Chapter 5 184 other. We visualized the relationships of these homologs based on sequence similarities by generating maximum likelihood phylogenies (Figures 5.10-5.12). The phylogenies of the three TFs tend to align with the relationships between the plant species (Figure 5.1). The phylogenies also reflect the recent gene or even whole genome duplication events with occurrences of two closely related paralogs, for example in pea for each gene. For the qPCR quantification of gene expression, we collected whole internode and pith samples simultaneously at 6 h and 24 h time points for WL and WL+FR for all species except pea, where we harvested only the whole internode due to the challenging pith morphology (as depicted in Figure 5.9i). For the orthologs of Solyc07g053450 and Solyc08g080150, we observed a uniform FR-upregulation in both internode and pith in each member of the Solanaceae family (Figures 5.10, 5.12, S5.1, S5.3). Notably, for Solyc01g090760, we identified two bell pepper homologs (Figure 5.11). The homolog with lower sequence similarity had stronger correlation in expression patterns with tomato, while the other ortholog, we observed a non-FR-responsive expression pattern (Figure 5.11, S5.2). This indicates that even with high sequence similarity, homologs can have diverged function, especially if another homolog has retained its role. Then, outside of the Solanaceae family, we looked at two Brassicaceae species, Arabidopsis and Brassica nigra, and two legumes, pea and soybean. In these families, we found a prevalent downregulation of TF homologs in most instances (Figures 5.10-5.12). In pea we had observed limited stem growth under FR and different stem morphology that made it difficult to get pith data, and there we observed that the expression of three TF homologs consistently decreased. We identified multiple best homologs in pea, and none of them exhibited the expected FR-responsive function. This would indicate that during evolution these TFs potentially gained their FR-responsive behavior within the Solanaceae lineage, or lost it in the lineage leading to pea despite the expansion of the TF family. In the FR-responsive soybean, while there is not such a clear downregulation in FR, there is also no clear homolog that would match the tomato patterns. For the two soybean homologs of Solyc08g080150 we observed a strong FR-induction specifically in pith at 6h (Figure 5.12), so potentially the role of these two homologs is retained to some extent in soybean. So, we found that the FR-responsive expression patterns for these genes resembles each other in Solanaceae, and it is distinct from Brassicaceae and Fabaceae. Even though some Brassicaceae and Fabaceae species exhibit similar elongation responses to Solanaceae, the TF expression pattern is not conserved.
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