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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 We have previously shown that the accumulation of Sl-IAA3 transcripts is enhanced by ethylene treatment in mature green fruit (Jones et al., 2002). In this work, we further characterized the dynamics of the ethylene responsiveness of the gene. A time-course analysis of Sl-IAA3 transcript accumulation in response to ethylene revealed a similar pattern to that shown by the well-characterized ethylene-responsive gene, E8 (Lincoln et al., 1987). The highest levels of Sl-IAA3 transcripts in tomato plants were found in the fruit at the orange/red stages of ripening. In tomato, autocatalytic ethylene production leads to high levels of endogenous ethylene during fruit ripening (Lelievre et al., 1997). In breaker stage fruit, when autocatalytic ethylene is already actively driving the ripening process, the ethylene inhibitor, 1-MCP, sharply reduced Sl-IAA3 transcript levels. There was also a dramatic reduction in Sl-IAA3 transcript levels in the tomato mutants rin, nor and Nr that lack the capacity to respond to autocatalytic ethylene and to undergo normal ethylene-regulated ripening processes (Figure 2). Together, these data strongly suggest that this presumptive auxin response element is ethylene-responsive and integral to ethylene-regulated fruit ripening. In untreated fruit, the Sl-IAA3 promoter drove GUS expression strictly at the boundary between the placenta and pericarp tissues. These observations suggested that down-regulation of Sl-IAA3 in transgenic lines may have resulted in a fruit ripening phenotype. Nevertheless, no changes were observed in the fruit ripening of antisense lines including the timing of the onset, levels of climacteric ethylene production and pigment accumulation examined in our study. Though we cannot exclude the possibility that other ripening aspects may have been altered, our data suggest that either there is some functional redundancy or that residual levels of Sl-IAA3 were sufficient to drive the fruit processes that rely on the protein. The data do, however, indicate that Sl-IAA3 lies at the crossroads of the auxin and ethylene responses during tomato fruit ripening. Trainotti et al. (2007) have recently demonstrated a network of interactions between auxin and ethylene during ripening in peaches. Our data show that modification of the auxin response is one of the suite of ethylene driven processes that together constitute climacteric fruit ripening. Two other phenotypes in the AS-IAA3 lines, the exaggerated apical hook formation and reduced epinasty, indicated that Sl-IAA3 is involved in physiological responses to ethylene. An exaggerated apical hook is formed in etiolated 73

Chapitre II: Sl-IAA3, a Tomato Aux/IAA at the Crossroads of Auxin and Ethylene Signalling seedlings in response to exogenous ethylene. This process forms the classical ethylene triple response together with reduced hypocotyl and root elongation (Bleecker et al., 1988; Ecker, 1995). An apical hook is formed during early seedling growth to protect the fragile shoot meristem from damage as the seedling grows through the soil (Darwin and Darwin, 1896). A complex pattern of coordinated cell elongation is needed to establish and maintain the apical hook structure (Silk and Erickson, 1978). The involvement of both ethylene and auxin in this differential cell elongation has been demonstrated through the analysis of ethylene and auxin signalling mutants that are altered in the process of hook formation (Lehman et al., 1996; Li et al., 2004). In Arabidopsis, mutants that are defective in ethylene perception and signalling, such as etr1-1, ein2, ein3, do not form an exaggerated hook in response to ethylene treatment. By contrast, the constitutive ethylene response mutant, ctr1, develops an exaggerated hook in the absence of ethylene (Guzman and Ecker, 1990; Kieber et al., 1993). Auxin promotes hypocotyl cell elongation and is unequally distributed in the apical hook (Schwark and Schierle, 1992). The axr1 mutant, which is altered in auxin responses, lacks a normal apical hook and the inhibition of auxin transport disrupts formation of the hook (Lincoln et al., 1990). Clearly, the apical hook is established and maintained by interplay between ethylene and auxin. The exaggerated apical hook phenotype in the AS-IAA3 lines provides direct evidence that Sl-IAA3 is important in physiological processes that rely on both auxin and ethylene. Active ethylene signalling is essential for the appearance of the exaggerated hook phenotype since blocking ethylene perception with 1-MCP prevents hook formation in the AS-IAA3 plants. Noteworthy, apart from the exaggerated hook, other aspects of the triple response like hypocotyl elongation and thickening and root elongation were not altered in AS-IAA3 lines indicating that Sl-IAA3 is rather involved in differential growth processes. Ethylene treatment of etiolated seedlings increased the ProIAA3:GUS expression in the inner surface of the apical hook (Figure 9). Likewise, ProIAA3:GUS staining was also clearly delimited in epinastic petioles suggesting that the ethylene-induced gradient of Sl- IAA3 expression is involved in the differential growth associated with both apical hook formation and the petiole epinastic response. Whereas, down-regulation of Sl-IAA3 resulted in an exaggerated ethylene-response of etiolated seedlings, it conferred reduced ethylene sensitivity in light-grown plants. The ability of 74

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