Resolving the contribution of the uncoupled phycobilisomes to cyanobacterial pulse-amplitude modulated (PAM) fluorometry signals.

Alonso M Acuña,Ivo H M Van Stokkum,Rienk Van Grondelle,Michal Gwizdala,Diana Kirilovsky,Joris J Snellenburg

doi:10.1007/s11120-015-0141-x

Abstract

Pulse-amplitude modulated (PAM) fluorometry is extensively used to characterize photosynthetic organisms on the slow time-scale (1–1000 s). The saturation pulse method allows determination of the quantum yields of maximal (FM) and minimal fluorescence (F0), parameters related to the activity of the photosynthetic apparatus. Also, when the sample undergoes a certain light treatment during the measurement, the fluorescence quantum yields of the unquenched and the quenched states can be determined. In the case of cyanobacteria, however, the recorded fluorescence does not exclusively stem from the chlorophyll a in photosystem II (PSII). The phycobilins, the pigments of the cyanobacterial light-harvesting complexes, the phycobilisomes (PB), also contribute to the PAM signal, and therefore, F0 and FM are no longer related to PSII only. We present a functional model that takes into account the presence of several fluorescent species whose concentrations can be resolved provided their fluorescence quantum yields are known. Data analysis of PAM measurements on in vivo cells of our model organism Synechocystis PCC6803 is discussed. Three different components are found necessary to fit the data: uncoupled PB (PBfree), PB–PSII complexes, and free PSI. The free PSII contribution was negligible. The PBfree contribution substantially increased in the mutants that lack the core terminal emitter subunits allophycocyanin D or allophycocyanin F. A positive correlation was found between the amount of PBfree and the rate constants describing the binding of the activated orange carotenoid protein to PB, responsible for non-photochemical quenching.Electronic supplementary materialThe online version of this article (doi:10.1007/s11120-015-0141-x) contains supplementary material, which is available to authorized users.

Highlights

Chlorophyll a (Chl a) fluorescence carries important information about the primary photophysical processes taking place in the thylakoid membrane, including nonphotochemical quenching (NPQ) (Baker 2008; Krause and Weis 1991; Papageorgiou and Govindjee 2004; van Grondelle 1985; van Grondelle et al 1994)
We focus on cyanobacterial Pulse-amplitude modulated (PAM) studies in which the PB-related contribution to the PAM signal must first be resolved for, as long as it remains neglected, no solid statements on the photosystem II (PSII)-specific activity can be formulated
An FM level decrease has been reported due to NPQ induced by the saturation pulses; this has been observed in Chlamydomonas and Synechocystis (Schreiber et al 1995) so extra attention should be paid to this effect when increasing the spf in quenching experiments and analysis on both plants and cyanobacteria

Summary

Introduction

Chlorophyll a (Chl a) fluorescence carries important information about the primary photophysical processes taking place in the thylakoid membrane, including nonphotochemical quenching (NPQ) (Baker 2008; Krause and Weis 1991; Papageorgiou and Govindjee 2004; van Grondelle 1985; van Grondelle et al 1994). When the sample undergoes a certain light treatment during the measurement, the fluorescence quantum yields of the unquenched and the quenched states can be determined.

Results

Conclusion