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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 IV:

Chapitre IV: Functional Characterization of Tomato Hookless Genes hook curvature in response to exogenous ethylene treatment. After 5 days of dark-growth and 24h of ethylene treatment (0.1 µL.L -1 ), more than 80% of the overexpressing seedlings displayed a full hook whereas only 48% of wild type did (Figure 7A). This indicates that overexpression of tomato hookless genes in wild type Arabidopsis confers hypersensitivity to ethylene. Sl-HLS1 and Sl-HLS2 overexpressing lines displayed a clear greening phenotype. After 6 days growth in the dark 100% of the overexpressing lines opened green cotyledons while under the same conditions 85 % wild type control seedlings failed to green (Figure 7B). In glucose assay, we showed previously that complementation of hls1 with Sl- HLS1 and Sl-HLS2 resulted in recovery of glucose–induced growth inhibition similar to that shown by wild type. We carried out a comparison between the hookless overexpressing lines and wild type plants at different concentrations of glucose. No overt difference in cotyledon development was found in the absence Figure 8. Over-expression of Tomato Sl-HLS1 and Sl-HLS2 Genes in Arabidopsis Confers Hypersensitivity to Glucose. A. seeds of WT and overexpressed hooklees lines were germinated and grown on MS medium containing 0%, 5.5%, 6 or 6.5% glucose for 10 days. B. The Numbers of green seedlings in panel A were quantitatively expressed by examinating a large number of seedlings (n>30). 110

Chapitre IV: Functional Characterization of Tomato Hookless Genes of exogenous glucose (Figure 8A). However, in the presence of 5.5% glucose, 55% of Sl-HLS2 overexpressing lines displayed developmental arrest while 100% of wild type seedlings underwent normal growth. At 6% glucose, up to 91.3% of Sl-HLS2 overexpressing lines displayed growth inhibition compared to only 55 % in wild type (Figure 8B). Similar data were obtained with Sl-HLS1 overexpressing lines indicating that both tomato genes play important role in germination and suncsequent cotyledon and true leaves development. Expression Pattern and Regulation of Sl-HLS1 and Sl-HLS2 in Tomato Sl-HLS2 Gene Expression Is Regulated by Ethylene and Auxin in Tomato RNA samples extracted from various tissues of tomato plants were analyzed for assessing the level of Sl-HLS1 and Sl-HLS2 mRNA accumulation using real-time quantitative PCR. Sl-HLS1 and Sl-HLS2 transcripts were detected in all tissues examined but showed the highest levels in ripening fruit tissue (Figure 9A and B). Sl-HLS1 and Sl-HLS2 transcript levels increased from immature green throughout breaker stage. In vegetative tissues, the highest levels of Sl-HLS1 and Sl-HLS2 transcript accumulation were found in leaves. ∆∆CT A 3,0 2,5 2,0 1,5 1,0 0,5 0 Sl-HLS1 IMG MG Br Or Re S L F R ∆∆CT B 3.0 2.5 2.0 1.5 1,0 0.5 0 Sl-HLS2 IMG MG Br Or Re S L F R Figure 9. Tissue-specific Expression of Sl-HLS1 and Sl-HLS2 in Tomato. The levels of transcripts were addressed by real-time quantitative PCR. Sl-HLS1 and Sl-HLS2 mRNA accumulation was monitored in reproductive tissues including immature green (IMG), mature green (MG), breaker fruit (Br), Orange (Or), Red (R), Flower (F), and in vegetative tissue including Stem (S), leaf (L), and Roots (R). ∆∆CT on the y axis refers to the fold difference in the Sl-HLS1 (A) and Sl-HLS2 (B) expression relative to the immature green fruit. 111

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