Essential oil constituents derived from different organs of a relictual conifer Wollemia nobilis

The chemical composition of the essential oil of leaves (0.9%, w/v) and twigs (0.33%, w/v) of Wollemia nobilis (Araucariaceae) – a remnant species thought to have been extinct for 65 million years – was investigated by GC/MS. The main constituents of both leaf- and twig-derived oil samples were 16-kaurene (61.8% and 38.2%, respectively) and germacrene D (9.9% and 22%). The principal difference was a considerably more pronounced sesquiterpene presence in the twig-oil, amounting to 33.5%, than in its folial counterpart (23.4%). On the contrary, while remaining the dominant group in both oil samples under investigation, diterpenoids were relatively more abundant in leaf-derived oil constituting 65.3%, versus 41.7% detected in twigs. To our knowledge, this is the first report dealing with the essential oil composition of Wollemi pine twigs, as opposed to the leaf-derived volatiles.     

Cold acclimation and photoinhibition of photosynthesis in Scots pine

Cold acclimation of Scots pine did not affect the susceptibility of photosynthesis to photoinhibition. Cold acclimation did however cause a suppression of the rate of CO₂ uptake, and at given light and temperature conditions a larger fraction of the photosystem II reaction centres were closed in cold-acclimated than in nonacclimated pine. Therefore, when assayed at the level of photosystem II reaction centres, i.e. in relation to the degree of photosystem closure, cold acclimation caused a significant increase in resistance to photoinhibition; at given levels of photosystem II closure the resistance to photoinhibition was higher after cold acclimation. This was particularly evident in measurements at 20° C. The amounts and activities of the majority of analyzed active oxygen scavengers were higher after cold acclimation. We suggest that this increase in protective enzymes and compounds, particularly Superoxide dismutase, ascorbate peroxidase, glutathione reductase and ascorbate of the chloroplasts, enables Scots pine to avoid excessive photoinhibition of photosynthesis despite partial suppression of photosynthesis upon cold acclimation. An increased capacity for light-induced de-epoxidation of violaxanthin to zeaxanthin upon cold acclimation may also be of significance.Abbreviations APX ascorbate peroxidase - DHA dehydroascorbate - DHAR dehydroascorbate reductase - Fm maximal fluorescence when all reaction centres are closed - Fv/Fm maximum photochemical yield of PSII - GR glutathione reductase - GSH reduced glutathione - Je rate of photosynthetic electron transport - MDAR monodehydroascorbate reductase - q_N nonphotochemical quenching of fluorescence - q_P photochemical quenching of fluorescence - SOD superoxide dismutase This work was supported by the Swedish Natural Science Research Council and the National Natural Science Foundation of China.     

Lateral root formation in pine seedlings

The role of assimilates in lateral root development was studied in Pinus pinea seedlings grown in a nutrient solution. Seedlings were treated with ¹⁴CO₂ for 2 h following removal of the tap root tip at various times prior to the application of ¹⁴CO₂ or removal of a different number of cotyledons at one time. In seedlings with intact root systems most of the radioactivity accumulated in the lower section of the root containing the tap root apex. When the tap root tip was removed, the pattern of radioactivity accumulation along the root was affected by the presence and the stage of lateral root development. Removing the tap root tip of young seedlings (with no lateral roots) resulted in an almost equal distribution of radioactivity along the root. About 50% of the total radioactivity was found in the section showing the highest lateral root growth. Removing the tap root tip of mature seedlings (with lateral roots in the upper section) resulted in an immediate increase in the radioactivity accumulation in the upper section. When lateral roots appeared in the middle section, the pattern of radioactivity distribution was similar to that found in root decapitated young seedlings. Removal of cotyledons of mature seedlings somewhat increased the transport of radioactivity to the lower root section at the expense of the radioactivity in the lateral roots of the upper section. The present study suggests that competition within the root system between the tap root apex and the lateral roots may play an important role in determining the morphology of the root system.     

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     

Photosynthetic acclimation and photoinhibition on exposure to high light in shade-developed leaves of Fagus crenata seedlings

The physiological response of leaves developed in low light (L) on Fagus crenata seedlings exposed to different levels of high light (H: high light, M: medium light) was studied. Measurements were conducted on potted seedlings in the F. crenata forest understory. The seedlings with leaves developed in L were transferred to H (L–H) and M (L–M) in summer. On exposure to high light, the photochemical efficiency of dark-adapted PSII (F_v/F_m) immediately decreased and was followed by a subsequent recovery in both L–H and L–M leaves. The mean value of F_v/F_m in L–H leaves was lower than that in L–M leaves through experiments, indicating that the degree of photoinhibition in L–H leaves was greater than that in L–M leaves. About 1 month after transfer, 37% and 5% of leaves had fallen in L–H and L–M seedlings, respectively. This result also indicated the greater photoinhibition in L–H leaves. Moreover, the photosynthetic capacity (P_Nmax) of L–H leaves decreased. In contrast, the P_Nmax of L–M leaves increased, although the P_Nmax was lower than that of M control leaves. An increase in the xanthophyll cycle pool (VAZ), indicating an increase of the photoprotective function, was found in both L–H and L–M leaves. Especially, the VAZ pool in L–M leaves was higher than that in M leaves by the end of experiments. L–M leaves may avoid photoinhibition effectively by the decrease in excess light with the increase of the P_Nmax or VAZ pool, compared to L–H leaves. Thus, the physiological acclimation on exposure to high light depended on the degree of high light. To achieve successful photosynthetic acclimation with slight photoinhibition, the variation of light intensity before and after exposure to high light would be an important factor because of the difference in excess light.     

Fluorescence quenching during photosynthesis and photoinhibition of Ulva rotundata blid.

The relationships between photoinhibition and photoprotection in high and low-light-grown Ulva were examined by a combination of chlorophyll-fluorescence-monitoring techniques. Tissues were exposed to a computer-controlled sequence of 5-min exposures to red light, followed by 5-min darkness, with stepwise increases in photon flux. Coefficients of chlorophyll fluorescence quenching (1?q_P and NPQ) were calculated following a saturating pulse of white light near the end of each 5-min light treatment. Dark-adapted chlorophyll fluorescence parameters (F₀ and F_V/F_M) were calculated from a saturating pulse at the end of each 5-min dark period. Low-light-grown Ulva showed consistently higher 1?q_P, i.e. higher reduction status of Q (high primary acceptor of photosystem II), and lower capacity for nonphotochemical quenching (NPQ) at saturating light than did high-light-grown plants. Consequently, low-light plants rapidly displayed photoinhibitory damage (increased F₀) at light saturation in seawater. Removal of dissolved inorganic carbon from seawater also led to photoinhibitory damage of high-light-grown Ulva at light saturation, and addition of saturating amounts of dissolved inorganic carbon protected low-light-grown plants against photoinhibitory damage. A large part of NPQ was abolished by treatment with 3 mM dithiothreitol and the processes so inhibited were evidently photoprotective, because dithiothreitol treatment accelerated photoinhibitory damage in both low- and high-light-grown Ulva. The extent of photoinhibitory damage in Ulva was exacerbated by treatment with chloramphenicol (1 mM) without much effect on chlorophyll-quenching parameters, evidently because this inhibitor of chloroplast protein synthesis reduced the rate of repair processes.     

Reversible photoinhibition of unhardened and cold-acclimated spinach leaves at chilling temperatures

The photoinhibition of photosynthesis at chilling temperatures was investigated in cold-acclimated and unhardened (acclimated to +18° C) spinach (Spinacia oleracea L.) leaves. In unhardened leaves, reversible photoinhibition caused by exposure to moderate light at +4° C was based on reduced activity of photosystem (PS) II. This is shown by determination of quantum yield and capacity of electron transport in thylakoids isolated subsequent to photoinhibition and recovery treatments. The activity of PSII declined to approximately the same extent as the quantum yield of photosynthesis of photoinhibited leaves whereas PSI activity was only marginally affected. Leaves from plants acclimated to cold either in the field or in a growth chamber (+1° C), were considerably less susceptible to the light treatment. Only relatively high light levels led to photoinhibition, characterized by quenching of variable chlorophyll a fluorescence (F_V) and slight inhibition of PSII-driven electron transport. Fluorescence data obtained at 77 K indicated that the photoinhibition of cold-acclimated leaves (like that of the unhardened ones) was related to increased thermal energy dissipation. But in contrast to the unhardened leaves, 77 K fluorescence of cold-acclimated leaves did not reveal a relative increase of PSI excitation. High-light-treated, cold-acclimated leaves showed increased rates of dark respiration and a higher light compensation point. The photoinhibitory fluorescence quenching was fully reversible in low light levels both at +18° C and +4° C; the recovery was much faster than in unhardened leaves. Reversible photoinhibition is discussed as a protective mechanism against excess light based on transformation of PSII reaction centers to fluorescence quenchers.Abbreviations F_O initial fluorescence - F_M maximal fluorescence - F_V devariable fluorescence (f_m-f_o) - PFD photon flux density - PS photosystem - SD standard deviation The authors thank the Deutsche Forschungsgemeinschaft and the Academy of Finland for financial support.     

Effect of Auxins on Adventitious Root Development from Single Node Cuttings of Camellia sinensis (L.) Kuntze and Associated Biochemical Changes

An attempt was made to induce rooting from single node cuttings of Camellia sinensis var. TV-20 under controlled conditions and study its biochemical changes during rooting. The nodal cuttings were pretreated with different concentrations of IAA, NAA and IBA and kept in a growth chamber (25 ±2 °C, 16 h photoperiod (55 μ mol m⁻² s⁻¹) with cool, white fluorescent lamps and 65% relative humidity) for 12 h. Among the three auxins used for pretreatment, IBA showed more positive response on rooting as compared to IAA and NAA within 2 weeks of transfer to potting medium. Among four concentrations of IBA tested, 75 ppm gave maximum percentage of rooting, number of roots and root length. Therefore, IBA was used further in experiments for biochemical investigation. The adventitious rooting was obtained in three distinct phases i.e. induction (0–12 days), initiation (12–14 days) and expression (14–18 days). IAA-oxidase activity of IBA-treated cuttings increased slightly as compared to control. The activity was found to decrease during induction and initiation phases and increase during expression phase. The peroxidase activity in IBA-treated cuttings increased up to initiation phase and declined at the expression phase. Polyphenoloxidase activity increased both in IBA-treated and control cuttings during induction and initiation phase but declined slowly during expression phase. Total phenolic content was higher in IBA-treated cuttings, particularly in initiation and expression phases and it also correlated with peroxidase activity. Phenolics might be playing key role for induction of adventitious rooting, and phenolic compounds can be used as rooting enhancer in tea plant.     

The kinetics of photoinhibition and its recovery in the red alga Porphyridium cruentum

When Porphyridium cruentum cells were illuminated with high fluence rate between 1900 and 4800 mol photons m^-2s^-1, a decrease in the photosynthetic activity of the cells was observed. Within the time frame of 20 min, and under the fluence rates studied, the sum of photons to be absorbed by cells (mg of chlorophyll (Chl), sufficient to initiate photoinhibition was calculated to be 9235.8 mol. The minimal specific light absorption rate to initiate photoinhibition in P. cruentum ranges between 2.29 and 4.26 mol photons s^-1 mg^-1 chl.a. There was a linear relationship between the specific rate of photoinhibition and the specific light absorption rate. A photon number of 2.56×10⁴ mol mg^-1 chl.a photoinhibited photosynthesis instantaneously. At 15°C, no photoinhibitory effect was observed at 2300 mol photons m^-2 s^-1 even after 45 min of illumination. At the other extreme of 35°C, 84% inhibition of photosynthetic activity was observed within 10 min of exposure to 2300 mol photons m^-2 s^-1. Between 20 and 30°C, the photoinhibitory effect was comparable. Photoinhibited P. cruentum cells recovered readily when transferred to low light (90 mol photons m^-2 s^-1) and darkness, and the specific rate of recovery was independent of the light intensity to which the cells were exposed, during the photoinhibitory treatment.Abbreviations Chlorophyll QL, specific light absorption rate Publication No. 28 of the Microalgal Biotechnology Laboratory     

Kinetic correlation of recovery from photoinhibition and zeaxanthin epoxidation

The generation of non-photochemical fluorescence quenching under photoinhibitory illumination and its relaxation under subsequent low light illumination in leaves from intermittent-light-grown pea (Pisum sativum L.) plants (IML-plants) has been investigated. In parallel, we studied (i) the activity of the xanthophyll cycle with emphasis on zeaxanthin formation and reconversion to violaxanthin and (ii) the degradation rate of D1 protein. In comparison to control plants grown in continuous light, IML-plants were much more susceptible to photoinhibition as determined from the increase of slowly (halftimes > 20 min) relaxing quenching (qI) of variable chlorophyll fluorescence. The relaxation (recovery) kinetics of qI (under weak light) in both types of plant depended on the photon flux density, temperature and duration of pre-illumination. The recovery time generally increased with an increasing degree of qI. In IML-plants, relaxation of qI was kinetically closely related to the epoxidation of zeaxanthin. At high degrees of photosystem II inhibition the kinetics resembled those of D1 degradation. The results are discussed in terms of the mechanisms of photosystem II inactivation in vivo.     

Nodulation of actinorhizal plants byFrankia strains capable of both root hair infection and intercellular penetration

Summary A morphological analysis of the initiation and development of root nodules ofElaeagnus angustifolia andMyrica cerifera inoculated with pure-culturedFrankia strains DDB 011610 or DDB 020110 was undertaken. From ultrastructural observations it was determined that both of theseFrankia strains can infectElaeagnus by an intercellular penetration mechanism andMyrica by the root hair infection mechanism. This indicates that both of these strains have the ability to infect host plant roots by either of two mechanisms. The reverse, thatElaeagnus orMyrica could be infected by both mechanisms, was not observed. The infection and nodule development processes of these two plants in combination with these strains were similar to observations made in previous studies (Miller andBaker 1985,Torrey andCallaham 1979). However, one exception was identified in the development of the prenodule ofMyrica when infected with strain 011610, in that endophytic hyphae developed vesicles within the cells of the prenodule. This event has not been described before for any of the actinorhizal genera and may be an indication of less than optimal compatibility between the host plant and the symbiont.Contribution no. 876 of the Battelle-Kettering Laboratory.     

Effect of high-light treatments in inducing photoinhibition of photosynthesis in intact leaves of low-light grown Phaseolus vulgaris and Lastreopsis microsora

Photoinhibition studies, using gas-exchange techniques, were conducted with leaflets of Phaseolus vulgaris L. plants that were grown under low photonfluence rates. Comparative measurements were made on attached, intact leaflets and in subsequently isolated chloroplasts. Photoinhibition studies were also conducted with attached fronds of the deep-shade fern Lastreopsis microsora (Endl.) Tindale. Leaflets of lowlight-grown Phaseolus vulgaris and fronds of the shade fern were found to be subject to similar photoinhibition when exposed to photon-fluence rates in excess of those at which they were grown. Photoinhibition following exposure to a photon fluence-rate approximating full sunlight is manifested as a reduction in the capacity for both light-saturated and light-limited carbon uptake and is reflected at the chloroplast level as substantial inhibition of electron flow through photosystem (PS) II, with little effect on PS I. The extent of photoinhibition is markedly dependent on the length of exposure to a high-light regime and on the actual photon-fluence rate maintained during treatment. A greater degree of photoinhibition is evident if carbon metabolism is prevented by the removal of CO₂ than when maximum rates of CO₂ uptake prevail throughout the exposure to a high photonfluence rate. Apparently a certain level of CO₂ turnover is beneficial in providing a sink for photochemically generated energy. When leaf material is exposed to photon-fluence rates well in excess of the rate present during growth apparently the potentials of the various biophysical and photochemical means of dissipating excitation energy are exceeded and photoinhibition of photosynthesis results.Abbreviation PFR photon fluence rate     

The temperature dependance of photoinhibition in the tropical basidiomycete lichen Cora pavonia E. Fries

Summary The response of net photosynthesis (NP) and dark respiration to periods of high insolation exposure was examined in the tropical basidiomycete lichen Cora pavonia. Photoinhibition of NP proved quite dependant on temperature. Rates of light saturated NP were severely impaired immediately after pretreatment high light exposure at temperatures of 10, 20 and 40°C, while similar exposure at 30°C resulted in only minimal photoinhibition. Apparent quantum yield proved an even more sensitive indicator of photoinhibition, reduced in all temperature treatments, although inhibition was again greatest at low and high temperatures. Concurrent exposure to reduced O₂ tensions during high light exposure mitigated some of the deleterious effects of high light exposure at 10 and 20°C, suggesting an interaction of O₂ with the inactivation of photosynthetic function. This represents the first reported instance of light dependant chilling stress in lichens, and may be an important limitation on the distribution of this and other tropical lichen species. This narrow range of temperatures within which thalli of C. pavonia can withstand periods of high insolation exposure coincides with that faced by hydrated thalli during rare periods of high insolation exposure within the cloud/shroud zone on La Soufrière, and points to the necessity of considering periods of atypical or unusual climatic events when interpreting patterns of net photosynthetic response, both in tropical and in north temperate lichen species.     

Mechanism of photosystem-I photoinhibition in leaves ofCucumis sativus L.

It was recently shown that the site of photoinhibition in leaves of Cucumis sativus L. at low temperatures is Photosystem I (PSI), not PSII (I. Terashima et al. 1994, Planta 193, 300–306). In the present study, the mechanisms of this PSI photoinhibition in vivo were examined. By lowering the photon flux density during the photoinhibitory treatment of leaves at 4°C for 5 h to less than 100 mol·m^–2s^–1, we were able to separate the steps of the destruction of the electron-transfer components. Although P-700, the reaction-center chlorophyll, was almost intact in this low-light treatment, the quantum yield of the electron transfer through PSI and photochemically induced absorption change at 701 nm were markedly inhibited. This, along with the results from the measurements of the light-induced absorption changes in the presence of various concentrations of methyl viologen, an artificial electron acceptor, indicates that the component on the acceptor side of the PSI, A₁ or F_x, is the first site of inactivation. When the photon flux density during the treatment was increased to 220 mol·m^–2s^–1, the destruction of P-700 itself was also observed. Furthermore, the partial degradation of the chlorophyll-binding large subunits was observed in photoinhibited leaves. This degradation of the subunits was not detected when the treatment was carried out under nitrogen atmosphere, the condition in which the electron transfer is not inhibited. Thus, the photoinhibitory processes in the reaction center of PSI go through three steps, the inactivation of the acceptor side, the destruction of the reaction-center chlorophyll and the degradation of the reaction center subunit(s). The similarities and the differences between the mechanisms of PSI photoinhibition and those of PSII photoinhibition are discussed.Abbreviations DAD 2,3,5,6-tetramethyl-p-phenylenediamine - LHCI, LHCII light-harvesting chlorophyll-a/b proteins associating with photosystems I and II, respectively - PFD photon flux density We are grateful to Dr. I. Enami (Department of Biology, Faculty of Science, Science University of Tokyo) and Drs. H. Matsubara and H. Oh-oka (Department of Biology, Faculty of Science, Osaka University) for generous gifts of antisera used in the present work. 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.     

Estimation of the effect of photoinhibition on the carbon gain in leaves of a willow canopy

The occurrence of photoinhibition of photosynthesis in leaves of a willow canopy was examined by measuring the chlorophyll-a fluorescence ratio of F _V/F _M (F_M is the maximum fluorescence level of the induction curve, and F_V is the variable fluorescence, F _V=F _M–F ₀, where F₀ is the minimal fluorescence). The majority of the leaves situated on the upper parts of peripheral shoots showed an afternoon inhibition of this ratio on clear days. This was the consequence of both a decrease in F _M and a rise in F _O. In the same leaves the diurnal variation in intercepted photosynthetic photon flux density (PPFD) was monitored using leaf-mounted sensors. Using the multivariate method, partial least squares in latent variables, it is shown that the dose of PPFD, integrated and linearly weighted over the last 6-h period, best predicts photoinhibition. Photoinhibition occurred even among leaves that did not intercept PPFDs above 1000 mol·m^–2·s^–1. Exposure of leaves to a standard photoinhibitory treatment demonstrated that the depression in the F _V/F _M ratio was paralleled by an equal depression in the maximal quantum yield of CO₂ uptake and a nearly equal depression in the rate of bending (convexity) of the light-response curve of CO₂ uptake. As a result, the rate of net photosynthesis is depressed over the whole natural range of PPFD. By simulating the daily course in the rate of net photosynthesis, it is estimated that in the order of one-tenth of the potential carbon gain of peripheral willow shoots is lost on clear days as a result of photoinhibition. This applies to conditions of optimal temperatures. Photoinhibition is even more pronounced at air temperatures below 23° C, as judged from measurements of the F_V/F_M ratio on clear days: the afternoon inhibition of this ratio increased in a curvilinear manner from 15% to 25% with a temperature decrease from 23° to 14° C.Abbreviations and Symbols F_O minimum fluorescence - F_V variable fluorescence - F_M maximum fluorescence - PLS partial least squares in latent variables - PPFD photosynthetic photon flux density - VPD water vapour-pressure deficit This study was supported by the Swedish Natural Science Research Council. We are indebted to Dr. Jerry Leverenz (Department of Plant Physiology, University of Umeå, Sweden) for guidance with the modelling of the photosynthesis data.     

Exotic pine plantations and indigenous Lepidoptera in Australia

Temperate regions of Australia have extensive commercial plantations of Monterey pine, Pinus radiata D. Don. Replacement of indigenous forests by P. radiata is likely to have significant effects on assemblages of native Lepidoptera, and has been considered a major threat to native fauna through displacing native species. However, many species of Lepidoptera, including ennomine geometrid moths in the genus Chlenias Guenèe, have adopted P. radiata as a larval host. Comparisons were made of oviposition preferences and nutritional ecology of Chlenias auctaria Guenée on P. radiata and two native host plants [Acacia mearnsii De Wild. (Mimosaceae) and Eucalyptus obliqua LHérit (Myrtaceae)]. Females showed no significant oviposition preference for any of the three hosts. Growth of sixth instar larvae was significantly less on P. radiata than on the native hosts. Pupal weights were significantly lower, suggesting that the fitness of moths reared on P. radiata will be significantly reduced. The life history strategy of C. auctaria, which includes dispersal of first instar larvae by ballooning, may predispose this species to feed on poor quality hosts, and this may be common to other polyphagous species found feeding on P. radiata in southern hemisphere plantations. The impact of P. radiata may have a long term effect on lepidopteran communities, beyond the simple replacement of indigenous host plants leading to extirpations of feeding specialists.     

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.     

Effect of long-term photoinhibition on growth and photosynthesis of cold-hardened spring and winter wheat

The effect of repeated exposure to high light (1200 mol · m^–2 · s^–1 photosynthetic photon flux density, PPFD) at 5° C was examined in attached leaves of cold-grown spring (cv. Katepwa) and winter (cv. Kharkov) wheat (Triticum aestivum L.) over an eight-week period. Under these conditions, Kharkov winter wheat exhibited a daily reduction of 24% in F_V/F_M (the ratio of variable to maximal fluorescence in the dark-adapted state), in contrast to 41% for cold-grown Katepwa spring wheat. Both cultivars were able to recover from this daily suppression of F_V/F_M such that the leaves exhibited an average morning F_V/F_M of 0.651 ± 0.004. Fluorescence measurements made under steady-state conditions as a function of irradiance from 60 to 2000 mol · m^–2 · s^–1 indicated that the yield of photosystem II (PSII) electron transport under light-saturating conditions was the same for photoinhibited and control cold-grown plants, regardless of cultivar. Repeated daily exposure to high light at low temperature did not increase resistance to short-term photoinhibition, although zeaxanthin levels increased by three- to fourfold. In addition, both cultivars increased the rate of dry-matter accumulation, relative to control plants maintained at 5° C and 250 mol · m^–2 · s^–1 PPFD (10% and 28% for Katepwa and Kharkov, respectively), despite exhibiting suppressed f_v/f_m and reduced photon yields for O₂ evolution following daily high-light treatments. Thus, although photosynthetic efficiency is suppressed by a longterm, photoinhibitory treatment, light-saturated rates of photosynthesis are sufficiently high during the high-light treatment to offset any reduction in photochemical efficiency of PSII. We suggest that in these cold-tolerant plants, photoinhibition of PSII may represent a longterm, stable, down-regulation of photochemistry to match the overall photosynthetic demand for ATP and reducing equivalents.Abbreviations and Symbols Chl chlorophyll - HL high light - PPFD photosynthetic photon flux density - F_O minimum fluorescence in the dark-adapted state - F_M maximum fluorescence in the dark-adapted state - F_V maximum variable fluorescence in the dark-adapted state (F_M-F_O) - F_V/F_V photosynthetic efficiency of the dark-adapted state - f_V/f_M photosynthetic efficiency of the light-adapted steady state - q_P photochemical quenching parameter - q_N non-photochemical quenching parameter - _e yield of electron transport and equals q_P · f_V/f_M - 1-q_O F_O quenching parameter - _app apparent photon yield. The assistance of Amy So is gratefully acknowledged. This research was supported by a Natural Sciences and Engineering Research Council of Canada (NSERCC) Operating Grant to N.P.A.H. G.Ö. was supported by an NSERCC International Exchange Award and the Swedish Natural Sciences Research Council.     

Reduced sensitivity to photoinhibition following frost-hardening of winter rye is due to increased phosphate availability

The possibility of a role for phosphate metabolism in the photosynthetic regulation that occurs during frost hardening was investigated in winter rye (Secale cereale L. cv. Musketeer). Leaves of frost-hardened and non-hardened winter rye were studied during photosynthetic induction, and at steady state after being allowed to take up 20 mM orthophosphate through the transpiration stream for 3 h. At the growth irradiance (350 mol·m^-2·s^-1) frost-hardening increased the stationary rate of CO₂-dependent O₂ evolution by 57% and 25% when measured at 5 and 20° C, respectively. Frosthardening also reduced the lag phase to stationary photosynthesis by 40% at 5° C and decreased the susceptibility of leaves to oscillations during induction and after interruption of the actinic beam during steady-state photosynthesis. These responses are all indicative of increased phosphate availability in frost-hardened leaves. As reported previously by Öquist and Huner (1993, Planta 189, 150–156), frost-hardening also decreased the reduction state of Q_A, the primary, stable quinone acceptor of PSII, and decreased the sensitivity of winter rye to photoinhibition of photosynthesis. Non-hardened rye leaves fed orthophosphate also showed an increased photosynthetic capacity (25% at 20° C and light saturation), lower reduction state of Q_A, a reduced sensitivity to photoinhibition and lower susceptibility to oscillations resulting from a brief interruption of the actinic light. Thus, the data indicate that phosphate metabolism plays a key role in photosynthetic acclimation of winter rye to low temperatures.Abbreviations F_o and F_o minimal fluorescence when all PSII reaction centres are open in dark-and light-acclimated leaves, respectively - F_m and F_m maximal fluorescence when all PSII reaction centres are closed in dark-and light-acclimated leaves, respectively - F_v variable fluoresence (F_m -F_o) in dark-acclimated leaves - F_v variable fluorescence (F_m-F_o) in light-acclimated leaves - PCR photosynthetic carbon reduction - PPFD photosynthetic photon flux density - Q_A the primary, stable quinone acceptor of PSII - q_P photochemical quenching of fluorescence - q_N non-photochemical quenching of fluorescence This work was supported by the Swedish Natural Sciences Research Council. The authors are indebted to Dr. N. Huner, Department of Plant Sciences, UWO, London, Canada, for helpful discussions during the initiation of this work and for the gift of rye seeds.