Lipid peroxidation and superoxide dismutase activity in relation to photoinhibition induced by chilling in moderate light

The effect of a chilling stress, at a moderate photon flux density for a few hours, on the peroxidation of membrane lipids and on superoxide dismutase (SOD) activity was compared in leaf slices of chilling-sensitive and chilling-insensitive plants. The aim was to determine if susceptibility to chill-temperature photoinhibition could be related to either damage to membrane lipids by superoxide and-or a decrease in activity of chloroplast SOD. Plants used were Nerium oleander L., grown at 45° C, and Cucumis sativus L., both susceptible to chill-temperature photoinhibition, and N. oleander, grown at 20° C and Spinacia oleracea L., both insensitive to chill-temperature photoinhibition. Lipid peroxidation was assessed by measuring the concentration of malondialdehyde (MDA). Leaf slices from all plants showed a basal level of MDA which decreased by about 15% when the leaf slices were chilled in the light. The level of MDA was not increased by the addition of either KHCO₃ or methyl viologen during chilling but it was increased, up to threefold, by the addition of Rose Bengal, which produces singlet oxygen. Chloroplast SOD activity was assessed in leaf extracts as the cyanide-sensitive production of H₂O₂ in a system which produced superoxide. Activity of SOD was similar in all the plants and was altered little by chilling. The results show that for the plants tested, chilling at a moderate photon flux density for 5 h does not increase the susceptibility of cell membranes to peroxidative damage nor does it decrease the activity of SOD. It was concluded that the susceptibility of chilling-sensitive plants to chill-temperature photoinhibition cannot be explained on the basis of differences in the vulnerability of membrane lipids to damage by superoxide or differences in SOD activity.Abbreviations Chl chlorophyll - MDA malondialdehyde - MV methyl viologen - O ₂ ^- superoxide - 20°-oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - PFD photon flux density - SOD superoxide dismutase Deceased     

Inhibition of photosynthesis by chilling in moderate light: a comparison of plants sensitive and insensitive to chilling

Photosynthetic activity, in leaf slices and isolated thylakoids, was examined at 25° C after preincubation of the slices at either 25° C or 4° C at a moderate photon flux density (PFD) of 450 mol·m^–2·s^–1, or at 4° C in the dark. The plants used wereSpinacia oleracea L.,Cucumis sativus L. andNerium oleander L. which was acclimated to growth at 20° C or 45° C. The plants were grown at a PFD of 550 mol·m^–2·s^–1. Photosynthesis, measured as CO₂-dependent O₂ evolution, was not inhibited in leaf slices from any plant after preincubation at 25° C at a moderate PFD or at 4° C in the dark. However, exposure to 4° C at a moderate PFD induced an inhibition of CO₂-dependent O₂ evolution within 1 h inC. sativus, a chilling-sensitive plant, and in 45° C-grownN. oleander. The inhibition in these plants after 5 h reached 80% and 40%, respectively, and was independent of the CO₂ concentration but was reduced at O₂ concentrations of less than 3%. Methyl-viologen-dependent O₂ exchange in leaf slices from these plants was not inhibited. There was no photoxidation of chlorophyll, in isolated thylakoids, or any inhibition of electron transport at photosystem (PS)II, PSI or through both photosystems which would account for the inhibition of photosynthesis. The conditions which inhibit photosynthesis in chilling-sensitive plants do not cause inhibition inS. oleracea, a chilling-insensitive plant, or in 20° C-grownN. oleander. The CO₂-dependent photosynthesis, measured at 5° C, was reduced to about 3% of that recorded at 25° C in chilling-sensitive plants but only to about 30% in the chilling-insensitive plants. Methyl-viologen-dependent O₂ exchange, measured at 5° C, was greater than 25% of the activity at 25° C in all the plants. The results indicate that the mechanism of the chilling-induced inhibition of photosynthesis does not involve damage to PSII. That inhibition of photosynthesis is observed only in the chilling-sensitive plants indicates it is related, in some way, to the disproportionate decrease in photosynthetic activity in these plants at chilling temperatures.Abbreviations Chl chlorophyll - DPIPH reduced form of 2,6-dichlorophenol-indophenol - DMQ 2,5-dimethyl-p-benzoquinone - MV methyl viologen - 20°-oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - PFD photon flux density (photon fluence rate) - PSI and PSII photosystem I and II, respectively     

Chloroplast thylakoid membrane fluidity and its sensitivity to temperature

In order to investigate membrane fluidity, the hydrophobic probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), has been incorporated into intact isolated thylakoids and separated granal and stromal lamellae obtained from the chloroplasts of Pisum sativum. The steady-state polarization of DPH fluorescence was measured as a function of temperature and indicated that at physiological values the thylakoid membrane is a relatively fluid system with the stromal lamellae being less viscous than the lamellae of the grana. According to the DPH technique, neither region of the membrane, however, showed a sharp phase transition of its bulk lipids from the liquid-crystalline to the gel state for the temperature range -20° to 50° C. Comparison of intact thylakoids isolated from plants grown at cold (4°/7°C) and warm (14°/17° C) temperatures indicate that there is an adaptation mechanism operating which seems to maintain an optimal membrane viscosity necessary for growth. Using a modified Perrin equation the optimal average viscosity for the thylakoid membrane of the chill-resistant variety used in the study (Feltham First) is estimated to be about 1.8 poise.Abbreviations DPH 1,6-diphenyl-1,3,5-hexatriene - Hepes N-(2-hydroxyethyl)-1-piperazineethanesulphonic acid     

Superoxide production by thylakoids during chilling and its implication in the susceptibility of plants to chilling-induced photoinhibition

Factors influencing the rate of superoxide (O ₂ ^- ) production by thylakoids were investigated to determine if increased production of the radical was related to injury induced by chilling at a moderate photon flux density (PFD). Plants used were Spinacia oleracea L., Cucumis sativus L. and Nerium oleander L. grown at either 200° C or 45° C. Superoxide production was determined by electron-spin-resonance spectroscopy of the (O ₂ ^- )-dependent rate of oxidation of 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH) to the corresponding oxazolidinoxyl radical, OXANO ·. For all plants, the steady-state rate of O ₂ ^- production by thylakoids, incubated at 25° C and 350 mol photon · m^–2 · s^–1 (moderate PFD) with added ferredoxin and NADP, was between 7.5 and 12.5 mol · (mg chlorophyll)^–1 · h^–1. Incubation at 5° C and a moderate PFD, decreased the rate of O ₂ ^- production 40% and 15% by thylakoids from S. oleracea and 20° C-grown N. oleander, chillinginsensitive plants, but increased the rate by 56% and 5% by thylakoids from C. sativus and 45° C-grown N. oleander, chilling-sensitive plants. For all plants, the addition of either ferredoxin or methyl viologen increased the rate of O ₂ ^- -production at 25° C by 75–100%. With these electron acceptors, lowering the temperature to 5° C caused only a slight decrease in O ₂ ^- production. In the absence of added electron acceptors, thylakoids produced O ₂ ^- at a rate which was about 45% greater than that when ferredoxin and NADP were present. The addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea reduced O ₂ ^- production under all conditions tested. The results show that the rate of O ₂ ^- production increases in thylakoids when the rate of electron transfer to NADP is reduced. This could explain differences in the susceptibility of thylakoids from chilling-sensitive and chilling-insensitive plants to chilling at a moderate PFD, and is consistent with the proposal that O ₂ ^- production is involved in the injury leading to the inhibition of photosynthesis induced under these conditions.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophen-yl)-1,1-dimethylurea - Fd ferredoxin - MV methyl viologen - 20°oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - OXANOH 2-ethyl-1-hydroxy-2,5,5-tri-methyl-3-oxazolidine - PFD photon flux density (photon fluence rate) - TEMED tetramethyl ethylenediamine We would like to thank R.T. Furbank, R.S.B.S., Australian National University, Canberra, A.C.T., and C.B. Osmond, now of Duke University, Durham, N.C., USA, for the gift of ferredoxin, R.A.J.H. was supported by a Commonwealth Postgraduate Research Award.     

Ultrastructure of cell wall and thylakoid membranes of the thermophilic cyanobacterium Synechococcus lividus under the influence of temperature shifts

The ultrastructure of the cell wall and the thylakoid membranes of the thermophilic cyanobacterium Synechococcus lividus was studied by freezefracture electron microscopy after temperature shifts. Different fracture faces of the outer, the cytoplasmic and the thylakoid membranes were demonstrated when the preparation-temperature was in the range of the optimal growth temperature at 52°C or after fixation at 52°C. In the outer membrane of the cell wall two fracture faces with holes and 7.5 nm intramembrane particles were detected. On both the outer (EF) and inner (PF) leaflet of the cytoplasmic membrane randomly distributed particles were demonstrated. The particle density on the PF-face was approx. three times that of the EF-face. The EF-face of the thylakoid membrane exposed rows of particles with an average diameter of 10 nm. The spacing between the particle rows was 35–50 nm. This regular particle arrangement on the EF-face was demonstrated only in a few cases. Mostly the intramembrane particles were distributed randomly on the thylakoid fracture faces. The particle density of thylakoids with a random distribution was approx. in the same range both on the EF-and PF-face. The EF-particles fall into four groups of 9,10,11, and 12.5 nm. The main particle class was the 10 nm class. The PF-face exposed smaller particles with two maxima at 8.5–9 nm and 10 nm. When Synechococcus lividus OH-53s was chilled to temperatures below 30–35°C before the freeze-etch preparation a phase transition took place after the temperature shift. On the fracture faces of the thylakoid and cytoplasmic membranes particle depleted areas occurred. The size of the areas were different in both membranes and dependent on the velocity of cooling. Contrary to Synechococcus lividus OH-53s in the mesophilic Synechococcus strain 6910 the phase transition point was 15°C. The lower phase transition point may be due to a higher content of unsaturated fatty acids.Dedicated to Prof. D. Peters (Hamburg) on the occasion of the 65th anniversary of his birthday     

The site of photoinhibition in leaves of Cucumis sativus L. at low temperatures is photosystem I,not photosystem II

Maximum quantum yields (QY) of photosynthetic electron flows through PSI and PSII were separately assessed in thylakoid membranes isolated from leaves of Cucumis sativus L. (cucumber) that had been chilled in various ways. The QY(PSI) in the thylakoids prepared from the leaves treated at 4° C in moderate light at 220 mol quanta·m^–2·s^–1 (400–700 nm) for 5 h, was about 20–30% of that in the thylakoids prepared from untreated leaves, while QY(PSII) decreased, at most, by 20% in response to the same treatment. The decrease in QY(PSI) was observed only when the leaves were chilled at temperatures below 10° C, while such a marked temperature dependency was not observed for the decrease in QY(PSII). In the chilling treatment at 4° C for 5 h, the quantum flux density that was required to induce 50% loss of QY (PSI) was ca. 50 umol quanta·m^–2·s^–1. When the chilling treatment at 4° C in the light was conducted in an atmosphere of N₂, photoinhibition of PSI was largely suppressed, while the damage to PSII was somewhat enhanced. The ferricyanide-oxidised minus ascorbate-reduced difference spectra and the light-induced absorbance changes at 700 nm obtained with the thylakoid suspension, indicated the loss of P700 to extents that corresponded to the decreases in QY(PSI). Accordingly, the decreases in QY(PSI) can largely be attributed to destruction of the PSI reaction centre itself. These results clearly show that, at least in cucumber, a typical chillingsensitive plant, PSI is much more susceptible to aerobic photoinhibition than PSII.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - P700 primary electron donor of PSI - PPFD photosynthetically active photon flux density - QY quantum yield We are grateful to invaluable comments by Prof. S. Katoh, K. Hikosaka and the members of our laboratory. We also thank A. Aoyama for technical assistance. This work was partly supported by the grants from the Ministry of Education, Science, and Culture, Japan, to I. Terashima (#03740342 and #04640621).     

Degradation of leaf polar lipids during chilling and post-chilling rewarming of Zea mays genotypes reflects differences in their response to chilling stress. The role of galactolipase

Degradation of leaf polar lipids [monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG)] and chlorophyll (Chl) were studied in four Zea mays genotypes differing in chilling susceptibility following dark chilling and post-chilling rewarming at original growth conditions. Assessment of visual chilling injury symptoms during post-chilling rewarming differentiated maize inbred lines into chiling-sensitive (CS) CM7 and Co151 lines and chillingtolerant (CT) S215 and EP1 lines. Severity of chilling injury in CS and CT inbreeds were correlated with the extent of Chl and polar lipids degradation. Chilling for either 4 or 6 days followed by 4 days of rewarming caused more extensive degradation of total polar lipids content in CS than in CT lines. MGDG decreased mostly during chilling whereas DGDG dropped during rewarming only. Chl content was not affected during chilling but its large decrease, greater in CS than in CT lines, was observed upon rewarming. Extent of polar lipids breakdown in CS and CT inbreeds during chilling and post-chilling rewarming is correlated with galactolipase activity in chloroplasts (Kaniuga et al., 1998) and visual assessment of chilling injury. In view of the data it is likely that contribution of galactolipase activity induced during low-temperature stress of CS plants is an important factor responsible for thylakoid lipid degradation and development of chilling injury as postulated previously (Kaniuga 1997). It is suggested that genetically engineered reduction of galactolipase activity or elimination of the factors(s) involved in induction/stimulation of its activity during chilling might increase tolerance of CS species to chilling stress.     

Photoinhibition of photosynthesis: effect on chlorophyll fluorescence at 77K in intact leaves and in chloroplast membranes of Nerium oleander

The effect of exposing intact leaves and isolated chloroplast membranes of Nerium oleander L. to excessive light levels under otherwise favorable conditions was followed by measuring photosynthetic CO₂ uptake, electron transport and low-temperature (77K=-196°C) fluorescence kinetics. Photoinhibition, as manifested by a reduced rate and photon (quantum) yield of photosynthesis and a reduced electron transport rate, was accompanied by marked changes in fluorescence characteristics of the exposed upper leaf surface while there was little effect on the shaded lower surface. The most prominent effect of photoinhibitory treatment of leaves and chloroplasts was a strong quenching of the variable fluorescence emission at 692 nm (F_v,692) while the instantaneous fluorescence (F_o,692) was slightly increased. The maximum and the variable fluorescence at 734 nm were also reduced but not as much as F_M,692 and F_v,692. The results support the view that photoinhibition involves an inactivation of the primary photochemistry of photosystem II by damaging the reaction-center complex. In intact leaves photoinhibition increased with increased light level, increased exposure time, and with decreased temperature. Increased CO₂ pressure or decreased O₂ pressure provided no protection against photoinhibition. With isolated chloroplasts, inhibition of photosystem II occurred even under essentially anaerobic conditions. Measurements of fluorescence characteristics at 77K provides a simple, rapid, sensitive and reproducible method for assessing photoinhibitory injury to leaves. The method should prove especially useful in studies of the occurrence of photoinhibition in nature and of interactive effects between high light levels and major environmental stress factors.Abbreviations and symbols PFD photon flux area density - PSI, PSII photosystem I, II - F_M, F_O, F_V maximum, instantaneous, variable fluorescence emission C.I.W.-D.P.B. Publication No. 773     

Gravity-regulated formation of the peg in developing cucumber seedlings

It has been proposed that peg formation in the vascular transition region (TR zone) between the hypocotyl and the root in Cucurbitaceae seedlings is a gravimorphogenetic phenomenon. Initiation of the peg became visible 36 h after imbibition when cucumber (Cucumis sativus L. cv. Burpee Hybrid II) seeds were germinated in a horizontal position at 24°C in the dark. Simultaneously, sedimented amyloplasts (putative statoliths) were apparent in the sheath cells surrounding the vascular strands, and in the cortical cells immediately adjacent to them, in the TR zone. In contrast, the other cortical cells, some of which were destined to develop into the peg, contained amyloplasts which were not sedimented. These results suggest that the graviperception mechanism for peg formation may be like that of statoliths in shoot gravitropism. By 48 h following imbibition, the cells of the TR zone still had sedimented amyloplasts but had lost their sensitivity to gravity, possibly because of their maturation.     

The role of ethylene in the prevention of chilling injury in nectarines

Woolliness is a chilling injury phenomenon occurring in nectarines held at low temperatures for extended periods. It is a disorder marked by altered cell wall metabolism during ripening leading to a dry, woolly texture in the fruit. Two treatments were found to alleviate this disorder. One was holding the fruits for 2 days at 20 °C before 0 °C storage (delayed storage) and the second was having ethylene present during cold storage (ethylene). Immediately stored fruit (control) had 88 percnt; woolliness while 7 percnt; of delayed storage and 15 percnt; of ethylene fruit showed woolliness. The severity of the injury in individual fruits was closely related to inhibition of ethylene evolution. Woolly fruit had higher levels of 1-aminocyclopropane-1-carboxylic acid (ACC) and less 1-aminocyclopropane-1-carboxylic acid oxidase (ACO, EC 1.4.3) activity than healthy fruit. It is suggested that ethylene is essential for promoting the proper sequence of cell wall hydrolysis necessary for normal fruit softening. This is in contrast to chilling injury in other fruits, whereby ethylene is often a sign of incipient damage. Respiration was also found to be associated with chilling injury, in that fruit with woolliness had a depressed respiration.     

The effect of vesicular-arbuscular mycorrhizae and chilling on two hybrids of Zea mays L.

In order to investigate the effect of vesicular-arbuscular mycorrhizae on the chilling resistance of Zea mays, seeds of two hybrids (Pioneer 3902 and Pride 5) were grown in soil inoculated with Glomus mosseae. Germination tests at 10° C and 25° C showed that Pride 5 was more resistant to chilling than Pioneer 3902. Plants grown at 25° C for 6 weeks were given a 1-week chilling treatment at 10° C and the responses of mycorrhizal and nonmycorrhizal plants of the two hybrids were compared. At 10° C, the mycorrhizal plants had greater biomass, carbohydrate, and protein content than the nonmycorrhizal plants.     

The effect of the environment on the permeability and composition of Citrus leaf cuticles

The constituents of the soluble cuticular lipids (SCL) of the leaf blades of Citrus aurantium L. were identified by gas chromatography-mass spectrometry and quantified. Major components were 1-alkanols (C₂₄ to C₄₀), n-alkyl esters (C₃₆ to C₅₆), n-alkanoic acids (C₂₈ to C₃₄), n-alkanes (C₂₂ to C₄₀) and triterpenones, while n-alkanals (C₂₉ to C₃₈), sterols, and alkyl benzenes (molecular weights 260, 274 and 288) made minor contributions. Leaf age and side significantly affected the quantitative composition of SCL. Increased day temperature during the development of leaves led to decreased amounts per unit area of n-alkanes, 1-alkanols, n-alkanoic acids and n-alkyl esters while increased night temperatures resulted in increased amounts of n-alkanes n-alkanoic acids and 1-alkanols. Relative humidity had no effect on the amounts or composition of SCL. The permeability of cuticular membranes to water (described in part I of this paper) and the composition of SCL were not related. A model for the molecular structure of the transport-limiting barrier of plant cuticles and for the transport of water across it is proposed.Abbreviations CM cuticular membrane - GC gas chromatogra-phy - MS mass spectroscopy - TLC thin-layer (planar) chromatography - SCL soluble cuticular lipids The authors are indebted to Dr. R. Winkler and H. Krause, Laboratorium für Strukturchemie des Fachbereichs Chemie, Biologie und Geowissenschaften, Technische Universität München, FRG, for performing the GC-MS analyses and their valuable help in the identification of SCL constituents. This work has been supported by the Deutsche Forschungsgemeinschaft and the Bayerische Staatsministerium für Wissenschaft und Kunst.     

Correlations between the thermal stability of chloroplast (thylakoid) membranes and the composition and fluidity of their polar lipids upon acclimation of the higher plant,Nerium oleander,to growth temperature

The thermal stability of excitation transfer from pigment proteins to the Photosystem II reaction center of Nerium oleander adjusts by 10 Celsius degrees when cloned plants grown at 20°C/15°C, day/night growth temperatures are shifted to 45°C/32°C growth temperature or vice versa. Concomitant with this adjustment is a decrease in the fluidity of thylakoid membrane polar lipids as determined by spin labeling. The results are consistent with the hypothesis that there is a limiting maximum fluidity compatible with maintenance of native membrane structure and function. This limiting fluidity was about the same as for a number of other species which exhibit a range of thermal stabilities. Inversely correlated shifts in lipid fluidity and thermal stability occurred during the time course of acclimation of N. oleander to new growth temperatures. Thus, the temperature at which the limiting fluidity was reached changed during acclimation while the limiting fluidity remained constant. Although the relative proportion of the major classes of membrane polar lipids remained constant during adjustments in fluidity, large changes occured in the abundance of specific fatty acids. These changes were different for the phospho- and galacto-lipids suggesting that the fatty acid composition of these two lipid classes is regulated by different mechanisms. Comparisons between membrane lipid fluidity and fatty acid composition indicate that fluidity is not a simple linear function of fatty acid composition.     

Dithiothreitol,an inhibitor of violaxanthin de-epoxidation,increases the susceptibility of leaves ofNerium oleander L. to photoinhibition of photosynthesis

Leaves ofNerium oleander L. plants, which had been previously kept in a shaded glasshouse for at least two months, were fed 1 mM dithiothreitol (DTT) through their petioles, either for 12h in darkness (overnight) or for 2h in low light (28 μmol photons·m⁻²·s⁻¹), in each case followed by a 3-h exposure to high light (1260 μmol photons·m⁻²·s⁻¹). During exposure to high light, violaxanthin became converted to zeaxanthin in control leaves, to which water had been fed, whereas zeaxanthin did not accumulate in leaves treated with DTT. Total carbon gain was not reduced by DTT during the photoinhibitory treatment. Exposure to high light led to a decrease in the photochemical efficiency of photosystem II, measured as the ratio of variable over maximum fluorescence emission,F _v/F _M, at both 298 K and 77K. The decrease was much more pronounced in the presence of DTT, mainly owing to a sustained increase in the instantaneous fluorescence,F _o. By contrast, in the control leaves,F _o determined immediately after the high-light treatment showed a transient decrease below theF _o value obtained before the onset of the photoinhibitory treatment (i.e. after 12 h dark adaptation), followed by a rapid return (within seconds) to this original level ofF _o during the following recovery period in darkness. Incubation of leaves with DTT led to large, sustained decreases in the photon-use efficiency of photosynthetic O₂ evolution by bright light, whilst the capacity of photosynthetic O₂ evolution at light and CO₂ saturation was less affected. In the control leaves, only small reductions in the photon yield and in the photosynthetic capacity were observed. These findings are consistent with previous suggestions that zeaxanthin, formed in the xanthophyll cycle by de-epoxidation of violaxanthin, is involved in protecting the photosynthetic apparatus against the adverse effects of excessive light.     

The relationships between growth temperature,fatty acid composition and the physical state and fluidity of membrane lipids in Yersinia enterocolitica

The relationship between membrane lipid composition and membrane lipid phase transitions was investigated in Yersinia enterocolitica cells grown at 5, 22 and 37°C. The total phospholipid concentrations were 9.4, 7.3 and 6.3% of the cell dry weight for cells grown at 5, 22 and 37°C, respectively. The relative concentrations of the three major phospholipids, phosphatidylethanolamine (73–76%), phosphatidylglycerol (9–11%) and cardiolipin (11–13%) were essentially the same at all three growth temperatures. The ratios of unsaturated to saturated fatty acids were 2.2, 1.1 and 0.4 for cells grown at 5, 22 and 37°C, respectively. This change in the fatty acid composition in response to temperature changes is similar to the patterns reported for other organisms. Reversible thermotropic phase transitions were detected by calorimetric analysis in both pure lipid preparations and membrane preparations. The mid-points of the thermotropic phase transitions were at ?13, ?9 and 1°C for membranes from cells grown at 5, 22 and 37°C, respectively. The phase transitions of the membranes from cells grown at the three different temperatures occurred below the lowest growth temperature (5°C). The alternations in the fatty acid composition in Y. enterocolitica did not, therefore, appear to be required to adjust membrane fluidity but might rather be required for some other membrane function.     

Immunological studies on chlorophyll-a/b proteins and their distribution in thylakoid membrane domains

The immunological relationships between chlorophyll-a/b proteins from higher-plant thylakoid membranes have been studied by assaying purified chlorophyll proteins (CPs) with polyclonal and monoclonal antibodies. Although low levels of cross-reactions were observed between all light-harvesting proteins, the peripheral antennae (LHCII) were largely distinct from the inner antennae (CP 26 and CP 29). Chlorophyll-protein 24 and LHCI-680 have been proposed to have a role in connecting the inner and outer antennae, respectively, in photosystems I and II, and were closely related. The immunological relationships closely corresponded to the spectral properties. Antibodies were also used for locating chlorophyll-a/b proteins in grana, stroma and bundle-sheath membranes showing a strong lateral heterogeneity, which was maintained following State I State II transition. The only exception to this pattern was a specific LHCII population enriched in State-II stroma membranes. Chlorophyll proteins from bundlesheath chloroplasts, that have only cyclic electron flow, had epitopes distinct from those of their mesophyll homologues.     

Thermal phase transitions in the polar lipids of plant membranes. Their induction by disaturated phospholipids and their possible relation to chilling injury

The phase behaviour of leaf polar lipids from three plants, varying in their sensitivity to chilling, was investigated by differential scanning calorimetry. For the lipids from mung bean (Vigna radiata L. var. Berken), a chilling-sensitive plant, a transition exotherm was detected beginning at $\text{10 ± 2°}\text{C}$. No exotherm was evident above 0°C with polar lipids from wheat (Triticum aestivum cv. Falcon) or pea (Pisum sativum cv. Massey Gem), plants which are insensitive to chilling. The enthalpy for the transition in the mung bean polar lipids indicated that only about 7% w/w of the lipid was in the gel phase at ?8°C. The thermal transition of the mung bean lipids was mimicked by wheat and pea polar lipids after the addition of 1 to 2% w/w of a relatively high melting-point lipid such as dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol or dimyristoylphosphatidylcholine. Analysis of the polar lipids from the three plants showed that a dipalmitoylphosphatidylglycerol was present in mung bean (1.7% w/w) and pea (0.3% w/w) but undetected in wheat, indicating that the transition exotherm temperature of 10°C in mung bean, 0°C in pea and about ?3°C in wheat correlates with the proportion of the high melting-point disaturated component in the polar lipids. The results indicate that the transition exotherm, observed at temperatures above 0°C in the membranes of chilling-sensitive plants, could be induced by small amounts of high melting-point lipids and involves only a small proportion of the membrane polar lipids.     

Photoacoustic and fluorescence measurements of the chilling response and their relationship to carbon dioxide uptake in tomato plants

The response of tomato plants to various chilling treatments was studied using two approaches for the measurement of photosynthetic activity. One involved the use of a portable fluorometer for the measurement of in-vivo chlorophyll fluorescence, while the other employed a newly introduced photoacoustic system which allowed changes in oxygen evolution to be followed in a leaf disc. A strong correlation was found between results obtained by each system and those obtained by a conventional open gas-exchange system for the determination of CO₂ uptake. Both systems of measurements could readily distinguish between the effects of chilling in the dark (at 3° C for 18 h) and chilling at high photon flux density (2000 mol m^-2 s^-1 for 5h at 5° C). Chilling in the dark had practically no effect on the quantum yield of oxygen evolution, chlorophyll fluorescence or CO₂ uptake, while chilling at excessively high photon flux density resulted in a sharp reduction (50–70%) in the quantum yields obtained. The results support the view that photosystem II cannot be the primary site of damage by chilling in the dark, although it is significantly affected by chilling at high light intensity.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PA photoacoustic - PFD photon flux density - PSII photosystem II     

Correlation between thylakoid protein phosphorylation and molecular organization of the photosynthetic apparatus in a dynamic system

In-vitro thylakoid protein phosphorylation has been studied in synchronized cells of Scenedesmus obliquus at the 8- and 16-h of the life cycle, stages which are characterized by the maximum and minimum photosynthetic activities, respectively. The stage of maximum photosynthetic activity (8-h) is characterized by the highest protein phosphorylation in vitro and in vivo, by the largest proportion of the heavy subfraction of thylakoids, and by maximum oligomerization of the light-harvesting chlorophyll a/b-protein complex, altogether creating the highest energy charge of the thylakoid membranes. Protein phosphorylation in vitro decreases the amount of the heavy subfraction and increases the amount of oligomerization of the antenna of photosystem I (PSI) (increase of chlorophyll b in the light fraction). Concomittantly, PSII units become smaller (longer time for the rise in fluorescence induction) and photosynthetic efficiency increases (decrease of fluorescence yield). In-vivo protein phosphorylation is controlled mainly endogenously during the 8-h of the life cycle but is exogenously modulated by light to optimize the photosynthetic activity by redistribution of pigment-protein complexes. In-vitro protein phosphorylation seems to restore partially the conditions prevalent in vivo and lost during the preparation of membranes. The effect is greater in 16-h cells which have less-stable membranes. The regulatory mechanism between membrane stabilization and oligomerization on the one hand and redistribution of the light-harvesting chlorophyll a/b-protein complex from PSII to PSI on the other hand remains unexplained. We have confirmed that the mechanism of protein phosphorylation is regulated via plastohydroquinone, but experiments with the plastohydroquinone analogue 2,3,5,6-tetramethyl-p-benzoquinone demonstrated that plastohydroquinone is not solely responsible for the differences in protein phosphorylation of 8- and 16-h thylakoids. The inhibitory effect of ADP and the distinct rates of kinase reaction indicate that the adenylate energy charge and changes in the organization of the photosynthetic apparatus also contribute to the observed differences in protein phosphorylation. Phosphorylation in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea indicated that the 32-kDa phosphoprotein and the herbicide-binding Q_B protein may be the same. These experiments also indicated that 3-(3,4-dichlorophenyl)-1,1-dimethylurea-binding reduces kinase activity directly and not only by inhibiting electron transport.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHCP light-harvesting chlorophyll a/b-protein complex - PSI, II photosystem I, II - TMQ 2,3,5,6-tetramethyl-p-benzoquinone Dedicated to Professor Dr. W. Nultsch on the occasion of his 60th brithday