Quantitative proteomic comparison of salt stress in Chlamydomonas reinhardtii and the snow alga Chlamydomonas nivalis reveals mechanisms for salt-triggered fatty acid accumulation via reallocation of carbon resources

Abstract

Background: Chlamydomonas reinhardtii is a model green alga strain for molecular studies; its fully sequenced genome has enabled omic-based analyses that have been applied to better understand its metabolic responses to stress. Here, we characterised physiological and proteomic changes between a low-starch C. reinhardtii strain and the snow alga Chlamydomonas nivalis, to reveal insights into their contrasting responses to salinity stress.

Results: Each strain was grown in conditions tailored to their growth requirements to encourage maximal fatty acid (as a proxy measure of lipid) production, with internal controls to allow comparison points. In 0.2 M NaCl, C. nivalis accumulates carbohydrates up to 10.4% DCW at 80 h, and fatty acids up to 52.0% dry cell weight (DCW) over 12 days, however, C. reinhardtii does not show fatty acid accumulation over time, and shows limited carbohydrate accumulation up to 5.5% DCW. Analysis of the C. nivalis fatty acid profiles showed that salt stress improved the biofuel qualities over time. Photosynthesis and respiration rates are reduced in C. reinhardtii relative to C. nivalis in response to 0.2 M NaCl. De novo sequencing and homology matching was used in conjunction with iTRAQ-based quantitative analysis to identify and relatively quantify proteomic alterations in cells exposed to salt stress. There were abundance differences in proteins associated with stress, photosynthesis, carbohydrate and lipid metabolism proteins. In terms of lipid synthesis, salt stress induced an increase in dihydrolipoyl dehydrogenase in C. nivalis (1.1-fold change), whilst levels in C. reinhardtii remained unaffected; this enzyme is involved in acetyl CoA production and has been linked to TAG accumulation in microalgae. In salt-stressed C. nivalis there were decreases in the abundance of UDP-sulfoquinovose (- 1.77-fold change), which is involved in sulfoquinovosyl diacylglycerol metabolism, and in citrate synthase (- 2.7-fold change), also involved in the TCA cycle. Decreases in these enzymes have been shown to lead to increased TAG production as fatty acid biosynthesis is favoured. Data are available via ProteomeXchange with identifier PXD018148.

Conclusions: These differences in protein abundance have given greater understanding of the mechanism by which salt stress promotes fatty acid accumulation in the un-sequenced microalga C. nivalis as it switches to a non-growth state, whereas C. reinhardtii does not have this response.

Keywords: Biofuel; Chlamydomonas nivalis; Chlamydomonas reinhardtii; Fatty acid production; Quantitative proteomics; Salt stress.

Fig. 1

Growth curves, chlorophyll, carbohydrate and FAME content of each strain during salt stress. Growth curves are based on cell count (A and B), chlorophyll a content as a percentage of biomass (C and D), carbohydrate content as a percentage of biomass (E and F) and total FAME content as a percentage of biomass (G and H), for C. reinhardtii grown in 0 and 0.2 M NaCl (A, C, E, G) and C. nivalis grown in 0 and 0.2 M NaCl (B, D, F, H). For all experiments n = 3. Error bars show SEM. Arrows indicate the proteomic sampling points (blue arrows: 0 h salt stress; red arrows: early stage salt stress; green arrows: control cultures mid-log phase; yellow arrows: mid-log salt stress). Note that different scales are used for the y axis of the comparative figures

Fig. 2

Relative percentages of FAME chain types in each strain during salt stress. FAME chain type relative percentages (in terms of degree of unsaturation) are shown for each time-course experiment for 0 M (A) and 0.2 NaCl (B) in C. reinhardtii and 0 M (C) and 0.2 M (D) NaCl in C. nivalis (n = 3). SFAs = saturated fatty acids; PUFAs = polyunsaturated fatty acids; MUFAs = monounsaturated fatty acids

Fig. 3

Quantitative proteomic analysis experimental set-up using 8-plex iTRAQ. Two 8-plex iTRAQ experiments were performed for C. reinhardtii (A) and C. nivalis (B). Four conditions (time 0 salt stress, early log-phase salt stress, mid-log phase salt stress, and time-matched mid-log control), two biological replicates of each, were compared for each species. The assigned iTRAQ labels are shown for each condition and replicate. Time points were selected at which C. nivalis was producing carbohydrate (early log) and fatty acids (mid-log) in response to 0.2 M NaCl stress, with the equivalent points of the growth curve selected for C. reinhardtii

Fig. 4

Photosynthetic activity and respiration rates in each strain during salt stress. Photosynthesis and respiration rates were measured by rate of oxygen evolution and uptake in C. reinhardtii (A) and C. nivalis (B) grown in 0 and 0.2 M NaCl. T-tests were carried out to identify statistically significant changes between treatments, "*" indicates a significant result (p < 0.05) in the comparison of 0 and 0.2. Error bars show SEM (n = 3)