Profiling of Small Molecular Metabolites in Nostoc flagelliforme during Periodic Desiccation

Abstract

The mass spectrometry-based metabolomics approach has become a powerful tool for the quantitative analysis of small-molecule metabolites in biological samples. Nostoc flagelliforme, an edible cyanobacterium with herbal value, serves as an unexploited bioresource for small molecules. In natural environments, N. flagelliforme undergoes repeated cycles of rehydration and dehydration, which are interrupted by either long- or short-term dormancy. In this study, we performed an untargeted metabolite profiling of N. flagelliforme samples at three physiological states: Dormant (S1), physiologically fully recovered after rehydration (S2), and physiologically partially inhibited following dehydration (S3). Significant metabolome differences were identified based on the OPLS-DA (orthogonal projections to latent structures discriminant analysis) model. In total, 183 differential metabolites (95 up-regulated; 88 down-regulated) were found during the rehydration process (S2 vs. S1), and 130 (seven up-regulated; 123 down-regulated) during the dehydration process (S3 vs. S2). Thus, it seemed that the metabolites' biosynthesis mainly took place in the rehydration process while the degradation or possible conversion occurred in the dehydration process. In addition, lipid profile differences were particularly prominent, implying profound membrane phase changes during the rehydration-dehydration cycle. In general, this study expands our understanding of the metabolite dynamics in N. flagelliforme and provides biotechnological clues for achieving the efficient production of those metabolites with medical potential.

Keywords: LC-MS; Nostoc flagelliforme; cyanobacteria; metabolic profiling; metabolites; rehydration and dehydration.

Figure 1

The overview of N. flagelliforme in response to the rewetting–drying cycle. (A) a descriptive model for native environmental adaptation. (B) the changes in PSII activity (in terms of Fv/Fm), and (C) the relative water contents of the samples, respectively, during the rewetting and drying processes. The data shown in (B) are means ± S.D. (n = 5). S1, S2, and S3—three sampling points.

Figure 2

The PCA analysis of the LS-MS metabolomic profiles in positive mode (A), negative mode (B), and the total ion chromatograms (C). The three groups of samples from the three time points (S1, S2, and S3) and pooled QC samples were shown.

Figure 3

A summary of the differentially regulated metabolites between the samples from the three time points.

Figure 4

The top 20 significantly differential metabolites relatively upregulated in the rewetting stage and then reduced in the drying stage. * significant difference (p-value < 0.05), as compared to the S1 stage. 1—Ethenodeoxyadenosine; 2—TG(12:0/12:0/20:2(11Z,14Z))[iso3]; 3—3-Hexadecanoyloleanolic acid; 4—Hericenone E; 5—PA(12:0/15:1(9Z)); 6—Bacteriohopane-32,33,34-triol-35-carbamate; 7—Norselic acid E; 8—PG(18:4(6Z,9Z,12Z,15Z)/15:1(9Z)); 9—PE(17:0/20:4(5Z,8Z,11Z,14Z)); 10—PG(13:0/20:3(8Z,11Z,14Z)); 11—PS(O-16:0/18:3(9Z,12Z,15Z)); 12—PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:4(5Z,8Z,11Z,14Z))[U]; 13—PS(O-16:0/18:4(6Z,9Z,12Z,15Z)); 14—MIPC(t18:0/22:0(2OH)); 15—PE(18:0/20:4(5Z,8Z,10E,14Z)(12OH[S])); 16—SQDG(16:0/16:0); 17—PS(12:0/21:0); 18—Mucronine A; 19—PE(16:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)); 20—PI(20:2(11Z,14Z)/22:4(7Z,10Z,13Z,16Z)).

Figure 5

The significantly differential metabolites relatively upregulated in the drying stage or both the rewetting and drying stages. * significant difference (p-value < 0.05), as compared to the S1 stage. 1—2-Ethylacrylylcarnitine; 2—PC(O-12:0/O-1:0); 3—Dihydroskullcap flavone I; 4—Neuraminic acid; 5—(E)-Resveratrol 3-glucoside 4’-sulfate; 6—Echinenone/ (Myxoxanthin); 7—PE-Cer(d14:2(4E,6E)/24:1(15Z)(2OH)); 8—(S)-2,3-Dihydro-3,5-dihydroxy-2-oxo-3-indoleacetic acid 5-[glucosyl-(1->4)-b-D-glucoside]; 9—Fenothiocarb; 10—6-Methoxy-9H-carbazole-3-carboxaldehyde; 11—Levomethadyl acetate; 12—3-Indolebutyric acid; 13—CerP(d18:1/12:0); 14—PI(O-16:0/12:0); 15—PC(15:0/19:3(9Z,12Z,15Z))[U]; 16—Taxiphyllin; 17—PG(16:0/22:5(4Z,7Z,10Z,13Z,16Z)); 18—Phosphohydroxypyruvic acid.

Figure 6

The metabolic pathway analysis of N. flagelliforme in response to the rewetting (A) and drying (B) processes. The impacted pathways are shown as circles. The size of the circle corresponds to the pathway impact score, and their colors are based on p-value.