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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 Down-regulation of Sl-IAA3 Specifically impacts the Expression of Selected Auxin and Ethylene Transcription Factors To provide mechanistic insights into how SI-IAA3 functions to bring about the observed phenotypes, we attempted to identify putative target genes controlled by SI-IAA3. Taking into account that down-regulation of SI-IAA3 mainly resulted in auxin and ethylene-related phenotypes, we analysed the expression of transcription factors known to mediate auxin and ethylene responses. The expression of tomato Aux/IAA (14), ARF (10) and ERF (12) genes was ∆∆CT A ∆∆CT 2 1.5 1 0.5 C 0 2 1.5 1 0.5 0 ARFs U317693 ERFs U329695 U329695 U314233 U313488 U320946 U320682 U319581 U329134 U324457 U319706 U319318 U315233 U327712 U315595 U327976 U317007 U317007 U327759 U321808 WT AS-IAA3 U321591 U315194 U315194 WT AS-IAA3 U327266 U327266 ∆∆CT U317071 U317071 B 2 1.5 1 0.5 0 Aux/IAAs WT AS-IAA3 U323670 U316052 U320280 U318191 U317606 U313802 U322787 U322644 U318434 U332300 U323974 U320412 U322175 U320261 ∆∆CT D 1.2 1 0.8 0.6 0.4 0.2 0 WT AS-IAA3-1 Sl-HLS Figure 11. Impact of Sl-IAA3 Down-regulation on the Expression of Auxin and Ethylene Response Genes. The expression of members of the ARF (A), Aux/IAA (B), and ERF (C) gene families of transcription factors as well as the Sl-HLS gene (D) was assessed by qRT-PCR in 5-day-old dark-grown WT and AS-IAA3 seedlings. The gene names correspond to the SGN annotation. Primers used are listed in Supplemental Table 2. ∆∆CT on the y axis refers to the fold difference in expression of each gene relative to that in WT seedlings taken as reference tissues. The data presented correspond to mean values of 3 replicates ± SE. 69

Chapitre II: Sl-IAA3, a Tomato Aux/IAA at the Crossroads of Auxin and Ethylene Signalling assessed by qRT-PCR in 5-day-old dark-grown wild type and AS-IAA3 seedlings (Figure 11). While most of the genes showed similar expression in wild type and transgenic lines, there was a clear down-regulation of the putative tomato ortholog of Arabidopsis ARF2 (SGN-U314233) and conversely a significant up-regulation of the ARF8 (SGN-U327976) transcript (Figure 11A). The down-regulation of ARF2 observed in the transgenic lines is in keeping with the data reporting that the hookless phenotype of hls1 Arabidopsis mutants can be partially reversed by a loss-of-function mutation in the ARF2 gene (Li et al., 2004). Notably, the expression of IAA29 (SGN-U320261) and Pti4 (SGN-U317071), a tomato ERF gene, were also significantly up-regulated in the transgenic lines (Figure 11B and 11C) indicating that down-regulation of Sl-IAA3 altered the expression of specific auxin and ethylene transcriptional mediators. DISCUSSION Aux/IAA proteins are critical components of the auxin response. In Arabidopsis, dominant gain-of-function mutations in individual Aux/IAAs have provided telling insights into the roles played by the various family members in eliciting specific auxin responses. We show here that Sl-IAA3, a tomato Aux/IAA, is an integral component of both auxin and ethylene response pathways. Indeed, transcripts for the gene accumulate in response to both of the hormones and its down-regulation results in auxin- and ethylene-related phenotypes. Phenotypic responses to Sl- IAA3 down-regulation include alterations to the classical auxin regulated processes of apical dominance and hypocotyl elongation, and to the typical ethylene responses such as apical hook formation in etiolated seedlings and leaf epinasty in light-grown plants. Sl-IAA3 and a number of other partial tomato Aux/IAA clones were initially isolated from fruit tissues (Jones et al., 2002). The Sl-IAA3 gene has strong sequence and structural similarities with its putative Arabidopsis orthologs, At- IAA4 and At-IAA3. An Arabidopsis mutant for At-IAA4 has an insertion in the first exon but shows no obvious growth phenotype (Overvoorde et al., 2005). In fact, although loss-of-function mutations have been identified in Arabidopsis for several Aux/IAA genes, the only phenotypes reported are subtle changes in the 70

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