The xanthophyll cycle and NPQ in diverse desert and aquatic green algae

Photosynthesis Research

Claire K. Lunch, Amy M. LaFountain, Suzanne Thomas, Harry A. Frank, Louise A. Lewis, and

Zoe G. Cardon

Corresponding author, current address:

Claire K. Lunch

clunch@neoninc.org

National Ecological Observatory Network, 1685 38th St, Boulder, CO 80301

Supplementary Information

In addition to the O2 microelectrode measurements described in the main text, we carried

out an experiment using planar optode sensors (Larsen et al. 2011, Limnology and

Oceanography: Methods, 9: 348-60) to quantify oxygen concentrations in liquid cultures of

algae dark-adapted overnight in fluorescence cuvettes. Planar optodes were made following the

procedure described in Larsen et al. 2011, except that we used the fluorophore PtTFPP

(platinum(II) meso-tetra (pentafluorophenyl) porphine) instead of the fluorophore PtOEP

(platinum(II) octaethylporphyrin) because it photodegrades less over time (M. Larsen, pers.

comm.). Briefly, the oxygen-sensitive fluorophore, PtTFPP (Frontier Scientific, Logan, Utah),

was dissolved in chloroform with polystyrene beads (Goodfellow, Huntingdon, England) along

with a reference fluorophore, Macrolex Yellow (Lanxess, Birmingham, NJ). The mixture was

spread very thinly and evenly on 0.125mm thick polyethylene terephthalate film (Goodfellow,

Huntingdon, England). After drying, a very thin layer of black silicone (GE, Huntersville, NC)

dissolved in hexane was spread evenly on top, and dried. Optodes were cut into small pieces that

fit snugly inside, against one face of, each of 4 fluorescence cuvettes, with the layered chemistry

oriented toward the inside of the cuvette.

To quantify O2 in the cuvettes, blue excitation light (~450 nm peak, delivered from a

Royal-Blue LED CoolBase Assembly, www.luxeonstar.com, equipped with a 475 nm short pass

filter, NT64-609, Edmund Optics, Barrington, NJ) was delivered in an even field through the

side of the cuvettes where the optodes were lodged, and fluorescence was captured using a

Canon camera (with near IR blocking filter removed and replaced with optically clear glass of

the same thickness), fitted with a 530 nm long pass filter (NT46-060, Edmund Optics,

Barrington, NJ ). A custom LED triggerbox with built-in Canon control software (available from

B.Grunwald@fish-n-chips.de) timed the excitation and image acquisition through the front faces

of four cuvettes. Because the excitation light used with the optodes peaks at ~450 nm, it also

excites chlorophyll fluorescence which can contribute substantially to the red portion of the

fluorescence signal detected by the imaging camera. During calculations of oxygen

concentration, this chlorophyll-related enhancement in the red artificially can decrease the

calculated oxygen concentration. For this reason, and because overnight dark adaptation was not

done with stirring during our chlorophyll fluorescence experiments, we did not stir the algae, and

we used the rapidly sinking aquatic Cylindrocystis as our test case. To calculate O2 concentration

in the cuvette liquid, we used only the portion of the image above the layer of settled algae. After

imaging was finished, temperatures of all solutions were measured, and calibration of the

optodes was conducted at those temperatures (following Larsen et al. 2011).

In the representative data shown below (Fig. S7), high and low densities of aquatic

Cylindrocystis were prepared as fluorometer samples had been prepared previously. Nine ml of

algae were removed from the growth flask at 2:00 pm, allowed to rest in a test tube until 4:00

pm, then 1 ml pipetted out into fluorometer cuvettes at high and low densities. Two cuvettes of

algae at each density were placed in the dark overnight. Fluorescence from the O2-sensitive

optodes in each cuvette was imaged every 15 minutes, during a brief flash of blue light, for ~ 18

hours. Results are expressed as a percent of saturated O2 concentration in the solutions (taking

into account solution temperature). Though the initial %O2 saturation was noticeably lower in the

high- than the low-density cultures, it was never below 50% saturation (~ 4 mg/L O2),

confirming measurements using the oxygen microelectrode presented in the main paper text.

Figure captions

Figure S1. A representative room temperature fluorescence trace, in this case from desert Cylindrocystis, showing maximal fluorescence during saturating pulses increasing in very low light, depression of Fm’ as light intensity increased, and recovery in the dark. Algae were dark-adapted overnight.

Figure S2. Example HPLC chromatogram showing pigments detected in desert Stichococcus sampled at high light exposure. Pigments include neoxanthin (N), violaxanthin (V), lutein (L), zeaxanthin (Z), chlorophyll a and b (Chl a, Chl b), and beta carotene ().

Figure S3. Non-photochemical quenching, expressed as NPQ, throughout light exposure and recovery, for all five species grown and measured on porous glass beads. Gray shading indicates actinic light level. n = 1 for all species except Klebsormidium sp., n = 2.

Figure S4. Non-photochemical quenching, expressed as qN, during the first three hours of the time course, for all five species grown and measured on porous glass beads. Gray bar indicates

where actinic was on at 4 mol photons m-2 s-1. Error bars are one standard deviation; stars indicate significant difference from zero with p<0.05. n = 4 for all species except Klebsormidium sp., n = 5.

Figure S5. Absolute values of 1/Fo and 1/Fm throughout the time course, for all five species grown and measured on porous glass beads. Dotted lines indicate the first 10 seconds of dark recovery. n = 1 for all species except Klebsormidium sp., n = 2.

Figure S6. 77K chlorophyll fluorescence spectra for all five species exposed to five light treatments. Points are means +/- standard errors at 0.5 nm intervals.

Figure S7. Percent saturation of oxygen in representative algal solutions during dark adaptation.

Table S1. Pigments identified in HPLC analysis and the mean percent content in each species in a dark-adapted state.

Entransia Klebsormidium Cylindro (A) Cylindro (D) Stichococcus

Neoxanthin 7.04 4.23 6.43 4.63 6.42Violaxanthin 5.14 4.37 1.95 2.25 2.83Antheraxanthin 0.28 1.17 1.45 0.55 0.20Luteoxanthin 0 0.39 0 0 0Zeaxanthin 0.68 1.12 2.6 1.88 0.41Lutein 4.86 13.17 14.05 10.63 6.18Chlorophyll a 57.1 55.79 51.83 59.3 52.19Chlorophyll b 23.6 18.33 19.33 17.4 30.42 carotene 1.34 1.467 2.65 3.35 1.31

Figure S1

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Figure S7