Supplementary material

This supplementary material contains two sections:

(i)details about experimental conditions

(ii) comparison of streak-camera DAS obtained from C.meneghinianain open and closed state upon 400 nm excitation

(i)Experimental conditions

Measurements using streak-camera setup

For streak-camera measurements, the sample was kept at 287 K in a flow cuvette and a sample reservoir (3-4 ml).In order to minimize the contribution of closed reaction centers and of annihilation, the cellswere flowing from the reservoir to the cuvette and back, with a flow speed of ~2.5 ml/sec. Excitation light was vertically polarized, the spot size diameter was typically∼100 μm, and the laser repetition rate was 250kHz while the excitation power was 15-35µW.The detector polarizer was set at magic angle orientation. For the experiments we used a cuvette with optical path length of 1 mm.To avoid significant self-absorption the OD at the excitation wavelength was set to ~0.1/cm (for 400nm:OD680≈0.06, for 535 nm: OD680≈0.13). In this way during the time (~3μs) needed to travel through the excitation spot 1 out of ~900 to 2200 pigments is excited. Supposing equal excitation of PSI and PSII, and a PSII antenna size of ~300 pigments/RC(for the estimations the number of pigments in PSII supercomplex of higher plants was used[1], it follows that 20±10 % of the RCs are excited while passing through the excitation beam.

In order to be able to resolve the components in the region from ~5 ps to ~2 ns two time windows were used: 800 ps and 2 ns.Each experiment was repeated 3 times on different generations. The streak-images ofdifferent samplesgrown in the same light conditions are very similar in case of the independent cultivation and are shown in Fig. S1.

Figure S1.Fluorescence 2D images obtained for 3 different generations ofC. meneghiniana LL as measured with the streak-camera setup upon 400nm excitation. The time window was 0.8 ns (A,B,C) and 2.0 ns(D,E,F). False colors reflect the fluorescence intensity.

TCSPC measurements

For the TCSPC measurements the pumping system as well as temperature conditions were kept the same asfor the streak-camera measurements (see above).But in case of the TCSPC setupthe spot size diameter was typically 3mm, and the laser repetition rate was 3.8MHz, while the excitation power was 0.1-5µW,which is up to 350 times lower than in case of streak camera. The optical path length of the cuvette used for the TCSPC experiments was 3 mm; to avoid significant self-absorption the OD at the excitation wavelength was set to ~0.06/cm (for 400nm:OD680≈0.04, for 535 nm: OD680≈0.08). In this way each laser pulse excites 1 out of ~5*107 to 2*109Chls. In the time (~4 ms) to travel through the excitation spot ~1*104pulses hit the sample, exciting 1 out of ~4*103to 1*105pigments. Supposing equal excitation of PSI and PSII, and a PSII antenna size of ~300 pigments/RC, it follows that while passing through the excitation beam, pigments associated with~0.2% -7.0% of the RCs are excited.So the probability to have an excitation on a RC in case of TCSPC measurements is on average 5 times lower than in case of streak-camera measurements.In order to be sure that the laser power as well as the measuring procedure did not introduce any changes in the results, the final experiment of a measuring series was always a repetition of the first experiment. The resulting decay curves were indistinguishable.Each sample was measured 3-5 times for different generations of cells. The fluorescence decay curves for every sample appeared to be very similar in case of independent generations.Fig.S2 shows the C.meneghiniana LL fluorescence kinetics excited at 400 nm, and detected at 689 nm in the beginning and in the end of measuring series(Fig.S2 A) and cells grown in different generations(Fig.S2 B).

Figure S2.Fluorescence kinetics of C. meneghinianaexcited at 400nm and detected at 679 nm, measured on the same sample in the beginning(black line) and in the end(red line) of the measuring series(A) and for three independent generations(B)

(ii)Comparison of streak-camera DAS obtained from C.meneghiniana LL under 400 nm excitation without and in the presence of DCMU

A rough estimate of the relative PSI contribution to the 76 ps component resolved in streak-camera measurements of C.meneghinana LL cells upon 400 nm excitation (Fig. 1A) was performed in the following way:streak-camera measurements of the samplegrown in the same light conditions were performed in the presence of 100µM DCMU.In this case a substantial increase of “closed” PSII reaction centers should be observed, thereby increasing the corresponding PSII lifetimes, while the lifetimes of PSI are hardly affected [2, 3]. Streak-camera data of C.meneghinina LL cells in the presence of DCMU was fitted globally and compared with the results obtained for the open state(Fig. 1A). The 76 ps component was also resolved in case of closed RCs with the spectral properties similar to the “open” state case(Fig S3).Generally the area under a positive DAS scales linearly with the number of pigments contributing to the component.Because the closing of RC should not affect the proportion of pigments attached to the photosystems, the relative contribution of the area assigned to the PSI component should notexperiencesignificant changes as well.Indeed the calculated relative contribution of the 76 ps DAS to the total area (the sum of the DAS areas of all components) stayed ~40% in both cases, while the total contribution of two longest components was evidently ~60 %. As expected the PSII average lifetime increased substantially from ~ 0.5 ns to 1.0 ns.These numbers are not very accurate because of the limited time window of the setup.

Figure S3.Comparison of the results obtained from C.meneghiniana LL by streak-camera measurements in the presence of DCMU (“closed” state)(red lines) and in the open state(black lines).The DAS in both cases were normalized to the 76 ps component.

[1] B. van Oort, M. Alberts, S. de Bianchi, L. Dall'Osto, R. Bassi, G. Trinkunas, R. Croce, H. van Amerongen, Effect of antenna-depletion in Photosystem II on excitation energy transfer in Arabidopsis thaliana, Biophys J, 98 (2010) 922-931.

[2] E. Wientjes, R. Croce, PMS: Photosystem I electron donor or fluorescence quencher, Photosynthesis Research, 111 (2012) 185-191.

[3] M. Byrdin, I. Rimke, E. Schlodder, D. Stehlik, T.A. Roelofs, Decay kinetics and quantum yields of fluorescence in photosystem I from Synechococcus elongatus with P700 in the reduced and oxidized state: are the kinetics of excited state decay trap-limited or transfer-limited?, Biophys J, 79 (2000) 992-1007.