The Role of SlARF8 Transcription Factor in Tomato Fruit Development

Globally, tomato (Solanum lycopersicum) is generally considered to be an important economic crop. As a fleshy fruit, tomato displays developmental traits related to fruit set, fruit growth, and fruit repining. Among the developmental processes of making a fleshy fruit, the fruit set process is considered an essential flower to fruit transition, which involves many physiological and structural changes in plants. During this important process, the plant hormone auxin is widely known to participate in triggering and coordinating those changes. Auxin Response Factors (ARFs) are transcriptional regulators that involve the auxin signaling and regulating auxin-responsive genes by specifically binding with the Auxin Response Elements (AuxREs) located in their promoters. Based on the previous work of analyzing the expression patterns of twenty-two characterized SlARF genes in tomatoes, SlARF8 was found to exhibit high expression levels in the fruit set transition. The main objective of this thesis is to gain more insight into the roles of SlARF8 in fruit set by reverse genetics. Two homologue genes, SlARF8A and SlARF8B, were identified and isolated. The subcellular localization studies revealed that SlARF8A and SlARF8B localized in the nucleus of the cells. The expression pattern analysis by RT-qPCR revealed that SlARF8A and SlARF8B displayed different expression levels before and after pollination and fertilization. A notable increase in SlARF8A transcript was displayed upon flower pollination, while a decrease in SlARF8B expression level was shown after pollination. The knockout mutants of SlARF8A and SlARF8B were generated by performing the CRISPR/Cas9 genome editing system, with the single mutant slarf8a-cr, slarf8b-cr, and double mutant slarf8a&b-cr were obtained and validated by sequencing. The slarf8a-cr, slarf8b-cr, and slarf8a&b-cr mutants displayed pleiotropic phenotypes, including dwarf plants, smaller and parthenocarpic fruits. Besides, histological analysis revealed the development of placenta, pericarp, locular tissue, and ovules were impaired in these mutants. The expression profiling at the genome level during fruit set process was studied by performing the RNA-Seq approach in these mutants. Among the significantly enriched functional categories in slarf8b-cr, many of which are related first to auxin, then with other hormones known for their role in fruit set, including gibberellin (GA). Besides, by analyzing the AuxRE motif in their promoters which is known to bind with ARFs, an in silico analysis was performed on the promoters of differentially expressed genes (DEGs) in these mutants to identify the potential target genes. It is interesting to notice that among those DEGs, several SAURs and IAAs display high expression levels in fruit set process and be potential target genes of SlARF8B. In parallel with this work and given the importance of identifying the auxin-related genes, a useful tool called Auxiscan was developed. This tool combines with different computer processes and analytical methods, which achieves the fast identification and functional annotation of auxin-related genes starting with a list of gene sequences or even an entire genome. The identification of auxin-related genes is mainly based on searching for conserved functional domains that characterizes each family. It also search for possible targets for miRNA and tasiRNA that are known to regulate some auxin-related genes. Altogether, the present study provides a comprehensive characterization of the SlARF8A and SlARF8B genes and a better understanding of their functions in regulating the fruit set process in tomato