Measuring P700 Absorbance Changes in the Near Infrared Spectral Region with a Dual Wavelength Pulse Modulation System

Abstract

Monitoring the oxidation state of the primary donor P700 of photosystem I (PS I) via near infra red absorption spectroscopy has been providing valuable information on PSI-driven photosynthetic electron transport in vivo (1, 2, 3). Redox changes of P700 are preferentially detected at a single wavelength band in the region of the P700⁺ cation radical absorption band peaking at 820 nm. NIR-measuring light > 800 nm is not absorbed by chlorophyll, does not generate chlorophyll fluorescence and has no actinic effect on photosynthesis of green plants. It can be applied at high intensity with the advantage of a high S/N ratio. In order to discriminate the measuring light from continuous background light the former usually is modulated. Modulation up to MHz frequencies can be easily achieved by infra-red emitting diodes (IRED). Such IREDs display bandwidths of 30–50 nm which is suitable for the broad P700⁺ absorption band. Single wavelength systems based on a modulated WED (e.g. ED-800T, Walz, Effeltrich) have been successfully used in monitoring light induced P700 redox changes in intact leaves (1, 2). Superimposed less wavelength-specific changes (as scattering changes) in most cases are slow and may be separated from P700⁺ by application of saturation pulses (4) or by switching off the actinic light from time to time and observing the rapid signal relaxation. However, the low selectivity of the single wavelength measurement becomes a major problem with suspensions of isolated chloroplasts or algae. Unspecific optical changes caused by shrinking, swelling and settling of the chloroplasts or cells lead to serious distortions of the much smaller P700⁺ signal, and artefacts caused by chemical additions and sample stirring preclude the observation of bathochromic absorption changes.