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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 shy2 mutant (Tian and Reed, 1999). Interestingly, the expression of At-IAA3, one of the putative orthologs of tomato Sl-IAA3, was shown to be altered in Shy2 mutant. Double or triple mutants of closely related Aux/IAA genes, such as iaa8- 1/iaa9-1 or iaa5-1/iaa6-1/iaa19-1 also exhibit WT phenotypes, indicating extensive functional redundancy among Arabidopsis Aux/IAA family members (Overvoorde et al., 2005). Arabidopsis gain-of-function Aux/IAA mutants result from genetic changes that lead to alterations in amino acids in the highly conserved domain II. This stabilizes the proteins and results in a variety of gene- specific auxin-related phenotypes (Reed, 2001). We have previously shown that down-regulation of a tomato Aux/IAA gene, Sl-IAA9, resulted in pleiotropic phenotypes including altered leaf architecture and parthenocarpic fruit, consistent with a pivotal role for auxin in tomato fruit set and leaf morphogenesis (Wang et al., 2005). In the present study we show that the down-regulation of Sl-IAA3 (AS- IAA3) also leads to well defined phenotypes in transgenic tomato lines. We were able to rule out that the observed changes might result from a lack of specificity of our antisense strategy by verifying that the expression of closely related Aux/IAA genes was not altered in the AS-IAA3 transgenic lines. These data strongly support the hypothesis that different members of the Aux/IAA family are involved in distinctive developmental processes. Sl-IAA3 Mediates Auxin-Dependent Gene Transcription and Auxin- Associated Phenotypes Aux/IAA genes were originally identified based on their rapid induction by auxin in etiolated soybean (Glycine max) and pea (Pisum sativum) tissues (Walker and Key, 1982; Theologis et al., 1985). Many Arabidopsis auxin responsive genes contain the canonical auxin response elements (AuxRE), TGTCTC or GAGACA in their promoters (Guilfoyle and Hagen, 2007). Our in silico search led to the identification of two degenerate AuxRE elements in the Sl-IAA3 promoter that may be responsible for the auxin responsiveness observed in this study (Figure 1 and Figure 3). Sl-IAA3 transcript levels varied dramatically among the different tomato tissues. The highest and lowest levels were found in the fruit and roots, respectively. Analyses of tomato lines expressing the Sl-IAA3 promoter fused to the GUS 71

Chapitre II: Sl-IAA3, a Tomato Aux/IAA at the Crossroads of Auxin and Ethylene Signalling reporter gene revealed that basal levels of expression were spatially restricted within organs. In the root, Sl-IAA3-driven GUS expression was restricted to the root cap and lateral root meristems. In the leaves it was restricted to the vasculature, and in the fruit in a narrow band defining the junction between placenta and pericarp. Interestingly, the precise tissue-specific expression patterns were abolished by exogenous auxin. Auxin treatment led to GUS staining throughout the whole pericarp in fruit and to all parts of the leaves and roots. These auxin responsive expression patterns are in agreement with previous data (Jones et al., 2002) but differed considerably from those seen with the artificial auxin-responsive promoter, DR5 (Figure 4). This suggests that a combination of promoter elements, including the ethylene-responsive element (ERE), contribute to the maintenance of the precise tissue-specific pattern of Sl- IAA3 expression. In most cases in Arabidopsis, Aux/IAA gain-of-function mutations are associated with phenotypes reminiscent of reduced auxin responsiveness (Tian et al., 2002; Nagpal et al., 2000; Rogg et al., 2001). Arabidopsis Aux/IAAs have been shown to repress the DR5-driven transcription (Ulmasov et al., 1997; Tiwari et al., 2001). This repression is thought to occur via interactions between the Aux/IAA proteins and their DNA-binding ARF partners (Guilfoyle and Hagen, 2007). As we were able to show that Sl-IAA3 also has the capacity to repress the activity of DR5 in vivo, we hypothesized that the down-regulation of Sl-IAA3 would reduce the level of auxin-responsive gene repression and ultimately lead to enhanced auxin responses. Unexpectedly, the AS-IAA3 lines have many phenotypes consistent with a reduced auxin sensitivity (Figure 8). This suggests that, even though in vivo assays showed that Sl-IAA3 has the capacity to repress auxin-responsive gene expression, in planta the protein seems to act as a positive regulator of auxin responses. One possible explanation for this apparent discrepancy is that in planta, Sl-IAA3 may repress the expression of some negative regulators of auxin responses. Two ARFs (ARF2 and ARF8) and one Aux/IAA (IAA29) that were differentially regulated in the AS-IAA3 lines, may contribute to the reduced auxin- responsiveness. Ethylene Related Expression and Phenotypes 72

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