Metabolite profiling of Chlamydomonas reinhardtii under nutrient deprivation

Abstract

A metabolite profiling technique for Chlamydomonas reinhardtii cells for multiparallel analysis of low-molecular weight polar compounds was developed. The experimental protocol was optimized to quickly inactivate enzymatic activity, achieve maximum extraction capacity, and process large sample quantities. As a result of the rapid sampling, extraction, and analysis by gas chromatography coupled to time-of-flight mass spectrometry, more than 800 analytes from a single sample could be measured, of which more than 100 could be identified. Analyte responses could be determined mostly with ses less than 10%. Wild-type cells of C. reinhardtii strain CC-125 subjected to nitrogen-, phosphorus-, sulfur-, or iron-depleted growth conditions develop highly distinctive metabolite profiles. Individual metabolites undergo marked changes in their steady-state levels. Compared to control conditions, sulfur-depleted cells accumulated 4-hydroxyproline more than 50-fold, whereas the amount of 2-ketovaline was reduced to 2% of control levels. The contribution of each compound to the differences observed in the metabolic phenotypes is summarized in a quantitatively rigorous way by principal component analysis, which clearly discriminates the cells from different growth regimes and indicates that phosphorus-depleted conditions induce a deficiency syndrome quite different from the response to nitrogen, sulfur, or iron starvation.

Figure 1.

Extraction capacity of MCW. Changing the ratio of MCW from 10:4:1 (v/v/v; white squares) to a ratio of 10:3:1 (black circles) increases extraction capacity 4-fold, as assessed by extraction of cell chlorophyll. In direct comparison with standard acetone extraction of chlorophyll (white circles), chlorophyll extraction with MCW 10:3:1 performs equally well. Extraction mix volume relates to the use of 3 mL of extraction mix for 1.2×10⁹ cells. Standard errors of triplicate experiments are indicated.

Figure 2.

Analytical signal for C. reinhardtii polar phase extract after GC-TOF measurement. A, Total ion chromatogram of wild-type strain CC-125. In a retention time window from 180 s to 1,200 s after sample injection, fragment ion intensities for all m/z in the mass range from 85 to 500 are recorded. The total ion chromatogram is a composite signal of the sum of all m/z values in each recorded spectrum. Identity of prominent peaks is indicated. B, Retention profiles of unique ions of deconvoluted peaks in the retention time interval between 591 and 597 s of the same chromatogram. Peak apices are indicated by black vertical lines.

Figure 3.

Sample scores for the first and second principal components extracted from the normalized peak response data of the five experimental groups. Each group is represented by five samples. The first and second principal components account for 35.2% and 19.1% of the total sample variance, respectively.

Figure 4.

Loading plots of the first (A) and second (B) principal components. For each principal component, the metabolites with the five largest absolute loading values are indicated. Peaks that could not be identified by library comparison are marked by “yCR” plus ID number. High positive loadings point to a more intense response of the corresponding metabolites in samples that have a high score value for this principal component. Samples with a small negative score value contain comparatively less of the metabolites with large positive loadings and more of those with small negative loadings.