3.4 Water deprivation caused extensive transcriptional reprogramming in both WT and Fld-expressing leaves
Gene expression levels were determined after 3 days of water restriction and transcripts displaying 2-fold or higher differences compared to control well-watered conditions were classified as DE. Distribution of these transcripts between treatments and genotypes (depicted as Venn diagrams in Figure 4) indicates that 999 genes were induced by drought in both lines, 1530 genes only in the non-transformed plants, and 698 uniquely in Stpfld 252 leaves (FC > 2, FDR < 0.05). In contrast, 1505 genes were repressed by drought in both lines, 1667 only in the wild type and 895 exclusively inStpfld 252 plants. In summary, drought induced 2529 genes in WT plants against 1697 in Stpfld 252 siblings, and repressed 3172 genes in the wild type versus 2400 in the transformant. Hence, the overall effect of Fld presence was to mitigate the changes in gene expression driven by the drought treatment; either induction or repression.
Functional enrichment analysis identified pathways and functional categories that were significantly affected in response to water withdrawal in the two genotypes. Among the categories that showed drought-dependent induction in both WT and Stpfld 252 plants, abiotic stress was over-represented. The opposite situation,e.g. , pathways repressed by water limitation in the two lines, is exemplified by cell wall metabolism and kinase-dependent protein modification and signaling (Figure 4, Supplementary Table S3).
Several DE genes showed differential stress responses in only one genotype. Drought-dependent inactivation of photosynthesis in WT plants (Figure 1b-d) was paralleled at the transcript level by widespread down-regulation of genes encoding photosynthetic components. Repression particularly impaired light reactions, and was largely prevented by chloroplast Fld (Figure 4, Supplementary Table S3), correlating with the protection of photosynthetic activities in the transformant (Figure 1b-d).
While the effect of drought on leaf gene expression was predominantly repressive (Figure 4, Supplementary Table S3), stress-dependent induction was also important in the wild type with more than 2500 genes affected. Enriched functional categories included protein metabolism with 98 DE genes (mostly ribosomal proteins), biotic stress and amino acid synthesis (Figure 4, Supplementary Table S3). Differential induction of all these pathways was totally or partially abolished by Fld expression in chloroplasts, suggesting once again that the water deficit regime had less impact in the transgenic plants than in their WT siblings.
Finally, some functional categories were specifically over-represented in the transformant, including drought-associated up-regulation of membrane transport, sulfur assimilation and flavonoid synthesis (Figure 4, Supplementary Table S3).