FIGURE LEGENDS
Figure 1. Maximum growth rate (μMAX) of
exponentially growing algal cultures at various temperatures.
(a) Chlamydomonas sp. UWO241 (b) Chlamydomonasreinhardtii . Data are the means ± SD of at least six biological
replicates
Figure 2: Principal component analysis (PCA) of the primary
metabolome of the two Chlamydomonas species acclimated to
different steady-state temperatures. C. reinhardtii was grown at
10°C (magenta; CR_10), 15°C (orange; CR_15), and 28°C (red; CR_28).
UWO241 was grown at 4°C (cyan; UWO241_04), 10°C (blue; UWO241_10) and
15°C (green; UWO241_15). The analysis includes all 771 quantified
metabolites separated along the first two principal components that
explained the largest degree of variation in the datasets, and the 95%
confidence interval for each treatment.
Figure 3. Differences in the primary metabolome of C.
reinhardtii and UWO241, acclimated at different steady-state
temperatures. (a ) Heat map showing the relative changes in
metabolite abundances between growth temperatures in the two algal
species. Only metabolites which are significantly different are shown
(392 metabolites, ANOVA, P<0.01). In each treatment, three
biological replicates are represented using a color based metabolite
profile as indicated (red – increase in abundance; blue – decrease in
abundance). Hierarchical clustering is based on Euclidean distances and
Ward’s linkage. A cluster of metabolites present in both species at the
lowest temperature is highlighted by a *. (b ) Relative
abundance of metabolites classified based on their chemical nature. Only
metabolites which were positively identified based on their GC-MS
spectra and retention times were taken into consideration. In this
analysis, the metabolite abundance corresponding to C.
reinhardtii grown at 28°C was arbitrarily set to 1 and all other
treatments were compared to this sample.
Figure 4. Kinetics of cell death in UWO241 (a,b) andC. reinhardtii (c,d) acclimated to different growth
temperatures and exposed to non-permissive conditions (24°C and 42°C,
respectively). Cell death was estimated as the loss of chlorophyll in
cells exposed to heat (a,c) or as a percentage of algal cells
stained with 0.5% Evans Blue that accumulates in cells with damaged
membranes (b,d) . Algal cells treated with 1% v/v chloroform
were taken as a positive control and used to calculate 100% cell death.
Data are means ± SD of at least three independent experiments and
analyzed by two-way ANOVA followed by Bonferroni post-test comparing
each treatment with 4°C (UWO241) and 28°C (C. reinhardtii ).
Statistical significance (P<0.01) is indicated as *
Figure 5. Light microscope images of UWO241 acclimated to
different steady state temperatures (4°C, 10°C, 15°C) and exposed to
non-permissive temperature (24°C) for 24h, 48h and 72h. Algae are
present as single cells or palmelloid colonies. Scale bar = 15 μm (400x
total magnification)
Figure 6. Principal component analysis (PCA) of the primary
metabolome of UWO241 acclimated to different steady-state temperatures
and subsequently exposed to non-permissive temperature for 6 hours.
UWO241 was grown at 4°C (blue, UWO241_4) and exposed to 24°C (yellow,
UWO241_4_HS); grown at 10°C (cyan; UWO241_10) and exposed to 24°C
(orange, UWO241_10_HS); and grown at 15°C (green; UWO241_15) and
exposed to 24°C (red, UWO241_15_HS). The analysis includes all
quantified metabolites separated along the first two principal
components and the 95% confidence interval for each treatment.
Figure 7. Differences in the primary metabolome in UWO241
acclimated at different temperatures (4°C, 10°C, 15°C) and subsequently
exposed to heat stress (24°C) for 6 hours. (a ) Heat map showing
the relative changes in metabolite abundances between control samples at
each steady-state growth temperature (C) and heat-treated samples (HS).
Only metabolites that are significantly different are shown (314
metabolites, ANOVA, P<0.01). Three biological replicates are
represented using a color-based metabolite profile as indicated (red –
high abundance; blue – low abundance). Hierarchical clustering is based
on Euclidean distances and Ward’s linkage (b ) Relative
abundance of metabolites classified based on their chemical nature. Only
metabolites which were positively identified based on their GC-MS
spectra and retention times were taken into consideration. In this
analysis, the metabolite abundance in algae grown at the three different
steady state temperatures were arbitrarily set to 1 (black bars) and all
the heat stress treatments were compared to the corresponding control
sample (blue bars).
Figure 8: (a) UWO241 grown at temperatures closest to its
natural environment in Lake Bonney has an active central metabolism and
constitutively accumulates metabolites important for life at low
temperatures, including soluble sugars, antioxidants and compounds
involved in nucleic acid protection. This simplified pathway map shows
key metabolites that are increased (red) or decreased (blue) in UWO241
at 4°C, when compared to C. reinhardtii at 28°C. Metabolites
shows in black did not change significantly, and those in gray were not
detected in this study. We propose that this metabolic state provides
UWO241 with the ability to cope with environmental stress. (b)When UWO241 is exposed to short-term heat stress at 24°C, many
metabolites characteristic for cold adaptation, including soluble sugars
and antioxidants, are maintained or even increased. The maintenance of
energy metabolism could provide the energy to drive the production of
protective compounds during heat stress. We show key metabolites that
are increased (red) or decreased (blue) in, when compared to UWO241 at
4°C. Metabolites shows in black did not change significantly, and those
in gray were not detected in this study.