Chapter 3 62 types and four timepoints, altogether 24 sample types, with 3-4 replicates each. The barcodes were assigned to each sample and separated in 4 individual lanes for sequencing. The samples were sequenced to the depth of at least 3 million raw reads and then mapped to SL4.0 tomato genome (https://solgenomics.net/) and ITAG4.1 annoqtation. 3.2.2 The two cultivars’ gene expression profiles were similar Before differential expression (DE) analysis, we firstly ran a quality check with samples using principal component analysis (PCA, Figure S3.1). From the PCA analysis, the samples from the two cultivars, MM and M82, did not separate from each other when compared to the other variables. DE analysis of MM vs M82 (Figure S3.2) shows no differentially expressed genes (DEGs) in WL at any timepoint, and the only distinguishable difference between the cultivars appeared at 6h WL+FR treatment. Combining with the phenotyping evidence from previous chapter and Figure 3.4, we assume these cultivars can be taken as replicates. Based on these data, we decided to put these two cultivars together in the following analysis, without no more considering cultivar differences. 3.2.3 Differential gene expression was induced already at our earliest FR timepoint To identify DEGs, we utilized the limma voom package (Law et al., 2014; Ritchie et al., 2015) for comparing WL+FR transcriptomes to WL at each different timepoint and tissue type (Figure 3.5a). The most pronounced gene regulation emerged at the initial timepoint, after 6 hours of FR treatment, highlighting the rapid adjustment of gene expression within the stem due to FR light. Notably, the DEGs in first internode exhibited a gradual decline throughout the FR treatment duration. Pith showed comparable DEG numbers to internode 1 at 30h and 48h timepoints (Figure 3.5b), The overall number of DEGs remained modest, but pith and internode do present different DEGs (Figure 3.6). Notably, FR treatment reveals a higher number of DEGs when comparing morning to afternoon, both upregulated and downregulated. This suggests that FR can augment diurnal changes (Figure 3.5 c). The sectioning protocol was carefully carried out to remove all outer layers to ensure the precision of pith harvest. Notably, pith-specific expression, when compared to internode expression, demonstrated minimal upregulation (pith-enriched gene expression) and predominantly showcased downregulation (Figure S3.2b). This could indicate the role of pith being a “blank slate” without any outstanding specialized processes. Consequently, this pattern did not captivate our interest to a significant degree.
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