Chapter 4 138 4.2.14 Comparative analysis of gene expression patterns in timepoints and tissues Finally, we wanted to verify how the different hormones induced gene expression in the internode, and if these changes resembled the supplemental FR-induced changes. Building on previous results from Chapter 3, we had observed that pith tissue responded differently to FR enrichment compared to the whole internode 1. In order to further characterize these differences and investigate earlier timepoints, we first analyzed FRresponsive gene expression changes with qRT-PCR in both pith tissue and the first internode tissue at 1, 2, and 6 hours after applying supplementary far-red light. For the first qRT-PCR analysis, we selected transcription factors and hormone-related and hormone-responsive genes that were identified as DEGs in the transcriptome data at the six hour timepoint. We found distinct FR-responses in the two tissues (Figure 4.22). Overall, we observed the clearest changes in gene expression at the two hour timepoint. As certain transcription factors showed only mild effects, they were excluded from subsequent experiments. 4.2.15 The effect of different hormones on gene expression patterns After confirming the FR-induced differences between the two tissues and discovering that the largest changes were observed at two hours of WL+FR treatment, we decided to sample the hormone qPCR experiment at this time point. In the hormone experiment, we harvested the plant material after two hours of treatment with WL, GA, IAA, GA+IAA, BR, BR+GA+IAA, and WL+FR, and performed a second gene expression study on the entire internode 1, as well as the pith tissues of this internode (Figures 4.22, 4.23). We found that GA induced expression of most of the studied genes in the internode. Research has shown that ELONGATED HYPOCOTYL5 (HY5) is a shade marker gene that is dependent on Phytochrome A, which gets upregulated in low R/FR (Ciolfi et al., 2013). Here, we also found an upregulation of HY5 in both tissues of FR-enriched plants. GA and IAA treated plants also show upregulation of HY5 but only in the whole internode tissue. Interestingly, the combined GA+IAA treatment shows downregulation of HY5. Furthermore, we found that INDOLE-3-ACETIC ACID INDUCIBLE 19 (IAA19), IAA22, PIN-FORMED 3 (PIN3) and SMALL AUXIN UPREGULATED RNA (SAUR) were upregulated in supplemental FR light and a similar pattern was found for the GA treated plants. In contrast, this pattern was not found in the IAA or GA + IAA treated plants. IAA19 and IAA22 are auxin responsive genes, There is an overall up-regulation of
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