Extracellular ice and cell shape in frost-stressed cereal leaves: A low-temperature scanning-electron-microscopy study

Low-temperature scanning electron microscopy was used to examine transverse fracture faces through cereal leaf pieces subjected to frost. Specimens were studied before and after sublimation of the ice. The position of extracellular ice in the leaf was inferred from the difference between the specimen before and after sublimation and from ridges and points which occurred in the extracellular ice during sublimation. Steps in the fracture surface indicated that the fracture plane passed through the extracellular ice crystals as well as through cells and also helped identify extracellular ice. The cells in controls were turgid and extracellular ice was absent. Leaf pieces from hardened rye were excised and frost-stressed to-3.3°,-21° and-72°C, cooling at 2–12°·h^-1. Cell collapse and extracellular ice were evident at-3.3°C and increased considerably by-21° C. At-21° and-72°C the leaf pieces were mainly filled with extracellular ice and there were few remaining gas spaces. The epidermal and mesophyll cells were laterally flattened, perpendicular to their attachment to adjacent cells, and phloem and vascular sheath cells were more irregularly deformed. Leaf pieces from tender barley were cooled at 2°C·min^-1 to-20° C; they were then mainly filled with extracellular ice, and the cells were highly collapsed as in the rye. In rye leaves frozen to-3.6° C before excision, ice crystals occurred in peri-vascular, sub-epidermal and intervening mesophyll spaces. In rye leaf pieces frozen to-3.3° C after excision or to-3.6° C before excision, mesophyll cells were partly collapsed even when not covered by ice, indicating that collapse of the cell wall, as well as the enclosed protoplast, was driven by dehydration. No gas or ice-filled spaces were found between wall and the enclosed protoplast. It is suggested that this can be explained without invoking chemical bonding between cell wall and plasma membrane: when the wall pores are filled by water, the pore size would reduce vapour pressure so making penetration of the wall by ice or gas less likely.Abbreviations SEM scanning electron microscopy     

Wheat tissues freeze-etched during exposure to extracellular freezing: distribution of ice

Pieces excised from leaf bases and laminae of seedlings of Triticum aestivum L. cv. Lennox were slowly frozen, using a specially designed apparatus, to temperatures between 2° and 14° C. These treatments ranged from non-damaging to damaging, based on ion-leakage tests to be found in the accompanying report (Pearce and Willison 1985, Planta 163, 304–316). The frozen tissue pieces were then freeze-fixed by rapidly cooling them, via melting Freon, to liquid-nitrogen temperature. The tissue was subsequently prepared for electron microscopy by freeze-etching. Ice crystals formed during slow freezing would tend to be much larger than those formed during subsequent freeze-fixation. Ice crystals surrounding the excised tissues were much larger in the frozen than in the control tissues (the latter rapidly freeze-fixed from room temperature). Large ice crystals were present between cells of frozen laminae and absent from controls. Intercellular spaces were infrequent in control leaf bases and no ice-filled intercellular spaces were found in frozen leaf bases. Intracellular ice crystals were smaller in frozen tissues than in controls. It is concluded that all ice formation before freeze-fixation was extracellular. This extracellular ice was either only extra-tissue (leaf bases), or extra-tissue and intercellular (laminae). Periplasmic ice was sometimes present, in control as well as slowly frozen tissues, and the crystals were always small; thus they were probably formed during freeze-fixation rather than during slow freezing. The plasma membrane sometimes showed imprints of cell-wall microfibrils. These were less abundant in leaf bases at 8° C than in controls, and were present on only a minority of plasma membranes from laminae. Therefore, extracellular ice probably did not compress the cells substantially, and changes in cell size and shape were possibly primarily a result of freezing-induced dehydration. Fine-scale distortions (wrinkles) in the plasma membrane, while absent from controls, were present, although only rarely, in both damaged and non-damaged tissues; they were therefore ice-induced but not directly related to the process of damage.     

Water droplets in intercellular spaces of barley leaves examined by low-temperature scanning electron microscopy

Low-temperature scanning electron microscopy was used to examine fracture faces in leaf blades taken from well-watered or drought-stressed barley (Hordeum vulgare L. cv. Mazurka) seedlings. The leaf blades were freeze-fixed while hydrated and were examined with or without gold-coating. There were droplets (with a smooth surface at the resolution achieved) on the surface of cell walls in leaf blades (0.91 g^-1 water content) from well-watered seedlings grown in an environment of 67% relative humidity. These were mainly on the vascular bundle sheath, the guard and subsidiary cells, and on some mesophyll cells around the substomatal cavity and between the stoma and vascular bundle. The droplets occurred, more abundantly, in the same places in seedlings from 100% relative humidity. They occurred on a few guard cells from wilting leaf blades (0.81 g·g^-1 water content) and were absent from severely drought-stressed leaf blades (0.15 g·g^-1 water content). The droplets sublimed at the same moment as both water which was in leaf cells and water which was allowed to condense (after freeze-fixation) on the wall surface. It is suggested that the droplets are aqueous. Their possible origin and importance is discussed.     

Water stress,temperature, and light effects on the capacity for isoprene emission and photosynthesis of kudzu leaves

Kudzu (Pueraria lobata (Willd) Ohwi.) is a vine which forms large, monospecific stands in disturbed areas of the southeastern United States. Kudzu also emits isoprene, a hydrocarbon which can significantly affect atmospheric chemistry including reactions leading to tropospheric ozone. We have studied physiological aspects of isoprene emission from kudzu so the ecological consequences of isoprene emission can be better understood. We examined: (a) the development of isoprene emission as leaves developed, (b) the interaction between photon flux density and temperature effects on isoprene emission, (c) isoprene emission during and after water stress, and (d) the induction of isoprene emission from leaves grown at low temperature by water stress or elevated temperature. Isoprene emission under standard conditions of 1000 mol photons·m^-2·s^-1 and 30°C developed only after the leaf had reached full expansion, and was not complete until up to two weeks past the point of full expansion of the leaf. The effect of temperature on isoprene emission was much greater than found for other species, with a 10°C increase in temperature causing a eight-fold increase in the rate of isoprene emission. Isoprene emission from kudzu was stimulated by increases in photon flux density up to 3000 mol photons·m^-2·s^-1. In contrast, photosynthesis of kudzu was saturated at less than 1000 mol·m^-2·s^-1 photon flux density and was reduced at high temperature, so that up to 20% of the carbon fixed in photosynthesis was reemitted as isoprene gas at 1000 mol photons·m^-2·s^-1 and 35°C. Withholding water caused photosynthesis to decline nearly to zero after several days but had a much smaller effect on isoprene emission. Following the relief of water stress, photosynthesis recovered to the prestress level but isoprene emission increased to about five times the prestress rate. At 1000 mol photons·m^-2·s^-1 and 35°C as much as 67% of the carbon fixed in photosynthesis was reemitted as isoprene eight days after water stress. Leaves grown at less than 20°C did not make isoprene until an inductive treatment was given. Inductive treatments included growth at 24°C, leaf temperature of 30°C for 5 h, or witholding water from plants. With the new information on temperature and water stress effects on isoprene emission, we speculate that isoprene emission may help plants cope with stressful conditions.     

A freeze-etch study of the effects of extracellular freezing on cellular membranes of wheat

Seedlings of Triticum aestivum L. cv. Lennox were grown in different environments to obtain different hardiness. Pieces of laminae and leaf bases were slowly cooled to sub-zero temperatures and the damage caused was assessed by an ion-leakage method. Comparable pieces of tissue were slowly cooled to temperatures between 2° and-14°C and were then freeze-fixed and freeze-etched. Membranes generally retained their lamellar structures indicated by the abundance of typical membrane fracture faces in all treatments, and some membrane fracture faces had patches which lacked the usual scattering of intramembranous particles (IMP). These IMP-free areas were present in the plasma membrane of tissues given a damaging freezing treatment, but were absent from the plasma membrane of room-temperature controls, of supercooled tissues, and of tissues given a non-damaging freezing treatment. The frequency of IMP-free areas and the proportion of the plasma membrane affected increased with increasing damage. In the most damaged tissue (79% damage; leaf bases exposed to-8°C), 20% of the plasma membrane was IMP-free. The frequencies of IMP at a distance from the IMP-free areas were unaffected by freezing treatments. There was a patchy distribution of IMP in other membranes (nuclear envelope, tonoplast, thylakoids, chloroplast envelope), but only in the nuclear envelope did it appear possible that their occurrence coincided with damage. The IMP-free areas of several membranes were sometimes associated together in stacks. Such membranes lay both to the outside and inside of the plasma membrane, indicating that at least some of the adjacent membrane fragments arose as a result of membrane reorganization induced by the damaging treatment. Occasional views of folded IMP-free plasma membrane tended to confirm this conclusion. The following hypothesis is advanced to explain the damage induced by extracellular freezing. Areas of plasma membrane become free of IMP, probably as a result of the freezing-induced cellular dehydration. The lipids in these IMP-free patches may be in the fluid rather than the gel phase. The formation of these IMP-free patches, especially in the plasma membrane, initiates or involves proliferation and possibly fusion of membranes, and during or following this process, the cells become leaky.Abbreviations EF exoplasmatic fracture face - IMP intramembranous particles - PF protoplasmatic fracture face     

Temperature-dependent feedback inhibition of photosynthesis in peanut

Arachis hypogaea L. is a tropical crop that is slow-growing at temperatures below 25°C. Unadapted CO₂-assimilation rate (A) showed insufficient variation between 15 and 30°C in the short term (hours) to explain this marked reduction in growth. However, at longer periods (12 d), A was depressed as were growth rate and leafproduction rate. To examine the possible relationship between growth, A and sink demand plants were transferred from 30°C, which is near the optimum for growth, to a suboptimal temperature (19°C). In the first 2 d of cooling, A decreased by 50–70%, the stomata stayed open, and the intercellular CO₂ concentration (c_i) rose, i.e. the decrease in A of the cooled plants was the result of non-stomatal factors. Changes in dark respiration did not account for the decline in A.Clear evidence was obtained of sink control of A by independently manipulating the temperature of different leaves on the plant. Cooling (to 19°C) most of the plant (the sink) led to a 70% decline in A of the remaining leaves at 30°C after 3 d, whereas the converse treatments (30°C sink, 19°C source) resulted in small changes (17%). In plants at 19°C which were exposed to low CO₂ concentration to prevent photosynthesis, A was not reduced when measured at normal CO₂ concentrations, indicating that carbohydrate accumulation was responsible for the decline in A. Dry-matter build-up at suboptimal temperature was also consistent with end-product inhibition of photosynthesis.Abbreviations and symbols A (mol·m^-2·s^-1) rate of net CO₂ assimilation - C_i (l·l^-1) substomatal CO₂ concentration - DW (g) dry weight - g (mol·m^-2·s^-1) stomatal conductance to diffusion of water vapour - PFD (mol·m^-2·s^-1) photon flux density     

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     

Intracellular ice formation and growth in MCF-7 cancer cells

Direct cell injury in cryosurgery is highly related to intracellular ice formation (IIF) during tissue freezing and thawing. Mechanistic understanding of IIF in tumor cells is critical to the development of tumor cryo-ablation protocol. In aid of a high speed CMOS camera system, the events of IIF in MCF-7 cells have been studied using cryomicroscopy. Images of ‘darkening’ type IIF and recrystallization are compared between cells frozen with and without ice seeding. It is found that ice seeding has significant impact on the occurrence and growth of intracellular ice. Without ice seeding, IIF is observed to occur over a very small range of temperature (∼1 °C). The crystal dendrites are indistinguishable, which is independent of the cooling rate. Ice crystal grows much faster and covers the whole intracellular space in comparison to that with ice seeding, which ice stops growing near the cellular nucleus. Recrystallization is observed at the temperature from −13 °C to −9 °C during thawing. On the contrary, IIF occurs from −7 °C to −20 °C with ice seeding at a high subzero temperature (i.e., −2.5 °C). The morphology of intracellular ice frozen is greatly affected by the cooling rate, and no ‘darkening’ type ice formed inside cells during thawing. In addition, the intracellular ice formation is directional, which starts from the plasma membrane and grows toward the cellular nucleus with or without ice seeding. These results can be used to explain some findings of tumor cryosurgery in vivo, especially the causes of insufficient killing of tumor cells in the peripheral area near vessels.     

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).     

Photoinhibition of photosynthesis in intact bean leaves: role of light and temperature,and requirement for chloroplast-protein synthesis during recovery

Photoinhibition of photosynthesis was induced in intact leaves of Phaseolus vulgaris L. grown at a photon flux density (PFD; photon fluence rate) of 300 mol·m^-2·s^-1, by exposure to a PFD of 1400 mol·m^-2·s^-1. Subsequent recovery from photoinhibition was followed at temperatures ranging from 5 to 35°C and at a PFD of either 20 or 140 mol·m^-2·s^-1 or in complete darkness. Photoinhibition and recovery were monitored mainly by chlorophyll fluorescence emission at 77K but also by photosynthetic O₂ evolution. The effects of the protein-synthesis inhibitors, cycloheximide and chloramphenicol, on photoinhibition and recovery were also determined. The results demonstrate that recovery was temperature-dependent with rates slow below 15°C and optimal at 30°C. Light was required for maximum recovery but the process was light-saturated at a PFD of 20 mol·m^-2·s^-1. Chloramphenicol, but not cycloheximide, inactivated the repair process, indicating that recovery involved the synthesis of one or more chloroplast-encoded proteins. With chloramphenicol, it was shown that photoinhibition and recovery occurred concomitantly. The temperature-dependency of the photoinhibition process was, therefore, in part determined by the effect of temperature on the recovery process. Consequently, photoinhibition is the net difference between the rate of damage and the rate of repair. The susceptibility of chilling-sensitive plant species to photoinhibition at low temperatures is proposed to result from the low rates of recovery in this temperature range.Abbreviations and symbols Da Dalton - Fo, Fm, Fv instantaneous, maximum, variable fluorescence emission - PFD photon flux density - PSII photosystem II - photon yield C.I.W.-D.P.B. Publication No. 871     

Cryo-scanning electron microscopic study on freezing behaviors of tissue cells in dormant buds of larch (Larix kaempferi)

The freezing behavior of dormant buds in larch, especially at the cellular level, was examined by a Cryo-SEM. The dormant buds exhibited typical extraorgan freezing. Extracellular ice crystals accumulated only in basal areas of scales and beneath crown tissues, areas in which only these living cells had thick walls unlike other tissue cells. By slow cooling (5 °C/day) of dormant buds to −50 °C, all living cells in bud tissues exhibited distinct shrinkage without intracellular ice formation detectable by Cryo-SEM. However, the recrystallization experiment of these slowly cooled tissue cells, which was done by further freezing of slowly cooled buds with LN and then rewarming to −20 °C, confirmed that some of the cells in the leaf primordia, shoot primordia and apical meristem, areas in which cells had thin walls and in which no extracellular ice accumulated, lost freezable water with slow cooling to −30 °C, indicating ability of these cells to adapt by extracellular freezing, whereas other cells in these tissues retained freezable water with slow cooling even to −50 °C, indicating adaptation of these cells by deep supercooling. On the other hand, all cells in crown tissues and in basal areas of scales, areas in which cells had thick walls and in which large masses of ice accumulated, had the ability to adapt by extracellular freezing. It is thought that the presence of two types of cells exhibiting different freezing adaptation abilities within a bud tissue is quite unique and may reflect sophisticated freezing adaptation mechanisms in dormant buds.     

Effects of freezing on membranes and proteins in LNCaP prostate tumor cells

Fourier transform infrared spectroscopy (FTIR) and cryomicroscopy were used to define the process of cellular injury during freezing in LNCaP prostate tumor cells, at the molecular level. Cell pellets were monitored during cooling at 2 °C/min while the ice nucleation temperature was varied between − 3 and − 10 °C. We show that the cells tend to dehydrate precipitously after nucleation unless intracellular ice formation occurs. The predicted incidence of intracellular ice formation rapidly increases at ice nucleation temperatures below − 4 °C and cell survival exhibits an optimum at a nucleation temperature of − 6 °C. The ice nucleation temperature was found to have a great effect on the membrane phase behavior of the cells. The onset of the liquid crystalline to gel phase transition coincided with the ice nucleation temperature. In addition, nucleation at − 3 °C resulted in a much more co-operative phase transition and a concomitantly lower residual conformational disorder of the membranes in the frozen state compared to samples that nucleated at − 10 °C. These observations were explained by the effect of the nucleation temperature on the extent of cellular dehydration and intracellular ice formation. Amide-III band analysis revealed that proteins are relatively stable during freezing and that heat-induced protein denaturation coincides with an abrupt decrease in α-helical structures and a concomitant increase in β-sheet structures starting at an onset temperature of approximately 48 °C.     

Phase transitions and thermal expansion coefficients of plant cuticles

The temperature-induced volume expansion of enzymatically isolated cuticular membranes of twelve plant species was measured. All cuticular membranes exhibited distinct second-order phase transitions in the temperature range of about 40 to 50° C. Increases in the volumes of fruit cuticles (Lycopersicon, Cucumis, Capsicum, Solanum and Malus) were fully reversible, while leaf cuticular membranes (Ficus, Hedera, Nerium, Olea, Pyrus, Picea and Citrus) underwent irreversible structural changes. Below the phase-transition temperature, volumetric expansion coefficients ranged from 0.39·10^–6 m³·kg^–1·K^–1 to 0.62·10^–6 m³·kg^–1·K^–1, and above from 0.60·10⁶ m³·kg^–1·K^\-1 to 1.41· 10^–6 m³·kg^–1·K^–1. Densities of cuticles at 25° C ranged from 1020 kg·m^–3 to 1370 kg·m^–3. Expansion coefficients and phase transitions were characteristic properties of the polymer matrix as a composite material, rather than of cutin alone. It is argued that the sudden increase of water permeability above the transition temperature, is caused by an increase of disorder at the interface between the polymer matrix and the soluble cuticular lipids. Possible ecological and physiological consequences of these results for living plants are discussed.Abbreviations CM Cuticular membrane - CU cutin - MX polymer matrix - SCL soluble cuticular lipids (waxes) The authors greatfully acknowledge stimulating discussions with Drs. H. Gruler (Exp. Physik 3, Universität Ulm, FRG) and M. Riederer (Institut für Botanik und Mikrobiologie, Technische Universität München, München, FRG) and financial support by the Deutsche Forschungsgemeinschaft.     

Metabolism and thermoregulation in the eastern hedgehogErinaceus concolor

Body temperature and oxygen consumption were measured in the eastern hedgehog,Erinaceus concolor Martin 1838, during summer at ambient temperatures (T _a) between-6.0 and 35.6°C.E. concolor has a relatively low basal metabolic rate (0.422 ml O₂·g^-1·h^-1), amounting to 80% of that predicted from its body mass (822.7 g). Between 26.5 and 1.2°C, the resting metabolic rate increases with decreasing ambient temperature according to the equation: RMR=1.980-0.057T _a. The minimal heat transfer coefficient (0.057 ml O₂·g^-1·h^-1·°C^-1) is higher than expected in other eutherian mammals, which may result from partial conversion of hair into spines. At lower ambient temperature (from-4.6 to-6.0° C) there is a drop in body temperature (from 35.2 to 31.4° C) and a decrease in oxygen consumption (1.530 ml O₂·g^-1·h^-1) even though the potential thermoregulation capabilities of this species are significantly higher. This is evidenced by the high maximum noradrenaline-induced non-shivering thermogenesis (2.370 ml O₂·g^-1·h^-1), amounting to 124% of the value predicted. The active metabolic rate at ambient temperatures between 31.0 and 14.5° C averages 1.064 ml O₂·g^-1·h^-1; at ambient temperatures between 14.5 and 2.0° C AMR=3.228-0.140T _a.Abbreviations AMR active metabolic rate - bm body mass - BMR basal metabolic rate - h heat transfer coefficient - NA noradrenaline - NST non-shivering thermogenesis - NST_max maximum rate of NA-induced non-shivering thermogenesis - RMR resting metabolic rate - RQ respiratory quotient - STPD standard temperature and pressure (25°C, 1 ATM) - T _a ambient temperature - T _b body temperature     

Paraheliotropic leaf movement in Siratro as a protective mechanism against drought-induced damage to primary photosynthetic reactions: damage by excessive light and heat

Damage to primary photosynthetic reactions by drought, excess light and heat in leaves of Macroptilium atropurpureum Dc. cv. Siratro was assessed by measurements of chlorophyll fluorescence emission kinetics at 77 K (-196°C). Paraheliotropic leaf movement protected waterstressed Siratro leaves from damage by excess light (photoinhibition), by heat, and by the interactive effects of excess light and high leaf temperatures. When the leaves were restrained to a horizontal position, photoinhibition occurred and the degree of photoinhibitory damage increased with the time of exposure to high levels of solar radiation. Severe inhibition was followed by leaf death, but leaves gradually recovered from moderate damage. This drought-induced photoinhibitory damage seemed more closely related to low leaf water potential than to low leaf conductance. Exposure to leaf temperatures above 42°C caused damage to the photosynthetic system even in the dark and leaves died at 48°C. Between 42 and 48°C the degree of heat damage increased with the time of exposure, but recovery from moderate heat damage occurred over several days. The threshold temperature for direct heat damage increased with the growth temperature regime, but was unaffected by water-stress history or by current leaf water status. No direct heat damage occurred below 42°C, but in water-stressed plants photoinhibition increased with increasing leaf temperature in the range 31–42°C and with increasing photon flux density up to full sunglight values. Thus, water stress evidently predisposes the photosynthetic system to photoinhibition and high leaf temperature exacerbates this photoinhibitory damage. It seems probable that, under the climatic conditions where Siratro occurs in nature, but in the absence of paraheliotropic leaf movement, photoinhibitory damage would occur more frequently during drought than would direct heat damage.Abbreviations and symbols PFD photon flux area density - PSI, PSII photosyntem I, II - F _M, F _O, F _V maximum, instantaneous, variable fluorescence emission - PLM paraheliotropic leaf movement; all data of parameter of variation are mean ± standard error     

Temperature dependence of violaxanthin de-epoxidation and non-photochemical fluorescence quenching in intact leaves ofGossypium hirsutum L. andMalva parviflora L.

The temperature dependence of the rate of de-epoxidation of violaxanthin to zeaxanthin was determined in leaves of chilling-sensitive Gossypium hirsutum L. (cotton) and chilling-resistant Malva parviflora L. by measurements of the increase in absorbance at 505 nm (A ₅₀₅) and in the contents of antheraxanthin and zeaxanthin that occur upon exposure of predarkened leaves to excessive light. A linear relationship between A ₅₀₅ and the decrease in the epoxidation state of the xanthophyll-cycle pigment pool was obtained over the range 10–40° C. The maximal rate of de-epoxidation was strongly temperature dependent; Q₁₀ measured around the temperature at which the leaf had developed was 2.1–2.3 in both species. In field-grown Malva the rate of de-epoxidation at any given measurement temperature was two to three times higher in leaves developed at a relatively low temperature in the early spring than in those developed in summer. Q₁₀ measured around 15° C was in the range 2.2–2.6 in both kinds of Malva leaves, whereas it was as high as 4.6 in cotton leaves developed at a daytime temperature of 30° C. Whereas the maximum (initial) rate of de-epoxidation showed a strong decrease with decreased temperature the degree of de-epoxidation reached in cotton leaves after a 1–2 · h exposure to a constant photon flux density increased with decreased temperature as the rate of photosynthesis decrease. The zeaxanthin content rose from 2 mmol · (mol chlorophyll)^–1 at 30° C to 61 mmol · (mol Chl)^–1 at 10° C, corresponding to a de-epoxidation of 70% of the violaxanthin pool at 10° C. The degree of de-epoxidation at each temperature was clearly related to the amount of excessive light present at that temperature. The relationship between non-photochemical quenching of chlorophyll fluorescence and zeaxanthin formation at different temperatures was determined for both untreated control leaves and for leaves in which zeaxanthin formation was prevented by dithiothreitol treatment. The rate of development of that portion of non-photochemical quenching which was inhibited by dithiothreitol decreased with decreasing temperature and was linearly related to the rate of zeaxanthin formation over a wide temperature range. In contrast, the rate of development of the dithiothreitol-resistant portion of non-photochemical quenching was remarkably little affected by temperature. Evidently, the kinetics of the development of non-photochemical quenching upon exposure of leaves to excessive light is therefore in large part determined by the rate of zeaxanthin formation. For reasons that remain to be determined the relaxation of dithiothreitolsensitive quenching that is normally observed upon darkening of illuminated leaves was strongly inhibited at low temperatures.Abbreviations and Symbols Chl chlorophyll - DTT dithiothreitol - EPS epoxidation state - NPQ non-photochemical chlorophyll fluorescence quenching - PFD photon flux density - PSII photosystem II - F, F_m fluorescence emission at the actual, full closure of the PSII centers C.I.W.-D.P.B. Publication No. 1092We thank Connie Shih for skillful assistance in growing the plants, for conducting the HPLC analyses, and for preparing the figures. A Carnegie Institution Fellowship and a Feodor-Lynen-Fellowship by the Alexander von Humboldt-Foundation to W.B. is gratefully acknowledged. This work was supported by Grant No. 89-37-280-4902 of the Competitive Grants Program of the U.S. Department of Agriculture to O.B.     

Long-term chilling of young tomato plants under low light and subsequent recovery

The influence of unfavourable climatic conditions at the onset of the growth period on chilling-sensitive tomato (Lycopersicon esculentum Mill., cv. Abunda) was studied by exposing young plants to combinations of low temperature and low light (60–100 mol quanta · m^–2 · s^–1) for several weeks. When the temperature did not decrease below a critical point (8 ° C) no loss of developmental capacity of the plants was detected. However, while new leaves were readily formed upon return to normal growth conditions (22/18 °C, day/night, in a greenhouse), net accumulation of biomass showed a lag phase of approximately one week. This delay was accompanied by a strong, irreversible inhibition of photosynthesis in the fully expanded leaves which had been exposed to the chilling treatment. When plants were subjected to temperatures below 8 ° C, survival rates decreased after three weeks at 6 ° C and irreversible damage of apical meristematic tissue occurred. Drought-hardening prior to chilling ensured survival at 6 ° C and protected the plants against meristem loss.Abreviation Chl chlorophyll Thanks are due to G.P. Telkamp for technical assistance. This research is financially supported by the Netherlands Technology Foundation (STW, Utrecht, The Netherlands), and is coordinated by the Foundation for Biological Research (BION, 's-Gravenhage, The Netherlands).     

Are sucrosyl-oligosaccharides synthesized in mesophyll protoplasts of mature leaves of Cucumis melo?

Biosynthesis of sucrosyl-oligosaccharides (raffinose, stachyose) was traced in source leaves of Cucumis melo after ¹⁴C-photoassimilation. The main carbon compound exported was ¹⁴C-labeled stachyose. No oligosaccharide synthesis was detected in young, importing leaves. Mesophyll protoplasts, isolated from mature leaves which had previously photosynthesized ¹⁴CO₂, did not contain ¹⁴C-oligosaccharides but contained [¹⁴C]-sucrose and ¹⁴C-hexoses. Isolated minor-vein-enriched fractions from the same leaves, however, showed nearly 30% of the ¹⁴C of the neutral fraction to be in oligosaccharides. Isolated, viable mesophyll protoplasts incubated with NaH¹⁴CO₃ also failed to incorporate radioactivity into oligosaccharides, although sucrose and galactinol synthesis was unimpaired. Galactinolsynthase activity in leaf extracts and in mesophyll protoplasts was 16.8 mol·h^-1·mg^-1 protein and 13.8 mol·h^-1·mg^-1 protein, respectively. Galactosyltransferase (EC 2.4.1.67), which synthesizes stachyose from raffinose and galactinol, had an activity of 50 nmol·h^-1·mg^-1 protein in leaf extracts and was also present in the minor-vein-enriched fraction, but could not be detected in mesophyll protoplast lysates. The results indicate that mesophyll cells may not be the site of stachyose synthesis although precursor compounds like sucrose and galactinol are synthesized there.Abbreviation HPLC high-performance liquid chromatography     

Phototropic fluence-response relations for Avena coleoptiles on a clinostat

Phototropism of Avena sativa L. has been characterized using a clinostat to negate the gravitropic response. The kinetics for development of curvature was measured following induction by a single pulse of blue light (BL), five pulses of BL at 20-min intervals, and this same pulsed-light regime following a 2-h red light (RL) pre-irradiation. A final curvature of about 14° is expressed within 180 min following the single pulse; a final curvature of about 62° in about 240 min following five pulses without pre-irradiation; and a curvature of over 125° in 360 min following five pulses after the RL pre-irradiation. For seedlings not pre-irradiated, the final curvature to five pulses of BL at a total fluence of 9.4 pmol·cm^-2 increases with time of darkness between pulses up to 15 min; with seedlings pre-irradiated with RL, curvature increased more slowly with time of darkness between pulses to a maximum at 35 min. The final curvature induced by a constant fluence of 9.4 pmol·cm^-2 increases linearly with time between the first pulse and last pulse of a five-pulse sequence. The curvature induced by a single BL pulse with a 5-min RL co-irradiation increases with fluence to a maximum of about 60° at about 10 pmol·cm^-2, and then decreases to 0° at about 200 pmol·cm^-2. Curvature induced by five BL pulses following a 2-h RL pre-irradiation increased with fluence from a threshold of about 0.05 pmol·cm^-2 to a maximum of 90° at about 10 pmol·cm^-2, and then gradually decreased with fluence to 50° at 1 000 pmol·cm^-2. Based on these data, it is concluded that the initial photoproduct formed by a BL pulse has a limited lifetime, that there is a kinetic limitation downstream of the photoreceptor pigment for phototropism, and that the additivive effect of pulsed BL is distinct from the potentiating effect of RL on phototropism. Thus, any degree of curvature from 0° to over 90° may be induced by a fluence in the ascending arm of what is traditionally called the first positive phototropic response.Abbreviations BL blue light - RL red light     

Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with “antisense” rbcS

Tobacco (Nicotiana tabacum L.) plants transformed with antisense rbcS to decrease the expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) have been used to investigate the contribution of Rubisco to the control of photosynthesis in plants growing at different irradiances. Tobacco plants were grown in controlled-climate chambers under ambient CO₂ at 20°C at 100, 300 and 750 mol·m^–2·s^–1 irradiance, and at 28°C at 100, 300 and 1000 mol·m^–2·s^–1 irradiance. (i) Measurement of photosynthesis under ambient conditions showed that the flux control coefficient of Rubisco (C _infRubisco ^supA ) was very low (0.01–0.03) at low growth irradiance, and still fairly low (0.24–0.27) at higher irradiance. (ii) Short-term changes in the irradiance used to measure photosynthesis showed that C _infRubisco ^supA increases as incident irradiance rises, (iii) When low-light (100 mol·m^–2·s^–1)-grown plants are exposed to high (750–1000 mol·m^–2·s^–1) irradiance, Rubisco is almost totally limiting for photosynthesis in wild types. However, when high-light-grown leaves (750–1000 mol·m^–2·s^–1) are suddenly exposed to high and saturating irradiance (1500–2000 mol·m^–2·s^–1), C _infRubisco ^supA remained relatively low (0.23–0.33), showing that in saturating light Rubisco only exerts partial control over the light-saturated rate of photosynthesis in sun leaves; apparently additional factors are co-limiting photosynthetic performance, (iv) Growth of plants at high irradiance led to a small decrease in the percentage of total protein found in the insoluble (thylakoid fraction), and a decrease of chlorophyll, relative to protein or structural leaf dry weight. As a consequence of this change, high-irradiance-grown leaves illuminated at growth irradiance avoided an inbalance between the light reactions and Rubisco; this was shown by the low value of C _infRubisco ^supA (see above) and by measurements showing that non-photochemical quenching was low, photochemical quenching high, and NADP-malate dehydrogenase activation was low at the growth irradiance. In contrast, when a leaf adapted to low irradiance was illuminated at a higher irradiance, Rubisco exerted more control, non-photochemical quenching was higher, photochemical quenching was lower, and NADP-malate dehydrogenase activation was higher than in a leaf which had grown at that irradiance. We conclude that changes in leaf composition allow the leaf to avoid a one-sided limitation by Rubisco and, hence, overexcitation and overreduction of the thylakoids in high-irradiance growth conditions, (v) Antisense plants with less Rubisco contained a higher content of insoluble (thylakoid) protein and chlorophyll, compared to total protein or structural leaf dry weight. They also showed a higher rate of photosynthesis than the wild type, when measured at an irradiance below that at which the plant had grown. We propose that N-allocation in low light is not optimal in tobacco and that genetic manipulation to decrease Rubisco may, in some circumstances, increase photosynthetic performance in low light.Abbreviations A rate of photosynthesis - C _infRubisco ^supA flux control coefficient of Rubisco for photosynthesis - c_i internal CO₂ concentration - qE energy-dependent quenching of chlorophyll fluorescense - qQ photochemical quenching of chlorophyll fluorescence - NADP-MDH NADP-dependent malate dehydrogenase - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - RuBP ribulose-1,5-bisphosphate This work was supported by the Deutsche Forschungsgemeinschaft (SFB 137).