Influence of structural and physical properties of the thylakoid membrane on QA - oxidation

Using isolated pea thylakoids, the relative rate of Q_A ^- oxidation has been estimated under various conditions, from the restoration of the induction curves following a dark period and from light 1-induced changes in modulated chlorophyll fluorescence excited by light 2.Alterations of _QinfA ^sup– oxidation rates were observed under conditions which affected the degree of thylakoid stacking, the lipid fluidity and the integrity of the membranes. The results are discussed in terms of the interactions between Q_A ^- and the plastoquinone pool with particular emphasis on lateral diffusion.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EDTA Ethylenediaminetetracetate - Hepes N-2-hydroxyethyl piperazine-N-2-ethanesulphonic acid - NADP nicotinamide adenine dinucleotide phosphate     

Simulation of grana stacking in a model membrane system. Mediation by a purified light-harvesting pigment-protein complex from chloroplasts

An isolated light-harvesting pigment-protein complex contains polypeptides which bind chlorophyll a and b. The individual complexes can be purified from detergent-solubilized membranes. The isolated light-harvesting complex, when dialyzed to remove detergents, was examined by freeze-fracture electron microscopy. The material consisted of planar sheets of 80-Å subunits which interacted via an edge-to-edge contact. Addition of cations caused the planar light-harvesting complex sheets to become tightly appressed in multilamellar stacks, with distinct subunits still visible within each lamellar sheet. A transition of particle organization from random to crystalline occurred in parallel with the cation-induced lamellar association. Treatment of the dialyzed light-harvesting complex subunits with low levels of the proteolytic enzyme trypsin removed a 2000 molecular weight segment of the major polypeptide of the light-harvesting complex and blocked all subsequent cation-induced changes in structural organization of the isolated light-harvesting complex lamellar sheets.To gain further evidence for mechanisms of cation effects upon the organization of the light-harvesting complex in native membranes, the light-harvesting complex was incorporated into uncharged (phosphatidylcholine) lipid vesicles. The protein complexes spanned the lipid bilayer and were arranged in either a random pattern or in hexagonal crystalline lattices. Addition of either monovalent or divalent cations to ‘low-salt’ (20 mM monovalent cation) vesicles containing light-harvesting complex caused extensive regions of membrane appression to appear. It is concluded that this cation-induced membrane appression is mediated by surface-exposed segments of the light-harvesting complex since (a) phosphatidylcholine vesicles themselves did not undergo cation-induced aggregation, and (b) mild trypsin digestion of the surface-exposed regions of the light-harvesting complex blocked cation-induced lamellar appression. The particles in the appressed vesicle membranes tended to form long, linear arrays of particles, with occasional mixed quasi-crystalline arrays with an angular displacement near 72°. Surface-mediated interactions among light-harvesting complex subunits of different membranes are, therefore, related to changes in structural organization and interaction of the particles within the lipid phase of the membrane.Numerous previous studies have implicated the involvement of the light-harvesting complex in mediating grana stocking in intact chloroplast membranes. The data presented herein provide a simulation of the membrane appression phenomena using a single class of chloroplast-derived membrane subunits. The data demonstrate that specific surface-localized regions of the light-harvesting complex are involved in membrane-membrane interactions.     

The paradoxical effects of nutrient ratios and supply rates on an outbreaking insect herbivore, the Australian plague locust

1. The Australian plague locust, Chortoicetes terminifera, develops following rainfall in an environment dominated by two host plants, the annual Dactyloctenium radulans and the perennial Astrebla lappacea. This simple system provides an ideal opportunity to explore the relationship between plant quality, individual herbivore performance and population responses. 2. We compared the two grasses chemically and structurally, and the behavioural, physiological and developmental responses of locust nymphs to these diets. 3. The grasses appeared to be of similar nutritional quality in terms of their chemical composition, although they differed in their physical properties. Early instar nymphs performed equally well on both grasses. However, older nymphs consuming D. radulans developed faster, survived better and attained a higher body weight compared with those consuming A. lappacea. 4. The differences in performance by the older nymphs related to the rate and ratio of supply of carbohydrate and protein from the two grasses, with less carbohydrate being assimilated from A. lappacea than D. radulans per unit of protein assimilated. Experiments showed that these differences arose as a direct result of the physical barrier to nutrient extraction provided by cell walls and indirectly through the amount of water contained within each cell. Paradoxically, nitrogen did not limit performance in the traditional sense through shortage,but rather its relative excess in A. lappacea appeared to impede intake and assimilation of adequate carbohydrate. 5. As a consequence, we predict that the length of time D. radulans remains available following rainfall will influence plaguing dynamics, although not for the reasons previously thought. 6. The results highlight the need to consider nutrient balance and actual rates of supply (rather than simply measuring the chemical composition of the plant) when attempting to understand herbivore nutritional ecology.     

Low growth temperatures modify the efficiency of light use by photosystem II for CO2 assimilation in leaves of two chilling-tolerant C4 species, Cyperus longus L. and Miscanthus x giganteus

Two C4 plants, Miscanthus x giganteus and Cyperus longus L., were grown at suboptimal growth temperatures and the relationships between the quantum efficiencies of photosynthetic electron transport through photosystem II (PSII) (PSII operating efficiency; Fq'/Fm') and CO2 assimilation (phiCO2) in leaves were examined. When M. x giganteus was grown at 10 degrees C, the ratio of the PSII operating efficiency to phiCO2 increased relative to that found in leaves grown at 14 and 25 degrees C. Similar increases in the Fq'/Fm': phiCO2 occurred in the leaves of two C. longus ecotypes when the plants were grown at 17 degrees C, compared to 25 degrees C. These elevations of Fq'/Fm': phiCO2 at low growth temperatures were not attributable to the development of anthocyanins, as has been suggested for maize, and were indicative of the operation of an alternative sink to CO2 assimilation for photosynthetic reducing equivalents, possibly oxygen reduction via a Mehler reaction, which would act as a mechanism for protection of PSII from photoinactivation and damage. Furthermore, in M. x giganteus grown at 10 degrees C, further protection of PSII was effected by a 20-fold increase in zeaxanthin content in dark-adapted leaves, which was associated with much higher levels of non-photochemical quenching of excitation energy, compared to that observed in leaves grown at 14 and 25 degrees C. These differences may explain the long growing season and remarkable productivity of this C4 plant in cool climates, even in comparison to other C4 species such as C. longus, which occur naturally in such climates.     

Photosynthetic Utilization of Radiant Energy by Temperate Lettuce Grown Under Natural Tropical Condition with Manipulation of Root-Zone Temperature

Photosynthetic utilization of radiant energy was studied by chlorophyll (Chl) fluorescence and maximum photosynthetic O₂ evolution (P _max) in temperate lettuce (Lactuca sativa L.) grown under natural tropical fluctuating ambient temperatures but with their roots exposed to two different root-zone temperatures (RZTs): a constant 20 °C-RZT (RZT₂₀) and a fluctuating ambient RZT (RZT_a) from 23 to 40 °C. On a sunny day, irrespective of RZT, F/_Fm [ratio of the variable to maximal fluorescence under irradiation (the maximal photosystem 2 quantum yield with actinic light)] decreased and non-photochemical quenching (NPQ) increased parallel to the increase of photosynthetic photon flux density (PPFD). However, RZT_a plants showed lower F/F_m and higher NPQ than RZT₂₀ plants. The electron transport rate (ETR) was much higher in RZT₂₀ plants than in RZT_a plants especially during moderately sunny days. There were no significant diurnal changes in P _max although these values of RZT₂₀ plants were much higher than those of RZT_a plants. On cloudy days, no significant diurnal changes in F/F_m and NPQ occurred, but F/F_m was higher and NPQ was lower in RZT₂₀ plants than in RZT_a plants. Diurnal changes in ETR were also observed in all plants while P _max values throughout the whole cloudy days in both RZT₂₀ and RZT_a plants were constant. Again, RZT₂₀ plants had much higher values of P _max than RZT_a plants. During RZT transfer period, all Chl fluorescence parameters measured at midday fluctuated with PPFD. Impact of RZT on these parameters was observed 2–3 d after RZT transfer. ETR and P _max measured with saturating PPFD in the laboratory did not vary with the fluctuating PPFD in the greenhouse but the effects of RZT on these two parameters were observed 3–4 d after RZT transfer. Thus RZT affects photosynthetic utilization of photon energy in temperate lettuce grown under natural tropical condition.     

Remote Sensing of Solar-excited Plant Fluorescence as a Measure of Photosynthetic Rate

Leaf level net photosynthetic rates (P _N) of laurel oak (Quercus hemispherica) juveniles grown under contrasting nutrient and CO₂ regimes were negatively correlated with red to far-red ratios, R/FR (690/760 nm), steady-state, solar-excited fluorescence ratios (r ² = 0.66, n = 12) measured across 12 plant canopies. Laurel oak juveniles that had been subjected to nitrogen stress over a period of a year demonstrated higher R/FR than their counterparts that had been provided with sufficient nitrogen. Plants that had been grown at elevated CO₂ concentrations, EC [700 mol (CO₂) mol^-1] also exhibited significantly higher R/FR when subjected to normal ambient carbon dioxide concentrations than their counterparts grown under ambient concentrations, AC [380 mol (CO₂) mol^-1]. All fluorescence measurements were obtained by observing a multi-plant canopy using a unique solar-blind passive sensor. This sensor, which utilizes Fraunhofer-line discrimination techniques, detects radiation at the cores of the lines comprising the atmospheric oxygen A- and B-bands, centered at 762 and 688 nm, respectively. These results support the use of solar-excited steady-state plant fluorescence as a potential tool for remote measurement of canopy radiation use efficiency.     

Enhancement of the light-triggered electrical response in plant cells following their de-energization with uncouplers

Light-triggered membrane potential changes in cells of a liverwort Anthoceros are greatly enhanced by the ionophorous uncouplers nigericin and monesin. Stimulation of the light-triggered electrical response (LTER) by nigericin occurred concomitantly with inhibition of a slow decline in the chlorophyll fluorescence, which suggests that the transmembrane pH gradient in thylakoids is not essential for generation of LTER at the plasma membrane. The extent of monensin-stimulated LTER remained high under a diminished driving force for the ionophore-induced proton-cation exchange across the plasma membrane (elevation of the external Na⁺ concentration from 1 to 50 m M ), which indicates that energy uncoupling in chloroplasts is more related to the electric response enhancement than the induction of the H⁺/K⁺(Na⁺) exchange at the plasma membrane. Enhancement of LTER by ionophores occurs in parallel with stimulation of light-triggered pH changes (alkalinization) in the vicinity of the cell surface, which suggests an association of trans-membrane H⁺ fluxes with LTER. The results are consistent with the hypothesis that illumination produces a temporary inhibition of the plasma membrane H⁺ pump with a subsequent activation of gated channels and transient rapid depolarization of the cell.     

Identification of possible signal transduction components mediating light induction of the Gsa gene for an early chlorophyll biosynthetic step in Chlamydomonas reinhardtii

 Light-induced expression of the Gsa gene encoding the heme and chlorophyll biosynthetic enzyme glutamate 1-semialdehyde aminotransferase in Chlamydomonas reinhardtii was previously shown to involve Ca²⁺ and calmodulin (CaM) (C. lm et al. 1996, Plant Cell 8: 2245–2253). To further analyze the signal transduction pathway for light-induced Gsa expression, the effects of several pharmacological agents were examined. Treatment of light-dark synchronized cells with the heterotrimeric G-protein agonist Mas-7 caused partial induction of Gsa in the dark. The phospholipase C inhibitor U73122 inhibited light induction of Gsa. Exposure of cells to light caused a sustained 3-fold increase in cellular d-inositol 1,4,5-trisphosphate (InsP₃) concentration. KN-93, a specific inhibitor of Ca²⁺/CaM-dependent protein kinase II, inhibited light induction of Gsa. In contrast, cyclosporin A, a specific inhibitor of the Ca²⁺/CaM-dependent phosphoprotein phosphatase calcineurin, did not affect light induction of Gsa. These results, together with the earlier results, suggest the involvement of a canonical signal transduction pathway for light-regulated Gsa expression that involves a heterotrimeric G-protein activation, phospholipase C-catalyzed InsP₃ formation, InsP₃-dependent Ca²⁺ release, and activation of a downstream signaling pathway through a Ca²⁺/CaM-dependent protein kinase. Received: 21 October 1999 / Accepted: 3 December 1999     

Magnesium chelatase subunit D from pea: characterization of the cDNA,heterologous expression of an enzymatically active protein and immunoassay of the native protein

Mg-chelatase catalyzes the insertion of Mg into protoporphyrin and lies at the branchpoint of heme and (bacterio)chlorophyll synthesis. In prokaryotes, three genes – BchI, D and H – encode subunits for Mg-chelatase. In higher plants, homologous cDNAs for the I, D and H subunits have been characterized. Since the N-terminal half of the D subunit is homologous to the I subunit, the C-terminal portion of the pea D was used for antigen production. The antibody recognized the chloroplast D subunit and was used to demonstrate that this subunit associated with the membranes in the presence of MgCl₂. The antibody immunoprecipitated the native protein and inhibited Mg-chelatase activity. Expression in Escherichia coli with a construct for the full-length protein (minus the putative transit peptide) resulted in induction of 24.5 kDa (major) and 89 kDa (minor) proteins which could only be solubilized in 6 M urea. However, when host cells were co-transformed with expression vectors for the full-length D subunit and for the 70 kDa HSP chaperonin protein, a substantial portion of the 89 kDa protein was expressed in a soluble form which was active in a Mg-chelatase reconstitution assay.