The phenomenon of non-photochemical quenching (NPQ) was studied in spinach chloroplasts using pulse amplitude modulated (PAM) fluorometry. We present a new analysis method which describes the observed fluorescence quantum yield as the sum of the product of four different states of PSII and their corresponding quantum yields. These four distinct states are PSII in the quenched or unquenched state, and with its reaction center either open or closed depending upon the reduction of the QA site. With this method we can describe the dynamics of the NPQ induction and recovery as well as quantify the percentage of photoinactivated RC throughout the measurement. We show that after one cycle of quenching followed by a period of recovery, approximately 8-9% of the RC are photoinactivated, after two cycles of illumination this number becomes 1-5-17%. The recovery from the quenching appeared with rates of (50 s)(-1) and (1 hr)(-1). The new analysis method presented here is flexible, allowing it to be applied to any type of PAM fluorometry protocol. The method allows to quantitatively compare qualitatively different PAM curves on the basis of statistically relevant fitting parameters and to quantify quenching dynamics and photoinactivation. Moreover, the results presented here demonstrate that the analysis of a single PAM fluorometry quenching experiment can already provide information on the relative quantum yield of the four different states of PSII for the intact chloroplasts- something no other form of spectroscopy could provide in a single measurement.
|Number of pages||11|
|Journal||Biochimica et Biophysica Acta (BBA) - Bioenergetics|
|Publication status||Published - Oct 2017|
- Fluorescence quenching analysis
- Non-photochemical quenching
- Parameter estimation
- Time-resolved fluorescence spectroscopy