Photoinactivation and protection of glycolate oxidase in vitro and in leaves.

Glycolate oxidase that was partially purified from pea leaves was inactivated in vitro by blue light in the presence of FMN. Inactivation was greatly retarded in the absence of O2. Under aerobic conditions H2O2 was formed. The presence of catalase, GSH or dithiothreitol protected glycolate oxidase against photoinactivation. Less efficient protection was provided by ascorbate, histidine, tryptophan or EDTA. The presence of superoxide dismutase or of hydroxyl radical scavengers had no, or only minor, effects. Glutathione suppressed H2O2 accumulation and was oxidized in the presence of glycolate oxidase in blue light. Glycolate oxidase was also inactivated in the presence of a superoxide-generating system or by H2O2 in darkness. In intact leaves photoinactivation of glycolate oxidase was not observed. However, when catalase was inactivated by the application of 3-amino-1,2,4-triazole or depleted by prolonged exposure to cycloheximide a strong photoinactivation of glycolate oxidase was also seen in leaves. In vivo blue and red light were similarly effective. Furthermore, glycolate oxidase was photoinactivated in leaves when the endogenous GSH was depleted by the application of buthionine sulfoximine. Both catalase and antioxidants, in particular GSH, appear to be essential for the protection of glycolate oxidase in the peroxisomes in vivo.

cDNA cloning of cytoplasmic ribosomal protein S7 of winter rye (Secale cereale) and its expression in low-temperature-treated leaves.

A cDNA clone (ScRPS7) encoding the cytoplasmic ribosomal protein S7 was isolated from a rye leaf cDNA library and sequenced. The deduced protein of 192 amino acids with M(r) 22189 shows identity of 52% or 47%, respectively, relative to S7 proteins of human or yeast. A RPS7 mRNA accumulation is higher in the meristematic zone at the leaf base than in the non-meristematic middle and upper section of leaves. Short periods of cold stress sharply reduce the mRNA level while leaves of cold hardened plants contain normal levels of ScRPS7 transcripts.

Slow turnover of the D1 reaction center protein of photosystem II in leaves of high mountain plants.

The D1 reaction center protein of photosystem II usually exhibits a rapid turnover in light. The D1 protein turnover was compared in three species of alpine plants, Homogyne alpina, Ranunculus glacialis, Soldanella alpina, and in the lowland plant Taraxacum officinale by radioactive labeling in light and subsequent chase experiments. The D1 protein of alpine plants could also be recognized by its more rapid labeling, relative to other membrane proteins. However, compared to T. officinale the turnover of the D1 protein was considerably slower in the alpine plants. The potential advantage of a slow D1 turnover for adaptation to the environmental conditions of high mountain plants is discussed.

Characterization of a plastid-specific HSP90 homologue: identification of a cDNA sequence, phylogenetic descendence and analysis of its mRNA and protein expression.

The isolation of cDNAs is described which encode the complete sequence of a precursor protein for a HSP90 homologue consisting of an N-terminal transit peptide of 5850 Da and a mature protein (cpHSP82) of 82 260 Da, located in the plastids of rye leaves (Secale cereale). Hybridization analysis indicated the presence of a single gene in the DNA of rye and a transcript size of 2.8 kb. A phylogenetic tree constructed on the basis of sequence comparisons for HSP90 homologues from different species and compartments indicated that the plastidic HSP82 from rye was more closely related to an eubacterial protein than to HSP90 homologues of the cytosol or ER from both plants and animals. The results suggest that during chloroplast evolution the gene for cpHSP82 was transferred to the nucleus from a prokaryotic endosymbiont. Immunoblots with specific antibodies and Percoll gradient-purified organelles confirmed the location of cpHSP82 in chloroplasts or non-green plastids. In green rye leaves cpHSP82 was constitutively expressed and equally distributed among tissues of different age. The expression of cpHSP82 was enhanced within 2 h by exposure to 42 degrees C. The cpHSP82 transcript and protein were much more strongly expressed in non-green tissues, such as etiolated, 70S ribosome-deficient 32 degrees C-grown, or herbicide-bleached, than in normal green leaves. Also chromoplasts from the pericarp of tomato fruits contained high levels of a HSP90 polypeptide while a photosynthetic protein, the large subunit of ribulose-1,5-bisphosphate carboxylase was largely degraded during ripening.

Comparison of the expression of a plastidic chaperonin 60 in different plant tissues and under photosynthetic and non-photosynthetic conditions.

A partial cDNA which codes for the beta-subunit of a plastidic chaperonin 60 (cpn60-beta) from rye (Secale cereale L.) leaves was identified and sequenced, except for 46 amino acids of the N-terminus of the mature protein and the transit sequence. This is the first cpn60-beta sequence determined for a monocotyledonous plant. Specific antibodies against cpn60-beta were affinity-purified from an antiserum raised against the total soluble protein fraction of ribosome-deficient plastids. The localization of cpn60-beta in chloroplasts or non-green plastids was confirmed by immunodetection in Percoll gradient-purified organelles. The expression and occurrence of cpn60-beta was analysed by immunoblotting with the specific antibodies and Northern hybridization. The cpn60-beta protein was constitutively expressed in various green and non-green tissues. It was evenly distributed along the major part of a rye leaf, while highest transcript levels occurred in the youngest and oldest leaf sections. The expression of the cpn60-beta protein was not enhanced by a heat-shock treatment at 42 degrees C. The cpn60-beta transcript and protein were more strongly expressed in various non-green, for instance etiolated, 70S-ribosome-deficient 32 degree C-grown, or herbicide-bleached tissues, than in green leaves of rye. A rapid increase in the cpn60-beta transcript level was also observed when green leaves were transferred from light to darkness while the protein level was not affected. The dark-induced increase in the cpn60-beta transcript was totally suppressed in the presence of 2% sucrose. Inhibitor treatments suggested that the change in cpn60-beta transcript level was not related to changes of the ATP supply of the tissue. While the large subunit of the photosynthetic protein ribulose-1,5-bisphosphate carboxylase was largely degraded during ripening of tomato fruits, high levels of cpn60-beta were detected in tomato chromoplasts and in the yellow flower petals of Narcissus. Low levels of cpn60-beta were detected in root tissue.

Analysis of the primary structure of the chloroplast isozyme of triosephosphate isomerase from rye leaves by protein and cDNA sequencing indicates a eukaryotic origin of its gene.

The primary structure of the chloroplast isozyme of triosephosphate isomerase from rye leaves was identified by protein and cDNA sequencing and compared to the deduced amino acid sequence of a cDNA for the cytosolic isozyme. The mature cytosolic and chloroplast isozyme proteins share 64% amino acid sequence identity. The cDNA for the chloroplast isozyme codes for a precursor protein consisting of an N-terminal transit peptide of Mr 4351 and a mature subunit of Mr 27,282. Southern blot analysis indicates that the two rye isozymes are encoded by two independent single genes. Amino acid residues or sequence regions of basic functional relevance in known triosephosphate isomerases are strictly conserved in the chloroplast isozyme. The chloroplast isozyme contains 6 cysteine residues, instead of 4 in the cytosolic isozyme. A cysteine at position 143 of the chloroplast isozyme appears to be modified. Phylogenetic trees constructed on the basis of sequence comparisons for triosephosphate isomerases from different species of all major taxonomic groups indicate that the chloroplast isozyme is much more closely related to eukaryotic cytosolic enzymes than to eubacterial enzymes. The results indicate that the nuclear gene for the chloroplast isozyme originated with that for the cytosolic isozyme through duplication of an ancestral eukaryotic gene, rather than through gene transfer from a prokaryotic endosymbiont.

Identification of the nuclear-encoded chloroplast ribosomal protein L12 of the monocotyledonous plant Secale cereale and sequencing of two different cDNAs with strong codon bias.

Two different cDNA clones (SCL12-1 and SCL12-2) encoding precursors of a chloroplast ribosomal protein with homology to L12 from Escherichia coli were isolated from rye leaf cDNA libraries and sequenced. The corresponding polypeptide of rye chloroplast ribosomes was identified. The sequences for the mature proteins of M(r) 13,447 and 13,609 share 85% amino acid identity. The mature polypeptide of clone SCL12-1 has an amino acid identity of 71%, 72% or 44%, respectively, relative to L12 proteins from spinach, tobacco, or E. coli. Codon usage of the rye L12 cDNAs shows a high preference (97% and 82%) for G or C in the third base position.

Light dependence of catalase synthesis and degradation in leaves and the influence of interfering stress conditions.

The enzyme catalase (EC 1.11.1.6) is light sensitive and subject to a rapid turnover in light, similar to the D1 reaction center protein of photosystem II. After 3 h of preadaptation to darkness or to different light intensities (90 and 520 mumol m(-2) s(-1) photosynthetic photon flux density), sections of rye leaves (Secale cereale L.) were labeled for 4 h with l-[(35)S]methionine. From leaf extracts, catalase was immunoprecipitated with an antiserum prepared against the purified enzyme from rye leaves. Both incorporation into catalase and degradation of the enzyme polypeptide during a subsequent 16-h chase period increased with light intensity. At a photon flux density of 520 mumol m(-2) s(-1), the apparent half-time of catalase in rye leaves was 3 to 4 h, whereas that of the D1 protein was much shorter, about 1.5 h. Exposure to stress conditions, such as 0.6 m NaCl or a heat-shock temperature of 40 degrees C, greatly suppressed both total protein synthesis and incorporation of the label into catalase and into the D1 protein. Immunoblotting assays indicated that in light, but not in darkness, steady-state levels of catalase and of the D1 protein strongly declined during treatments with salt, heat shock, or translation inhibitors that block repair synthesis. Because of the common property of rapid photodegradation and the resulting dependence on continuous repair, declines in catalase as well as of the D1 protein represent specific and sensitive indicators for stress conditions that suppress the translational activities of leaves.

Photoinactivation of Catalase Occurs under Both High- and Low-Temperature Stress Conditions and Accompanies Photoinhibition of Photosystem II.

Severe photoinactivation of catalase (EC 1.11.1.6) and a decline of variable fluorescence (F(v)), indicating photoinhibition of photosynthesis, were observed as rapid and specific symptoms in leaves exposed to a high heat-shock temperature of 40 degrees C as well as in leaves exposed to low chilling temperatures in white light of only moderately high photosynthetic photon flux density of 520 muE m(-2) s(-1). Other parameters, such as peroxidase (EC 1.11.1.7), glycolate oxidase (EC 1.1.3.1), glutathione reductase (EC 1.6.4.2), or the chlorophyll content, were hardly affected under these conditions. At a compatible temperature of 22 degrees C, the applied light intensity did not induce severe photoinactivations. In darkness, exposures to high or low temperatures did not affect catalase levels. Also, decline of F(v) in light was not related to temperature sensitivity in darkness. The effective low-temperature ranges inducing photoinactivation of catalase differed significantly for chilling-tolerant and chilling-sensitive plants. In leaves of rye (Secale cereale L.) and pea (Pisum sativum L.), photoinactivation occurred only below 15 degrees C, whereas inactivation occurred at 15 degrees C in cucumber (Cucumis sativus L.) and maize (Zea mays L.). The behavior of F(v) was similar, but the difference between chilling-sensitive and chilling-tolerant plants was less striking. Whereas the catalase polypeptide, although photoinactivated, was not cleaved at 0 to 4 degrees C, the D1 protein of photosystem II was greatly degraded during the low-temperature treatment of rye leaves in light. Rye leaves did not exhibit symptoms of any major general photodamage, even when they were totally depleted of catalase after photoinactivation at 0 to 4 degrees C, and catalase recovered rapidly at normal temperature. In cucumber leaves, the decline of catalase after exposures to bright light at 0 to 4 degrees C was accompanied by bleaching of chlorophyll, and the recovery observed at 25 degrees C was slow and required several days. Similar to the D1 protein of photosystem II, catalase differs greatly from other proteins by its inactivation and high turnover in light. Inasmuch as catalase and D1 protein levels depend on continuous repair synthesis, preferential and rapid declines are generally to be expected in light whenever translation is suppressed by stress actions, such as heat or chilling, and recovery will reflect the repair capacity of the plants.

Genetics and evolution of chloroplast isozymes of triosephosphate isomerase.

Triosephosphate isomerase is an ubiquitous and highly conservative dimeric enzyme, consisting of subunits of Mr 26,000-27,000. Plants usually contain one cytosolic and one plastid isozyme. While 2x wheats also contain one plastid isozyme, 4x wheats contain 3, and 6x cultural wheats contain five plastid isozymes. The multiplicity of the isozyme pattern in 6x wheats is explainable by the presence of three different genomes (AABBDD), each contributing a distinct triosephosphate isomerase gene (alpha',beta,delta), and by the formation of homodimeric and heterodimeric isozyme forms. While the beta beta-form was, as expected, also found in Aegilops speltoides which is regarded as donor of the B genome, the descent of the other genes for plastid triosephosphate isomerase did not occur in accordance with common contentions on the evolution of 6x cultural wheat and its presumptive ancestors. In the reciprocal intergeneric hybrids between wheat and rye, Secalotricum and Triticale, the patterns of both the cytosolic and the plastid-specific triosephosphate isomerases were biparentally inherited, indicating also that the plastid isozyme was nuclear-encoded. Data which are available about amino acid sequences and gene organization and immunological observations show that the cytosolic triosephosphate isomerase of plants is strongly related to other eukaryotic animal triosephosphate isomerase genes. Multiple evidence has been presented that the plastid- specific isozyme represents a distinct polypeptide and is specified by a distinct gene, relative to the cytosolic isozyme. Immunological comparisons indicate that the plastid isozyme shares homologies with the cytosolic isozyme but is not related to the enzyme from prokaryotic cyanobacteria or bacteria. To enable a more precise comparison, plastid triosephosphate isomerase has been cloned from a cDNA library from rye, and cDNA clones are being sequenced. The plastid enzyme of triosephosphate isomerase appears to have evolved from a duplication on an ancestral nuclear gene of the primordial plant cell. For other plastid-specific isozymes evidence exists that their genes were incorporated into the nucleus by gene transfer from a prokaryotic endosymbiont.

Multiple coordinate controls contribute to a balanced expression of ribulose-1,5-bisphosphate carboxylase/oxygenase subunits in rye leaves.

In the leaves of rye (Secale cereale L.), control mechanisms acting at multiple molecular levels contribute to a coordinate expression of the subunit polypeptides of ribulose-1,5-bisphosphate carboxylase. The relevance and hierarchy of the different control steps were evaluated by comparing the time courses of changes in levels of translatable mRNA, rates of in vivo amino acid incorporation, and the turnover of subunit polypeptides after selective interference with translation at either cytoplasmic 80S ribosomes, or at the 70S ribosomes of the chloroplast, by compartment-specific inhibitors, or by the use of 70S-ribosome-deficient leaves. The latter were generated by growing the plants at a non-permissive elevated temperature of 32 degrees C. The rates of synthesis of the two ribulose-1,5-bisphosphate carboxylase subunits were most rapidly adapted to each other by translational controls. Within 0.5-2.5 h after selective inhibition of the synthesis of either subunit, that of the other subunit made in the unaffected compartment also declined by more than 90% without any marked change in its mRNA. After prolonged inhibition (24 h) of either cytoplasmic or chloroplast protein synthesis, the levels of mRNAs for both subunits were greatly diminished. In rye, the mRNA levels for both subunits changed under all experimental conditions tested in a closely parallel manner and appeared to be always maintained in a balanced, fairly constant ratio by strong coordinate controls. Even 70S-ribosome-deficient leaves contained mRNAs for both the small and the large subunits, although only in small amounts. The mRNAs for both subunits were also markedly further decreased in 70S-ribosome-deficient leaves after application of an inhibitor of cytoplasmic translation. MDMP [2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide], suggesting that the suppression of the large subunit mRNA in the plastids was not mediated through feedback effects of accumulating unassembled large subunits. Coordinate controls at both the mRNA and the translational level require a bidirectional exchange of regulatory signals between chloroplast and cytoplasm. However, these controls were not absolutely restrictive and allowed low rates of uncoupled synthesis of either large or small subunits. Large subunits made in the presence of MDMP were stable over 24 h. However, unassembled small subunits synthesized in 70S-ribosome-deficient leaves were degraded with a half-time of 10.5 h, in contrast to their behavior after integration into the holoprotein in normal leaves, where no turnover was detected. The proteolytic removal of surplus free small subunits is regarded as a final post-translational fine-tuning step to establish a balanced subunit stoichiometry in leaves.

Unassembled polypeptides of the plastidic ribosomes in heat-treated 70S-ribosome-deficient rye leaves.

The polypeptides of the subunits of 70S ribosomes isolated from rye (Secale cereale L.) leaf chloroplasts were analyzed by two-dimensional polyacrylamide gel electrophoresis. The 50S subunit contained approx. 33 polypeptides in the range of relative molecular mass (Mr) 13000-36000, the 30S subunit contained approx. 25 polypeptides in the range of Mr 13000-40500. Antisera raised against the individual isolated ribosomal subunits detected approx. 17 polypeptides of the 50S and 10 polypeptides of the 30S subunit in the immunoblotting assay. By immunoblotting with these antisera the major antigenic ribosomal polypeptides (r-proteins) of the chloroplasts were clearly and specifically visualized also in separations of leaf extracts or soluble chloroplast supernatants. In extracts from rye leaves grown at 32° C, a temperature which is non-permissive for 70S-ribosome formation, or in supernatants from ribosome-deficient isolated plastids, six plastidic r-proteins were visualized by immunoblotting with the anti-50S-serum and two to four plastidic r-proteins were detected by immunoblotting with the anti-30S-serum, while other r-proteins that reacted with our antisera were missing. Those plastidic r-proteins that were present in 70S-ribosome-deficient leaves must represent individual unassembled ribosomal polypeptides that were synthesized on cytoplasmic 80S ribosomes. For the biogenesis of chloroplast ribosomes the mechanism of coordinate regulation appear to be less strict than those known for the biogenesis of bacterial ribosomes, thus allowing a marked accumulation of several unassembled ribosomal polypeptides of cytoplasmic origin.

Photoinactivation of catalase in vitro and in leaves.

Purified catalase from bovine liver and catalase of isolated intact peroxisomes from rye leaves were inactivated in vitro by irradiation with visible light. During photoinactivation the protein moiety of pure catalase was not cleaved; however, the electrophoretic mobility of the native enzyme was decreased, and a major portion of enzyme-bound heme was dissociated. In a suspension of isolated chloroplasts photoinactivation of pure or peroxisomal catalase was mediated by light absorption in the chloroplasts. Both the direct and the chloroplast-mediated photoinactivation of catalase were affected little by the presence of D2O or superoxide dismutase but were greatly retarded by formate. In isolated peroxisomes substantial photoinactivation of catalase occurred only in the presence of nonphotosynthesizing but not in the presence of photosynthesizing isolated chloroplasts. Substantial and selective photoinactivation of catalase was also observed in vivo when leaf sections from various plant species (rye, pea, sunflower, cucumber, maize) were irradiated with light of high intensity in the presence of the translation inhibitors cycloheximide or 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide, while catalase activity was much less or not affected in 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated or untreated control sections. The extent of photoinactivation of catalase in leaves depended on light intensity and also occurred in red light. The results suggest that photoinactivation of catalase generally occurs in leaves under high light intensity, though it is not apparent under normal physiological conditions because it is compensated for by new synthesis. Apparent photoinactivation of catalase has to be regarded as an early indication of photodamage in leaves and conceivably enhances its progress.

Genetic control of triosephosphate isomerase isoenzymes in wheat and rye.

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.

Synthesis and degradation of unassembled polypeptides of the coupling factor of photophosphorylation CF1 in 70S ribosome-deficient rye leaves.

The formation of polypeptides of the coupling factor CF1 was investigated in 70S ribosome-deficient rye leaves generated by growing the plants at a non-permissive elevated temperature of 32 degrees C, in order to analyse mechanisms coordinating subunit accumulation. Antibodies were raised in rabbits against total CF1 as well as against its five individual subunits purified from chloroplast thylakoids from rye leaves. Several immunological techniques applying these antibodies (immunoprecipitation, immunoblotting, antibody affinity chromatography) were unable to detect the presence of any of the CF1 subunits in heat-treated 70S ribosome-deficient leaves. After in vivo labeling with L-[35S]methionine and subsequent immunoprecipitation, however, radioactivity was found to be incorporated into the subunits gamma and delta, but not into alpha, beta and epsilon, in 70S ribosome-deficient leaves, demonstrating the cytoplasmic synthesis of CF1-gamma and CF1-delta. Chase experiments after in vivo labeling with L-[35S]methionine indicated that the unassembled subunits gamma and delta were rapidly and preferentially degraded, while they were stabilized when integrated into the complete CF1 complex in normal green leaves from permissive growth conditions. The apparent half-times of the unassembled subunits were 2 h for CF1-gamma and 4 h for CF1-delta in 32 degrees C-grown leaves. Several other, stromal, plastid proteins of cytoplasmic origin were stable in 32 degrees C-grown leaves during the period of chase. In etiolated leaves total CF1, including all subunits, appeared to be less stable than in green leaves grown under permissive temperature conditions in light. Rapid degradation of the excess of unassembled subunits is regarded as an important mechanism ensuring a constant stoichiometry and apparently synchronous development of CF1 subunits.

Control of plastidic glycolipid synthesis and its relation to chlorophyll formation.

Mechanisms restricting the accumulation of chloroplast glycolipids in achlorophyllous etiolated or heat-treated 70S ribosome-deficient rye leaves (Secale cereale L. cv "Halo") and thereby coupling glycolipid formation to the availability of chlorophyll, were investigated by comparing [(14)C]acetate incorporation by leaf segments of different age and subsequent chase experiments. In green leaves [(14)C]acetate incorporation into all major glycerolipids increased with age. In etiolated leaves glycerolipid synthesis developed much more slowly. In light-grown, heat-bleached leaves [(14)C]acetate incorporation into glycolipids was high at the youngest stage but declined with age. In green leaves [(14)C]acetate incorporation into unesterified fatty acids and all major glycerolipids was immediately and strongly diminished after application of an inhibitor of chlorophyll synthesis, 4,6-dioxoheptanoic acid. The turnover of glyco- or phospholipids did not differ markedly in green, etiolated, or heat-bleached leaves. The total capacity of isolated ribosome-deficient plastids for fatty acid synthesis was not much lower than that of isolated chloroplasts. However, the main products synthesized from [(14)C]acetate by chloroplasts were unesterified fatty acids, phosphatidic acid, and diacylglycerol, while those produced by ribosome-deficient plastids were unesterified fatty acids, phosphatidic acid, and phosphatidylglycerol. Isolated heat-bleached plastids exhibited a strikingly lower galactosyltransferase activity than chloroplasts, suggesting that this reaction was rate-limiting, and lacked phosphatidate phosphatase activity.

Origin and developmental changes of envelope proteins and translocator activities from plastids of Secale cereale L.

To determine the sites of synthesis of chloroplast-envelope proteins, we have analysed several enzyme and translocator functions ascribed to the envelope membranes, and investigated the envelope polypeptide composition of plastids isolated from 70S ribosome-deficient leaves of rye (Secale cereale L.) generated by growing the plants at a temperature of 32°C. Since the ribosomedeficient plastids are also achlorophyllous in light-grown leaves, not only were chloroplasts from mature, green leaves used for comparison, but also those from yellowing, aged leaves as well as etioplasts from dark-grown leaves raised at a temperature of 22° C. A majority of the plastidenvelope polypeptides appeared to be of cytoplasmic origin. The envelopes of ribosome-deficient plastids possessed ATPase (EC 3.6.1.3) activity; this was not, however, dependent on divalent cations, in contrast to the Mn(2+)- or Mg(2+)-dependent ATPase which is associated with chloroplast envelopes. Adenylate kinase (EC 2.7.4.3) was present in the stromal fraction of ribosome-deficient plastids and the stromal form of this enzyme is, therefore, of cytoplasmic origin. In contrast to previous findings, adenylate kinase was not, however, specifically associated with the chloroplast-envelope membranes, either in rye or in spinach. Measurements of the uptake of L-[(14)C]-malate into ribosome-deficient plastids indicated the presence and cytoplasmic origin of the dicarboxylate translocator. Malate uptake into rye etioplasts was, however, low. The phosphate translocator was assayed by the uptake of 3-phospho-[(14)C]glycerate. While rapid 3-phosphoglycerate uptake was observed for rye chloroplasts and etioplasts, it was hardly detectable for ribosome-deficient, plastids and rather low for chloroplasts from aged leaves. A polypeptide of M r approx. 30000 ascribed to the phosphate translocator was greatly reduced in the envelope patterns of ribosome-deficient plastids and of chloroplasts from aged leaves.

The effects of caffeine on physiological functions and mental performance.

Polygraphic monitoring of several physiological variables was done throughout an experiment investigating the effects of caffeine on mental performance. The experiment started with a mental maze learning task. Then the subjects were given the test beverages according to the group design (CC group (N = 16): 300 mg caffeine in decaffeinated coffee, DC group (N = 16): decaffeinated coffee, WW group (N = 8): warm water, and NB group (N = 8): no beverage). The experiment continued with a letter cancellation task which was followed by a second mental maze learning task. The caffeine treated subjects differed from the other groups by increased regularity of letter cancellation performance, as indicated by decreases in intraindividual variance. They also differed from the other groups by a slight but significant acrodermal vasoconstriction. No intergroup differences were obtained for mental maze learning, heart rate, respiration, muscle tension, and skin conductance. The results suggest therefore that the drug at this dose level improves behavioral routine and speed rather than cognitive functions and that the vegetative side effects are minimal.

Metabolism of activated oxygen in detached wheat and rye leaves and its relevance to the initiation of senescence.

The activities of several enzymes either generating or decomposing O 2 (-) or H2O2, were investigated during the course of senescence of detached wheat (Triticum aestivum L.) and rye (Secale cereale L.) leaves in light and in darkness. Most of the activities, although not in full synchrony, declined with the degradation of chlorophyll and protein. The decline was slower in light than in darkness (e.g. glycolate oxidase, EC 1.1.3.1; urate oxidase, EC 1.7.3.3.; catalase, EC 1.11.1.6) and was further retarded after application of kinetin. The activity of superoxide dismutase (EC 1.15.1.1) declined only very little or, in detached rye leaves, even remained unchanged. For lipoxygenase (EC 1.13.11.12) the decline was enhanced in light and not affected by kinetin. Total peroxidase (EC 1.11.1.7) activity strikingly increased after excision of the leaves. The increase was higher in the dark than in light and further enhanced by kinetin. Activity of L-amino-acid oxidase (EC 1.4.3.2) was not detected. The peroxide content of the detached leaves slowly increased during senescence, being higher in light than in darkness. The malondialdehyde content strongly increased in light, but not in darkness. Application of several chemicals known as scavengers for oxygen radicals (1,4-diazobicyclo(2,2,2)octane, α-tocopherol acetate, p-benzoquinone, D-penicillamine copper, 2-amino-2-(hydroxymethyl)-1,3-propanediol, formate) did not notably retard chlorophyll degradation in senescencing leaves. Thiourea and urate retarded chlorophyll breakdown in light, obviously because they were used as nitrogen sources. Chlorophyll breakdown was greatly accelerated by D2O, particularly in light, presumably by enhancing photooxidative damage. The results indicate that increased peroxide metabolism accompanies the senescence of detached leaves. They do not, however, support the free-radical theory that an accumulation of activated oxygen initiates leaf senescence.

Nature of photooxidative events in leaves treated with chlorosis-inducing herbicides.

Leaves of rye seedlings (Secale cereale L.) grown in the presence of four chlorosis-inducing herbicides under a low light intensity of 10 lux formed chlorophyll. When segments of such dim-light-grown leaves were exposed to 30,000 lux at either 0 degrees C or 30 degrees C, treatments with aminotriazole or haloxidine (group 1) showed no or only minor changes of their chlorophyll contents. In treatments with San 6706 or difunon (group 2), however, rapid photodestruction of chlorophyll occurred both at 0 degrees C and at 30 degrees C and was accompanied by an increase of malondialdehyde that was not seen in the presence of group 1 herbicides. Unlike the in vivo behavior, virtually equal rates of chlorophyll breakdown were observed for aminotriazole and San 6706 treatments in suspensions of isolated chloroplasts from 10 lux-grown leaves after exposure to strong light. The free radical scavengers p-benzoquinone and hydroquinone and the d-penicillamine copper complex exerting superoxide dismutating activity effectively prevented photooxidation of chlorophyll in 10 lux-grown herbicide-treated leaf segments or even restored an accumulation of chlorophyll at 30,000 lux. Ascorbate and several singlet oxygen or hydroxyl radical scavengers had no protective effects. Deuterium oxide and H(2)O(2) did not enhance the degradation of chlorophyll. Superoxide dismutase activity was decreased in leaves bleached in the presence of group 2 herbicides.