: Sl-IAA3, a Tomato Aux/IAA at the Crossroads of Auxin and Ethylene Signalling containing 1 µM NPA and the phenotypes affecting root and leaf growth were observed on young 19-day-old seedlings. For qRT-PCR expression studies, 21- day-old tomato seedlings were harvested and treated with auxin (20 µM IAA for two hours) in presence or absence of 1-MCP (Agrofresh, USA), the ethylene perception inhibitor (1 µL L -1 ) applied 16h prior to auxin treatment. The tissues were then immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. For GUS analysis, 21-day-old tomato seedlings and sections of mature green fruit (Vibratom, Leica VT 1000 S, Vetzlar, Germany) were incubated for 2 h in 50% MS buffer with or without 20 µM IAA. Tissues were then immediately incubated on GUS staining buffer. Ethylene treatments were performed for 5 h in sealed glass boxes. Tomato fruit at the mature green (MG) stage were treated with 50 µL L -1 ethylene and control fruits were exposed to air alone. 1-MCP (1 µL L -1 ) was applied to fruit at breaker stage (Br) for 16 h at room temperature. Following treatment, the tissues were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Ethylene treatment (10 µL L -1 ) was also performed on 5-day-old etiolated ProIAA3:GUS transformed seedlings. Non-treated seedlings grown under the same conditions as for the control. The samples were immediately incubated on GUS buffer. Ethylene treatment of light grown plants was performed by sealing the WT and AS-IAA3 plants in airtight chambers and injecting ethylene to a final concentration of 50 µL L -1 for 16 h. Histochemical GUS Analysis Transgenic lines expressing the GUS reporter gene under the control of either the Sl-IAA3 promoter (ProIAA3:GUS ), the Sl-HLS promoter (ProHLS:GUS) or the DR5 synthetic promoter (DR5:GUS) were incubated at 37°C for 5 to 15 hours with GUS staining solution containing 100 mM sodium phosphate buffer, pH 7.2, 10 mM EDTA, 0.1% Triton and 1 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid to reveal GUS activity. Following GUS staining, samples were washed several times with a graded ethanol series to extract chlorophyll. Assessment of Apical Hook Curvature 79

Chapitre II: Sl-IAA3, a Tomato Aux/IAA at the Crossroads of Auxin and Ethylene Signalling Sterilized seeds were placed on MS agar medium plates and left in the dark for 2 days at 4°C. The vernalized seeds were then placed at 25°C. The level of apical curvature was assessed on 5-day-old dark-grown seedlings using a scale ranging from stage 0, corresponding to total absence of hook, to stage 7 corresponding to maximal exaggerated hook. RNA Extraction and Quantitative RT-PCR RNAs were extracted from various tomato tissues according to Zegzouti et al. (1999). DNase-treated RNA (2 µg) was then reverse-transcribed in a total volume of 20 µl using the Omniscript Reverse Transcription Kit (Qiagen, Valencia, CA, USA). Quantitative Real-Time PCR was performed using cDNAs corresponding to 2.5 ng of total RNA in a 10 µl reaction volume using the SYBR Green PCR Master Mix (PE-Applied Biosystems, Foster City, CA, USA) on an ABI PRISM 7900HT sequence detection system. PRIMER EXPRESS software (PE-Applied Biosystems) was used to design gene-specific primers. The primer sequences are listed in supplemental Table 2. Actin was used as a reference gene with constitutive expression in various tissues. Real-Time PCR conditions were as follow: 50°C for 2 min, 95°C for 10 min, then 40 cy cles of 95°C for 15 s and 60°C for 1 min and finally one cycle at 95°C for 15 s an d 60°C for 15 s. For all Real- Time RT-PCR experiments, two biological replicates were made and each reaction was run in triplicate. For each sample, a Ct (threshold constant) value was calculated from the amplification curves by selecting the optimal ∆Rn (emission of reporter dye over starting background fluorescence) in the exponential portion of the amplification plot. Relative fold differences were calculated based on the comparative Ct method using the Sl-Actin-51 as an internal standard. To determine relative fold differences for each sample in each experiment, the Ct value for Sl-IAA3 gene was normalized to the Ct value for Sl- Actin-51 and was calculated relative to a calibrator using the formula 2 -∆∆ Ct . 80

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