Genomic and Biotechnology of the Fruit GBF

Auxin signalling and Fruit development

In flowering plants, seed set and fruit development normally occur in a coordinated manner following pollination of the stigma and subsequent double fertilization in the ovule inside the flower. Different parts of the flower can contribute to the final structure of dry and fleshy fruits. The final form of the fruit is dependent upon the contributing number and type of floral organ components, the position of the contributing organs, and how the different tissues within them grow and differentiate. The ovary of tomato expands after fertilization and the locule spaces around the developing seeds fill with pulp to form this familiar fleshy fruit.
Various phytohormones, including gibberellins (GAs), cytokinin, and auxin, are involved in the growth and development of both seeds and fruit.

In our laboratory, we study the role of AUX/IAA and ARF transcription factors in fruit development and quality.

The tomato AUX/IAA gene family

Corinne Delalande, Mondher Bouzayen,

The phytohormone auxin is involved in various developmental processes, including apical dominance, tropisms, vascular patterning and fruit set. Proteins encoded by members of the Aux/IAA gene family are involved in auxin action through the regulation of auxin-dependent gene expression and developmental processes. Partial Aux/IAA clones were isolated from tomato (Solanum lycopersicum) fruit using degenerate primers (Jones et al., 2002).

In order to better unravel the role of auxin during fruit development and ripening, we sought to isolate and characterize all the members of SlAux/IAA gene family in tomato.

Aux/IAA proteins are transcriptional regulators that mediate many aspects of plant responses to auxin by repressing ARFs (auxin response factors) function. The Arabidopsis genome encodes 29 Aux/IAA proteins. Putative tomato Aux/IAA genes were cloned and their spatio-temporal expression analysed by Quantitative Real-Time RT-PCR. Co-transfection experiments carried out in tobacco protoplasts with synthetic Auxin-responsive promoter fused to reporter gene and effector constructs encoding Aux/IAA proteins resulted in specific repression of the reporter gene indicating that Aux/IAA proteins were negative regulators of auxin-dependent gene transcription.

To address the function of all these proteins in planta, we generated suppressed antisense tomato lines and over-expressing tomato lines. Down-regulation of one of them (SlIAA9) resulted in pleiotropic phenotypes, consistent with its ubiquitous expression pattern. LeIAA9-inhibited lines have simple leaves instead of wild-type compound leaves, and fruit development is triggered prior to fertilization, giving rise to parthenocarpy. This indicates that LeIAA9 is a key regulator of fruit set and leaf morphogenesis (Wang et al., 2005).

Down-regulation of another Aux/IAA gene, Sl-IAA3 results in auxin and ethylene-associated phenotypes including altered apical dominance, lower auxin sensitivity, exaggerated apical hook curvature in the dark and reduced petiole epinasty in the light. The ethylene-related phenotypes in the antisense tomato lines (AS-IAA3) reveal new roles for Aux/IAAs genes and position Sl-IAA3 firmly at the crossroads of auxin and ethylene signalling in tomato (Chaabouni et al., under revision).

Large scale transcriptomic analyses by DNA microarrays aiming at identifying altered gene expression in the two antisense lines are underway.

The tomato ARF gene family

Mohamed Zouine, Isabelle Mila, Mondher Bouzayen

Auxin Response Factors (ARFs) are transcription factors encoded by a large multigene family in plants that bind the auxin-response elements found in the promoter of auxin-regulated genes. A comprehensive genome-wide and transcript analysis was carried out in this study to identify tomato ARF genes. Full-length cDNA clones were isolated for the identified tomato ARF genes (named Sl-ARFs) and a phylogenetic study generated by alignments of the full-length protein sequences established the relationship between the tomato Sl-ARFs and the 23 AtARFs described in Arabidobsis. The phylogenetic tree drawn from this study indicated that ARF gene family fall into three major sub-classes. Ten AtARF/Sl-ARF sister pairs were formed, showing different orthologous relationships between AtARFs and Sl-ARFs.
To gain better insight on the transcriptional activity of the tomato ARFs, the ability of the encoded proteins to activate or repress transcription from synthetic or native complex auxin-responsive promoters was investigated in a plant protoplast transfection assay. Our data indicate that Sl-ARFs can be repressors, activators or neutral on auxin-dependent gene transcription. Expression studies carried out by Quantitative Real-Time RT-PCR showed that members of the tomato ARF family display clear preferential expression in targeted plant tissues and organs with some genes specifically expressed in reproductive tissues. These data are consistent with those previously reporting the involvement of some ARFs in flower development and subsequent fruit set.
The outcome of the present study provides detailed structural and expression information on the tomato ARF gene family and a starting point for addressing their physiological role via a reverse genetic approach.