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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 Figure 5. Altered Vegetative Growth Phenotypes in Antisense Sl-IAA3 Plants. A. Down-regulation of Sl-IAA3 in transgenic tomato plants. The level of Sl-IAA3 transcripts in transgenic antisense lines (1 and 2) was assessed by qRT-PCR. ∆∆CT refers to the fold difference in Sl-IAA3 transcript levels relative to the WT. B. Reduced apical dominance in 7-week-old AS-IAA3 transgenic plants compared to WT plants. C. The number of lateral shoots branching from the first leaf node in WT and AS-IAA3 plants. The data are the mean ± SE of at least 30 plants and are representative of three independent experiments. D. Triple cotyledon phenotype occurring at higher frequency in AS-IAA3 transgenic lines compared to WT. Plants are light-grown in MS medium for 7 days. Three cotyledon structures are indicated by yellow arrows. E. Frequency of triplicate cotyledons occurring in AS-IAA3 and WT seedlings expressed as a % of the total population. Error bars represent mean ± SE of 40 plants. After two hours auxin treatment (Figure 6A), the WT hypocotyl segments elongated more than the AS-IAA3 plants at all auxin concentrations tested, indicating that Sl-IAA3 suppression reduces sensitivity to auxin in planta. To further investigate this, we examined the effects of the auxin transport inhibitor, N- 1-napthylphthalamic (NPA) on the growth of WT and AS-IAA3 seedlings. NPA treatment is known to alter the endogenous auxin gradient, leading to an over- accumulation of auxin in the root apical meristem which results in an inhibition of cell division and root growth. When grown in the presence of 1µM NPA, there was a marked reduction of primary root elongation and a complete suppression of lateral root formation in the WT seedlings (Figure 6B and 6C). In the absence of 61

Chapitre II: Sl-IAA3, a Tomato Aux/IAA at the Crossroads of Auxin and Ethylene Signalling NPA, 19-day-old AS-IAA3 seedlings are not morphologically different to the WT (Figure 6B). By contrast, NPA only weakly affected primary root growth in AS- IAA3 plants, although lateral root formation was inhibited similarly to the WT (Figure 6Band 6C). Leaf emergence was also strongly inhibited in NPA-treated WT seedlings, but not in the AS-IAA3 plants (arrow Figure 6B). Once again, these data are consistent with reduced auxin sensitivity in the antisense lines. Other auxin-related processes like hypocotyl and root gravitropism were not affected in Figure 6. Auxin-Associated Phenotypes of Sl-IAA3 Down-Regulated Lines. A. Auxin dose-response in hypocotyl segments. 8 mm hypocotyl fragments cut from 3-week-old light grown seedlings were incubated for 2 h in a solution containing the indicated concentration of NAA. Elongation is given as percentage increase in final length over the initial length. The results are representative of data obtained with two independent AS- IAA3 lines and with two replicates for each line. Standard errors are indicated (n ≥ 25). B. Effect of NPA treatment on the development of light-grown WT and AS-IAA3 seedlings. 19-day-old WT and AS-IAA3 tomato seedlings were grown in the presence or absence of 1 µM of NPA. The scale bar indicates 10 mm C. Primary root length upon NPA treatment of light-grown WT and AS-IAA3 lines. Primary root length was assessed following NPA treatment. Error bars represent mean ± SE (n ≥ 60). D. Expression of early-auxinresponse genes in AS-IAA3 and WT lines. Expression analysis of Sl-IAA4 (SGN- U316052), Sl-IAA14 (SGN-U318434), Sl-IAA17 (SGN-U323974), SAUR (SGN-U318031) and GH3 (SGN-U319351) genes were carried out by qRT-PCR in WT and AS-IAA3 hypocotyl fragments after auxin treatment (1µM NAA for 2h). The data are expressed in fold increase with respect to non treated tissues taken as reference values. 62

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