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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 ...

A C Chapitre II:

A C Chapitre II: Sl-IAA3, a Tomato Aux/IAA at the Crossroads of Auxin and Ethylene Signalling ProIAA3:GUS Air C 2H 4 DR5:GUS Air C 2H 4 ProIAA3:GUS ProHLS:GUS DR5:GUS B Air C 2 H 4 Figure 9. Expression of ProIAA3:GUS and ProHLS:GUS Associated with Differential Growth during Hook Formation and Epinastic Response. A. Tissuespecific expression of ProIAA3:GUS and DR5:GUS in etiolated seedlings. Etiolated seedlings expressing the GUS reporter gene driven either by the Sl-IAA3 or DR5 promoter were dark grown on MS media for five days and then treated with air or 10 µL L -1 ethylene for 48 hours. The left panel shows the ethylene-dependent GUS staining in the apical hook of transgenic tomato plants expressing the ProIAA3:GUS construct. The right panel shows GUS staining in the DR5:GUS transformed plants used for detection of active auxin signalling in the hook. Bars = 5 mm B. Expression pattern of ProIAA3:GUS in epinastic and non-epinastic petioles. Six-week-old light-grown plants were placed in airtight chambers for 16 h in the absence (upper panel) or presence of 50 µL L -1 ethylene (lower panel). The arrows indicate the expression of GUS in the leaf nodes of the petiole. C. Comparative expression patterns of ProIAA3:GUS (left panel) and ProHLS:GUS (middle panel) in the apical hook. The right panel shows the expression of GUS driven by DR5. Bars = 1 mm. Etiolated seedlings expressing the GUS reporter gene driven either by the Sl-IAA3, Sl-HLS or DR5 promoter were dark grown on MS media for five days and then treated with 10 µL L -1 ethylene for 48 hours. The images displayed are representative of at least three independent experiments with n >30 seedlings examined per experiment. potential interactions between Sl-HLS and Sl-IAA3 in controlling hook formation we analyzed the spatial expression of the Sl-HLS and Sl-IAA3 in the apical hook by native promoter-reporter constructs (Figure 9C). We isolated a 1.3 kb fragment of the Sl-HLS promoter and fused it to the GUS reporter gene. Etiolated seedlings expressing this construct were treated with ethylene. Remarkably, ProHLS: GUS staining was restricted to the outer side of the hook curvature, whereas the Sl- IAA3 promoter drove GUS staining exclusively on the inner side. These data suggest that Sl-IAA3 acts as a repressor of auxin/ethylene-mediated cell 67

Chapitre II: Sl-IAA3, a Tomato Aux/IAA at the Crossroads of Auxin and Ethylene Signalling elongation on the inner surface of the apical hook and/or conversely that SI-HLS1 is involved in promoting cell elongation on the outer surface. A ∆∆CT 2 1.5 1 0.5 ∆∆CT 0 Sl-IAA3 4 E8 3 2 1 0 0 0.1 0.5 1 [C2H4 ] µL L-1 [C2H4 ] µL L-1 0 0.1 0.5 1 [C2H4 ] µL L-1 [C2H4 ] µL L-1 ∆∆CT C D 2.5 Sl-IAA3 2.5 Sl-IAA3 2 1.5 1 0.5 ∆∆CT 0 4 E8 3 2 1 0 0 1 5 24 Times (hours) 0 1 5 24 Times (hours) Figure 10. Characterization of Ethylene Regulation of Sl-IAA3. Ethylene dose response (A) and time course (B) of Sl-IAA3 induction in 5-day-old dark-grown WT seedlings. Expression of the ethylene-responsive gene E8 was used as control for hormone treatment (C and D). ∆∆CT on the y axis refers to the fold difference in Sl-IAA3 expression relative to untreated seedlings. The data presented correspond to mean values of 3 replicates ± SE. To further characterise the ethylene regulation of Sl-IAA3 we determined the ethylene dose-response using 5-day-old dark-grown wild type seedlings. qRT- PCR analysis revealed that Sl-IAA3 transcript accumulation reached a maximum at 0.5 µL L -1 (Figure 10A), the same saturating concentration was found for the previously characterised ethylene-responsive gene, E8 (Figure 10C). A study of the time-course of ethylene induction (performed with 0.5 µL L -1 ) also indicated that Sl-IAA3 transcript accumulation mimicked that of E8 and reaching a maximum after 5 hours treatment and then decreasing at 24 hours (Figure 10B and 10D). B 68

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