 2 FC ≥ 1) with an FDR value of < 0.05 were identified as DEGs. PCAs of each type of tissue were performed individually, as leaves and roots have widely different patterns of gene expression Zhou et al., 2019). The PCA plots of leaves and roots showed different grouping of elp2-5 samples along with the components compared to Col-0 and met1-7 (Supplementary File 2 and Supplementary Figure 1). In the comparison of spaceflight to ground leaves, Col-0 had a total of 207 (123 upregulated and 84 down-regulated) DEGs, elp2-5 had 36 (26 upregulated and 10 down-regulated) DEGs, and met1-7 had 226 (160 up-regulated and 66 down-regulated) DEGs ( Figure 7A). In roots, a total of 147 (28 up-regulated and 119 down-regulated), 120 (50 up-regulated and 70 down-regulated), and 47 (39 upregulated and 8 down-regulated) DEGs were found in Col-0, elp2-5, and met1-7, respectively ( Figure 7B). Only a few DEGs overlapped between the genotypes in both leaf and root tissues (Figures 7C,D). In leaves, the majority of DEGs were unique to Col-0 and met1-7, whereas, in roots, the majority of DEGs were unique to Col-0 and elp2-5 (Figures 7C,D). The patterns of DEGs in all three genotypes were distinctly organ-specific, and of the 436 DEGs in leaves and 288 in roots, only 35 DEGs were common to both sets ( Figure 7E). Although there were overall fewer root-specific DEGs, a greater proportion of the root DEGs were also differentially methylated in response to spaceflight compared to the DEGs of leaves. In roots, 82% of the DEGs also exhibited DmCs, whereas in leaves, 31% of the DEGs also mapped to DmCs (Figure 7F and Supplementary File 5).
The composition of DmC-DEGs in elp2-5 compared to Col-0 was distinctive in each environmental and organ comparison (Figure 8). In leaves, elp2-5 vs Col-0 on the ground had a total of 2,119 DEGs (1,322 up-regulated and 797 downregulated) whereas in spaceflight there were a total of 2,149 DEGs (1,448 up-regulated and 701 down-regulated; Figure 8A). Of these, 996 DEGs were also differentially methylated in the GCs, and a total of 1,188 DmC-DEGs were observed in spaceflight (Figure 8B). A total of 497 DmCs-DEGs were shared between both environmental conditions. In roots, the pairwise comparison of elp2-5 and Col-0 revealed a total of 1,667 (725 up-regulated and 942 down-regulated) DEGs on the ground and 1,626 (724 up-regulated and 902 down-regulated) DEGs in spaceflight ( Figure 8C). Of these, 1,222 DEGs on  the ground were also associated with DmCs and 1,481 DEGs in spaceflight were associated with DmCs. Among these, 723 DmC-DEGs were shared in both spaceflight and GC conditions ( Figure 8D). A visual heat-map summary of the organ-specific distribution of DEGs and the DmC-DEGs for all genotypes is presented in the Supplementary Figure 2, which is annotated in Supplementary File 6.
Multiple metabolic processes appeared to be altered in elp2-5 compared to Col-0 ( Figure 8E). GO analysis of the DmC-DEGs of elp2-5 compared to Col-0 showed that elp2-5 was engaged with many processes characteristic of stress responses (Figure 8E). Processes shared as enriched between leaf and root DmC-DEGs included general responses to stimuli, oxidoreductase activities, and heme-binding. Leaf-specific DmC-DEGs were enriched in biosynthetic processes of flavonoids and pigments, jasmonic acid, and wounding responses in both GC and FT. DmC-DEGs in GC leaves were more specifically enriched in phenylpropanoid metabolism and the response to oxidative stress, as well as in further oxidoreducatase and peroxidase activities. DmC-DEGs in FT leaves were enriched with defense and hypoxia responses. DmC-DEGs annotated to the plasma membrane and extracellular structures were enriched among both GC and FT roots, in addition to transport processes and activities. Carbohydrate transport and binding were specifically enriched among root GC DmC-DEGs. Thus, elp2-5 shows differential expression and methylation of stress and hormone response pathways, as well as transport and metabolic pathways, when compared to the Col-0 wild-type line within each tissue and growth condition.
Relative to the Col-0 response, there were substantially more DEGs and DmCs in elp2-5 (Figure 8) than were seen in met1-7 (Figure 9). In leaves, elp2-5 vs Col-0 showed a total of 2,119 (1,322 up-regulated and 797 down-regulated) DEGs in GC, whereas 2,149 (1,448 up-regulated and 701 down-regulated) DEGs were observed in FT (Figure 8A). DmC-DEGs in the elp2-5 to Col-0 leaves comparison revealed 996 differentially methylated and expressed transcripts on the GC and 1,188 in FT, of which 499 were unique to GC, 691 unique to FT, and 497 were shared among both conditions (Figure 8B). In the roots, the pairwise comparison of elp2-5 and Col-0 in the GC revealed a total of 1,667 (725 up-regulated and 942 down-regulated) DEGs and a total of 1,626 (724 up-regulated and 902 down-regulated) DEGs in FT (Figure 8C). DmC-DEGs in the elp2-5 to Col-0 roots comparison revealed a total of 1,222 DmC-DEGs in GC roots and 1,481 DmC-DEGs in FT roots, of which 723 were shared in both conditions ( Figure 8D). In leaves, met1-7 vs Col-0 showed a total of 840 (355 up-regulated and 485 down-regulated) DEGs in GC, whereas in FT 637 (375 up-regulated and 262 down-regulated) DEGs were observed ( Figure 9A). DmC-DEGs in the met1-7 to Col-0 leaves comparison revealed 527 differentially methylated and expressed transcripts on the ground and 408 in flight, of which 261 were unique to GC, 142 unique to FT, and 266 were shared among both conditions (Figure 9B). In the roots, the pairwise comparison of met1-7 and Col-0 in the GC revealed a total of 779 (343 up-regulated and 436 down-regulated) DEGs and a total of 593 (369 up-regulated and 224 down-regulated) DEGs in spaceflight ( Figure 9C). DmC-DEGs in the met1-7 to Col-0 roots comparison revealed a total of 648 DmC-DEGs in GC roots and 430 DmC-DEGs in FT roots, of which 313 were shared in both conditions ( Figure 9D).
Gene ontology analysis of DmC-DEGs between met1-7 and the wild-type line yielded few term enrichments ( Figure 8E). Biological process enrichments associated with hypoxic responses and biosynthesis of anthocyanin-containing compounds were present in GC root DmC-DEGs. Molecular functions of ADP binding, sesquiterpene compound synthase activities, and cell wall constituents, and nutrient reservoir activity were enriched in leaf GC DmC-DEGs, leaf FT DmC-DEGs, and root GC DmC-DEGs, respectively.
Alignments of DmC-DEGs in each methylation context in the various genic regions were generated for each genotype and depicted as heatmaps (Figure 10). In leaves, a total of 59 DmC-DEGs were observed in wild-type Col-0, while in elp2-5 and met1-7, there were 11 and 72 DmC-DEGs, respectively. Correlations between differential expression and DNA methylation were used to organize the heatmap. The top bracketed sections of the heatmaps show negative correlations where up-regulated genes aligned with hypomethylated DmCs and down-regulated genes aligned with hypermethylated DmCs are grouped. The following bracketed section shows positive correlations where the converse relationship between DEGs and DmCs was highlighted. The unbracketed section at the bottom of the heatmap shows genes that have no distinguishable correlations between DEGs and DmCs. In Col-0 leaves, 41% (24 out of 59) of genes had a negative correlation and 49% (29 out of 59) had a positive correlation. In elp2-5 leaves, negative and positive correlation were evenly distributed at 45% (5 out of 11), whereas in met1-7 leaves, 26% (19 out of 72) of genes showed a negative correlation and 60% (43 out of 72) had a positive correlation. In roots, there were a total of 114 DmC-DEGs in Col-0, 101 DmC-DEGs in elp2-5, and 28 DmC-DEGs in met1-7 (Figure 11). 16% (19 out of 114) of the wild-type Col-0 root DmC-DEGs had a negative correlation between gene expression and methylation levels, whereas 66% (75 out of 114) were positively correlated. In elp2-5 roots, 54% (