Cold-hardening-induced resistance to photoinhibition of photosynthesis in winter rye is dependent upon an increased capacity for photosynthesis

Analyses of chlorophyll fluorescence and photosynthetic oxygen evolution were conducted to understand why cold-hardened winter rye (Secale cereale L.) is more resistant to photoinhibition of photosynthesis than is non-hardened winter rye. Under similar light and temperature conditions, leaves of cold-hardened rye were able to keep a larger fraction of the PS II reaction centres in an open configuration, i.e. a higher ratio of oxidized to reduced Q_A (the primary, stable quinone acceptor of PSII), than leaves of non-hardened rye. Three fold-higher photon fluence rates were required for cold-hardened leaves than for non-hardened leaves in order to establish the same proportion of oxidized to reduced Q_A. This ability of cold-hardened rye fully accounted for its higher resistance to photoinhibition; under similar redox states of q_a cold-hardened and non-hardened leaves of winter rye exhibited similar sensitivities to photoinhibition. Under given light and temperature conditions, it was the higher capacity for light-saturated photosynthesis in cold-hardened than in non-hardened leaves, which was responsible for maintaining a higher proportion of oxidized to reduced Q_A. This higher capacity for photosynthesis of cold-hardened leaves also explained the increased resistance of photosynthesis to photoinhibition upon cold-hardening.Abbreviations F_m and F'_m fluorescence when all PSII reaction centres are closed in dark- and light-acclimated leaves, respectively - F_o and F'_o fluorescence when all PSII reaction centres are open in darkness and steady-state light, respectively - F_v variable fluorescence (F'_m-F'_o) under steady-state light conditions - F_v/F_m the ratio of variable to maximum fluorescence as an expression of the maximum photochemical yield of PSII in dark-acclimated leaves - Q_A the primary, stable, quinone electron acceptor of PSII - q_N non-photochemical quenching of fluorescence due to high energy state (pH) - q_p photochemical quenching of fluorescence - RH cold-hardened rye - RNH non-hardened rye This work 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 by the Swedish Natural Science Research Council.     

Photoinhibition of isolated mesophyll cells from cold-hardened and non-hardened winter rye

Cold-hardened rye leaves have been shown to be more resistant to low temperature photoinhibition than non-hardened rye leaves. Isolated mesophyll cells from winter rye (Secale cereale L. cv. Musketeer) were exposed to photoinhibitory light conditions to estimate the importance of leaf morphology and leaf optical properties in the resistance of cold-hardened rye leaves to photoinhibition. Cold-hardened rye cells showed more resistance to photoinhibition than non-hardened rye cells when monitored with chlorophyll a variable to maximal fluorescence ratio (F_v/F_m). Thus, leaf morphology does not contribute to the resistance of cold-hardened rye leaves to low temperature photoinhibition. However, cold-hardened and non-hardened rye cells showed a similar extent of photoinhibition when photsynthetic CO₂ fixation rates were measured. They also showed the same capacity to recover from photoinhibition. During both photoinhibition and recovery, F_v/F_m and light limited CO₂ fixation rates showed different kinetics. We propose that inactivation and subsequent reactivation during recovery of some light activated Calvin cycle enzymes explain the greater extent of photoinhibition of light limited CO₂ fixation and its faster recovery compared to F_v/F_m kinetics during photoinhibition.     

Resistance of cold-hardened winter rye leaves (Secale cereale L.) to photo-oxidative stress

Catalase and photosystem II (PSII) were strongly inactivated during exposure to 4 °C and moderate light in 22 °C-grown non-hardened leaves (NHL) of winter rye (Secale cereale L.), but highly resistant to photo-inactivation at low temperature in 4 °C-grown cold-hardened leaves (CHL). Resistance of CHL to chilling-induced photo-inactivation of catalase and PSII depended partially on more efficient de novo synthesis at 4 °C and partially on improved protection. Lower rates of chloroplast-mediated inactivation of catalase in vitro indicated that less reactive oxygen was released by chloroplasts from CHL than by chloroplasts from NHL. The contents of xanthophyll cycle carotenoids, α-tocopherol, ascorbate, glutathione, the activities of superoxide dismutase and glutathione reductase, and the tolerance against paraquat-induced photo-oxidative damage were greatly increased in CHL, relative to NHL. Zeaxanthin-related thermal energy dissipation was only of minor importance for paraquat-tolerance and protection of catalase in CHL. When CHL were transferred to a higher temperature of 22 °C the increased resistance to photo-inactivation of catalase and PSII and the increased paraquat-tolerance were largely lost within 3 d, whereas most non-enzymic and enzymic antioxidants retained higher levels than in NHL. The decline of resistance to photodamage during dehardening was not related to concomitant changes of antioxidants or antioxidative enzymes.     

Investigations on heat resistance of spinach leaves

Exposure of spinach plants to high temperature (35° C) increased the heat resistance of the leaves by about 3° C. This hardening process occurred within 4 to 6 h, whereas dehardening at 20°/15° C required 1 to 2 days. At 5° C dehardening did not take place. Hardening and dehardening occurred in both the dark and the light. The hardiness was tested by exposure of the leaves to heat stress and subsequent measurements of chlorophyll fluorescence induction and light-induced absorbance changes at 535 nm on the leaves and of the photosynthetic electron transport in thylakoids isolated after heat treatment. Heat-induced damage to both heat-hardened and non-hardened leaves seemed to consist primarily in a breakdown of the membrane potential of the thylakoids accompanied by partial inactivation of electron transport through photosystem II. The increase in heat resistance was not due to temperature-induced changes in lipid content and fatty acid composition of the thylakoids, and no conspicuous changes in the polypeptide composition of the membranes were observed. Prolonged heat treatment at 35° C up to 3 days significantly decreased the total lipid content and the degree of unsaturation of the fatty acids of membrane lipids without further increase in the thermostability of the leaves. Intact chloroplasts isolated from heat-hardened leaves retained increased heat resistance. When the stroma of the chloroplasts was removed, the thermostability of the thylakoids was decreased and was comparable to the heat resistance of chloroplast membranes obtained from non-hardened control plants. Compartmentation studies demonstrated that the content of soluble sugars within the chloroplasts and the whole leaf tissue decreased as heat hardiness increased. This indicated that in spinach leaves, sugars play no protective role in heat hardiness. The results suggest that changes in the ultrastructure of thylakoids in connection with a stabilizing effect of soluble non-sugar stroma compounds are responsible for acclimatization of the photosynthetic apparatus to high temperature conditions. Changes in the chemical composition of the chloroplast membranes did not appear to play a role in the acclimatization.Abbreviations DGDG digalactosyl diglyceride - MGDG monogalactosyl diglyceride - PG phosphatidyl glycerol - PGA 3-phosphoglyceric acid Dedicated to Professor Wilhelm Simonis, Würzburg, on the occasion of his 70th birthday     

Cold-hardening results in increased activity of enzymes involved in carbon metabolism in leaves of winter rye (Secale cereale L.)

Light- and CO₂-saturated photosynthesis of nonhardened rye (Secale cereale L. cv. Musketeer) was reduced from 18.10 to 7.17 mol O₂·m^–2·s^–1 when leaves were transferred from 20 to 5°C for 30 min. Following cold-hardening at 5°C for ten weeks, photosynthesis recovered to 15.05 mol O₂·m^–2·s^–1,comparable to the nonhardened rate at 20°C. Recovery of photosynthesis was associated with increases in the total activity and activation of enzymes of the photosynthetic carbon-reduction cycle and of sucrose synthesis. The total hexose-phosphate pool increase by 30% and 120% for nonhardened and cold-hardened leaves respectively when measured at 5°C. The large increase in esterified phosphate in coldhardened leaves occurred without a limitation in inorganic phosphate supply. In contrast, the much smaller increase in esterified phosphate in nonhardened leaves was associated with an inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase and sucrose-phosphate synthase activation. It is suggested that the large increases in hexose phosphates in cold-hardened leaves compensates for the higher substrate threshold concentrations needed for enzyme activation at low temperatures. High substrate concentrations could also compensate for the kinetic limitations imposed by product inhibition from the accumulation of sucrose at 5°C. Nonhardened leaves appear to be unable to compensate in this fashion due to an inadequate supply of inorganic phosphate.Abbreviations DHAP dihydroxyacetone phosphate - Fru6P fructose-6-phosphate - Fru 1,6BP fructose-1,6-bisphosphate - Fru1,6BPase fructose-1,6-bisphosphatase - Glc6P glucose-6-phosphate - PGA 3-phosphoglycerate - PPFD photosynthetic photon flux density - CH cold-hardened rye grown at 5°C - NH nonhardened rye grown at 24°C - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SPS sucrose-phosphate synthase - UDPGlc uridine 5-diphosphoglucose This work was supported by operating grants from the Swedish Natural Sciences Research Council to G.Ö. and P.G.     

Mitochondria contribute to increased photosynthetic capacity of leaves of winter rye (Secale cereale L.) following cold-hardening

Cold-hardening of winter rye (Secale cereale L. cv. Musketeer) increased dark respiration from ?2.2 to ?3.9 μmol O₂ m^?2s^?1 and doubled light-and CO₂-saturated photosynthesis at 20°C from 18.1 to 37.0μmol O₂ m^?2 s^?1 We added oligomycin at a concentration that specifically inhibits oxidative phosphorylation to see whether the observed increase in dark respiration reflected an increase in respiration in the light, and whether this contributed to the enhanced photosynthesis of cold-hardened leaves. Oligomycin inhibited light- and CO₂-saturated rates of photosynthesis in non-hardened and cold-hardened leaves by 14 and 25%, respectively, and decreased photochemical quenching of chlorophyll a fluorescence to a greater degree in cold-hardened than in non-hardened leaves. These data indicate an increase both in the rate of respiration in the light, and in the importance of respiration to photosynthesis following cold-hardening. Analysis of metabolite pools indicated that oligomycin inhibited photosynthesis by limiting regeneration of ribulose-1,5-bisphosphate. This limitation was particularly severe in cold-hardened leaves, and the resulting low 3-phospho-glycerate pools led to a feed-forward inhibition of sucrose-phosphate synthase activity. Thus, it does not appear that oxidative phosphorylation supports the increase in photo-synthetic O₂ evolution following cold-hardening by increasing the availability of cytosolic ATP. The data instead support the hypothesis that the mitochondria function in the light by using the reducing equivalents generated by non-cyclic photosynthetic electron transport.     

Chloroplast biogenesis at cold-hardening temperatures. Development of photosystem I and photosystem II activities in relation to pigment accumulation

Chloroplast biogenesis during continuous illumination at either low, cold-hardening temperatures (5°C) or non-hardening temperatures (20°C) was examined by monitoring the etioplast-chloroplast transformation with respect to pigment accumulation and the development of PSI- and PSII-associated electron transport activities in winter rye (Secale cereale L. cv Puma). Generally, chlorophyll and carotenoid accumulation during greening at 20°C were characterized by rapid initial rates in contrast to pronounced, initial lag times during biogenesis at 5°C. Although greening temperature had no effect on the sequential appearance of PSI relative to PSII, greening temperature significantly altered the pattern of appearance of PSI relative to chlorophyll accumulation. Thylakoid biogenesis under continuous illumination at 20°C imposed a pattern whereby the development of PSI activity was antiparallel to chlorophyll accumulation. In contrast, the development of PSI activity under continuous illumination at 5°C was paralllel to chlorophylll accumulation. These developmental patterns were independent of the temperature experienced during etiolation. However, rye seedlings etiolated at 20°C and subsequently subjected to continuous illumination at 5°C exhibited a 70% reduction in the maximum PSII activity (100 mol DCPIP reduced.mg Chl^-1.h^-1) attained relative to that observed for similar etiolated seedlings greened at 20°C (300 mol DCPIP reduced.mg Chl^-1.h^-1). This low temperature-induced inhibition could be alleviated by an initial 2 h exposure to continuous light at 20°C prior to greening to 5°C. Rye seedlings etiolated at 5°C attained similar maximal PSII activities (300 mol DCPIP reduced.mg Chl^-1.h^-1) regardless of the greening temperature. We suggest that the altered kinetics for pigment accumulation, the low temperature-induced change in the pattern for the appearance of PSI activity relative to chlorophyll accumulation and the differential sensitivity of 20° and 5° etiolated seedlings to greening temperature reflect an alteration in membrane organization incurred as a consequence of thylakoid assembly at low temperature.Abbreviations RH cold-hardened rye - RNH non-hardened rye - MV methylviologen - ASC ascorbate - Chl chlorophyll - DCPIP dichlorophenol indophenol     

Membrane alterations in winter rye and Brassica napus cells during lethal freezing and plasmolysis

Abstract Cells fixed during freezing or plasmolysis were used to study membrane alterations in hardened and non-hardened Brassica napus suspension-cultured cells and rye leaf mesophyll cells. The plasmalemma in non-hardened rye mesophyll cells formed multilamellar vesicles during lethal freezing at high subzero temperatures (–5°C). These vesicles became highly condensed at lower subzero temperatures (–10°C). Conversely, cold-hardened rye mesophyll cells did not undergo membrane alterations at these temperatures. The results from plasmolysis of B. napus and rye mesophyll cells hardened by ABA at 25 °C and low temperature (2°C), respectively, verify the cell response to lethal freezing. Again there was a continuum of responses with 1 kmol m^?3 balanced salt causing multilamellar protrusions. Appression of the plasmalemma against the tonoplast to form multilamellar vesicles and the invagination of these vesicles into the tonoplast were also observed in rye cells undergoing lethal plasmolysis. Increasing the plasmolysing solution to 3 kmol m^?3 occasionally caused the formation of multilamellar vesicles on the cell surface of hardened rye mesophyll cells.     

Capacity for RNA synthesis in 70S ribosome-deficient plastids of heat-bleached rye leaves

In the leaves of rye seedlings (Secale cereale L.) grown at an elevated temperature of 32°C the formation of plastidic 70S ribosomes is specifically prevented. The resulting plastid ribosome-deficient leaves, which are chlorotic in light, represent a system for the identification of translation products of the 80S ribosomes among the chloroplastic proteins. Searching for the primary heat-sensitive event causing the 70S ribosome-deficiency, the thermostability of the chloroplastic capacity for RNA synthesis was investigated. The RNA polymerase activity of isolated normal chloroplasts from 22°-grown rye leaves was not inactivated in vitro at temperatures between 30° and 40°C. The ribosome-deficient plastids purified from bleached 32°-grown leaf parts contained significant RNA polymerase activity which was, however, lower than in functional chloroplasts. After application of [³H]uridine to intact leaf tissues [³H]uridine incorporation was found in ribosome-deficient plastids of 32°C-grown leaves. The amount of incorporation was similar to that in the control chloroplasts from 22°C-grown leaves. According to these results, it is unlikely that the non-permissive temperature (32°C) causes a general inactivation of the chloroplastic RNA synthesis in rye leaves.     

Effects of Cold-Hardening on Chilling-Induced Photoinhibition of Photosynthesis and on Xanthophyll Cycle Pigments in Sweet Pepper

Two cultivars of Capsicum annuum L. were acclimated for 5 d at sub-optimal temperature (14 °C) and irradiance of 250 µmol m^–2 s^–1. This cold-hardening resulted in some reduction in the extent of photoinhibition during an 8 h exposure to high irradiance at 4 °C. Obvious differences were observed between non-hardened leaves (NHL) and cold-hardened leaves (CHL) in the recovery under low irradiance at room temperature. The CHL of both cultivars recovered faster than NHL, especially during the initial fast phase of recovery. Compared with NHL, the total content of carotenoids (Cars), based on chlorophyll, Chl (a+b), and the proportions of xanthophyll cycle pigments referred to total Cars increased in CHL, mainly due to an increase of violaxanthin (V) + antheraxanthin (A) + zeaxanthin (Z) content per mol Chl (a+b). Faster development and a higher non-photochemical quenching (NPQ) of Chl fluorescence, related to a stronger deepoxidation of the larger xanthophyll cycle pool in NHL, could act as a major defence mechanism to reduce the formation of reactive oxygen species during severe chilling. This is suggested by higher content of Z or Z+A in photoinhibition as well as by its rapid decline during the initial fast phase of recovery. In contrast to the chilling-sensitive cv. 0004, the chilling-tolerant cv. 1141 did more easily acclimate its photosynthetic apparatus to low temperatures.     

Growth and development of winter rye at cold-hardening temperatures results in thylakoid membranes with increased sensitivity to low concentrations of osmoticum

Chloroplasts developed at cold-hardening (5°C) and non-hardening temperatures (20°C) were compared with respect to the stability of photosynthetic electron transport activities, the capacity to produce and maintain a H⁺ gradient and the capacity fat photophosphorylation as a function of resuspension in the presence or absence of osmoticum. The results for electron transport indicate that whole chain, photosystem I and pfaotosystem II activities in non-hardened chloroplast thyalkoids were unaffected by resuspension in the presence of high or low osmoticum. In contrast, the same electron transport activities in cold-hardened chloroplast thylakoids exhibited a 3- to 4-fold decrease in activity when resuspended in the presence of low osmoticum. Impairment of electron transport through photosystem II of cold-hardened thylakoids resuspended in the presence of low osmoticum was supported by room temperature fluorescence induction kinetics. Since the presence of Mn²⁺ partially overcame this inhibition, it is concluded that this osmotically-induced inhibition of PSII activity in cold-hardened chloroplast thylakoids may, in part, be due to damage to the H₂O-splitting side of photosystem II. Both the initial rate and the maximum capacity for cyclic photophosphorylation were significantly inhibited in cold-hardened as compared to non-hardened thylakoids upon resuspension in the presence of low concentrations of osmoticum. This was correlated with an inability of the cold-hardened chloroplast thylakoids to maintain a significant transrnembrane H⁺ gradient. The results indicate that cold-hardened thylakoid membranes required an osmotic concentration (0.8 M) twice as high as non-hardened thylakoids (0.4 M) to produce the same initial rate of H⁺ uptake. In addition, the capacity to produce a proton gradient in cold-hardened thylakoids was less stable than that in non-hardened thylakoids regardless of the osmotic concentration tested. It is concluded that development of rye thylakoid membranes at low temperature results in a differential sensitivity to low osmoticum and thus extreme caution should be exercised when comparing the structure and function of isolated thylakoids developed under contrasting thermal regimes.     

Formation of chloroplast pigments and sterols in rye leaves deficient in plastid ribosomes

Summary 1. In rye (Secale cereale L.) leaves the formation of plastidic ribosomes is sensitive to elevated growth temperatures. Parallel to the loss of 70S ribosomes, in leaves growing at 32° chlorophyll accumulation was also prevented. Except for the tips of the first leaves which still contained some 70S ribosomes, the leaves were chlorotic. The amount of chlorophyll formed at 32° depended on the light intensity and decreased with higher intensities. After return to normal temperature (22°) chlorotic parts of the first leaves greened to a varying extent while those parts of most 2. or 3. leaves which had been formed in light at 32° remained permanently bleached until they died. Those parts of 2. and 3. leaves which were newly formed at 22° became normally green again. — 2. Formation and distribution of total and individual carotenoids were compared after development at 22° and 32°. In dark-grown leaves the higher growth temperature had no marked influence on the quantity or composition of carotenoids. At 22° the content of total carotenoids was 5fold and that of -carotene 25fold increased by light. At 32° these light-induced increases were much lower. Only 41% of the total carotenoids and 18% of the -carotene formed at 22° in light were found at 32°. Of the carotenoids present at 32°, 76% were located in the light green tips of the leaves. In plastids isolated from completely chlorotic leaf parts, carotenoids were still present and were even the predominant pigments. — 3. The contents of total sterols, the fractions of free sterols, sterol glycosides and esters, and the composition of individual sterols were compared in rye leaves grown at 22° and at 32°, in light or darkness. Light had little effect on the total sterol contents per leaf. However, more than 2fold higher sterol contents were observed in leaves grown at 32°, as compared to those from 22°. The amounts of most sterol fractions and individual sterols were similarly increased at the higher temperature but the sterol glycosides being relatively more increased than the total sterols.     

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.     

Capacity for chlorophyll synthesis in heat-bleached 70S ribosome-deficient rye leaves

The leaves of young rye plants (Secale cereale L.) grown at 32° were deficient in chlorophyll and in chloroplastic rRNA as compared to those grown at 22°, which developed normally. Both chlorophyll accumulation and the formation of plastidic rRNA were largely restored at 32° when the plants were transfered several times for 1 h per day to 22°. In the chlorotic 32°-grown rye leaves the in vivo activity of -aminolevulinate synthetase was very low. Aminolevulinate dehydratase however, exhibited high activity in extracts from 32°-grown leaves and was localized in the plastid fraction isolated from the chlorotic leaf tissue. After application of -aminolevulinic acid to chlorotic parts of leaves growing at 32°, protochlorophyll(ide) was formed and accumulated in the dark. In the light, the protochlorophyll(ide) was photooxidized at 32°. The results suggest a cytoplasmic site of synthesis for the series of enzymes converting -aminolevulinate to protochlorophyll(ide). It is concluded that an inhibition of -aminolevulinate synthetase and the photooxidation of protochlorophyll(ide) or chlorophyll are responsible for the chlorosis of the leaves at 32°.Abbreviations ALA -aminolevulinic acid - ALAD -aminolevulinate dehydratase - ALAS -aminolevulinate synthetase     

Lipid polymers accumulate in the epidermis and mestome sheath cell walls during low temperature development of winter rye leaves

Summary Winter rye (Secale cereale L cv. Puma) was grown at 20 °C and at 5 °C and the development of epidermal and mestome sheath cells of leaves from plants grown at both temperatures was compared by electron microscopy. At 5 °C, the cells became densely packed with cytoplasm and small vacuoles after 41 days of growth. By day 56 at 5 °C, epidermal and mestome sheath cells were small in diameter and multivacuolate with asymmetrically thickened walls. By day 76 at 5 °C, a new developmental stage had been reached in epidermal and mestome sheath cells. The cells were larger in diameter although the thickened cell walls and multivacuolate cytoplasm were still present. As epidermal and mestome sheath cell walls thickened during low temperature growth of winter rye, an increase in cuticle thickness and the deposition of a lamellar layer could be observed in epidermal and mestome sheath cells, respectively. The lipid-derived polymers from the leaves of rye plants grown at 20 °C were shown by reductive depolymerization and GC-MS analysis to be comprised of 18-hydroxy-9, 10-epoxyoctadecanoic acid (47%) and dihydroxyhexa-decanoic acid (29%). The leaves of plants grown at 5 °C had two to four times as much lipid-derived polymeric material as those grown at 20 °C and the proportion of the major monomer, 18-hydroxy-9,10-epoxyoctadecanoic acid, increased to 73% of the polymeric material. Physical isolation of both epidermal tissue and vascular bundles followed by GC-MS analysis of the monomeric components released by reduction of the respective lipid polymers showed that 18-hydroxy-9,10 epoxyoctadecanoic acid was the major monomer in the polymer of both the epidermis and the mestome sheaths. The presence of this epoxide monomer in both the cuticles and mestome sheath cell walls of rye leaves was confirmed and visualized by using an epoxide-specific staining reaction.     

Genetic control of triosephosphate isomerase isoenzymes in wheat and rye

Summary The patterns of chloroplastic and cytosolic isoenzymes of triosephosphate isomerase were analysed by immunoblotting in leaves of rye, wheat, and some species of Aegilops or Agropyrum. While rye contained solely one chloroplastic and one cytosolic isoenzyme, wheat had a much more complex pattern which can be explained by the presence of three genomes in 6 x wheats (AABBDD) with distinct triosephosphate isomerase genes that provided different subunit species for the dimeric isoenzyme molecules. The 6 × wheats contained five, the 4 × wheats three, and the 2 × wheats only one chloroplastic isoenzyme band. The isoenzyme patterns were in accordance with a potential origin of one of the three chloroplastic triosephosphate isomerase genes of 6 × wheats from an Aegilops ancestor. The descent of the other two genes was, however, not in accordance with common contentions on the general evolution of cultural wheats. In the reciprocal intergeneric hybrids Secalotricum and Triticale both the chloroplastic and the cytosolic isoenzyme patterns of rye and wheat were biparentally inherited, indicating that both isoenzymes were controlled by nuclear genes. When monitored by immunoblotting the chloroplastic triosephosphate isomerase isoenzymes may provide useful genetic markers.     

Malate metabolism and reactions of oxidoreduction in cold-hardened winter rye (Secale cereale L.) leaves

In cold-hardened leaves (CHL) of winter rye (Secale cereale L.) much higher levels of malate were detected by (13)C-NMR than in non-hardened leaves (NHL). As this was not observed previously, malate metabolism of CHL was studied in more detail by biochemical assays. The activities of several enzymes of malate metabolism, NADP-malate dehydrogenase, NAD-malate dehydrogenase, phosphoenolpyruvate carboxylase, and NADP-malic enzyme, were also increased in CHL. Short exposures to low temperature of 1-3 d did not induce increases in the malate content or in the activities of enzymes of malate metabolism in mature NHL. The malate content and the enzyme activities declined within 1-2 d after a transfer of CHL from their growing temperature of 4 degrees C to 22 degrees C. The malate content was further increased when CHL were exposed to a higher light intensity at 4 degrees C. In CO(2)-free air the malate content of CHL strongly declined at 4 degrees C. Malate may thus serve as an additional carbon sink and as a CO(2)-store in CHL. It may further function as a vacuolar osmolyte balancing increased concentrations of soluble sugars previously observed in the cytosol of CHL. Malate was not used as a source of reductants when CHL were exposed to photo-oxidative stress by treatment with paraquat. However, the activities of enzymes of the oxidative pentose phosphate pathway were markedly increased in CHL and may serve as non-photosynthetic sources of NADPH and thus contribute to the previously observed superior capacity of CHL of winter rye to maintain their antioxidants in a reduced state in the presence of paraquat.     

Significance of antioxidants and electron sinks for the cold-hardening-induced resistance of winter rye leaves to photo-oxidative stress

The contents of ascorbate and glutathione and the activities of superoxide dismutase and glutathione reductase were increased to levels as high as those in cold-hardened leaves (CHL) by incubating non-hardened leaves (NHL) of winter rye (Secale cereale L.) with the precursor substrates L -galactonic acid-γ-lactone and 2-oxothiazolidine-4-carboxylate. Reduced glutathione was rapidly depleted from NHL after application of D , L -buthionine sulfoximine, an inhibitor of its biosynthesis. In spite of greatly divergent antioxidant contents the rates of photo-inactivation of photosystem II (PSII) and catalase observed in the presence of translation inhibitors did not differ greatly. The paraquat-induced catalase inactivation and chlorophyll degradation in light were reduced in NHL with increased antioxidant levels. Paraquat-induced photo-inactivation of PSII was, however, not mitigated. The CHL had a higher capacity to prevent paraquat-induced oxidation of ascorbate and glutathione than NHL with increased antioxidant contents. Increased antioxidant contents did not establish resistance to low temperature-induced photo-inactivation of PSII and catalase in NHL. The resistance of CHL to low temperature-induced photo-inactivation of PSII and catalase required repair at low temperature and active carbon assimilation but was only little affected when photorespiration was suppressed by phosphinothricin. Protection of PSII depended also on non-photochemical quenching of excitation energy.     

Resistance to low temperature photoinhibition is not associated with isolated thylakoid membranes of winter rye

In vivo measurements of chlorophyll a fluorescence indicate that cold-hardened winter rye (Secale cereale L. cv Musketeer) develops a resistance to low temperature-induced photoinhibition compared with nonhardened rye. After 7.2 hours at 5°C and 1550 micromoles per square meter per second, the ratio of variable fluorescence/maximum fluorescence was depressed by only 23% in cold-hardened rye compared with 46% in nonhardened rye. We have tested the hypothesis that the principal site of this resistance to photoinhibition resides at the level of rye thylakoid membranes. Thylakoids were isolated from cold-hardened and nonhardened rye and exposed to high irradiance (1000-2600 micromoles per square meter per second) at either 5 or 20°C. The photoinhibitory response measured by room temperature fluorescence induction, photosystem II electron transport, photoacoustic spectroscopy, or [¹⁴C]atrazine binding indicates that the differential resistance to low temperature-induced photoinhibition in vivo is not observed in isolated thylakoids. Similar results were obtained whether isolated rye thylakoids were photoinhibited or thylakoids were isolated from rye leaves preexposed to a photoinhibitory treatment. Thus, we conclude that increased resistance to low temperature-induced photoinhibition is not a property of thylakoid membranes but is associated with a higher level of cellular organization.