Abstract
Technical features and examples of application of a special emitter–detector module for highly sensitive measurements of the electrochromic pigment absorbance shift (ECS) via dual-wavelength (550–520 nm) transmittance changes (P515) are described. This device, which has been introduced as an accessory of the standard, commercially available Dual-PAM-100 measuring system, not only allows steady-state assessment of the proton motive force (pmf) and its partitioning into ΔpH and ΔΨ components, but also continuous recording of the overall charge flux driven by photosynthetic light reactions. The new approach employs a double-modulation technique to derive a continuous signal from the light/dark modulation amplitude of the P515 signal. This new, continuously measured signal primarily reflects the rate of proton efflux via the ATP synthase, which under quasi-stationary conditions corresponds to the overall rate of proton influx driven by coupled electron transport. Simultaneous measurements of charge flux and CO2 uptake as a function of light intensity indicated a close to linear relationship in the light-limited range. A linear relationship between these two signals was also found for different internal CO2 concentrations, except for very low CO2, where the rate of charge flux distinctly exceeded the rate of CO2 uptake. Parallel oscillations in CO2 uptake and charge flux were induced by high CO2 and O2. The new device may contribute to the elucidation of complex regulatory mechanisms in intact leaves.
Highlights
Progress in photosynthesis research has been driven to a large extent by the development of new measuring techniques and methodology
Technical features and examples of application of a special emitter–detector module for highly sensitive measurements of the electrochromic pigment absorbance shift (ECS) via dual-wavelength (550–520 nm) transmittance changes (P515) are described. This device, which has been introduced as an accessory of the standard, commercially available Dual-PAM-100 measuring system, allows steady-state assessment of the proton motive force and its partitioning into DpH and DW components, and continuous recording of the overall charge flux driven by photosynthetic light reactions
Analysis of dark-interval relaxation kinetics (DIRK) method has been advanced by Kramer and co-workers for non-intrusive measurement of the rate of electron flow via P700 (Sacksteder and Kramer 2000), for assessment of the DpH and DW components of overall pmf (Cruz et al 2001; Avenson et al 2004a) and for determination of the rate of proton efflux via the ATP-synthase (Sacksteder et al 2000; Kanazawa and Kramer 2002; Kramer et al 2003; Cruz et al 2005)
Summary
Progress in photosynthesis research has been driven to a large extent by the development of new measuring techniques and methodology. A different kind of approach for measuring in vivo absorbance changes was taken by Klughammer et al (1990), which was based on the Pulse-Amplitude-Modulation (PAM) method previously developed for measurements of chlorophyll fluorescence in natural daylight and assessment of various quenching parameters by the saturation pulse method (Schreiber 1986; Schreiber et al 1986) This approach employs continuous trains of 1 ls ML pulses generated by light emitting diodes (LED), the frequency of which can be adjusted over a wide range (depending on the rate of the investigated changes), and a special pulse signal amplifier. While the time resolution (1 ms) of this type of Kinetic LED Array Spectrophotometer (KLAS) cannot cope with that of the Joliot-type device (30 ls), the KLAS displays the practical advantage of absorbance being measured quasisimultaneously at 16 wavelengths In this way, changes can be measured continuously under close to natural conditions of illumination, during dark-light or light–dark induction and in the steady-state, very similar to chlorophyll fluorescence, rendering this device suited for in vivo studies. Deviations between the two signals can be interpreted in terms of alternative types of electron flow, regulatory changes in the conductivity of the reversible ATP synthase or of the H?/e- ratio (see Kramer et al 2004a, b for a reviews)
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