Investigating cytoskeletal function in chloroplast protrusion formation in the arctic-alpine plant Oxyria digyna

Arctic and alpine plants like Oxyria digyna have to face enhanced environmental stress. This study compared leaves from Oxyria digyna collected in the Arctic at Svalbard (78 degrees N) and in the Austrian Alps (47 degrees N) at cellular, subcellular, and ultrastructural levels. Oxyria digyna plants collected in Svalbard had significantly thicker leaves than the samples collected in the Austrian Alps. This difference was generated by increased thickness of the palisade and spongy mesophyll layers in the arctic plants, while epidermal cells had no significant size differences between the two habitats. A characteristic feature of arctic, alpine, and cultivated samples was the occurrence of broad stroma-filled chloroplast protrusions, 2 - 5 microm broad and up to 5 microm long. Chloroplast protrusions were in close spatial contact with other organelles including mitochondria and microbodies. Mitochondria were also present in invaginations of the chloroplasts. A dense network of cortical microtubules found in the mesophyll cells suggested a potential role for microtubules in the formation and function of chloroplast protrusions. No direct interactions between microtubules and chloroplasts, however, were observed and disruption of the microtubule arrays with the anti-microtubule agent oryzalin at 5 - 10 microM did not alter the appearance or dynamics of chloroplast protrusions. These observations suggest that, in contrast to studies on stromule formation in Nicotiana, microtubules are not involved in the formation and morphology of chloroplast protrusions in Oxyria digyna. The actin microfilament-disrupting drug latrunculin B (5 - 10 microM for 2 h) arrested cytoplasmic streaming and altered the cytoplasmic integrity of mesophyll cells. However, at the ultrastructural level, stroma-containing, thylakoid-free areas were still visible, mostly at the concave sides of the chloroplasts. As chloroplast protrusions were frequently found to be mitochondria-associated in Oxyria digyna, a role in metabolite exchange is possible, which may contribute to an adaptation to alpine and arctic conditions.     

Reversibility of cold- and light-stress tolerance and accompanying changes of metabolite and antioxidant levels in the two high mountain plant species Soldanella alpina and Ranunculus glacialis

Two high mountain plants Soldanella alpina (L.) and Ranunculus glacialis (L.) were transferred from their natural environment to two different growth conditions (22 degrees C and 6 degrees C) at low elevation in order to investigate the possibility of de-acclimation to light and cold and the importance of antioxidants and metabolite levels. The results were compared with the lowland crop plant Pisum sativum (L.) as a control. Leaves of R. glacialis grown for 3 weeks at 22 degrees C were more sensitive to light-stress (defined as damage to photosynthesis, reduction of catalase activity (EC 1.11.1.6) and bleaching of chlorophyll) than leaves collected in high mountains or grown at 6 degrees C. Light-stress tolerance of S. alpina leaves was not markedly changed. Therefore, acclimation is reversible in R. glacialis leaves, but constitutive or long-lasting in S. alpina leaves. The different growth conditions induced significant changes in non-photochemical fluorescence quenching (qN) and the contents of antioxidants and xanthophyll cycle pigments. These changes did not correlate with light-stress tolerance, questioning their role for light- and cold-acclimation of both alpine species. However, ascorbate contents remained very high in leaves of S. alpina under all growth conditions (12-19% of total soluble carbon). In cold-acclimated leaves of R. glacialis, malate represented one of the most abundant compounds of total soluble carbon (22%). Malate contents declined significantly in de-acclimated leaves, suggesting a possible involvement of malate, or malate metabolism, in light-stress tolerance. Leaves of the lowland plant P. sativum were more sensitive to light-stress than the alpine species, and contained only low amounts of malate and ascorbate.     

Photoacoustic analysis indicates that chloroplast movement does not alter liquid-phase CO2 diffusion in leaves of Alocasia brisbanensis

Light-mediated chloroplast movements are common in plants. When leaves of Alocasia brisbanensis (F.M. Bailey) Domin are exposed to dim light, mesophyll chloroplasts spread along the periclinal walls normal to the light, maximizing absorbance. Under high light, the chloroplasts move to anticlinal walls. It has been proposed that movement to the high-light position shortens the diffusion path for CO(2) from the intercellular air spaces to the chloroplasts, thus reducing CO(2) limitation of photosynthesis. To test this hypothesis, we used pulsed photoacoustics to measure oxygen diffusion times as a proxy for CO(2) diffusion in leaf cells. We found no evidence that chloroplast movement to the high-light position enhanced gas diffusion. Times for oxygen diffusion were not shorter in leaves pretreated with white light, which induced chloroplast movement to the high-light position, compared with leaves pretreated with 500 to 700 nm light, which did not induce movement. From the oxygen diffusion time and the diffusion distance from chloroplasts to the intercellular gas space, we calculated an oxygen permeability of 2.25 x 10(-)(6) cm(2) s(-)(1) for leaf cells at 20 degrees C. When leaf temperature was varied from 5 degrees C to 40 degrees C, the permeability for oxygen increased between 5 degrees C and 20 degrees C but changed little between 20 degrees C and 40 degrees C, indicating changes in viscosity or other physical parameters of leaf cells above 20 degrees C. Resistance for CO(2) estimated from oxygen permeability was in good agreement with published values, validating photoacoustics as another way of assessing internal resistances to CO(2) diffusion.     

Changes in chloroplast ultrastructure in some high-alpine plants: adaptation to metabolic demands and climate?

Summary. The cytology of leaf cells from five different high-alpine plants was studied and compared with structures in chloroplasts from the typical high-alpine plant Ranunculus glacialis previously described as having frequent envelope plus stroma protrusions. The plants under investigation ranged from subalpine/alpine Geum montanum through alpine Geum reptans, Poa alpina var. vivipara, and Oxyria digyna to nival Cerastium uniflorum and R. glacialis. The general leaf structure (by light microscopy) and leaf mesophyll cell ultrastructure (by transmission electron microscopy [TEM]) did not show any specialized structures unique to these mountain species. However, chloroplast protrusion formation could be found in G. reptans and, to a greater extent, in O. digyna. The other species exhibited only a low percentage of such chloroplast structural changes. Occurrence of protrusions in samples of G. montanum and O. digyna growing in a mild climate at about 50 m above sea level was drastically reduced. Serial TEM sections of O. digyna cells showed that the protrusions can appear as rather broad and long appendices of plastids, often forming pocketlike structures where mitochondria and microbodies are in close vicinity to the plastid and to each other. It is suggested that some high-alpine plants may form such protrusions to facilitate fast exchange of molecules between cytoplasm and plastid as an adaptation to the short, often unfavorable vegetation period in the Alps, while other species may have developed different types of adaptation that are not expressed in ultrastructural changes of the plastids. Correspondence and reprints: Institute of Botany, University of Innsbruck, Sternwartestrasse 15, 6020 Innsbruck, Austria.     

Temperature-sensitive formation of chloroplast protrusions and stromules in mesophyll cells of Arabidopsis thaliana

Summary. In leaf mesophyll cells of transgenic Arabidopsis thaliana plants expressing GFP in the chloroplast, stromules (stroma-filled tubules) with a length of up to 20 μm and a diameter of about 400–600 nm are observed in cells with spaces between the chloroplasts. They appear extremely dynamic, occasionally branched or polymorphic. In order to investigate the effect of temperature on chloroplasts, we have constructed a special temperature-controlled chamber for usage with a light microscope (LM-TCC). This LM-TCC enables presetting of the temperature for investigation directly at the microscope stage with an accuracy of ±0.1 °C in a temperature range of 0 °C to +60 °C. With the LM-TCC a temperature-dependent appearance of chloroplast protrusions has been found. These structures have a considerably smaller length-to-diameter ratio than typical stromules and reach a length of 3–5 μm. At 5–15 °C (low temperatures), almost no chloroplast protrusions are observed, but they appear with increasing temperatures. At 35–45 °C (high temperatures), numerous chloroplast protrusions with a beaklike appearance extend from a single chloroplast. Interaction of stromules with other organelles has also been investigated by transmission electron microscopy. At 20 °C, transverse sections of stromules are frequently observed with a diameter of about 450 nm. A close membrane-to-membrane contact of stromules with the nucleus and mitochondria has been visualised. Golgi stacks and microbodies are found in the spatial vicinity of stromules. At 5 °C, virtually no chloroplast protrusions or stromules are observed. At 35 °C, chloroplast protrusions are present as broader thylakoid-free stroma-filled areas, resulting in an irregular chloroplast appearance. Correspondence and reprints: Department of Physiology and Cell Physiology of Alpine Plants, Institute of Botany, University of Innsbruck, Sternwartestrasse 15, 6020 Innsbruck, Austria.     

青海高原矮嵩草和珠芽蓼的光合适应性比较(英文)

对生长在两个海拔地带 (3 2 0 0m ,3 980m)的矮嵩草 (KobresiahumilisSerg.)和珠芽蓼 (PolygonumviviparumL .)叶片的叶绿素荧光特性及其叶绿体超微结构进行了比较研究。海拔升高 ,矮嵩草和珠芽蓼叶片的Fv/Fo、Fv/Fm和Rfd值均增大 ,且矮嵩草的Fv/Fo、Fv/Fm 和Rfd值均大于珠芽蓼。叶绿体超微结构的结果显示 ,海拔升高 ,珠芽蓼和矮嵩草的叶绿体都表现出一定程度的变形 ,但珠芽蓼的叶绿体变形和类囊体膜肿胀现象更为显著。研究表明 ,矮嵩草和珠芽蓼光合作用对高山胁迫环境具有很强的适应性 ,且矮嵩草的适应能力比珠芽蓼强。     

日光温室光温因子对黄瓜叶绿体超微结构及其功能的影响

在日光温室内,研究了光温因子对黄瓜叶绿体超微结构及其功能的影响．结果表明,因季节之间光、温条件不同,日光温室黄瓜叶片显微结构和叶绿体超微结构有一定差异,1月份光照弱叶肉细胞较大,而5月份光照强叶绿体数较多．在该试验条件下,未发现叶片光合速率与叶绿体超微结构之间有直接或密切的相关性．在各生长季节其光合速率均为第4叶>初展叶>基部叶,与叶龄及各叶位的受光量有关．如果将不同叶位叶放在相同的光照下,则差异明显减少．黄瓜叶片的叶肉细胞、叶绿体和淀粉粒的大小以及叶绿体数、基粒数、基粒厚度、基粒片层数都随叶位的下降而呈增加趋势。不同品种、同品种不同生长时期的叶片显微结构和叶绿体超微结构及其功能也有一定的差异．限制日光温室冬季黄瓜光合作用的主要因素是光照弱、有效光照时数少,而在晴天温度的限制作用相对较小。阴天因光照弱而导致的室内低温则是限制黄瓜生长的关键因素．     

Light-dependent reversal of dark-chilling induced changes in chloroplast structure and arrangement of chlorophyll-protein complexes in bean thylakoid membranes

Changes in chloroplast structure and rearrangement of chlorophyll-protein (CP) complexes were investigated in detached leaves of bean (Phaseolus vulgaris L. cv. Eureka), a chilling-sensitive plant, during 5-day dark-chilling at 1 degrees C and subsequent 3-h photoactivation under white light (200 mumol photons m(-2) s(-1)) at 22 degrees C. Although, no change in chlorophyll (Chl) content and Chl a/b ratio in all samples was observed, overall fluorescence intensity of fluorescence emission and excitation spectra of thylakoid membranes isolated from dark-chilled leaves decreased to about 50%, and remained after photoactivation at 70% of that of the control sample. Concomitantly, the ratio between fluorescence intensities of PSI and PSII (F736/F681) at 120 K increased 1.5-fold upon chilling, and was fully reversed after photoactivation. Moreover, chilling stress seems to induce a decrease of the relative contribution of LHCII fluorescence to the thylakoid emission spectra at 120 K, and an increase of that from LHCI and PSI, correlated with a decrease of stability of LHCI-PSI and LHCII trimers, shown by mild-denaturing electrophoresis. These effects were reversed to a large extent after photoactivation, with the exception of LHCII, which remained partly in the aggregated form. In view of these data, it is likely that dark-chilling stress induces partial disassembly of CP complexes, not completely restorable upon photoactivation. These data are further supported by confocal laser scanning fluorescence microscopy, which showed that regular grana arrangement observed in chloroplasts isolated from control leaves was destroyed by dark-chilling stress, and was partially reconstructed after photoactivation. In line with this, Chl a fluorescence spectra of leaf discs demonstrated that dark-chilling caused a decrease of the quantum yield PSII photochemistry (F(v)/F(m)) by almost 40% in 5 days. Complete restoration of the photochemical activity of PSII required 9 h post-chilling photoactivation, while only 3 h were needed to reconstruct thylakoid membrane organization and chloroplast structure. The latter demonstrated that the long-term dark-chilled bean leaves started to suffer from photoinhibition after transfer to moderate irradiance and temperature conditions, delaying the recovery of PSII photochemistry, independently of photo-induced reconstruction of PSII complexes.     

Fine Chloroplast Structure in Cucumber and Pea Leaves Developed under Red Light

Photosynthetic activity, the content of various photosynthetic pigments, and the chloroplast ultrastructure were examined in the leaves of cucumber (Cucumis sativus L.) and pea (Pisum sativum L.) plants of different ages grown under red light (600–700 nm, 100 W/m²). In pea leaves tolerant to red-light irradiation, chloroplast ultrastructure did not essentially change. In the first true leaves of cucumber plants susceptible to red-light irradiation, we observed a considerable increase in the number and size of plastoglobules, the appearance of chloroplasts lacking grana or containing only infrequent grana, and stromal thylakoids. In the upper leaves of 22-day-old cucumber plants, the chloroplast structure was essentially similar to that of the control chloroplasts in white light, and we therefore suppose that these plants have acclimated to red light.     

Circadian rhythms of chloroplast orientation and photosynthetic capacity in ulva

Ulva lactuca L. var. latissima (L.) Decandolle and var. rigida (C. Agardh) Le Jolis and U. mutabilis Foyn have a circadian rhythm of chloroplast orientation which results in large changes in the light-absorption properties of the thallus. During the day, the chloroplasts cover the outer face of the cells and absorbance is high. At night, the chloroplasts are along the side walls and absorbance is low. Enteromorpha linza (L.) J. Agardh, E. intestinalis (L.) Link, E. sp., and Monostroma grevillei (Thuret) Wittrock, members of the Ulvales, were not observed to have this rhythmic movement. Chloroplasts, when in the face position, could not be induced to move to the sides by high intensity light up to 80,000 lux. Unrelated to chloroplast position per se and light-absorption efficiency, there is a rhythm of photosynthetic capacity which peaks just before midday and which continues in constant darkness.     

Accumulation of overproduced ferritin in the chloroplast provides protection against photoinhibition induced by low temperature in tobacco plants

Wild-type tobacco plants (Nicotiana tabacum L. cv. Petit Havanna line SR1) and plants transformed with full-length alfalfa ferritin cDNA with the chloroplast transit peptide under the control of a Rubisco small subunit gene promoter (C3 and C8) were cold-treated at 0 degrees C with continuous light (250mumolm(-2)s(-1)). These transgenic plants had higher chlorophyll content and higher F(v)/F(m) chlorophyll-a fluorescence induction parameters than wild-type plants after 2 or 3d of cold treatment in C3 and C8 transgenic plants, respectively. Thermoluminescence studies on the high-temperature bands suggest that these plants suffered less oxidative damage in comparison to the wild-type genotype. The present experiments provide evidence that transgenic tobacco lines overexpressing alfalfa ferritin, which is accumulated in the chloroplasts, may show higher tolerance to various stress factors, generating ROS including low temperature-induced photoinhibition.     

Rapid-growth responses of corn root segments: Effect of citrate-phosphate buffer on elongation

Summary Chloroplasts from the alga, Vaucheria dichotoma (L.) Ag., are taken up into protoplasts of carrot (Daucus carota L.) during polyethylene-glycol treatment. Since chloroplasts are found with equal frequency in uni- and multinucleate protoplasts, chloroplast uptake does not depend on protoplast fusion. However, higher frequencies of chloroplast uptake are observed when experimental conditions favor greater aggregation of protoplasts. The intracellular localization of chloroplasts is confirmed by electron microscopy, and it is shown that the chloroplasts, once within the protoplasts, are not surrounded by a limiting membrane of carrot origin.     

Effect of growth temperature on chloroplast structure and activity in barley

Seedlings of barley (Hordeum vulgare L. cv. Abyssinian) were grown at constant temperature and light intensity and the properties and structure of chloroplasts in the primary leaf were examined. Seventeen growth temperatures ranging from 2 to 37 C were employed. Three major effects of the growth temperature were seen. (a) At very low and high growth temperatures chloroplast biogenesis was inhibited. This occurred in plants grown at temperatures above 32 C while growth at 2 C resulted in a mixed population of pale yellow, pale green, and green plants. (b) Chloroplasts were produced at all other temperatures tested but growth temperatures within a few degrees of those inhibitory to chloroplast development resulted in chloroplasts with abnormal properties and structure. Chloroplasts in the green plants grown at 2 and 5 C showed a number of structural peculiarities, including a characteristic crimping of granal thylakoids. Photoreductive activity, measured using ferricyanide as the Hill oxidant in the presence of gramicidin D, was high, but this activity in chloroplasts isolated from plants grown at 2 C showed thermal inactivation at temperatures 5 degrees lower than was the case with plants grown at higher temperatures. High growth temperatures (30 to 32 C) yielded chloroplasts with reduced photoreductive activity and a tendency toward the formation of large grana and disorientation of the lamellar systems with respect to one another. Chloroplasts of the most affected plants (grown at 32 C) frequently contained a very large elongated granum, with narrow intrathylakoid spaces. (c) Photoreductive activity was not constant at intermediate growth temperatures but steadily declined with decreasing growth temperatures between 27 and 11 C. Some alterations in chloroplast structure were also observed.

The changes in chloroplast activity and structure indicate that acclimation to temperature takes place over the entire temperature range in which chloroplast development is permitted.

     

NaCl胁迫对5个引自北美的树种叶肉细胞超微结构的影响

为明确美国白蜡(Fraxinus americana L.)、茶条槭(Aceginnala Maxim.)、红桤木(Alnus rubra Bong.)、水紫树(Nyssa aquatica L.)和美国皂荚(Gleditsia triacanthos L.)5个引自北美的树种的耐盐性,采用水培方法、利用透射电镜技术对0(对照)、4和8 g·L-1 NaCl胁迫处理后5个树种1年生苗叶肉细胞超微结构的变化进行了观察和比较.观察结果表明:正常条件(0g·L-1NaCl)下,5个树种叶肉细胞在叶绿体形态、嗜锇颗粒数量等方面略有差异,但均未发生质壁分离现象.经NaCl胁迫处理后,5个树种叶肉细胞中的叶绿体和细胞核受到不同程度的损伤,表现为叶绿体膜消失,类囊体片层结构肿胀,叶绿体降解,嗜锇颗粒增大或增多,细胞核的核膜消失、核染色质凝聚;且随NaCl质量浓度的提高,损伤程度均逐渐加剧.4和8g·L-1NaC1胁迫条件下,美国皂荚、茶条槭和水紫树的叶肉细胞发生质壁分离现象,而红桤木、美国白蜡和水紫树的叶肉细胞内出现环状片层.根据观察结果,推测红桤木和美国白蜡对NaCl胁迫的耐性较强,美国皂荚和茶条槭也有一定的耐性,而水紫树的耐性最弱.     

Possible association of actin filaments with chloroplasts of spinach mesophyll cells in vivo and in vitro

Summary. In palisade mesophyll cells of spinach (Spinacia oleracea L.) kept under low-intensity white light, chloroplasts were apparently immobile and seemed to be surrounded by fine bundles of actin filaments. High-intensity blue light induced actin-dependent chloroplast movement concomitant with the appearance of a couple of long, straight bundles of actin filaments in each cell, whereas high-intensity red light was essentially ineffective in inducing these responses. The actin organization observed under low-intensity white light has been postulated to function in anchoring chloroplasts at proper intracellular positions through direct interaction with the chloroplasts. Intact chloroplasts, which retained their outer envelopes, were isolated after homogenization of leaves and Percoll centrifugation. No endogenous actin was detected by immunoblotting in the final intact-chloroplast fraction prepared from the leaves kept under low-intensity white light or in darkness. In cosedimentation assays with exogenously added skeletal muscle filamentous actin, however, actin was detected in the intact-chloroplast fraction precipitated after low-speed centrifugation. The association of actin with chloroplasts was apparently dependent on incubation time and chloroplast density. After partial disruption of the outer envelope of isolated chloroplasts by treatment with trypsin, actin was no longer coprecipitated. The results suggest that chloroplasts in spinach leaves can directly interact with actin, and that this interaction may be involved in the regulation of intracellular positioning of chloroplasts. Correspondence and reprints: Department of Biology, Graduate School of Science, Osaka University, Machikaneyama 1-1, Toyonaka, Osaka 560-0043, Japan. Present address: Tsukuba Research and Development Center, Fuji Oil Co., Ltd., Tsukuba-gun, Ibaraki, Japan.     

Arabidopsis thaliana leaves with altered chloroplast numbers and chloroplast movement exhibit impaired adjustments to both low and high light

The effects of chloroplast number and size on the capacity for blue light-dependent chloroplast movement, the ability to increase light absorption under low light, and the susceptibility to photoinhibition were investigated in Arabidopsis thaliana. Leaves of wild-type and chloroplast number mutants with mean chloroplast numbers ranging from 120 to two per mesophyll cell were analysed. Chloroplast movement was monitored as changes in light transmission through the leaves. Light transmission was used as an indicator of the ability of leaves to optimize light absorption. The ability of leaves to deal with 3 h of high light stress at 10 degrees C and their capacity to recover in low light was determined by measuring photochemical efficiencies of PSII using chlorophyll a fluorescence. Chloroplast movement was comparable in leaves ranging in chloroplast numbers from 120 to 30 per mesophyll cell: the final light transmission levels after exposure to 0.1 (accumulation response) and 100 micromol photons m(-2) s(-1) (avoidance response) were indistinguishable, the chloroplasts responded quickly to small increases in light intensity and the kinetics of movement were similar. However, when chloroplast numbers per mesophyll cell decreased to 18 or below, the accumulation response was significantly reduced. The avoidance response was only impaired in mutants with nine or fewer chloroplasts, both in terms of final transmission levels and the speed of movement. Only mutants lacking both blue light receptors (phot1/phot2) or those with drastically reduced chloroplast numbers and severely impacted avoidance responses showed a reduced ability to recover from high light stress.     

几种高山植物叶绿体淀粉粒的变化特征

利用透射电镜对生长于青藏高原东北部达坂山（海拔3900m）的5种高山植物叶绿体超微结构进行了观察。结果发现,在所研究的5种高山植物叶绿体中,淀粉粒数量均较多,淀粉粒呈长椭圆形或圆形,沿叶绿体长轴分布。在珠芽蓼的叶绿体中,淀粉粒的电子密度内外不均匀,外周电子密度低,中央电子密度高。在其余4种高山植物中,淀粉粒的电子密度均较低。另外,在这5种高山植物叶绿体中还出现了脂质小球。其类囊体均出现了不同程度的膨大现象。研究表明,高山植物叶绿体中淀粉粒的这种变化是对逆境的一种适应,是青藏高原特殊生态条件长期胁迫的结果。     

Photorespiration and light act in concert to regulate the expression of the nuclear gene for chloroplast glutamine synthetase.

In Pisum sativum, distinct chloroplast and cytosolic forms of glutamine synthetase (GS) are encoded by homologous nuclear genes that are differentially expressed in vivo (Tingey, S. V., Tsai, F.-Y., Edwards, J. W., Walker, E. L., and Coruzzi, G. M. [1988]. J. Biol. Chem. 263, 9651-9657). In leaves, light selectively affects the expression of the nuclear gene for chloroplast GS2. Differences in the maximal levels of GS2 mRNA in etiolated plants treated with red or white light indicate that only part of the white-light-induced accumulation of GS2 mRNA is due to a phytochrome-mediated response. The kinetics of GS2 mRNA accumulation in response to white-light illumination of etiolated or dark-adapted green plants indicates that GS2 mRNA accumulates more rapidly in plants containing mature, photosynthetically competent chloroplasts. Other evidence that GS2 mRNA levels are affected by the metabolic status of chloroplasts concerns the selective induction of GS2 mRNA in plants grown under conditions that result in the production of photorespiratory ammonia. These results indicate that the light-induced accumulation of GS2 mRNA in leaves results from the action of phytochrome as well as light-induced changes in chloroplast metabolism.     

Laser-flash-activated electron paramagnetic resonance studies of primary photochemical reactions in chloroplasts.

Electron paramagnetic resonance studies of the primary reactants of Photosystems I and II have been conducted at cryogenic temperatures after laser-flash activation with monochromatic light.P-700 photooxidation occurs irreversibly in chloroplasts and in Photosystem I fragments after activation with a 730 nm laser flash at a temperature of 35 degrees K. Flash activation of chloroplasts or Photosystem II chloroplast fragments with 660 nm light results in the production of a free-radical signal (g = 2.002, linewidth approximately 8 gauss) which decays with a half-time of 5.0 ms at 35 degrees K. The half-time of decay is independent of temperature in the range of 10-77 degrees K. This reversible signal can be eliminated by preillumination of the sample at 35 degrees K with 660 nm light (but not by 730 nm light), by preillumination with 660 nm light at room temperature in the presence of 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea (DCMU) plus hydroxylamine, or by adjustment of the oxidation-reduction potential of the chloroplasts to - 150 mV prior to freezing. In the presence of ferricyanide (20-50 mM), two free-radical signals are photoinduced during a 660 nm flash at 35 degrees K. One signal decays with a half-time of 5 ms, whereas the second signal is formed irreversibly. These results are discussed in terms of a current model for the Photosystem II primary reaction at low temperature which postulates a back-reaction between P-680+ and the primary electron acceptor.     

Effect of temperature on cuticular transpiration of isolated cuticular membranes and leaf discs

Cuticular transpiration was measured in the temperature range between 10 degrees C and 55 degrees C using tritiated water and five species (Vinca major L., Prunus laurocerasus L., Forsythia intermedia L., Citrus aurantium L., and Hedera helix L.). Cuticular water permeabilities measured with isolated cuticular membranes were not different from cuticular water permeabilities measured with leaf discs. Depending on the species cuticular water permeabilities increased by factors between 12 (V. major) to 264 (H. helix) when temperature was increased from 10 degrees C to 55 degrees C. Arrhenius plots (lnP versus 1/T) of all investigated species were characterized by phase transitions occurring in the temperature range of 30-39 degrees C. Activation energies for water permeability across plant cuticles below and above the midpoint of phase transition were calculated from Arrhenius plots. Depending on the species they varied between 26 (F. intermedia) to 61 kJ mol(-1) (H. helix) below the phase transition and from 67 (V. major) to 122 kJ mol(-1) (F. intermedia) above the phase transition. Since the occurrence of phase transitions always lead to significantly increased rates of cuticular transpiration it is argued that temperatures higher than 35 degrees C caused structural defects to the transport-limiting barrier of the plant cuticles of all species investigated.