Zoran G. Cerovic

[15] Isolation of intact chloroplasts: General principles and criteria of integrity

Publisher Summary This chapter discusses method for the isolation of intact chloroplasts. The intact chloroplast comprises three distinct entities: (1) the thylakoid membranes, which contain the pigments, components of the electron-transport chain, and the coupling factor for photophosphorylation, (2) the stroma, which contains soluble proteins including those necessary for photosynthetic carbon reduction, plus metabolites and other salts, and (3) the envelope membranes, which enclose the organelle and also contain the metabolite translocators. Intact chloroplasts capable of high rates of photosynthesis can be prepared from many species, such as cereals, only by first digesting the leaves with cellulase and pectinase to obtain protoplasts and then rupturing these to release the chloroplasts. The metabolic activity of isolated chloroplasts cannot exceed that of the parent tissue, but in addition, healthy and rapidly growing plants generally have softer leaves that contain smaller amounts of interfering substances (such as phenolics, oxalate, etc.), and thus more readily yield intact chloroplasts with high rates of photosynthesis.

The role of photophosphorylation in SO2 and SO 3 2- inhibition of photosynthesis in isolated chloroplasts

Sulphur dioxide inhibits noncyclic photophosphorylation in isolated envelope-free chloroplasts. This inhibition was shown to be reversible and competitive with phosphate, with an inhibitor constant of Ki=0.8mM. The same inhibition characteristics were observed when phosphoglycerate (PGA)- or ribulose-1,5-bisphosphate (RuBP)-dependent oxygen evolution was examined in a reconstituted chloroplast system in the presence of SO32-. Using an ATP-regenerating system (phosphocreatine-creatine kinase), it was demonstrated that the inhibition of PGA-dependent oxygen evolution is solely the result of inhibited photophosphorylation. It is concluded that at low SO2 and SO32-concentrations the inhibition of photophosphorylation is responsible for the inhibition of photosynthetic oxygen evolution.

Slow Secondary Fluorescence Kinetics Associated with the Onset of Photosynthetic Carbon Assimilation in Intact Isolated Chloroplasts

Experiments with carefully isolated, largely intact chloroplasts, capable of fast rates of CO2-dependent O2 evolution, show that the fall in chlorophyll a fluorescence (from the early maxima reached immediately after illumination) is interrupted by a ‘shoulder’ which is associated with the exponential increase in the rate of O2 evolution. The length of this induction period was increased by storage, by decreased temperature, by increased orthophosphate concentration in the assay medium or by the presence of D, L-glyceraldehyde. It could also be shortened by the addition of 3-phosphoglycerate or dihydroxyacetonephosphate. In each treatment the shoulder in fluorescence shifted so that the association with the period of exponential increase was maintained. When illumination was re-started after a short dark interval, induction was minimal and no shoulder could be discerned, but both the lag in the onset of O2 evolution and the shoulder were restored when the chloroplasts were resuspended in fresh assay medium during the period of darkness. The relation between chlorophyll a fluorescence and the onset of photosynthetic carbon assimilation is discussed.

Photosynthetic oxygen evolution and chlorophyll fluorescence in intact isolated chloroplasts on a solid support: the influence of orthophosphate

We devised recently a method to trap intact isolated chloroplasts on a solid support consisting of membrane filters made of cellulose nitrate (Cerović et al., 1987, Plant Physiol. 84, 1249–1251). The addition of alkaline phosphatase to the reaction medium enabled continuous photosynthesis by spinach (Spinacia oleracea L.) chloroplasts to be sustained by hydrolysis of newly produced and exported triose phosphates and recycling of orthophosphate. In this system, simultaneous measurements of chlorophyll fluorescence and oxygen evolution were performed and their dependence on orthophosphate concentration was investigated. Optimal photosynthesis was obtained at a much higher initial orthophosphate concentration (2–4 mM) compared to intact chloroplasts in suspension. Secondary kinetics of chlorophyll fluorescence yield were observed and were shown to depend on the initial orthophosphate concentration.

Induction of oscillations in chlorophyll fluorescence by re-illumination of intact isolated pea chloroplasts

Oscillations in chlorophyll fluorescence yield were observed upon re-illumination of intact isolated pea (Pisum sativumL.) chloroplasts that had attained their maximal rate of photosynthesis and had spent a short period in darkness. The oscillations depended on the length of the previous dark period, the length of previous illumination, and the reaction temperature. This finding confirms the presence of an “oscillatory center” in the chloroplasts temselves.

Simultaneous Measurement of CHL α Fluorescence and Photosynthetic O2 Evolution in Systems of Decreasing Complexity (from the Leaf to the Reconstituted Chloroplast System)

Short term variations in the rate of photosynthesis by leaves have been frequently described. These observations, over many years, ranged from simple irregularities to dampening oscillations and were mostly observed when steady-state photosynthesis was perturbed by introducing a dark interval, a change in the gas phase surrounding the leaf, or both. In spinach and barley leaves, chlorophyll a (Chl a) fluorescence and O2 evolution have been measured simultaneously in an apparatus designed by Delieu and Walker which incorporates polarographic measurements of oxygen in the gas-phase. Irregularities in both Chl a fluorescence and oxygen signals (following re-illumination after a dark interval or when steady-state photosynthesis was perturbed by changes in the gasphase) were characterised. In high [CO2], both O2 and fluorescence can display marked dampening oscillations that are anti-parallel but slightly out of phase (a rise or fall in fluorescence anticipating a corresponding fall or rise in O2 by about 10–15 seconds), (Walker et al 1983). IRGA measurements showed that carbon dioxide uptake behaved like oxygen evolution both in the period of oscillation (about 1 minute) and in its relation to fluorescence (Fig.1).

Some Relationships between Photosynthetic Carbon Metabolism and Chlorophyll a Fluorescence

In their 1977 review, Lavorel and Etienne declared that, in vivo, chlorophyll fluorescence is “both a rich and ambiguous signal” and “no longer a subject for specialists alone”. Its richness means that the non-specialists, like ourselves, can look beyond picosecond fluorimetry and the large variety of events which are encompassed within the microscale and are thereby complete within O.l nsec. of illumination. We can even stride, without lingering more than a few milliseconds, through the photochemical era into the pattern of successive waves of fluorescence which constitute the Kautsky effect (1931). Towards the end of this period there are often peaks and troughs in fluorescence emission which have been labelled S (for quasi steady-state), M (for a secondary maximum) and T (for a terminal or true steady-state value). These usually occur within 0.5 to 1.5 minutes of illumination (Lavorel and Etienne, 1977, Walker 1981b) Accordingly, there is an obvious possibility that they might be related, in some manner, to the onset of photosynthetic carbon assimilation. After a period of darkness, carbon assimilation (and its associated O2 evolution) fails to re-start immediately upon illumination but only after a lag or induction period, which varies with the length of the preceding dark interval (and the temperature) but normally takes some minutes to complete (Walker 1981a). “Induction” as in “fluorescence induction” refers to all the changes in fluorescence which are induced by re-illumination after a period of darkness. The SMT transient could equally well be called “induction fluorescence”. Certainly it occurs when the complex events which govern the autocatalytic phase of carbon assimilation might be expected to have an increasing impact on the photochemistry and, thereby, on the fluorescence kinetics.

Oscillations of Photosynthesis in Intact Isolated PEA Chloroplasts in the Presence of DCMU and Antimycin A

The study of photosynthetic induction in intact isolated chloroplasts can contribute to a better understanding of photosynthetic regulatory mechanisms. In that respect, study of induced oscillations is of prime importance because they are an indication of the presence of regulated feed-back mechanisms. Oscillations of photosynthesis in intact isolated spinach chloroplasts were observed during the induction period, in non-optimal conditions, when the transport of phosphates was limited (1,2). Oscillations in both chlorophyll fluorescence yield and the rate of O2 evolution were also induced when intact isolated pea chloroplasts were re-illuminated at 27°C (3). Under these conditions, the kinetics of fluorescence quenching and the rate of O2 evolution were dependent on the length of the dark period, concentrations of bicarbonate, orthophosphate and Calvin cycle intermediates (3). The aim of the present work is to examine the effect of a deliberate change in the ratio of noncyclic and cyclic electron transport on fluorescence kinetics during oscillations. For that purpose, addition of DCMU and antimycin A, in catalytic amounts, was used.

The Function of the Photosystem II Reaction Center — An Alternative Model —

Current concepts on Photosystem II reaction center (PSII RC) function are strongly influenced by the elucidation of the three-dimensional molecular structure of RC of purple bacteria1, and the recognized homology of the two types of RC regarding their core proteins and chromophores2,3. This led to the proposal, in almost all of the published models (e.g. ref. 2 & 29), of charge separation in PSII RC along only one branch of the chromophore system, as was found in bacteria1. This concept, however, did not give a satisfactory answer to the following questions: how is the high redox potential needed to oxidize water, achieved; what is the role of the other branch of chromophores (B); what is the role of the ubiquitous cytochrome b-559; what is the origin of efficient thermal energy dissipation in the RC; how to explain a number of anomalies concerning the overlapping of the acceptor and donor side of the RC? I have proposed recently4,5 a model for the function of PSII RC designed to incorporate several phenomena related to the variable yield of chlorophyll fluorescence during photosynthetic induction. This alternative model seems to give an answer to most of the above mentioned questions because, in it, the same currently accepted and most probable structure of PSII RC is involved in charge separation that includes all the chromophores present, and a dual role is assigned to the QB binding site.

A Hypothetical Model for the Structure and Function of the Photosystem II Reaction Center

Chlorophyll fluorescence has extensively been used to investigate the function of Photosystem II reaction centers (PSII RC) of higher plants (see Ref. 1.). The assignment of variable chlorophyll fluorescence to the redox state of PSII RC2 has been of prime importance for these investigations. However, the interpretation of fluorescence signals is still encumbered by two major problems: the discrepancy between the measured and predicted Fm/Fo ratios3 and the presence under physiological conditions4 of the “energy-dependent” quenching5 (qg) in addition to photochemical quenching2 (qg). To account for these phenomena, the existence of a radiationless deactivation pathway in “closed” reaction centers was postulated3. It is considered4 to be of non-photochemical nature and to depend on ultrastructural changes of the thylakoid membrane.