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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 Isolation of the Sl-IAA3 Genomic Clone Genomic DNA was extracted from tomato leaf tissue using a DNA extraction Kit (Promega, Lyon, France) and was treated with RNase for 10 min at 37°C. PCR reactions were performed on genomic DNA using primers designed from the cDNA sequence. The amplified fragments were cloned and fully sequenced. Comparative analysis between the genomic clone and cDNA sequences allowed the delimitation of introns and exons. Isolation of the Sl-IAA3 Promoter The Universal Genome Walker Kit (Clontech Laboratories, Inc., Palo Alto, CA, USA) was used to isolate the Sl-IAA3 gene promoter region. Each tomato genomic DNA aliquot was digested with four restriction enzymes, DraI, EcoRV, PvuII and StuI. Adaptor DNA which contained two primer-binding sites for AP1 and AP2 primers provided by the Genome Walker Kit was linked to both ends of the restricted tomato DNA fragment at 16°C. Two pri mers, AP1 (5’- GTAATACGACTCACTATAGGGC-3’) and AP2 (5’-ACTATAGGGCACG CGTGGT-3’) paired with two Sl-IAA3 gene specific antisense primers were used for PCR amplification. The tomato genomic DNA fragment with adaptors at both ends was used as a template for the amplification of the promoter region. The PCR product was cloned into the pGEMT-easy vector (Promega) and fully sequenced. The Sl-IAA3 promoter was then fused to the GUS reporter gene in the plp100 binary vector (Szabados et al., 1995) and used for stable tomato transformation. DNA sequences were analyzed with BLAST network services at the National Center for Biotechnology Information (Altschul et al., 1997) and by PlantCARE (Lescot et al., 2002). Transient Expression Using a Single Cell System For nuclear localization of the Sl-IAA3 fusion protein, the coding sequence of Sl- IAA3 was cloned as a C-terminal fusion in frame with GFP (Green Fluorescent Protein) into the pGreen vector (Hellens et al., 2000) and expressed under the control of the 35S CaMV, a Cauliflower Mosaic Virus promoter. Protoplasts for 77

Chapitre II: Sl-IAA3, a Tomato Aux/IAA at the Crossroads of Auxin and Ethylene Signalling transfection were obtained from suspension-cultured tobacco (Nicotiana tabacum) BY-2 cells according to the method described previously (Leclercq et al., 2005). Protoplasts were transfected by a modified polyethylene glycol method as described by Abel and Theologis (1994). Typically, 0.2 mL of protoplast suspension (0.5 x 10 6 ) was transfected with 50 µg of sheared salmon sperm carrier DNA and 10 µg of either 35S-IAA3:GFP or 35S:GFP (control) plasmid DNA. Transfected protoplasts were incubated 16 h at 25°C and analyzed for GFP fluorescence by confocal microscopy. Light micrographs and fluorescence images are merged to illustrate the different location of the two proteins. For co-transfection assays, the coding sequence of Sl-IAA3 was cloned into the pGreen vector and expressed under the control of the 35S CaMV promoter. Aliquots of protoplasts (0.5 x 10 6 ) were transformed either with 10 µg of the reporter vector alone containing the DR5 synthetic auxin-response element fused to the GFP reporter gene (gift from Prof. K. Palme, Freiburg, Germany) or in combination with 10 µg of the effector plasmid, allowing the constitutive expression of the Sl-IAA3 protein. Transformation assays were performed in three independent replicates. After 16 h incubation in the presence or absence of 2,4-D (50 µM), GFP expression was analyzed and quantified by flow cytometry (FACS Calibur II instrument, BD Biosciences, San Jose, CA). For each sample, 100 to 1000 protoplasts were gated on forward light scatter and the GFP fluorescence per population of cells corresponds to the average fluorescence intensity of the population of cells above the background threshold (set arbitrarily based on a zero DNA transformed control, so that all control cells fall below this threshold). Data were analyzed using Cell Quest software. All transient expression assays were repeated at least three times with similar results. Auxin and Ethylene Treatment For auxin dose–response experiments, 8 mm long hypocotyl segments were cut from three-week-old light grown plantlets and were immediately floated in sucrose/MES buffer (1% sucrose [w/v] and 5 mM MES/KOH, pH 6.0). After 2 h pre-incubation, the hypocotyl segments were randomly distributed to fresh buffer solutions with or without NAA (0, 1, 10, 100 µM) and were measured following 2 h of incubation. For NPA treatment, the seeds were sown on MS medium 78

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