3.3 Effect of Cu on gene expression
The transcript expression profiles of the plants were significantly affected by Cu exposure in the two studied populations. The effect of Cu was greater in the most tolerant population (Sc3) as shown by: (i) the greatest separation between control and Cu-treated samples of Sc3 in the multidimensional scaling plot (Fig. 1C1); (ii) the greater number of DETs in Sc3 than in Sc4, i.e. 1,710 DETs in Sc3 - 7.4% of the total - and 259 DTEs in Sc4 - 1.1% of the total; and (iii) the greater expression changes shown by DETs in Sc3 compared to the same DETs in Sc4 (Fig. 1C2; Fig. S6). The majority of the DETs found in each population were down-regulated, i.e. 99% and 95% in Sc3 and Sc4 respectively (Table S6), indicating that these transcripts were constitutively expressed in control plants, and their expression was significantly repressed in response to Cu. The direction of the expression change, i.e. up- or downregulation, of the 220 DETs that were common to both populations was the same; the intensity of the change, however, was ≥ 2 times higher in Sc3 for 62% of the common DETs (on average, FC in this fraction of DETs was 9x higher in Sc3 compared to Sc4); the opposite was true in only 9% of the common DETs (with an average FC in this fraction of DETs 3.4x higher in Sc4 than Sc3) (Fig. 1C2, S6, Table S6).
In total, 74% and 76% of the DETs were annotated in Sc3 and Sc4 respectively (Table S6). The results of the Fisher’s exact test showed 117 over-represented and 13 under-represented GO terms in Sc3, and 11 over-represented GO terms in Sc4 in the lists of DETs compared to the full list of transcripts (Table S7). The most over-represented molecular functions in Sc3 included terms related to RNA metabolism and ribosomal structure (RNA binding, GO:0003723; structural constituent of ribosome, GO:0003735), protein molecular interactions (protein-containing complex binding GO:0044877; protein homodimerization activity; GO:0042803; protein N-terminus binding, GO:0047485), glycosidic bond hydrolysis (hydrolase activity, hydrolyzing O-glycosyl compounds, GO:0004553), protein hydrolysis and its regulation (exopeptidase activity, GO:0008238; serine-type endopeptidase activity, GO:0004252; endopeptidase inhibitor activity, GO:0004866), redox activity (oxidoreductase activity, GO:0016715), and Ca signaling/regulation (Ca ion binding, GO:0005509) (Fig. 2A). These functions were only represented within the down-regulated DETs list (except Ca ion binding, also present within the up-regulated DETs; Table S7). Over-represented molecular functions in Sc4 consisted of protein hydrolysis (cysteine-type endopeptidase activity, GO:0004197; serine-type endopeptidase activity, GO:0004252), chitin binding (GO:0008061), and structural molecule activity (GO:0005198) (Fig. 2B), all of which were only represented in the down-regulated DETs list (Table S5). The GO terms related to biological process, molecular function, and cellular component associated to all DETs of each population are shown in Fig. S7.
We classified down-regulated DETs from Sc3 and Sc4 into 13 different functional groups according to their annotation descriptions (Table S8; Fig. 2C,D) which included transcripts related to: i) protein and ribosome biosynthesis, including structural constituents of ribosomal subunits as well as transcripts involved in protein transcription, translation and elongation; ii) protein degradation and/or turnover, including many proteases, transcripts involved in protein ubiquitination, and structural components and regulators of the proteasome; iii) transcripts with reduction-oxidation (redox) activity, including components of the reactive oxygen species (ROS) scavenging systems (e.g. glutathione S-transferase, ascorbate peroxidase, catalase); iv) protein repair and proper protein folding activity, mostly comprised by protein chaperones; v) Ca homeostasis and signal transduction including Ca-transporting ATPases, Ca calmodulin-dependent kinases, and calmodulin; vi) energy metabolism including enzymes involved in glycolysis, pentose-phosphate, and tricarboxylic acid cycle pathways; vii) transmembrane transport like V-type proton ATPases, ABC transporters, or zinc finger proteins; viii) RNA metabolism, including RNA helicases, mRNA splicing factors, and rRNA processing factors. The remaining groups included transcripts related to vesicle trafficking, fatty acid oxidation, chromatin organization, polyamine biosynthesis and degradation, and purine metabolism. For Sc4, all transcripts except those involved in vesicle trafficking, fatty acid oxidation, and chromatin organization, were represented within the down-regulated DETs list, although the number of transcripts within each group was significantly lower (Fig. 2D; Table S8).
Up-regulated transcripts in Sc3 included enzymes involved in the regulation of the redox state of the cells (probable 2-oxoglutarate-dependent dioxygenase ANS, L-ascorbate oxidase-like, cytokinin hydroxylase, and peroxidase P7-like), and transcripts were involved in cellular signaling processes (EG45-like domain containing protein) and lipid hydrolysis (GDSL esterase/lipase). Three transcripts were up-regulated both in Sc3 and Sc4: allene oxide cyclase, an enzyme catalyzing the most important step in the jasmonic acid (JA) biosynthetic pathway; CML25, a member of the calmodulin-like protein group; and L-ascorbate oxidase-like protein, involved in the regulation of the redox status of the cells. Uniquely up-regulated DETs in Sc4 included the metal homeostasis factor ATX1-like, a copper chaperone that delivers Cu to heavy metal P-type ATPases, two probable copper-transporting ATPases (HMA5), involved in copper transport across membranes, two dehydrogenases with oxidoreductase activity (alcohol dehydrogenase 1-like; and aldehyde dehydrogenase family 2 member B7, mitochondrial-like), and one primary amine oxidase-like, involved in polyamine degradation (descriptions as well as expression changes of all DETs are present in Table S6).
Finally, we found 100 DETs between control plants from Sc3 and Sc4; 68 DETs were significantly upregulated in Sc3 whereas 32 were downregulated in these plants compared to those from Sc4. Annotated upregulated DETs showed a greater expression of components of the protein biosynthesis and degradation machinery and signal transduction, which were downregulated in response to Cu in both populations. Annotated downregulated DETs, on the other hand, were related to the energy metabolism and vacuole transport, among others (Table S6)