Integrated RNA-seq and Proteomic Studies Reveal Resource Reallocation towards Energy Metabolism and Defense in Skeletonema marinoi in Response to CO2 Increase

Abstract

Rising atmospheric CO₂ concentrations are causing ocean acidification (OA) with significant consequences for marine organisms. Because CO₂ is essential for photosynthesis, the effect of elevated CO₂ on phytoplankton is more complex and the mechanism is poorly understood. Here we applied RNA-seq and iTRAQ proteomics to investigate the impacts of CO₂ increase (from ∼400 to 1000 ppm) on the temperate coastal marine diatom Skeletonema marinoi We identified 32,389 differentially expressed genes (DEGs) and 1,826 differentially expressed proteins (DEPs) from elevated CO₂ conditions, accounting for 48.5% of total genes and 25.9% of total proteins we detected, respectively. Elevated pCO₂ significantly inhibited the growth of S marinoi, and the 'omic' data suggested that this might be due to compromised photosynthesis in the chloroplast and raised mitochondrial energy metabolism. Furthermore, many genes/proteins associated with nitrogen metabolism, transcriptional regulation, and translational regulation were markedly up-regulated, suggesting enhanced protein synthesis. In addition, S marinoi exhibited higher capacity of ROS production and resistance to oxidative stress. Overall, elevated pCO₂ seems to repress photosynthesis and growth of S marinoi, and through massive gene expression reconfiguration induce cells to increase investment in protein synthesis, energy metabolism and antioxidative stress defense, likely to maintain pH homeostasis and population survival. This survival strategy may deprive this usually dominant diatom in temperate coastal waters of its competitive advantages in acidified environments.Importance Rising atmospheric CO₂ concentrations are causing ocean acidification with significant consequences for marine organisms. Chain-forming centric diatoms of Skeletonema is one of the most successful groups of eukaryotic primary producers with widespread geographic distribution. Among the recognized 28 species, S. marinoi can be a useful model for investigating the ecological, genetic, physiological, and biochemical characteristics of diatoms in temperate coastal regions. In this study, we found that the elevated pCO₂ seems to repress photosynthesis and growth of S. marinoi, and through massive gene expression reconfiguration induce cells to increase investment in protein synthesis, energy metabolism and antioxidative stress defense, likely to maintain pH homeostasis and population survival. This survival strategy may deprive this usually dominant diatom in temperate coastal waters of its competitive advantages in acidified environments.

FIG 1

Time courses for S. marinoi cell density (10⁵ cells ml⁻¹) and pH values. An arrow indicates when samples were collected for “omic” analyses.

FIG 2

Correlation of differential transcriptional and translational expression of genes between normal and elevated CO₂ treatments.

FIG 3

Top 30 ranking GO terms found in the transcriptome in the proteomic analysis. (A) Top 30 ranking GO terms found in the transcriptome. Red text indicates significantly enriched GO terms (Q value < 0.05), and the color strength represents the Q value. (B) Top 30 ranking GO terms found in the proteome. Red text indicates significantly enriched GO terms (P < 0.05), and the color strength represents the P value.

FIG 4

Significant KEGG enrichment pathways either in the transcriptomic (Q value < 0.05) or in the proteomic analysis (P < 0.05). Circle color strength represents the Q or P value, whereas the circle size indicates the enrichment magnitude. The font color of the process names on the left shows that either the transcriptional level, the translational level, or both levels are significant.

FIG 5

Transcript and protein levels of genes encoding components of the nitrogen metabolism pathway in S. marinoi. The heatmap on the right shows changes in the expression levels of genes or proteins involved in nitrogen metabolism. The color strength represents homogenized gene or protein expression values (log₂-fold change) on a column z-score, increasing from blue (lowest) to red (highest). T, transcript; P, protein; NRT, nitrate transporter; AAPT, amino acid/polyamine transporter; AAT, amino acid transporter; AMT, ammonium transporter; UT, urea transporter; NR, nitrate reductase; NiR, nitrite reductase (ferredoxin); CK, carbamate kinase; CPS, carbamoyl-phosphate synthase; CPA2, carbamoyl-phosphate synthase large subunit; CPA1, carbamoyl-phosphate synthase small subunit; AGM, agmatinase; ARG, arginase; ASUS, argininosuccinate synthase; ASL, argininosuccinate lyase; UAP, urease accessory protein; URE, urease; OCD, ornithine cyclodeaminase; ODC, ornithine decarboxylase; GS, glutamine synthetase; GLTD, glutamate synthase (ferredoxin); GLTB, glutamate synthase (NADPH/NADH); GDHB, glutamate dehydrogenase [NAD(P)⁺]; GDHA, glutamate dehydrogenase (NADP⁺); GS-GOGAT, glutamine synthetase/glutamate synthase; NA, not available.

FIG 6

Heatmap showing changes in expression levels of genes or proteins involved in the transport of nutrients, calcium signaling, ROS homeostasis, cell death, photosynthesis, and carbon fixation. The color scale represents normalized gene or protein expression values (log₂-fold change) on a column z-score, increasing from blue (lowest) to red (highest). NA, not available.

FIG 7

Significant expression changes of genes or proteins under the elevated CO₂ condition. (A) Summary of differentially expressed genes or proteins (DEGs/DEPs) involved in oxidative phosphorylation. (B) Summary of DEGs/DEPs involved in RNA polymerases I, II, and III, RNA degradation, eukaryotic translation initiation factors, protein processing in the ER, and ER-associated degradation. N, total DEGs number/total gene number; n, total DEPs number/total protein number. Detailed information is available in Tables S5 and S6.

FIG 8

Heatmap showing changes in expression levels of genes/proteins involved in glycolysis, the pentose phosphate pathway, the TCA cycle, fatty acid metabolism, and N-glycan biosynthesis. The heatmap color scale represents homogenized gene or protein expression values (log₂-fold change) on a column z-score, increasing from blue (lowest) to red (highest). NA, not available.

FIG 9

Schematic representation of basic biological pathways in S. marinoi under the elevated CO₂ condition. RBCL, ribulose-bisphosphate carboxylase large chain; PSII, photosystem II reaction center; PSI, photosystem I reaction center; OEC, oxygen-evolving complex; Lhca, light-harvesting complex I chlorophyll a/b binding proteins; Cytb, cytochrome b₆-f complex; Fd, ferredoxin; FNR, ferredoxin-NADP⁺ reductase; ACL, ATP citrate (pro-S)-lyase; CS, citrate synthase; PK, pyruvate kinase; PDHA, pyruvate dehydrogenase E1 component alpha subunit; PDHB, pyruvate dehydrogenase E1 component beta subunit; I, NADH dehydrogenase; II, succinate dehydrogenase; III, cytochrome c reductase; IV, cytochrome c oxidase; ATPs, ATP synthase; Pol I, RNA polymerase I; Pol II, RNA polymerase II; Pol III, RNA polymerase III; eIFs, eukaryotic translation initiation factors; PABPC, polyadenylate-binding protein; TRAPα, translocon-associated protein subunit alpha; TRAPβ, translocon-associated protein subunit beta; Sec61, protein transport protein Sec61; Sec62, translocation protein Sec62; Sec63, translocation protein Sec63.