Charles J. Arntzen

Differentiation of chloroplast lamellae: Light harvesting efficiency and grana development

Abstract Incomplete development of chloroplast lamellae occurred when etiolated pea plants were greened under cycles of 2 min light, 118 min dark. Although the plastids had full photochemical activities, they were nearly agranal. They were also characterized by a high quantum requirement for whole chain electron transport in low light; this is thought to be the result of unequal light absorption by incompletely developed light-harvesting assemblies of photosystem I and II and a lack of regulation of excitation energy distribution between the two photosystems. Continuous illumination induced the final stages of membrane differentiation. These stages were primarily characterized by the appearance of grana stacking and an increase in photosynthetic unit size. A biphasic decrease in quantum requirement for whole chain electron transport correlated directly with the appearance of grana during the final steps of membrane assembly. Structural organization of the membrane may be related to the light-harvesting efficiency of the membrane.

Evidence for the role of surface-exposed segments of the light-harvesting complex in cation-mediated control of chloroplast structure and function.

Abstract Chloroplast membranes contain a light-harvesting pigment-protein complex (LHC) which binds chlorophylls a and b . A mild trypsin digestion of intact thylakoid membranes has been utilized to specifically alter the apparent molecular weights of polypeptides of this complex. The modified membrane preparations were analyzed for altered functional and structural properties. Cation-induced changes in room temperature fluorescence intensity and low temperature chlorophyll fluorescence emission spectra, and cation regulation of the quantum yield of photosystem I and II partial reactions at limiting light were lost following the trypsin-induced alteration of the LHC. Electron microscopy revealed that cations can neither maintain nor promote grana stacking in membranes which have been subjected to mild trypsin treatment. Freeze-fracture analysis of these membranes showed no significant differences in particle density or average particle size of membrane subunits on the EF fracture face; structural features of the modified lamellae were comparable to membranes which had been unstacked in a “low salt” buffer. Digitonin digestion of trypsin-treated membranes in the presence of cations followed by differential centrifugation resulted in a subchloroplast fractionation pattern similar to that observed when control chloroplasts were detergent treated in cation-free medium. We conclude that: (a) the initial action of trypsin at the thylakoid membrane surface of pea chloroplasts was the specific alteration of the LHC polypeptides, (b) the segment of the LHC polypeptides which was altered by trypsin is necessary for cation-mediated grana stacking and cation regulation of membrane subunit distribution, and (c) cation regulation of excitation energy distribution between photosystem I and II involves the participation of polypeptide segments of the LHC which are exposed at the membrane surface.

Redox reactions on the reducing side of photosystem II in chloroplasts with altered herbicide binding properties

Abstract Measurements of chlorophyll fluorescence have been used to monitor electron transfer from Q (the primary electron acceptor of photosystem II) to B (the bound quinone which serves as the secondary acceptor) in chloroplasts isolated from atrazine-susceptible and atrazine-resistant pigweed chloroplasts. The Q − → B electron transfer was at least 10-fold slower in the plastids from resistant plants. Binary oscillations in the rate of Q − decay after a series of flashes were of opposite phase in the two types. The data are interpreted to indicate that the apoprotein of B is altered in the photosytem II complex of the two types of plants—this is correlated to altered binding affinity of herbicides to this component and may be related to altered redox properties of the bound quinone cofactor.

The mode of Action of Photosystem II-Specific Inhibitors in Herbicide-Resistant Weed Biotypes

Abstract This report reviews studies which provide evidence defining the mode of action and site of action of photosystem II (PS II) herbicides; the involvement of the secondary electron carrier on the reducing side of PS II (called B) is indicated as the target site for these compounds. These studies of the action of PS II-inhibitors were performed in chloroplasts of various weed species in order to define the mechanism which is responsible for herbicide tolerance at the level of chloroplast membranes in newly discovered triazine-resistant weed biotypes. Many species of triazine-resistant weed biotypes have been collected in North America and Europe. Where data is available, these plants have been found to share the following common features: a) they were discovered in areas where triazine herbicides had been used repeatedly, b) resistance to the triazines is extreme; it is not due to a minor shift in herbicidal response, c) no changes in herbicide uptake, translocation or metabolism - as compared to susceptible biotypes - can be detected, d) resistance is selective for only certain classes of photosynthetic herbicides, and, e) chloroplasts isolated from triazine-resistant weeds display high preferential resistance to the triazines in assays of photosystem II partial reactions. To focus on the mechanism which regulates preferential herbicide activity, we have characterized susceptible and resistant chloroplasts in the presence and absence of herbicides. Properties of the PS II complex of chloroplasts from several different triazine-resistant weed biotypes share the following traits: a) the herbicide binding site (as measured by direct binding of radiolabeled herbicides or by inhibition experiments) is modified such that the affinity for triazines is dramatically reduced. b) alterations in response to many PS II-herbicides occur such that the triazine-resistant chloroplasts are very strongly resistant to all symmetrical triazines, strongly resistant to assymmetrical triazinones, partially resistant to pyridazones and uracils, only slightly resistant to ureas or amides, and increasingly susceptible to nitrophenols, phenols and the herbicide bentazon (all as compared to susceptible chloroplasts), c) there is a change in the reaction kinetics of the electron transport step between the primary and secondary electron acceptors (referred to as Q and B ), and d) in two examples, specific small changes in a membranepolypeptide can be detected in the resistant thylakoids. We suggest that certain amino acids or segments of the apoprotein of B (the bound quinone which acts as the secondary electron carrier) are modified or deleted in these chloroplasts. Such a polypeptide change could affect both the redox poising of the Q-/B reaction pair, and the specific binding of herbicides.

Inhibition of photophosphorylation by tentoxin, a cyclic tetrapeptide.

Abstract Tentoxin, a fungal toxin which is known to cause seedling chlorosis, was found to inhibit cyclic photophosphorylation but not reversible proton accumulation by isolated chloroplasts. The toxin, at low concentrations, also inhibited coupled electron flow (in the presence of ADP and phosphate) but did not effect basal electron flow (in the absence of ADP and phosphate) or uncoupled electron transport. It is suggested that tentoxin is an energy transfer inhibitor which acts at the terminal steps of ATP synthesis.

Lactoperoxidase-catalyzed iodination of chloroplast membranes. II. Evidence for surface localization of Photosystem II reaction centers

Abstract The enzyme lactoperoxidase was used to specifically iodinate the surface-exposed proteins of chloroplast lamellae. This treatment had two effects on Photosystem II activity. The first, occurring at low levels of iodination, resulted in a partial loss of the ability to reduce 2,6-dichlorophenolindophenol (DCIP), even in the presence of an electron donor for Photosystem II. There was a parallel loss of Photosystem II mediated variable yield fluorescence which could not be restored by dithionite treatment under anaerobic conditions. The same pattern of inhibition was observed in either glutaraldehyde-fixed or unfixed membranes. Analysis of the lifetime of fluorescence indicated that iodination changes the rate of deactivation of the excited state chlorophyll. We have concluded that iodination results in the introduction of iodine into the Photosystem II reaction center pigment-protein complex and thereby introduces a new quenching. The data indicate that the reaction center II is surface exposed. At higher levels of iodination, an inhibition of the electron transport reactions on the oxidizing side of Photosystem II was observed. That portion of the total rate of photoreduction of DCIP which was inhibited by this action could be restored by addition of an electron donor to Photosystem II. Loss of activity of the oxidizing side enzymes also resulted in a light-induced bleaching of chlorophyll a680 and carotenoid pigments and a dampening of the sequence of O2 evolution observed during flash irradiation of treated chloroplasts. All effects on electron transport on the oxidizing side of Photosystem II could be eliminated by glutaraldehyde fixation of the chloroplast lamellae prior to lactoperoxidase treatment. It is concluded that the electron carriers on the oxidizing side of Photosystem II are not surface localized; the functioning of these components is impaired by structural disorganization of the membrane occurring at high levels of iodination. Our data are in agreement with previously published schemes which suggest that Photosystem II mediated electron transport traverses the membrane.

Inhibition of ion accumulation in maize roots by abscisic acid

SummaryAn inhibition of root growth, a decrease in the amount of potassium (as 86Rb) and phosphate (32P) accumulation by the root, and a partial depolarization of transmembrane electropotential were observed to develop with a similar time course and to a similar extent when intact maize (Zea mays L.) roots were treated with 10-5 M abscisic acid (ABA). Potassium uptake was inhibited by ABA when excised, low-salt roots were bathed in KCl, KH2PO4, or K2SO4. ABA did not affect the ATP content of the tissues, the activity of isolated mitochondria, nor the activity of microsomal K+-stimulated ATPases.

Uniparental inheritance of a chloroplast Photosystem II polypeptide controlling herbicide binding

The ability of atrazine to inhibit Photosystem II electron transport and the rate of electron transfer from the primary to the secondary quinone electron acceptors in the photosystem II complex were examined in triazine-resistant and -susceptible parental biotypes of Brassica campestris L. and their F1 progeny derived from reciprocal crosses. The lack of herbicide inhibitory activity and the presence of functional properties which decreased the Q- to B electron transport rate constant were inherited in parallel through the maternal parent. We conclude that the herbicide receptor protein is uniparentally inherited through the female parent. These data are discussed in relation to other studies which indicate that the binding site is a 32 000-dalton polypeptide which determines the functional properties of B (the secondary Photosystem II electron acceptor).

Reversible inactivation of photosystem II reaction centers in cation-depleted chloroplast membranes

Abstract Isolated pea chloroplasts were washed once in 10 m m NaCl and were then suspended in “low-salt” medium. Approximately one-half of the photosystem II reaction centers of these salt-depleted membranes were found to be photochemically inactive. These units became active in the presence of low concentrations of divalent cations (5–10 m m Mg 2+ ) or high concentrations of monovalent cations (150–200 m m Na + ), as evidenced by a twofold increase in the steady-state flash yield of oxygen evolution under short (~10-μs) saturating repetitive flashes (two per second). The half-maximal increase in flash yield occurred at ~2 mM Mg 2+ or ~75 m m Na + . The flash yield of hydroxylamine oxidation in these low-salt chloroplasts increased twofold after Mg 2+ addition, indicating that the cation action was close to the reaction-center chlorophyll complex. The relation between flash yield and dark time between flashes was not changed significantly by Mg 2+ , indicating that the rate-limiting step of the overall electron transport (H 2 0 —→ ferricyanide) was not affected significantly. When the rate-limiting step was bypassed using silicomolybdate as the photosystem II electron acceptor (in the presence of diuron), the reduction rate doubled in the presence of Mg 2+ , even under continuous, saturating light. In glutaraldehyde-fixed chloroplasts, Mg 2+ did not increase the flash yield of O 2 evolution; this suggests that protein conformational changes in the chloroplast membranes were involved in Mg 2+ activation of photosystem II centers.

Structural analysis of the isolated chloroplast coupling factor and the N,N'-dicyclohexylcarbodiimide binding proteolipid.

Negative staining of purified spinach dicyclohexylcarbodiimide (DCCD) sensitive ATPase revealed a population of 110 A subunits attached by stalks to short string-like aggregates. The interpretation of these data is that 110 A CF1 are attached by stalks to an aggregate of CF0. The CF1-CF0 complex was incorporated into phospholipid vesicles; freeze-fracture analysis of this preparation revealed a homogeneous population of particles spanning the lipid bilayer; those averaged 96 A in diameter. The DCCD binding proteolipid (apparent molecular weight 7500), an integral component of CF0, was isolated from membranes by butanol extraction and was incorporated into phospholipid vesicles. Freeze-fracture analysis of the DCCD-binding proteolipid/vesicle preparation revealed a population of particles averaging 83 A in diameter suggesting that the DCCD-binding proteolipid self-associates in lipid to form a stable complex. This complex may be required for proton transport across chloroplast membranes in vivo. The size difference between CF0 and DCCD-proteolipid freeze-fracture particles may be related to differences in polypeptide composition of the two complexes.