Colette A. Sacksteder

How acidic is the lumen

Proton motive force (pmf), established across the thylakoid membrane by photosynthetic electron transfer, functions both to drive the synthesis of ATP and initiate processes that down-regulate photosynthesis. At the same time, excessively low lumen pH can lead to the destruction of some lumenal components and sensitization of the photosynthetic apparatus to photoinhibition. Therefore, in order to understand the energy budget of photosynthesis, its regulation and responses to environmental stresses, it is essential to know the magnitude of pmf, its distribution between ΔpH and the electric field (Δϕ) as well as the relationships between these parameters and ΔGATP, and down-regulatory and inhibitory processes. We review past estimates of lumen pH and propose a model that can explain much of the divergent data in the literature. In this model, in intact plants under permissive conditions, photosynthesis is regulated so that lumen pH remains mod erate (between 5.8 and 6.5), where it modulates the activity of the violaxanthin deepoxidase, does not significantly restrict the turnover of the cytochrome b6f complex, and does not destabilize the oxygen evolving complex. Only under stressed conditions, where light input exceeds the capacity of both photosynthesis and down-regulatory processes, does lumen pH decrease below 5, possibly contributing to photoinhibition. A value of n = 4 for the stoichiometry of protons pumped through the ATP synthase per ATP synthesized, and a minor contribution of Δϕ to pmf, will allow moderate lumen pH to sustain the observed levels of ΔGATP.

Dark-interval relaxation kinetics (DIRK) of absorbance changes as a quantitative probe of steady-state electron transfer.

We introduce a new, non-invasive technique to measure linear electron transfer in intact leaves under steady-state illumination. Dark-interval relaxation kinetic or ‘DIRK’ analysis is based on measurements of the initial rates of relaxation of steady-state absorbance signals upon a rapid light-dark transition. We show that estimates of electron flux by DIRK analysis of absorbance signals, reflecting redox changes in the photosynthetic electron transfer chain, can yield quantitative information about photosynthetic flux when the light-dependent partitioning of electrons among redox components of the electron transfer chain are considered. This concept is modeled in computer simulations and then demonstrated in vivo with tobacco plants under non-photorespiratory conditions resulting in linear relationships between DIRK analysis and gross carbon assimilation (AG). Estimation based on DIRK analysis of the number of electrons transferred through the photosynthetic apparatus for each CO2 fixed was within 20% of the theoretical value. Possible errors and future improvements are discussed. We conclude that the DIRK method represents a useful tool to address issues such as plant stress and photosynthetic regulation.

A portable, non-focusing optics spectrophotometer (NoFOSpec) for measurements of steady-state absorbance changes in intact plants.

Kinetically-resolved absorbance measurements during extended, or steady-state illumination are typically hindered by large, light-induced changes in the light-scattering properties of the material. In this work, a new type of portable spectrophotometer, the Non-Focusing Optical Spectrophotometer (NoFOSpec), is introduced, which reduces interference from light-scattering changes and is in a form suitable for fieldwork. The instrument employs a non-focusing optical component, called a compound parabolic concentrator (CPC), to simultaneously concentrate and homogeneously diffuse measuring and actinic light (from light-emitting diode sources) onto the leaf sample. Light passing through the sample is then collected and processed using a subsequent series of CPCs leading to a photodiode detector. The instrument is designed to be compact, lightweight and rugged for field work. The pulsed measuring beam allows for high sensitivity (typically < 100 ppm noise) and time resolution (∼ 10 μs) measurements in the visible and near infrared spectral regions. These attributes allow high-resolution measurements of signals associated with energization of the thylakoid membrane (the electrochromic shifting of carotenoid pigments), as well as electron transfer, e.g., the 820-nm changes associated with electron transfer through Photosystem I (PS I). In addition, the instrument can be used as a kinetic fluorimeter, e.g., to measure saturation-pulse fluorescence changes indicative of Photosystem II (PS II) quantum efficiency. The instrument is demonstrated by estimating electron and proton fluxes through the photosynthetic apparatus in an intact tobacco leaf, using respectively the saturation-pulse fluorescence changes and dark-interval relaxation kinetics (DIRK) of the electrochromic shift. A linear relationship was found, confirming our earlier results with the laboratory-based diffused-optics flash spectrophotometer, indicating a constant H+/e− stoichiometry for linear electron transfer, and suggesting that cyclic electron flow around PS I is either negligible or proportional to linear electron flow. This type of measurement should be useful under field conditions for estimating the extent of PS I cyclic electron transfer, which is proposed to operate under stressed conditions.

H + /E - Ratios for Photosynthetic Electron Transfer in Intact Leaves in the Steady State.

The energy budget of a plant depends upon the ratio of protons pumped across the thylakoid membrane to electrons passed through photosynthetic electron transfer complexes (the H+/e- ratio) which determines the ratio of NADPH reduced to ATP phosphorylated. Thus it is the H+/e- that ultimately sets the stoichiometries for ATP and NADPH utilization in the Calvin cycle and other biochemical pathways. There has been a long-standing debate about the magnitude of H+/e- for green plant photosynthesis. In the generally accepted scheme, for each electron transferred through the linear pathway, one proton is released into the lumen at the level of water splitting, another is transported across the thylakoid membrane by the reduction and reoxidation of plastoquinone at the photosystem II (PSII) QB site and the cytochrome (cyt) b6f complex Qo sites, respectively. A third proton is pumped, at least under some conditions, by the turnover of a Q-cycle associated with the oxidation of plastoquinol at the b6f complex [1,2]. However, several groups have provided evidence that the H+/e- ratio for linear electron transport in isolated thylakoids changes from 3 to 2 with increasing light intensity [3-5] thus suggesting that the Q-cycle can be bypassed under some conditions. On the other hand, several authors have suggested that H+/e- ratio remains constant [6–8]. Much of this controversy may be due to the fact that past estimates of H+/e- have been measured in isolated thylakoids where light-induced acidification of the lumen can severely retard photosynthetic rates. In this work, we demonstrate non-invasive techniques for the measurement of H+/e- in intact plants and show that this ratio remains fixed during normal photosynthesis under non-stressed conditions.