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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 IAA3-down-regulated lines (see supplemental Figure 4 online). In addition, no alteration in root growth and vascular formation was observed in AS-IAA3 lines. We therefore investigated typical molecular responses to auxin by assessing the expression of three Aux/IAA genes (Sl-IAA4, Sl-IAA14, Sl-IAA17) and two other primary auxin-responsive genes, SAUR and GH3. RNA samples isolated from AS-IAA3 and WT hypocotyls incubated 2 h in presence or absence of 1 µM NAA were used in qRT-PCR experiments to monitor transcript accumulation for the five genes. Neither the basal, nor the auxin responsive transcript levels were altered by the down-regulation of Sl-IAA3 (Figure 6D). It is important to point out that Sl-IAA4, Sl-IAA14 and Sl-IAA17 were selected among the Aux/IAA genes because of their high sequence homology to Sl-IAA3 (See supplemental Table1 online). Therefore, since their expression remained unchanged in the AS-IAA3 plants it is most likely that Sl-IAA3 was exclusively directly targeted by the antisense strategy. Sl-IAA3 Suppression Results in Modified Ethylene Sensitivity The ethylene responsiveness of Sl-AA3 prompted us to examine the role of the encoded protein in two classical ethylene response processes, epinastic petiole curvature in light-grown plants and the formation of an apical hook in etiolated seedlings. Tomato leaf petioles typically curve downwards in response to exogenous ethylene (Kazemi et al., 1974). To investigate the impact of the down- regulation of Sl-IAA3 on this epinastic response, light-grown WT and AS-IAA3 tomato plantlets were treated with exogenous ethylene (50 µL L -1 ) for 16 h. The angle of the petioles was then measured in the first and second leaf-nodes as indicated in Figure 7B. In WT, ethylene treatment led to a leaf angle of 100°. By contrast in AS-IAA3 lines 1 and 2, the leaf angle after treatment was 87° and 75°, respectively (Figure 7A and Table 1), indicating an alteration in the ethylene- induced epinastic response in the AS-IAA3 plants. In the absence of ethylene treatment, both WT and AS-IAA3 lines display the same leaf angle (Table 1). One of the most striking phenotypes in the AS-IAA3 seedlings is the exaggerated apical hook formation in the absence of exogenous ethylene (Figure 7C). Exaggeration of the apical hook is one of the hallmarks of the classical ethylene triple response (Ecker, 1995). To better characterise this phenotype we first 63

Chapitre II: Sl-IAA3, a Tomato Aux/IAA at the Crossroads of Auxin and Ethylene Signalling defined different grades of hook formation ranging from stage 0, corresponding to total absence of the hook, to stage 7 corresponding to maximal exaggerated hook (Figure 8A). Treatment of seedlings with 1-MCP abolished apical hook formation Figure 7. Ethylene Associated Phenotypes of AS-IAA3 Lines. A. Petiole epinasty in WT and AS-IAA3 plants in response to ethylene. Five-week-old light-grown plants were placed in airtight chambers for 16 h in the presence of 50 µL L -1 ethylene. B. Diagram depicting the position of the first and the second leaf node in tomato plants. C. Hook curvature in 5-day-old WT (left panel) and AS-IAA3 (right panel) etiolated seedlings. The scale bar indicates 5 mm. in both the antisense and WT lines (Figure 8B). In air-grown seedlings, in the absence of exogenous ethylene, most AS-IAA3 seedlings displayed hook curvature corresponding to stage 4 (60%) and stage 3 (35%), while of wild type plants exhibited mainly stage 1 (60%) and stage 2 (35%) hook curvature (Figure 8C). Adding exogenous ethylene (0.1 µL L -1 ) increased hook curvature to stage 3 and 4 for wild type and to stages 4 and 5 for AS-IAA3 (Figure 8D). Under 1 µL L -1 exogenous ethylene, 90 % of WT seedlings displayed hook curvature from stage 5 and 6 and 80% of AS-IAA3 lines exhibited hook curvature from stage 6 and 7 (Figure 8E). These data indicate that the exaggerated apical hook curvature 64

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