PMS: photosystem I electron donor or fluorescence quencher

Abstract

Light energy harvested by the pigments in Photosystem I (PSI) is used for charge separation in the reaction center (RC), after which the positive charge resides on a special chlorophyll dimer called P700. In studies on the PSI trapping kinetics, P700(+) is usually chemically reduced to re-open the RCs. So far, the information available about the reduction rate and possible chlorophyll fluorescence quenching effects of these reducing agents is limited. This information is indispensible to estimate the fraction of open RCs under known experimental conditions. Moreover, it would be important to understand if these reagents have a chlorophyll fluorescence quenching effects to avoid the introduction of exogenous singlet excitation quenching in the measurements. In this study, we investigated the effect of the commonly used reducing agent phenazine methosulfate (PMS) on the RC and fluorescence emission of higher plant PSI-LHCI. We measured the P700(+) reduction rate for different PMS concentrations, and show that we can give a reliable estimation on the fraction of closed RCs based on these rates. The data show that PMS is quenching chlorophyll fluorescence emission. Finally, we determined that the fluorescence quantum yield of PSI with closed RCs is 4% higher than if the RCs are open.

Fig. 1

Rate of photo-oxidized P700 reduction by PMS. The 830 minus 875 nm absorption signal is monitored after P700 is oxidized by a 20 mmol/m²/s light pulse with a duration of 0.2 s. PMS/NaAsc concentrations were as in previous reports: 10 μM/10 mM (e.g., Ihalainen et al. 2005), 60 μM/40 mM (Slavov et al. 2008), and 150 μM/5 mM (Byrdin et al. 2000)

Fig. 2

P700⁺ build-up for different PMS concentrations. The P700⁺ formation upon illumination of PSI using 531 μmol/m²/s of actinic light (gray bar) was compared with that after a strong light pulse of 20 mmol/m²/s (white bar), the rest of the time the light was off (black bar) to allow for full re-opening of the RCs. PMS was reduced using NaAsc, at concentrations reported in the legend of Fig. 1

Fig. 3

Theoretical and empirical fractions of closed RCs. PMS concentrations were 10, 60, and 150 μM, light intensities were 53, 166, 531, and 1028 μmol/m²/s. For 150 μM of PMS, the lowest light intensity gave a P700⁺ fraction which was too low to quantify, therefore this data point is not reported

Fig. 4

Fluorescence emission of LHCII and PSI followed in time during the addition of PMS and NaAsc. For LHCII, the excitation was at 630 nm and the emission was detected at 680 nm; for PSI, the excitation was at 500 nm and the emission was detected at 725 nm. Excitation of PSI at 630 nm gave similar results

Fig. 5

Simultaneous detection of fluorescence emission and P700⁺ absorption of PSI. The fluorescence emission of PSI was followed during the photo-oxidation of P700 using 70 μmol/m²/s of actinic light (gray bar) and the re-opening of the RCs in the dark by 10 mM NaAsc (black bar). The maximum level of P700⁺ was determined by a 500 ms saturating light pulse of 8000 μmol/m²/s (white bar)