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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 In Arabidopsis and pea, ethylene enhanced the expression level of At-HLS1 and Ps-HLS1 respectiely in the apical hook (Lehmanet al., 1996; Du et al., 2001). To study the expression of tomato HLS in 5-day-old etiolated seedlings, different tissues were isolated from the seedlings as indicated in Figure 10A. Sl-HLS mRNA was detected in all tissues harvested (data not shown). Ethylene-induced expression of Sl-HLS2 was observed only in the hook of etiolated seedlings after 5 hours of treatment (Figure 10C). Auxin has also been implicated in regulating apical hook closure through the HLS1 gene in Arabidopsis (Li et al., 2004). Sl- HLS2 transcript is highly accumulated upon auxin treatment in the cotyledons and in the hook of dark-grown seedlings, however, auxin had no effect on the level of Sl-HLS2 expression in other tissues tested (Figure11E). cotyledon s hoo k hypocotyl roots ∆∆CT ∆∆CT 1.5 1.0 0.5 6 4 2 0 0 B Sl-HLS1 Air C 2 H 4 Air C 2 H 4 Air C 2 H 4 Air C 2 H 4 cotyledons hook hypocotyl root C Sl-HLS2 Air C 2 H 4 Air C 2 H 4 Air C 2 H 4 Air C 2 H 4 cotyledons hook hypocotyl root ∆∆CT ∆∆CT 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 16 14 12 10 8 6 4 2 0 0 D E Air IAA 112 Sl-HLS1 Air IAA Air IAA Air IAA cotyledons hook hypocotyl root Air IAA Sl-HLS2 Air IAA Air IAA Air IAA cotyledons hook hypocotyl root Figure10. Ethylene and auxin regulation of Sl-HLS1 and Sl-HLS2 genes in different tissues of etiolated tomato seedlings. A. Four days etiolated seedlings of tomato.Cotyledons, hook, hypocotyls and roots were harvest for RNA extraction and analyzed for level of Sl-HLS1 (B,D) and Sl-HLS2 (C,E) using real time quantitative PCR. X ppm of ethylene was applied on seedlings during 5 hours (B,C). Auxin treatment (100µM of IAA) was applied by spraying seedlings during X hours (D,E). B. ∆∆CT on the y axis refers to the fold difference in the Sl- HLS1 (A), and Sl-HLS2 (B) expression relative to air-treated cotyledons, hook, hypocotyl and root, respectively.

Chapitre IV: Functional Characterization of Tomato Hookless Genes The level of Sl-HLS1 transcripts accumulation was hunderds of times lower than that of Sl-HLS2 in all tissues tested (data not shown). Moreover, no significant differential regulation of this gene after ethylene and auxin treatment could be observed in any tissues (Figure10 B-D). This prompted us to further focus our interest on the characterization of Sl-HLS2 promoter. To better characterize the dynamics of ethylene responsiveness of Sl-HLS2 and particularly at the onset of ripening, we examined the Sl-HLS2 transcript accumulation on mature green fruit upon ethylene treatment, and breaker fruit upon MCP treatment. In tomaot fruit, Sl-HLS2 gene did not display ethylene regulation as revealed by treatment of mature green fruit by ethylene or by 1- MCP, the inhibitor of ethylene perception (data not shown). This result suggests that Sl-HLS2 is ethylene inductible only in the hook of etiolated seedlings and not in tomato fruit tissues. The Expression of Sl-HLS2 Is Tightly Regulated in Tomato Plant Tissues The tomato Sl-HLS2 genomic clone contains a 1.298 bp fragment upstream of the transcription site corresponding to the promoter region that is likely to harbor Figure12: Expression pattern of HLS2 revealed by the expression of the GUS reporter gene driven by the pHLS2 promoter. The GUS staining is analysed in leaves (A), flowers (B), mature green fruit (C) and etiolated seedlings (D). 113

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