Systems-level analysis of nitrogen starvation-induced modifications of carbon metabolism in a Chlamydomonas reinhardtii starchless mutant

Abstract

To understand the molecular basis underlying increased triacylglycerol (TAG) accumulation in starchless (sta) Chlamydomonas reinhardtii mutants, we undertook comparative time-course transcriptomics of strains CC-4348 (sta6 mutant), CC-4349, a cell wall-deficient (cw) strain purported to represent the parental STA6 strain, and three independent STA6 strains generated by complementation of sta6 (CC-4565/STA6-C2, CC-4566/STA6-C4, and CC-4567/STA6-C6) in the context of N deprivation. Despite N starvation-induced dramatic remodeling of the transcriptome, there were relatively few differences (5 × 10(2)) observed between sta6 and STA6, the most dramatic of which were increased abundance of transcripts encoding key regulated or rate-limiting steps in central carbon metabolism, specifically isocitrate lyase, malate synthase, transaldolase, fructose bisphosphatase and phosphoenolpyruvate carboxykinase (encoded by ICL1, MAS1, TAL1, FBP1, and PCK1 respectively), suggestive of increased carbon movement toward hexose-phosphate in sta6 by upregulation of the glyoxylate pathway and gluconeogenesis. Enzyme assays validated the increase in isocitrate lyase and malate synthase activities. Targeted metabolite analysis indicated increased succinate, malate, and Glc-6-P and decreased Fru-1,6-bisphosphate, illustrating the effect of these changes. Comparisons of independent data sets in multiple strains allowed the delineation of a sequence of events in the global N starvation response in C. reinhardtii, starting within minutes with the upregulation of alternative N assimilation routes and carbohydrate synthesis and subsequently a more gradual upregulation of genes encoding enzymes of TAG synthesis. Finally, genome resequencing analysis indicated that (1) the deletion in sta6 extends into the neighboring gene encoding respiratory burst oxidase, and (2) a commonly used STA6 strain (CC-4349) as well as the sequenced reference (CC-503) are not congenic with respect to sta6 (CC-4348), underscoring the importance of using complemented strains for more rigorous assignment of phenotype to genotype.

Figure 1.

Increased TAG but Not Starch in the sta6 Mutant. In all panels, CC-4349 is shown in red, sta6 (CC-4348) in black, and three strains complemented for STA6 (STA6-C2, STA6-C4, and STA6-C6) in blue. Chlorophyll content (A), cell density (B), starch content per cell (C), and TAG per cell (D). Error bars represent one sd calculated from three separate cultures (biological replicates). (E) shows an immunoblot of MLDP1 using protein extracts obtained from N-deprived cultures of sta6 and STA6-C2, STA6-C4, and STA6-C6, sampled at 0, 24, 48, and 96 h as indicated.

Figure 2.

Experimental Design for Sampling the Transcriptome. Cultures were grown to a density of 4 × 10⁶ cells mL⁻¹ in +N medium and washed in N-free medium before resuspending cells to a final density of 2 × 10⁶ cells mL⁻¹ in N-free medium. For the time-course experiment comparing CC-4349 and sta6, samples were taken in triplicate at 0’ (i.e., from +N medium before washing), 0, 0.5, 2, 4, 8, 12, 24, and 48 h after N deprivation as indicated with red arrows. In a separate experiment, sta6, STA6-C2, STA6-C4, and STA6-C6 were sampled at 0.5, 4, and 48 h after N deprivation, as indicated with blue arrows. The light-green color of an N-free culture at 48 h indicates the cells are chlorotic.

Figure 3.

Greater Upregulation of Genes Encoding Enzymes of Acetate Metabolism, Gluconeogenesis, and the Oxidative Pentose Phosphate Pathway in sta6 versus CC-4349 and STA6. Heat map showing fold change of RPKM (relative to 0 h in CC-4349), ranging from 0.18 (yellow) to 18 (violet), of genes involved in acetate assimilation, the glyoxylate cycle, glycolysis/gluconeogenesis, and the oxidative pentose phosphate pathway as indicated. Heat map was generated using Matrix2png (Pavlidis and Noble, 2003). RPKM values for complete pathways are shown in Supplemental Data Set 3 and Supplemental Figure 2 online.

Figure 4.

Upregulated Genes Encode Key Enzymes in Central Carbon Metabolism. Steps in the glyoxylate cycle (A), gluconeogenesis (B), and oxidative pentose phosphate pathway (C) are shown. The complete oxidative pentose phosphate pathway is shown in Supplemental Figure 11 online. For clarity, only relevant enzymes are shown.

Figure 5.

Expression Profiles of Genes Related to TAG Accumulation. DGTT2, encoding a type-2 diacylglycerolacyltransferase (A); DGAT3, encoding a putative soluble diacylglycerolacyltransferase (B); and MLDP1 encoding major lipid droplet protein (C). Expression level is expressed as RPKM for each mRNA at 0.5, 4, and 48 h. CC-4349 is in red, sta6 is in black, and STA6 is in blue.

Figure 6.

Resequencing of the sta6 Genome Reveals Extent of Gene Disruption. The reads were aligned to the C. reinhardtii reference strain (CC-503) using the Burrows-Wheeler Aligner and displayed via the Integrative Genomics Viewer. The depth of coverage, indicated in the figure by vertical gray bars on a scale from 0 to 100, drops off precipitously at the boundaries of the disrupted genes. The breakout tracks above and below display the left and right boundaries, respectively, magnified to the nucleotide level. Gene models for STA6 and RBO1 are shown with exons represented by thick bars and introns represented by thin bars. The nucleotide track indicates the reference sequence at these loci.

Figure 7.

Comparative Analysis of sta6 and STA6 Transcriptomes Identifies sta6-Dependent Expression Patterns. (A) Venn diagram showing genes differentially expressed between sta6 and STA6-C2, STA6-C4, and STA6-C6 at 0.5, 4, and 48 h after N deprivation. The central intersect indicates the differentially expressed genes common to all STA6-complemented strains relative to sta6. (B) The 521 differentially expressed genes resulting directly from STA6 were grouped according to their function in 25 categories, as assigned by MapMan ontology. For clarity, genes not assigned are not shown, but total 195.

Figure 8.

Increased Isocitrate Lyase and Malate Synthase Activities in sta6 versus STA6. Isocitrate lyase (A) and malate synthase (B) activities. C2, C4, and C6 represent the complemented strains (white), and sta6 is shown in black. Extracts were prepared from cells sampled 0, 24, 48, and 96 h after removal of N. Error bars indicate the sd of three independent biological repeats.

Figure 9.

Metabolite Profiles in sta6 and STA6-Complemented Strains Are Consistent with Predictions from Transcriptomes. (A) to (G) sta6 (black) was grown in six independent cultures, and STA6-C (white) comprises duplicate cultures each of STA6-C2, STA6-C4, and STA6-C6. Strains were cultured in +N to a density of 4 × 10⁶ cells mL⁻¹ before transferring to +N and –N cultures to a final cell density of 2 × 10⁶ cells mL⁻¹. Samples were taken at 48 h after transfer for metabolite measurement. Significance was assessed by pairwise Student’s t test. P value correction was performed using the Bonferroni correction method (*P < 0.05, **P < 0.01, and ***P < 0.001). Error bars represent one sd. (H) sta6 (black) and STA6-C2, STA6-C4, and STA6-C6 (white) were each grown in triplicate in +N to a density of 4 × 10⁶ cells mL⁻¹ before transferring to –N to a final cell density of 2 × 10⁶ cells mL⁻¹. Samples were taken at 0, 48, and 96 h after transfer for Glc-6-P measurement. Error bars represent one sd.

Figure 10.

Acetate Utilization Is Similar in All Strains. sta6 (black) and three independent STA6 strains (STA6-C2, STA6-C4, and STA6-C6; shown in white) were cultured in +N to a density of 4 × 10⁶ cells mL⁻¹ before transferring to –N cultures to a final cell density of 2 × 10⁶ cells mL⁻¹. Acetate levels in the supernatant were assessed at 0, 48, and 96 h after transfer to –N. Error bars represent one sd.