Supplementary material to Spijkerman et al. “Nutrient induced fluorescence
transients (NIFTs) provide a rapid measure of P and C (co)-limitation in a
green alga“
Supplementary figures.
Suppl. Figs 1-2. Extent of NIFT response to a Pi-spike (Fig. 1: 10 µM) and CO2-spike (Fig.
2: 100 µM) plotted versus the cellular P content (Fig. 1) and cellular C content (Fig. 2) of
Chlamydomonas acidophila cultured under 4 different combinations of Pi and CO2
conditions. The values of the inoculation culture were: 0.73 pg P cell-1 and 40 pg C cell-1; the
NIFT values at these cellular contents were measured directly after inoculation. In both
figures negative values were excluded for clarity.
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NIFT responses to different concentrations of Pi
At the end of the growth experiment (day 8 for -P+CO2, and day 12 for -P-CO2), NIFT
responses were recorded on Pi-limited cells by the addition of eight different Pi
concentrations to compare the NIFT response with Pi-uptake kinetics. A fresh sample was
taken for every measurement to avoid effects from prior additions. From the NIFT response 2
parameters were calculated. First, the relative maximal change in fluorescence output (ΔF%)
was calculated as the most reliable and useful measurement (see e.g. Roberts et al.,2008).
This was calculated by subtracting the minimum chl a fluorescence after the nutrient pulse
(Ft,min) from the Ft before the pulse (Ft,max) divided by Ft,max. Second, we calculated the rate of
change in fluorescence (ΔF% min-1) on the data from the experiment where we added
different Pi concentrations as this parameter reportedly has a direct positive correlation to Piuptake rates (Roberts et al. 2008). We calculated the half-saturation constant (Km) by
linearization of the NIFT response kinetics (as v, for uptake rate) following Hofstee (1952),
and compared the Km with that obtained for C. acidophila in a prior study using 33Pincorporation (Spijkerman 2007). As an average we took the data from the lowest two steady
state growth rates.
The measurements from adding different concentrations of Pi and the Pi-affinity
curves thereby obtained are shown in Fig. 3. In this case we also calculated the rate of relative
change in chlorophyll a fluorescence directly after each Pi-spike, and described the NIFT
response by the concentration of each Pi-spike following a Michaelis-Menten model
(resulting in a “maximum uptake rate”, “Vmax” and an affinity constant, Km). Based on the
rate of fluorescence change, the model estimated the Vmax to be higher and the Km to be lower
in the low CO2 than in the high CO2 cells (Table 1). A similar observation was made for Vmax
when the percentage fluorescence change was used in the calculation (extent of NIFT
response; Table 1).
In contrast, the estimated Km for high CO2 cells was the same by both calculations
(0.30 and 0.29 µM P), whereas the Km was 2-fold lower in the rate of change calculation (0.20
µM P) than by the relative change calculation (0.43 µM P) in the low CO2 cells (Table 1).
Independent of these differences, the Km values were much lower when measured by
fluorescence than when measured by 33P-incorporation (Table 1), suggesting that fluorescence
quenching is a faster response to a Pi-limitation than nutrient uptake. Thus, there is a more
pronounced, faster effect on photosynthesis than there is on pure nutrient uptake kinetics.
Most likely this reflects that uptake porters are slower in response than redox processes
involved in electron transport.
2
Supplementary Table 1 Pi-uptake kinetics of Chlamydomonas acidophila grown in Pilimiting semi-continuous cultures (33P uptake) or Pi-limited batch cultures (fluorescence; this
study) at 2 different CO2 concentrations
High CO2
Method (unit of Vmax)
33P
uptake (mmol P g Chl a-1 h-1)a
Fluorescence (ΔF% min-1)
Fluorescence (ΔF%)
a
Low CO2
Vmax
Km (µM P)
Vmax
Km (µM P)
157
1.30
88
0.79
84
0.29
99
0.20
0.33
0.30
0.47
0.43
Data calculated from 33P uptake data at the two lowest growth rates (Spijkerman 2007).
The value of Km for high CO2 cultures was the same when using the rate of fluorescence
change or the relative change in fluorescence. In contrast, Km was higher when the relative
decrease in Ft was used than when the rate of decrease in fluorescence was evaluated in low
CO2-grown cells (Table 1). A similar observation was made in Dunaliella tertiolecta (Roberts
et al. 2008). Compared with kinetics of 33P incorporation, a lower Km in the low CO2- than in
the high CO2-grown cells is expected. Thus the rate of fluorescence change indeed provides
the results most closely resembling the traditional uptake kinetics.
Suppl. Fig. 3 The rate of change in fluorescence (ΔF% min-1) of Chlamydomonas acidophila
grown in Pi-limited batch cultures and 2 different CO2 concentrations in relation to the
concentration of the Pi-spike. Values are the mean and SD of duplicates. Michaelis-Menten
modelling of the kinetics is additionally shown.
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References:
Hofstee, B. H. J. (1952). On the evaluation of the constants Vm and Km in enzyme reactions.
Science 116: 329-331.
Roberts, S. Shelly, K. Beardall, J. (2008). Interactions among phosphate uptake,
photosynthesis, and chlorophyll fluorescence in nutrient-limited cultures of the chlorophyte
microalga Dunaliella tertiolecta. Journal of Phycology 44: 662-669.
Spijkerman, E. (2007). Phosphorus acquisition by Chlamydomonas acidophila under
autotrophic and osmo-mixotrophic growth conditions. Journal of Experimental Botany 58:
4195-4202.
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