David Joly

Quantitative analysis of the experimental O-J-I-P chlorophyll fluorescence induction kinetics : Apparent activation energy and origin of each kinetic step

Fluorescence induction has been studied for a long time, but there are still questions concerning what the O–J–I–P kinetic steps represent. Most studies agree that the O–J rise is related to photosystem II primary acceptor (QA) reduction, but several contradictory theories exist for the J–I and I–P rises. One problem with fluorescence induction analysis is that most work done to date has used only qualitative or semiquantitative data analysis by visually comparing traces to observe the effects of different chemicals or treatments. Although this method is useful to observe major changes, a quantitative method must be used to detect more subtle, yet important, differences in the fluorescence induction trace. To achieve this, we used a relatively simple mathematical approach to extract the amplitudes and half‐times of the three major fluorescence induction phases obtained from traces measured in thylakoid membranes kept at various temperatures. Apparent activation energies (EA) were also obtained for each kinetic step. Our results show that each phase has a different EA, with EA O–J 

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.

Protective effect of active oxygen scavengers on protein degradation and photochemical function in photosystem I submembrane fractions during light stress

The protective role of reactive oxygen scavengers against photodamage was studied in isolated photosystem (PS) I submembrane fractions illuminated (2000 µE·m−2·s−1) for various periods at 4 °C. The photochemical activity of the submembrane fractions measured as P700 photooxidation was significantly protected in the presence of histidine or n‐propyl gallate. Chlorophyll photobleaching resulting in a decrease of absorbance and fluorescence, and a blue‐shift of both absorbance and fluorescence maximum in the red region, was also greatly delayed in the presence of these scavengers. Western blot analysis revealed the light harvesting antenna complexes of PSI, Lhca2 and Lhca1, were more susceptible to strong light when compared to Lhca3 and Lhca4. The reaction‐center proteins PsaB, PsaC, and PsaE were most sensitive to strong illumination while other polypeptides were less affected. Addition of histidine or n‐propyl gallate lead to significant protection of reaction‐center proteins as well as Lhca against strong illumination. Circular dichroism (CD) spectra revealed that the α‐helix content decreased with increasing period of light exposure, whereas β‐strands, turns, and unordered structure increased. This unfolding was prevented with the addition of histidine or n‐propyl gallate even after 10 h of strong illumination. Catalase or superoxide dismutase could not minimize the alteration of PSI photochemical activity and structure due to photodamage. The specific action of histidine and n‐propyl gallate indicates that 1O2 was the main form of reactive oxygen species responsible for strong light‐induced damage in PSI submembrane fractions.

Period-four Modulation of Photosystem II Primary Quinone Acceptor (QA) Reduction/Oxidation Kinetics in Thylakoid Membranes

Photosystem II (PSII), a multiprotein complex mainly coded by the chloroplast genome in higher plants and algae, contains the oxygen‐evolving complex with four manganese atoms responsible for the oxidation of water. After each absorption of a light quantum by pigment molecules in the light harvesting complexes of PSII, the Mn cluster advances in its oxidation states denoted from S0 to S4. The S4 state decays to S0 in the dark with the concurrent release of molecular oxygen. Therefore, the oxygen production in PSII exposed to successive single turnover excitations follows a period‐four oscillation pattern. The intensity of chlorophyll a fluorescence of PSII is also known to be influenced by the oxidation state of the Mn cluster. In the present work, fluorescence induction kinetics was measured in isolated thylakoids with various initial S‐state populations settled by preflashes. The shape of the fluorescence induction traces was strongly affected by preflashes. O‐J and J‐I phases of the induction followed a period‐four oscillation pattern. The results indicate that these changes reflect the influence of the oxidation rate of the Mn cluster on the reduction/oxidation kinetics of the primary quinone acceptor (QA) of PSII.

Redox state of the photosynthetic electron transport chain in wild-type and mutant leaves of Arabidopsis thaliana: Impact on photosystem II fluorescence

In addition to the photosynthetic linear electron transport, several alternative electron transport routes exist in thylakoids of higher plants. The plastoquinone (PQ) pool acts as a common electron carrier in these pathways. In the cyclic electron flow around photosystem I (PSI), reduced ferredoxin is used by the ferredoxin-quinone reductase (FQR) to reduce the PQ pool. Chlororespiratory pathway consists in the reduction of the PQ pool by the NAD(P)H dehydrogenase (NDH). These alternative pathways and their role in photosynthesis are still not fully understood. In the present study, the accumulation kinetics of quinone acceptors was measured by fluorescence induction in leaves of Arabidopsis thaliana wild-type and mutants altered in alternative electron pathways after various light- and dark-adaptation conditions. Results show that NDH activity can be probed by fluorescence induction during light-to-dark transition of plants. Also, the activity of FQR pathway did not affect directly the FI kinetics. However, the accumulation kinetics of reduced PQ under actinic light was dependant on the redox state of PSI acceptors prior to illumination.

Rapid Isolation of Intact Chloroplasts from Spinach Leaves

In this chapter, a rapid method to isolate intact chloroplasts from spinach leaves is described. Intact chloroplasts are isolated using two short centrifugation steps and avoiding the use of percoll gradient. Intactness of chloroplast is evaluated by the inability of potassium ferricyanide to enter inside the chloroplasts and to act as an electron acceptor for photosystem II.

Effect of moderate and high light on photosystem II function in Arabidopsis thaliana depleted in digalactosyl-diacylglycerol.

The response of the heat-sensitive dgd1-2 and dgd1-3 Arabidopsis mutants depleted in the galactolipid DGDG to photoinhibition of chloroplasts photosystem II was studied to verify if there is a relationship between heat stress vulnerability due to depletion in DGDG and the susceptibility to photoinhibitory damage. Non-photochemical quenching (NPQ) is known to dissipate excessive absorbed light energy as heat to protect plants against photodamage. The main component of NPQ is dependent of the transthylakoid pH gradient and is modulated by zeaxanthin (Zx) synthesis. These processes together with chlorophyll fluorescence induction were used to characterize the response of the genotypes. The mutants were more sensitive to photoinhibition to a small extent but this was more severe for dgd1-3 especially at high light intensity. It was deduced that DGDG was not a main factor to influence photoinhibition but other lipid components could affect PSII sensitivity towards photoinhibition in relation to the physical properties of the thylakoid membrane. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Photosystem II reconstitution into proteoliposomes and methodologies for structure-function characterization.

This chapter discusses the photosystem II (PSII) reconstitution into proteoliposomes. In the first part of the chapter, protocols are outlined for the preparation of lipid bilayer vesicles (liposomes) constituted of individual thylakoid lipids or their mixtures, for the preparation of PSII particles, and for the incorporation of the PSII particles into the liposomes. In the second part of the chapter, methodologies are described for the structure-function characterization of the PSII-lipid complexes (proteoliposomes). This includes the sodium dodecylsulfate-polyacrylamide gel electrophoresis determination of the PSII proteins, the measurement of oxygen-evolving activity of PSII in the proteoliposomes, the study of structural changes of the PSII proteins upon their incorporation into the lipid bilayers by Fourier transform infrared (FT-IR) spectroscopy, and the characterization of the PSII activity by fluorescence induction.