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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 exogenous auxin (20 µM for two hours) led to an increase in the range of GUS expressing tissues. After hormone treatment, GUS staining was detected throughout the pericarp and columella tissues but remained excluded from the placental tissues (Figure 4H). As a control for auxin responsiveness, we also transformed tomato plants with a construct containing the artificial auxin- responsive promoter, DR5, fused to the GUS reporter gene (DR5:GUS). Interestingly, this synthetic promoter used here as auxin sensor, drove GUS expression in the leaf midrib and root tips (Fig 4I to 4K), but not in the fruit (Figure 4L). Exogenous auxin treatment of DR5:GUS plants resulted in enhanced staining in vegetative tissues but in the fruit, expression remained restricted to the vascular tissues (Figure 4M to 4P). These data indicate that the transcriptional control of Sl-IAA3 is more complex than that of DR5, providing evidence that although it is auxin responsive, its tight tissue-specific regulation depends on a variety of factors. Sl-IAA3 Encodes a Nuclear Localized Protein that Acts In Vivo as a Repressor of Auxin-Responsiveness We investigated the sub-cellular localization of the Sl-IAA3 protein by expressing in tobacco cell protoplasts the green fluorescent protein (GFP) fused to Sl-IAA3 (Sl-IAA3:GFP). As expected, 35S Cauliflower Mosaic Virus (CaMV) promoter- driven GFP alone was present throughout the cytoplasm (see supplemental Figure 2A online) whereas the Sl-IAA3:GFP fusion protein was localized exclusively in the nucleus (see supplemental Figure 2B online). This nuclear localization is consistent with a transcriptional regulatory function for the native Sl- IAA3 protein. In order to determine the function of the Sl-IAA3 encoded protein and to address its ability to regulate in vivo the activity of auxin-responsive promoters, a DR5-driven GFP reporter construct was used (Ottenschlager et al., 2003). This reporter construct was co-transfected into tobacco protoplasts with an effector construct giving constitutive 35S-driven Sl-IAA3 protein expression. Transient expression experiments using this dedicated "single cell system" revealed that in the absence of the effector construct, DR5-driven GFP expression was enhanced 59

Chapitre II: Sl-IAA3, a Tomato Aux/IAA at the Crossroads of Auxin and Ethylene Signalling up to 10-fold by the auxin (2,4-D) treatment (see supplemental Figure 3 online). The presence of 35S-driven Sl-IAA3 in co-transfection assays, however, strongly reduced this auxin induction. A mock effector plasmid containing the 35S promoter but lacking the Sl-IAA3 coding sequence did not impact the auxin- induction of the DR5 promoter activity (see supplemental Figure 3 online). These data indicate that Sl-IAA3 acts in vivo as a repressor of auxin-dependent transcription and is consistent with Sl-IAA3 being a member of the Aux/IAA family. Sl-IAA3 Down-Regulation Results in Vegetative Growth Phenotypes We generated Sl-IAA3-suppressed antisense tomato lines (AS-IAA3) in order to address the function of the protein in planta. Several homozygous transgenic lines corresponding to independent transformation events were obtained and analyzed. Two representative lines (1 and 2) showing 6-fold and 10-fold reductions in Sl-IAA3 transcript levels, respectively, were selected for further study (Figure 5A). In these lines, down-regulation of Sl-IAA3 resulted in multiple vegetative growth phenotypes, including a dramatic reduction in apical dominance (Figure 5B and 5C). In determinate WT tomato plants (e.g. MicroTom), lateral shoots develop only after floral transition and their growth is initiated in an apical-basal sequence along the primary shoot axis. The first lateral shoot arises from the last leaf node just below the first inflorescence. By contrast, outgrowth of the axillary shoots in the AS-IAA3 plants began in the lowest leaf node (Figure 5B) and the number of lateral shoots was increased in the transgenic lines (Figure 5C). The AS-IAA3 lines also showed a higher frequency of ectopic cotyledons than the WT (Figure 5D and 5E). The frequency of polycotyledon structure reached 25 and 20% in AS-AA3-1 and AS-IAA3-2 lines, respectively, compared to only 5% in the WT (Figure 5E). Sl-IAA3 Suppression Results in Reduced Auxin Responsiveness To further investigate the role of the Sl-IAA3 in auxin responses, we assessed the elongation of hypocotyl sections after auxin treatment in WT and AS-IAA3 lines. 60

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