Proteomic analysis of metabolic pathways supports chloroplast-mitochondria cross-talk in a Cu-limited diatom

Abstract

Diatoms are one of the most successful phytoplankton groups in our oceans, being responsible for over 20% of the Earth's photosynthetic productivity. Their chimeric genomes have genes derived from red algae, green algae, bacteria, and heterotrophs, resulting in multiple isoenzymes targeted to different cellular compartments with the potential for differential regulation under nutrient limitation. The resulting interactions between metabolic pathways are not yet fully understood. We previously showed how acclimation to Cu limitation enhanced susceptibility to overreduction of the photosynthetic electron transport chain and its reorganization to favor photoprotection over light harvesting in the oceanic diatom Thalassiosira oceanica (Hippmann et al., 2017, 10.1371/journal.pone.0181753). In order to gain a better understanding of the overall metabolic changes that help alleviate the stress of Cu limitation, we have further analyzed the comprehensive proteomic datasets generated in that study to identify differentially expressed proteins involved in carbon, nitrogen, and oxidative stress-related metabolic pathways. Metabolic pathway analysis showed integrated responses to Cu limitation. The upregulation of ferredoxin (Fdx) was correlated with upregulation of plastidial Fdx-dependent isoenzymes involved in nitrogen assimilation as well as enzymes involved in glutathione synthesis, thus suggesting an integration of nitrogen uptake and metabolism with photosynthesis and oxidative stress resistance. The differential expression of glycolytic isoenzymes located in the chloroplast and mitochondria may enable them to channel both excess electrons and/or ATP between these compartments. An additional support for chloroplast-mitochondrial cross-talk is the increased expression of chloroplast and mitochondrial proteins involved in the proposed malate shunt under Cu limitation.

Keywords: Cu limitation; copper; cross‐talk; diatom; proteomics.

FIGURE 1

Overview of proteomics data for Thalassiosira oceanica CCMP 1003 (TO03) and 1005 (TO05) grown in Cu‐limiting conditions. (a–c) Venn diagrams of distinct identified proteins in the original datasets of TO03 and TO05: (a) All proteins; (b) significantly upregulated proteins; (c) significantly downregulated proteins. Note that even though only 50% of the proteins identified in TO05 were identified in TO03, in TO03, there were three times more significantly upregulated proteins and five times more significantly downregulated proteins than in TO05

FIGURE 2

Bar charts of second level Kegg Orthology (KO) identifiers associated with proteins of original TO03 dataset comparing proportions between all proteins and those that are significantly upregulated or downregulated. Numbers in brackets indicate absolute protein numbers in each set. Note that certain aspects of metabolism are most highly affected: Energy, amino acid and carbohydrate metabolism

FIGURE 3

Relative expression of proteins involved in the Calvin–Benson–Bassham and citrate (tricarboxylic acid [TCA]) cycle. Boxes indicate proteins with their abbreviated name and known Thalassiosira pseudonana (Tp) and Thalassiosira oceanica (To) homologs. The colors of the boxes indicate expression in T. oceanica TO03 under low Cu: Dark red, highly upregulated (>2‐fold, p < .05); light pink, upregulated by 1.3‐ to 2‐fold (p < .05); dark blue, highly downregulated (>2‐fold, p < .05); light blue, downregulated by 1.3‐ to 2‐fold (p < .05); white, expressed in TO03; gray border around box, found in T. oceanica T005 genome but not expressed in TO03 proteomic data; gray, dashed border around box, no putative homologs in the T. oceanica genome. Metabolites: 1,3‐bisPG, 1,3‐bisphosphateglycerate; DHAP, dihydroxyacetone phosphate; Ery‐4‐P, erythrose 4 phosphate; Fru‐1,6‐bis‐P, fructose 1,6‐bisphosphate; Fru‐6‐P, fructose 6‐phosphate; G3P, glucose‐3‐phosphate; GAP, glyceraldehyde 3‐phosphate; HCO₃ ⁻, bicarbonate; OAA, oxaloacetate; Rib‐1,5‐bisP, ribulose‐1,5‐bisphosphate; Sedo‐1,7‐bisP, sedoheptulose 1 7‐bisphosphate; TP, triose phosphate. For other abbreviations, see Table 1

FIGURE 4

Relative expression of proteins involved in glycolysis in the three compartments (chloroplast, mitochondrion, cytosol), including Entner–Dourdoroff pathway. Boxes indicate proteins with their abbreviated name and known Thalassiosira pseudonana (Tp) and Thalassiosira oceanica (To) homologs. Colors and shading as in Figure 3. Metabolites: 1,3‐ bisPG, 1,3‐bisphosphateglycerate; 2K3D‐PG, 2‐keto‐3‐deoxyphosphogluconate; 2PG, 2‐phosphoglycerate; 3PG, 3‐phosphoglycerate; 6PG, 6‐phosphogluconate; DHAP, dihydroxyacetone phosphate; Fru‐1,6‐bis‐P, fructose 1,6‐bisphosphate; Fru‐6P, fructose 6‐phosphate; GAP, glyceraldehyde 3‐phosphate; Glu 6‐P, glucose 6‐phosphate; HCO₃ ⁻, bicarbonate; OAA, oxaloacetate; PEP, phosphoenolpyruvate. Other abbreviations in Table 1

FIGURE 5

Relative expression of proteins involved in nitrogen metabolism. Boxes indicate proteins with their abbreviated name and known Thalassiosira pseudonana (Tp) and Thalassiosira oceanica (To) homologs. Colors and shading as in Figure 3. Metabolite abbreviations as in Figure 3 or Table 1

FIGURE 6

Relative expression of proteins involved in the malate shunt. Boxes indicate proteins with their abbreviated name and known Thalassiosira pseudonana (Tp) and Thalassiosira oceanica (To) homologs. Colors and shading as in Figure 3.Abbreviations: AAT, aspartate aminotransferase; MDH, malate dehydrogenase; ME, malic enzyme; OAA, oxaloacetate; PC, pyruvate carboxylase; PK, pyruvate kinase. Other abbreviations in Table 1

FIGURE 7

Relative expression of proteins involved in glutathione metabolism and response to reactive oxygen species (ROS). Boxes indicate proteins with their abbreviated name and known Thalassiosira pseudonana (Tp) and Thalassiosira oceanica (To) homologs. Colors and shading as in Figure 3. Protein and metabolite abbreviations: as in Table 1