Exploring conservation of cellular-level traits in shade avoidance syndrome among species 5 183 ► Figure 5.9. Pea (P. sativum) has mild stem elongation in response to FR. P. sativum seedlings were subjected to one week of WL+FR treatment versus WL treatment. (a) A comparison of pea growth phenotypes between WL and WL+FR conditions after 7 days of treatment. (b) Illustration of P. sativum growth after one week of treatment. This figure provides insights into each internode and leaf, revealing stem bending and a tendency for plants in both treatments to tip over. Phenotyping data for P. sativum is presented as follows (c) stem length, (d) internode 1 length, (e) internode 2 length, (f) internode 3 length, (g) internode 4 length, and (h) stem diameter. The data is expressed as mean values ± SEM, with different letters indicating significant differences between treatments based on ANOVA analysis with Tukey’s post hoc test (P<0.05). There are 6 biological replicates. The following microscopy analysis results aspects are examined: (i) microscopy example of each cell type from cross section in pea, (j) vasculature area from cross sections, (k) pith total area from cross sections, (l) ratio of pith area/total area from cross section, and (m) pith cell length from longitudinal sections. Asterisks in the figure indicate the significance of differences between WL and WL+FR treatments, with the following significance levels: * p≤0.05; ** p≤0.01; *** p≤0.001. Error bars represent the standard error (SE), and the sample size for each measurement is n=40. 5.2.5 FR-responsive tomato TFs have family-specific and tissuespecific expression patterns To investigate the molecular events underlying the internode and pith elongation responses, we delved into the behavior of the transcription factor (TF) encoding genes that we identified as our TFs of interest in Chapter 3, Solyc08g080150, Solyc01g090760, Solyc07g053450, across various species. In Chapter 3, we identified these three genes not only as DEGs (Table S3.5) upregulated in response to FR in the entire internode 1, but they also exhibited a significant upregulation in the pith after 30 hours of FR treatment (see Figure 3.18). We wanted to test if the FR-responsive gene expression, especially in pith, was conserved in our species panel. This approach aims to uncover the broader similarities and differences in TF expression patterns among dicot relatives. For this section, we selected species from our previous experiments (Figures 5.2-5.9) based on genome sequence availability. Hence, we worked with tomato, Arabidopsis, pepper, eggplant, black mustard, soybean, and pea to construct an expression analysis of TF homologs. First, we needed to identify homologs of the three TFs in each species to conduct an interspecies qPCR analysis. We conducted ortholog searches using default BLAST searches on various websites (details in methods) (Fernandez-Pozo et al., 2015; The Arabidopsis Information Resource, 2015; Chen et al., 2022a). For the other two Solanaceae species, pepper and eggplant, the top result was a single hit for each of the three genes (with one exception). In the case of Arabidopsis, we considered not only the BLAST result but also literature annotations to identify a homolog with a potentially conserved function. For pea, soybean, and black mustard, we selected genes based on the highest similarity results (85% and up), and we found the chosen genes closely resembled each
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