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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 fold after auxin (50 µM of 2,4-D) treatment (Figure 3B), clearly indicating that Sl- IAA3 expression is positively regulated by auxin. Since auxin is known to stimulate ethylene production (Abel et al, 1995), we sought to determine whether the auxin-responsiveness of Sl-IAA3 results from increased ethylene production. Light-grown seedlings were thus treated overnight with 1-MCP (1µL L -1 ) and then incubated in presence or absence of auxin. qRT-PCR analysis revealed that 1- MCP treatment completely abolished the expression of Sl-IAA3 gene in the absence of auxin but only partially reduced Sl-IAA3 transcript accumulation in the auxin-treated plants (Figure 3A). This indicates that the basal expression of Sl- IAA3 is ethylene-dependent and that the auxin-responsiveness of this gene is not mediated by ethylene. ∆∆CT A 4 3 2 1 0 Air IAA 1-MCP 1-MCP+IAA Mean fluorescence (a.u.) 200 150 100 50 0 B ProIAA3:GFP Figure 3. Auxin Responsiveness of the Sl-IAA3 Gene. A. RT-PCR analysis of Sl-IAA3 transcript levels in RNA samples extracted from three week-old light-grown control and auxin treated (20 µM IAA for two hours) seedlings in presence or absence of 1-MCP, the ethylene perception inhibitor (1 µL L -1 1-MCP applied 16h prior to auxin treatment). ∆∆CT on the y axis refers to the fold difference in SI-IAA3 transcript levels relative to the nontreated plantlets. B. Auxin responsiveness of the Sl-IAA3 promoter. Tobacco protoplasts were transformed by a chimeric construct consisting of 1668 bp of the Sl-IAA3 promoter fused to the GFP reporter gene (ProIAA3:GFP) and incubated in the presence or absence of 50 µM of 2,4-D. Transformation was performed in triplicate and in each experiment GFP fluorescence was measured by flow cytometry 16 h after transfection. Values are expressed in arbitrary units (a.u.) ± SE. Sl-IAA3 Is Differentially Expressed in Different Plant Organs and Displays Tightly Regulated Tissue-Specific Expression To gain further insights into Sl-IAA3 expression, we fused the Sl-IAA3 promoter to the GUS (β–glucuronidase) reporter gene (ProIAA3:GUS) and stably introduced it -2,4-D +2,4-D 57

Chapitre II: Sl-IAA3, a Tomato Aux/IAA at the Crossroads of Auxin and Ethylene Signalling into tomato plants. GUS activity driven by the Sl-IAA3 promoter was then assessed in lines homozygous for the chimeric construct. In untreated vegetative tissues, the Sl-IAA3 promoter drove GUS expression exclusively in the leaf vasculature and root tips (Figure 4A to 4C). However, a brief auxin treatment (20 µM for two hours) of light grown seedlings led to a dramatic increase in GUS expression throughout the roots and shoots (Figure 4E to 4G). In mature green fruit, GUS staining was restricted to a narrow band in the placenta exolayer at the junction between the placenta and pericarp tissues (Figure 4D). Once again, ProIAA3:GUS - IAA ProIAA3:GUS ProIAA3:GUS + IAA IAA DR5:GUS DR5:GUS +IAA A B C E F I J M N O P Figure 4. Tissue-Specific Expression of Sl-IAA3 Monitored by GUS Reporter Gene Activity Driven by the Sl-IAA3 Promoter (ProIAA3:GUS). The construct was stably transformed into tomato and GUS staining was assessed in various tissues of homozygous plants. The expression pattern was assessed in 3-week-old seedlings (A), leaves (B), roots (C) and MG fruit (D). E, F, G and H correspond to the same tissues treated for 2 hours with 20 µM IAA. I, J, K and L correspond to the same tissues expressing the DR5 auxin-responsive promoter fused to the GUS reporter gene (DR5:GUS) used as sensor for active auxin signalling. M, N, O and P correspond to DR5:GUS treated with 20 µM IAA. The images displayed are representative of at least three independent experiments with n>20 seedlings examined per experiment. G K D H L 58

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