Characterization of CRF domain containing ERF genes- Solanum lycopersicum Cytokinin Response Factors SlCRF3 and SlCRF5 in tomato development

Abstract

Cytokinin is an important hormone involved in numerous aspects of plant growth and development. A newly identified group of transcription factors- Cytokinin Response Factors (CRFs) has been included as a side branch to cytokinin signaling pathway. CRFs constitute a subset of the AP2/ERF family of transcription factor proteins found in all land plants. Within the ethylene response factor (ERF) subfamily, CRFs are defined by the presence of a group-specific domain, known as the CRF domain, several of which were originally identified in Arabidopsis microarray experiments as induced by cytokinin. Initial studies of CRF mutants have also linked these genes to normal leaf/cotyledon development in Arabidopsis. In this study, two transcription factor genes SlCRF3 and SlCRF5 (Solanum lycopersicum Cytokinin Response Factors) were identified and characterized to determine their involvement in regulating cytokinin responses in tomato development. Additionally, the first transcriptome analysis of cytokinin and auxin response in tomato roots was conducted and several novel hormone regulated genes were identified through Illumina RNA-seq technology. This project was initiated with the following objectives: 1) To characterize SlCRF3 and SlCRF5 and their pattern of regulation by cytokinin and abiotic stresses, 2) To determine the roles of SlCRF5 in tomato development, 3) To characterize novel genes involved in cytokinin and auxin regulation in tomato root. For the first two objectives, previous unknown full length DNA sequences for SlCRF3 and SlCRF5 were determined, cloned into different expression vectors, then transformed into plants to study where and when these genes are expressed as well as the effects these genes produced in tomato development. Expression analysis using GUS reporter transgenic lines revealed that these genes are targeted to the vascular tissue, more specifically in the phloem of leaf, stem, root, and floral parts. Knockdown studies, using antisense lines indicated the potential involvement of SlCRF5 in various aspects of tomato development, including leaf size and primary root length as well as the number of lateral roots, and number of flowers, fruits, and seeds produced. SlCRF3 and SlCRF5 were also examined at the protein level. Bimolecular florescence (BiFC) experiments using a split YFP system in protoplasts revealed the ability of these two proteins to form both homo and hetero dimers. In addition cellular localization experiments indicated that these proteins were nuclear localized in planta. In order to investigate the responses of SlCRF3 and SlCRF5 to hormones and stress, tomato plants were treated with cytokinin and other stress hormones and were exposed to abiotic stress conditions. These results revealed that SlCRF3 and SlCRF5 are regulated by cytokinin and also by some abiotic stresses. For the third objective, Illumina RNA-sequencing was utilized to analyze the transcriptome of tomato roots with the main focus on the spatial patterning and regulation of genes by the hormones cytokinin and auxin. The analysis revealed that a number of genes involved in mechanisms such as defense, stress response, cytokinin signaling, hormonal crosstalk, and metabolism were regulated. Together, these findings indicate that SlCRF3 and SlCRF5 are potential regulators of tomato developmental processes associated with cytokinin and abiotic stresses and these results set a foundation for future research directed towards tomato development.