Johanne Harnois

Kinetic analyses of the OJIP chlorophyll fluorescence rise in thylakoid membranes

N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) was previously used to study the kinetics of the OJIP chlorophyll fluorescence rise. The present study is an attempt to elucidate the origin of TMPD-induced delay and quenching of the I–P step of fluorescence rise. For this purpose, we analyzed the kinetics of OJIP rise in thylakoid membranes in which electron transport was modified using ascorbate, methyl viologen (MV), and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). In the absence of TMPD, the OJIP kinetics of fluorescence induction (FI) was not altered by ascorbate. However, ascorbate eliminated the I–P rise delay caused by high concentrations of TMPD. On the other hand, neither ascorbate nor DBMIB, which blocks the electron release from Photosystem II (PS II) at the cytochrome b6/f complex, could prevent the quenching of I–P rise by TMPD. In control thylakoids, MV suppressed the I–P rise of FI by about 60. This latter effect was completely removed if the electron donation to MV was blocked by DBMIB unless TMPD was present. When TMPD intercepted the linear electron flow from PS II, re-oxidation of TMPD by photosystem I (PS I) and reduction of MV fully abolished the I–P rise. The above is in agreement with the fact that TMPD can act as an electron acceptor for PS II. With MV, the active light-driven uptake of O2 during re-oxidation of TMPD by PS I contributes towards an early decline in the I–P step of the OJIP fluorescence rise.

Reconstruction of the Water-Oxidizing Complex in Manganese-Depleted Photosystem II Preparations Using Mononuclear Manganese Complexes

Abstract— The water‐oxidizing complex of chloroplast photosystem II is composed of a cluster of four manganese atoms that can accumulate four oxidizing redox equivalents. Depletion of manganese from the water‐oxidizing complex fully inhibits oxygen evolution. However, the complex can be reconstituted in the presence of exogenous manganese in a process called photoactivation. In the present study, mononuclear manganese complexes with ligands derived from either nitrosonaphthol and ethylenediamine (Niten) or from diaminohexane and salicylaldehyde (Salhxn) are used in photoactivation experiments. Measurements of photoinduced changes of chlorophyll fluorescence yield, thermal dissipation using photoacoustic spectroscopy, photoreduction of 2,6‐dichorophenolindophenol and oxygen evolution in manganese‐depleted and in reconstituted photosystem II preparations demonstrate that photoactivation is more efficient when Niten and Salhxn complexes are used instead of MnCl2. It is inferred that the aromatic ligands facilitate the interaction of the manganese atoms with photosystem II. The addition of CaCl2 and of the extrinsic polypeptide of 33 kDa known as the manganese‐stabilizing protein during photoactivation further enhances the recovery of electron transport and oxygen evolution activities. It is proposed that mononuclear manganese complexes are able to contribute to re‐constitution of the water‐oxidizing complex by sequential addition of single ions similarly to the current model for assembly of the tetranuclear manganese cluster and that these complexes constitute suitable model systems to study the assembly of the water‐oxidizing complex.

Demonstration of thermal dissipation of absorbed quanta during energy-dependent quenching of chlorophyll fluorescence in photosynthetic membranes

When plant leaves or chloroplasts are exposed to illumination that exceeds their photosynthetic capacity, photoprotective mechanisms such as described by the energy‐dependent (non‐photochemical) quenching of chlorophyll fluorescence are involved. The protective action is attributed to an increased rate constant for thermal dissipation of absorbed quanta. We applied photoacoustic spectroscopy to monitor thermal dissipation in spinach thylakoid membranes together with simultaneous measurement of chlorophyll fluorescence in the presence of inhibitors of opposite action on the formation of ΔpH across the thylakoid membrane (tentoxin and nigericin/valinomycin). A linear relationship between the appearance of fluorescence quenching during formation of the ΔpH and the reciprocal variation of thermal dissipation was demonstrated. Dicyclohexylcarbodiimide, which is known to prevent protonation of the minor light‐harvesting complexes of photosystem II, significantly reduced the formation of fluorescence quenching and the concurrent increase in thermal dissipation. However, the addition of exogenous ascorbate to activate the xanthophyll de‐epoxidase increased non‐photochemical fluorescence quenching without affecting the measured thermal dissipation. It is concluded that a portion of energy‐dependent fluorescence quenching that is independent of de‐epoxidase activity can be readily measured by photoacoustic spectroscopy as an increase in thermal deactivation processes.

How does iron deficiency disrupt the electron flow in photosystem I of lettuce leaves

The changes observed photosystem I activity of lettuce plants exposed to iron deficiency were investigated. Photooxidation/reduction kinetics of P700 monitored as ΔA820 in the presence and absence of electron transport inhibitors and acceptors demonstrated that deprivation in iron decreased the population of active photo-oxidizable P700. In the complete absence of iron, the addition of plant inhibitors (DCMU and MV) could not recover the full PSI activity owing to the abolition of a part of P700 centers. In leaves with total iron deprivation (0μM Fe), only 15% of photo-oxidizable P700 remained. In addition, iron deficiency appeared to affect the pool size of NADP(+) as shown by the decline in the magnitude of the first phase of the photooxidation kinetics of P700 by FR-light. Concomitantly, chlorophyll content gradually declined with the iron concentration added to culture medium. In addition, pronounced changes were found in chlorophyll fluorescence spectra. Also, the global fluorescence intensity was affected. The above changes led to an increased rate of cyclic electron transport around PSI mainly supported by stromal reductants.

Isolation of Photosystem I Submembrane Fractions

In this chapter, we describe a method to prepare photosystem I (PSI) submembrane fractions derived from the chloroplast stroma lamellae of spinach chloroplasts. These preparations retain the cytochrome b6/f complex and a pool of about 11 plastoquinones per P700. The PSI submembrane fractions are thus able to perform both cyclic and linear electron transport reactions from various artificial electron donors to oxygen or methylviologen. They are useful to study both PSI and cytochrome b6/f complex activities in a nearly native form without interference from photosystem II.

INHIBITION OF OXYGEN EVOLUTION IN CHLOROPLAST PHOTOSYSTEM II BY THE PROTEIN‐MODIFYING AGENT TETRANITROMETHANE

Abstract— The protein‐modifying agent tetranitromethane (TNM) reacts with tyrosine residues and ‐SH groups. It was found to inhibit photo synthetic electron transport on the water splitting side of photosystem II (P. V. Sane and U. Johanningmeier, Z. Naturforsch. 35c, 293–297, 1979). In the present work the inhibition by TNM is studied in detail using photosystem II submembrane fractions. It is shown that the action of TNM with membrane‐bound proteins could imply the modification of tyrosine residues. At concentrations below 30 μM and with short incubation periods (<2 min), TNM produces the release of the extrinsic polypeptides involved in the stabilization of the water‐splitting complex, this being correlated with inhibition of electron transport at a site prior to H2O2 electron donation even though the inhibition cannot be prevented by the addition of Cl or Ca2+, which are known cofactors for oxygen evolution. As the incubation period or the concentration of TNM is increased, photosynthetic pigments are bleached, starting with aggregates absorbing at relatively long wavelengths. The inhibition by low concentrations of TNM differs from the effect of most of the previously reported inhibitors acting at the oxygen‐evolving complex of photosystem II.

Effect of Cations on Heat Stimulation of Photosystem I in Stroma Lamellae Preparations

Higher plants are yery sensitive to heat stress. The integrity of the thylakoid membrane constituents is first to be affected, well before other stromal or cellular components (1). Short exposure of photosynthetic membranes to temperatures in the range of 35–50°C results in a loss of grana stacking with concurrent dissociation of the peripheral antenna complex of PSII (2). However, the primary site of thermal damaging seems to be located at the PSII reaction center complex (3,4).