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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 displayed by members of the gene family suggest that individual Aux/IAAs have precise and distinct functions during normal plant growth and development. We have previously shown that members of the Aux/IAA gene family can be regulated by ethylene in the tomato (Jones et al., 2002) and that down-regulation of the tomato Aux/IAA, Sl-IAA9, results in a pleiotropic phenotype, including altered leaf architecture and parthenocarpy (Wang et al., 2005). Similarly, Kloosterman et al. (2006) have shown that suppression of St-IAA2 in potato results in distinctive phenotypes including increased plant height, petiole hyponasty and curvature of growing leaf primordia in the shoot apex. In Arabidopsis, it has been reported that a mutation in an ARF reverses the ethylene-related hookless phenotype in the hls1 mutant (Li et al., 2004). This suggests that auxin related transcription factors have evolved as points of intersection between ethylene and auxin responses. We report here on the isolation and functional characterization of the tomato Aux/IAA, Sl-IAA3 that defines one of the mechanisms by which auxin and ethylene signaling pathways converge. Both expression data and the analysis of transgenic plants down-regulated for Sl-IAA3 support the hypothesis that Sl-IAA3 is a key molecular link between ethylene and auxin responses in tomato plants. RESULTS Isolation and Structure of the Sl-IAA3 Gene Sl-IAA3 and other partial tomato Aux/IAA clones were initially isolated from tomato fruit using gene family-specific degenerate primers designed from conserved Aux/IAA sequences (Jones et al., 2002). Sl-IAA3 corresponds to a previously isolated, partial tomato clone named IAA3 (Nebenführ et al., 2000). Sl- IAA3, formerly named DR3, was reported to be ethylene inducible and differentially expressed during fruit ripening (Jones et al., 2002). We isolated the full length Sl-IAA3 cDNA (U 320812, now available from the Solanaceae Genome Network Database, http://www.sgn.cornell.edu) and determined the transcription start site by 5’ Race-PCR. An open reading frame of 558 bp encoding a putative protein of 185 amino acids was identified. The predicted protein comprises the 53

Chapitre II: Sl-IAA3, a Tomato Aux/IAA at the Crossroads of Auxin and Ethylene Signalling four conserved domains (I to IV) characteristic of Aux/IAA proteins and falls into sub-family I of the four Aux/IAA sub-families (Wang et al., 2005). We also isolated a genomic fragment of 2723 bp in order to identify the structure of the tomato Sl- IAA3 gene. The sequence comprised 1668 bp of promoter and 1055 bp of transcribed sequence composed of three exons and two introns (Figure 1) and matched that of its closest Arabidopsis homologs, At-IAA3 (AT1G04240) and IAA4 (AT5G43700) consistent with the phylogenetic analysis (see Supplemental Figure 1 online). The Sl-IAA3 nucleotide coding and predicted amino acid sequences display 65.8 % and 56 % identity, respectively with At-IAA3 and 65.4 % and 56.3 % identity, respectively with At-IAA4. Features of the Sl-IAA3 Promoter The 1668 bp promoter fragment was analyzed in silico using PlantCare software (Lescot et al., 2002). The program identified several putative cis-acting elements, including two degenerate auxin-response elements (TGTCNC) at positions -216 and -175, an ethylene-response element ERE (ATTTCAAA) at position -1174, three methyl-jasmonate response elements (CGTCA) at positions -434, -969, and -1018, a heat-response element HSE (AAAAAATTTC) at position -934 and an TC rich repeat - 1406 ERE - 1174 Me JA element - 1018 Me JA element - 969 HSE - 934 EIRE - 656 Promoter Me JA element - 434 TC rich repeat - 385 - 1668 Aux RE -216 Aux RE -175 Sl-IAA3 (2723 bp) 82 260 355 560 654 826 1055 UTR E1 E2 E3 UTR 438 Antisense construct Figure 1. Genomic Structure of the Tomato Sl-IAA3 Gene. The black portion represents the promoter region, the gray lines the introns, the gray boxes the exons and the white boxes the untranslated regions (UTR). The putative cis-acting elements found in the promoter region are indicated by black bars. The black arrow represents the antisense construct used to generate the silenced lines. 54

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