Overexpression of 18S rRNA methyltransferase CrBUD23 enhances biomass and lutein content in Chlamydomonas reinhardtii

Abstract

Post-transcriptional modification of nucleic acids including transfer RNA (tRNA), ribosomal RNA (rRNA) and messenger RNA (mRNA) is vital for fine-tunning of mRNA translation. Methylation is one of the most widespread post-transcriptional modifications in both eukaryotes and prokaryotes. HsWBSCR22 and ScBUD23 encodes a 18S rRNA methyltransferase that positively regulates cell growth by mediating ribosome maturation in human and yeast, respectively. However, presence and function of 18S rRNA methyltransferase in green algae are still elusive. Here, through bioinformatic analysis, we identified CrBUD23 as the human WBSCR22 homolog in genome of the green algae model organism Chlamydonomas reinhardtii. CrBUD23 was a conserved putative 18S rRNA methyltransferase widely exited in algae, plants, insects and mammalians. Transcription of CrBUD23 was upregulated by high light and down-regulated by low light, indicating its role in photosynthesis and energy metabolism. To characterize its biological function, coding sequence of CrBUD23 fused with a green fluorescence protein (GFP) tag was derived by 35S promoter and stably integrated into Chlamydomonas genome by glass bead-mediated transformation. Compared to C. reinhardtii wild type CC-5325, transgenic strains overexpressing CrBUD23 resulted in accelerated cell growth, thereby leading to elevated biomass, dry weight and protein content. Moreover, overexpression of CrBUD23 increased content of photosynthetic pigments but not elicit the activation of antioxidative enzymes, suggesting CrBUD23 favors growth and proliferation in the trade-off with stress responses. Bioinformatic analysis revealed the G1177 was the putative methylation site in 18S rRNA of C. reinhardtii CC-849. G1177 was conserved in other Chlamydonomas isolates, indicating the conserved methyltransferase activity of BUD23 proteins. In addition, CrTrm122, the homolog of BUD23 interactor Trm112, was found involved in responses to high light as same as CrBUD23. Taken together, our study revealed that cell growth, protein content and lutein accumulation of Chlamydomonas were positively regulated by the 18S rRNA methyltransferase CrBUD23, which could serve as a promising candidate for microalgae genetic engineering.

Keywords: 18S rRNA methyltransferase; Chlamydomonas reinhardtii; CrBUD23; CrTrm112; biomass; lutein; overexpression.

FIGURE 1

CrBUD23 is a 18S rRNA methyltransferase. (A) Structure of CrBUD23 (Cre09. g398104). (B) Conserved motifs in BUD23 homologs. (C) Phylogenetic trees of BUD23 homologs.

FIGURE 2

Transcription of CrBUD23 is upregulated by high light. (A) qRT-PCR analysis of CrBUD23 under high light and low light. CK, 25 μmol photons/m²s. High light, 75 μmol photons/m²s. Low light, 8 μmol photons/m²s. (B) qRT-PCR analysis of CrBUD23 under modulate (0.1 M NaCl) and severe (0.2 M NaCl) salt stresses. (C) qRT-PCR analysis of CrBUD23 under heavy metal stress. Error bars represent the mean value from three independent experiments. Statistically significant differences were determined by Student’s t-test (**p < 0.01, *p < 0.05).

FIGURE 3

Generation of C. reinhardtii transgenic strains overexpressing CrBUD23. (A). Schematic diagram of CrBUD23 overexpression cassettes in pCAMBIA2300-CaMV35S:CrBUD23-GFP. (B). Genomic PCR of the wild-type (WT) and transgenic strains integrated with CrBUD23-GFP. M, Marker; WT, CC-5325; Arabic numerals, transformants with pCAMBIA2300-CaMV35S:CrBUD23-GFP. (C). qRT-PCR analysis of transgenic strains OE-CrBUD23-10, OE-CrBUD23-11, OE-CrBUD23-15.

FIGURE 4

Overexpression of CrBUD23 enhances cell growth and biomass. (A). Dry weight of WT, transgenic strains OE-CrBUD23-10 and OE-CrBUD23-11. (B). Cell density of WT, transgenic strains OE-10. (C). Total protein content in WT, transgenic strains OE-10. (D). Chlorophyll a content in WT, transgenic strains OE-10 and OE-11. (E). Chlorophyll b content in WT, transgenic strains OE-10 and OE-11. (F). Glucose content in WT, transgenic strains OE-10 and OE-11. Different letters above the bars indicate significant difference at p < 0.01. Error bars indicate SD (n = 6). Letters above the bars indicate significant differences (p < 0.01). Similar results were obtained in three independent experiments.

FIGURE 5

Overexpression of CrBUD23 increases lutein content. (A) Lutein content in WT, transgenic strains OE-10 and OE-11. (B) β-carotene content in WT, transgenic strains OE-10 and OE-11. (C) Zeaxanthin content in WT, transgenic strains OE-10 and OE-11.

FIGURE 6

Overexpression of CrBUD23 maintains redox homeostasis. (A). CAT activity of WT, transgenic strains OE-CrBUD23-10 and OE-CrBUD23-11. (B). NO content in WT, transgenic strains OE-10 and OE-11. (C). Cell density of WT, transgenic strains OE-10 and OE-11 under heavy metal stresses.

FIGURE 7

Conserved motif in 18S rRNA in Chlorophyte. (A) alignment of 18S rRNAs of H. sapiens, C. renhardtii and A. thaliana. (B) conserved G1177 in 41 C. renhardtii isolates.

FIGURE 8

Transcription of CrTrm112 is upregulated by high light. (A). Phylogenetic trees of Trm112 homologs. (B). Alignment of Trm112 homologs. (C). qRT-PCR assay of CrTrm112 under high light, low light, modulate (0.1 M NaCl) and severe (0.2 M NaCl) salt stresses.