Transcriptome analysis reveals the molecular mechanism of differences in growth between photoautotrophy and heterotrophy in Chlamydomonas reinhardtii

Abstract

Background: The green alga Chlamydomonas reinhardtii can grow photoautotrophically utilizing light and CO₂, and heterotrophically utilizing acetate. The physiological and biochemical responses of autotrophy and heterotrophy are different in C. reinhardtii. However, there is no complete understanding of the molecular physiology between autotrophy and heterotrophy. Therefore, we performed biochemical, molecular and transcriptome analysis of C. reinhardtii between autotrophy and heterotrophy.

Results: The cell growth characterization demonstrated that heterotrophic cell had enhanced growth rates, and autotrophic cell accumulated more chlorophyll. The transcriptome data showed that a total of 2,970 differentially expressed genes (DEGs) were identified from photoautotrophy 12h (P12h) to heterotrophy 12h (H12h). The DEGs were involved in photosynthesis, the tricarboxylic acid cycle (TCA), pyruvate and oxidative phosphorylation metabolisms. Moreover, the results of qRT-PCR revealed that the relative expression levels of malate dehydrogenase (MDH), succinate dehydrogenase (SDH), ATP synthase (ATPase), and starch synthase (SSS) were increased significantly from P12h and H12h. The protein activity of NAD-malate dehydrogenase (NAD-MDH) and succinate dehydrogenase (SDH) were significantly higher in the H12h group.

Conclusion: The above results indicated that the high growth rate observed in heterotrophic cell may be the effects of environmental or genetic regulation of photosynthesis. Therefore, the identification of novel candidate genes in heterotrophy will contribute to the development of microalga strains with higher growth capacity and better performance for biomass production.

Keywords: C. reinhardtii; heterotrophy; metabolic pathways; photoautotrophy; transcriptome.

Figure 1

The growth characteristics of C. reinhardtii in photoautotrophy and heterotrophy. (A) Number of cells cultured under photoautotrophy and heterotrophy at 0, 2, 4, 6, 8, and 10 days; (B) OD₇₅₀ variation curves; (C) Fv/Fm variation curves; (D) Total chlorophyll content; (E) Number of cells between P12h and H12h; (F) OD₇₅₀ from P12h to H12h; (G) Fv/Fm between P12h and H12h; (H) Total chlorophyll content between P12h and H12h. Significance was determined by t-test analysis: ** indicated P < 0.01.

Figure 2

RNA-seq data expression of C. reinhardtii in photoautotrophy and heterotrophy. (A) PCA analysis, PC1 indicated the first principal component and PC2 indicated the second principal; (B) Venn diagram showing expressed genes in P12h vs. H12h; (C) The volcanic map of DEGs between P12h and H12h; (D) Heatmap analysis of the DEGs in H12h and P12h.

Figure 3

Functional analysis of DEGs. (A) Top 20 GO enrichments with DEGs; (B) Top 20 KEGG pathways enriched with DEGs.

Figure 4

Differentially expressed genes (DEGs) between P12h and H12h were involved in the photosynthesis and carbon fixation metabolic pathways. (A) Schematic overview of photosynthesis and carbon fixation in chloroplast of C. reinhardtii, PEP: carboxylate phosphoenolpyruvate, PEPC: phosphoenolpyruvate carboxylase, OAA: oxalo acetic acid, Rubisco: ribulose bisphosphate carboxylase, PGA: 3-phosphoglyceric acid, PGAL: 3-phosphoglyceraldehyde, CAM: crassulacean pathway, red words indicated up-regulated DEGs and blue words indicated down-regulated DEGs; (B) The heatmap of relative expression of DEGs.

Figure 5

Differentially expressed genes (DEGs) between P12h and H12h were involved in glycolysis and the TCA cycle metabolic pathways. (A) Scheme of glycolysis and the TCA cycle in mitochondria of C. reinhardtii. 1,3-DPG, 1,3-diphosphoglyceric acid; Ac-CoA, acetyl-CoA; (B) The heatmap of relative expression of DEGs.

Figure 6

Differentially expressed genes (DEGs) between P12h and H12h were involved in pyruvate metabolic pathway. (A) Scheme of pyruvate metabolic network in mitochondria of C. reinhardtii. (B) The heatmap of relative expression of DEGs.

Figure 7

Differentially expressed genes (DEGs) between P12h and H12h were involved in oxidative phosphorylation. (A) Schematic diagram showing ATP synthase. ATP synthase structural model was constructed (Ge et al., 2021). (B) The heatmap of relative expression of DEGs related to oxidative phosphorylation.

Figure 8

qPCR expression profile of marker genes RBCL, MDH, SDH, Atpase, and SSS between the P12h and the H12h groups. (A) Expression changes of RBCL; (B) Expression changes of MDH; (C) Expression changes of SDH; (D) Expression changes of ATPase; (E) Expression changes of SSS. Data are representative of three independent experiments. Significance was determined by t-test analysis: ** indicated P < 0.01.

Figure 9

The key enzyme activities Rubisco, NAD-MDH, SSS, PK, and SDH in the P12h and the H12h groups. (A) Rubisco activity in P12h and H12h. (B) NAD-MDH activity; (C) SSS activity; (D) PK activity; (E) SDH activity. Data are representative of 3 independent experiments. Significance was determined by t-test analysis: * indicated P < 0.05.