Cytosolic calcium, hydrogen peroxide and related gene expression and protein modulation in Arabidopsis thaliana cell cultures respond immediately to altered gravitation: parabolic flight data.

Callus cell cultures of Arabidopsis thaliana (cv. Columbia) were exposed to parabolic flights in order to assess molecular, short-term responses to altered gravity fields. Using transgenic cell lines, hydrogen peroxide (H2 O2 ) and cytosolic Ca(2+) were continuously monitored. In parallel, the metabolism of samples was chemically quenched (RNAlater, Ambion for RNA; acid/base for NADPH, NADP) at typical stages of a parabola [1 g before pull up; end of pull up (1.8 g), end of microgravity (20 s) and end of pull out (1.8 g)]. Cells exhibited an increase in both Ca(2+) and H2 O2 with the onset of microgravity, and a decline thereafter. This behaviour was accompanied by a decrease of the NADPH/NADP redox ratio, indicating Ca(2+) -dependent activation of a NADPH oxidase. Microarray analyses revealed concomitant expression profiles. At the end of the microgravity phase, 396 transcripts were specifically up-, while 485 were down-regulated. Up-regulation was dominated by Ca(2+) - and ROS-related gene products. The same material was also used for analysis of phosphopeptides with 2-D SDS PAGE. Relevant spots were identified by liquid chromatography-MS. With the exception of a chaperone (HSP 70-3), hypergravity (1.8 g) and microgravity modified different sets of proteins. These are partly involved in primary metabolism (glycolysis, gluconeogenesis, citrate cycle) and detoxification of ROS. Taken together, these data show that both gene expression and protein modulation jointly respond within seconds to alterations in the gravity field, with a focus on metabolic adaptation, signalling and control of ROS.

Microgravity-related changes in gene expression after short-term exposure of Arabidopsis thaliana cell cultures.

Cell cultures of Arabidopsis thaliana (cv. Columbia) were used to screen for early alterations in gene expression as a response to altered gravitational fields. Genes of interest were selected from a larger group whose expression was altered under hypergravity (microarray study; M. Martzivanou and R. Hampp, Physiol. Plant. 118: 221-231, 2003). Transcriptional changes of these genes were studied within a time frame of up to 10 min of exposure to microgravity in a sounding-rocket experiment, to clinorotation (random positioning machine), and to hypergravity (8 g). We could identify a set of nine genes (mainly components of signaling chains) with increased transcript levels after about 6 min of exposure to microgravity. As clinorotation and hypergravity treatment did not alter the respective transcript amounts, we assume that the identified genes could be involved in a microgravity-related response.

Carbon balance in leaves of young poplar trees.

In the present study, important components of carbon metabolism of mature leaves of young poplar trees (Populus x canescens) were determined. Carbohydrate concentrations in leaves and xylem sap were quantified at five different times during the day and compared with photosynthetic gas exchange measurements (net assimilation, transpiration and rates of isoprene emission). Continuously measured xylem sap flow rates, with a time resolution of 15 min, were used to calculate diurnal balances of carbon metabolism of whole mature poplar leaves on different days. Loss of photosynthetically fixed carbon by isoprene emission and dark respiration amounted to 1% and 20%. The most abundant soluble carbohydrates in leaves and xylem sap were glucose, fructose and sucrose, with amounts of approx. 2 to 12 mmol m(-2) leaf area in leaves and about 0.2 to 15 mM in xylem sap. Clear diurnal patterns of carbohydrate concentration in xylem sap and leaves, however, were not observed. Calculations of the carbon transport rates in the xylem to the leaves were based on carbohydrate concentrations in xylem sap and xylem sap flow rates. This carbon delivery amounted to about 3 micromol C m(-2) s(-1) during the day and approx. 1 micromol C m(-2) s(-1) at night. The data demonstrated that between 9 and 28 % of total carbon delivered to poplar leaves during 24 h resulted from xylem transport and, hence, provide a strong indication for a significant rate of carbon cycling within young trees.

Carbon dioxide concentration and nitrogen input affect the C and N storage pools in Amanita muscaria-Picea abies mycorrhizae.

We studied the influence of elevated atmospheric CO2 concentration ([CO2]) on the vacuolar storage pool of nitrogen-containing compounds and on the glycogen pool in the hyphal sheath of Amanita muscaria (L. ex Fr.) Hooker-Picea abies L. Karst. mycorrhizae grown with two concentrations of ammonium in the substrate. Mycorrhizal seedlings were grown in petri dishes on agar containing 5.3 or 53 mg N l(-1) and exposed to 350 or 700 microl CO2 l(-1) for 5 or 7 weeks, respectively. Numbers and area of nitrogen-containing bodies in the vacuoles of the mycorrhizal fungus were determined by light microscopy linked to an image analysis system. The relative concentration of nitrogen in the vacuolar bodies was measured by electron energy loss spectroscopy (EELS). Glycogen stored in the cytosol was determined at the ultrastructural level by image analysis after staining the sections (PATAg test). Shoot dry weight, net photosynthesis and relative amounts of N in vacuolar bodies were greater at the higher N and CO2 concentrations. The numbers and areas of vacuolar N-containing bodies were significantly greater at the higher N concentration only at ambient [CO2]. In the same treatment the percentage of hyphae containing glycogen declined to nearly zero. We conclude that, in the high N/low [CO2] treatment, the mycorrhizal fungus had an insufficient carbohydrate supply, partly because of increased amino acid synthesis by the non-mycorrhizal rootlets. When [CO2] was increased, the equilibrium between storage of glycogen and N-containing compounds was reestablished.

Recent advances in exploring physiology and biodiversity of ectomycorrhizas highlight the functioning of these symbioses in ecosystems.

Ectomycorrhizas, the dominating mycorrhizal symbiosis in boreal, temperate and some tropical forests, are formed by 5000-6000 species of the asco- and basidiomycetes. This high diversity of fungal partners allows optimal foraging and mobilisation of various nitrogen and phosphorus forms from organic soil layers. In this review, two approaches to study the functioning of this multitude of symbiotic associations are presented. On selected culture models, physiological and molecular investigations have shown that the supply of hexoses has a key function in controlling the plant-fungus interaction via partner-specific regulation of gene expression. Environmental factors which affect fungal carbon supply, such as increased nitrogen availability, also affect mycorrhiza formation. Based on such laboratory results, the adaptative capability of ectomycorrhizas to changing field conditions is discussed. The second approach consists of analysing the distribution of mycorrhizas in ecosystem compartments and to relate distribution patterns to variations of ecological factors. Recent advances in identification of fungal partners in ectomycorrhizas by analysing the internal transcribed spacer of ribosomal DNA are presented, which can help to resolve sampling problems in field studies. The limits of the laboratory and the field approaches are discussed. Despite some problems, this combined approach is the most promising. Direct investigation of gene expression, which has been introduced for soil bacteria, will be difficult in the case of mycorrhizal fungi which constitute organisms with functionally varying structures.

Sugar- and nitrogen-dependent regulation of an Amanita muscaria phenylalanine ammonium lyase gene.

The cDNA of a key enzyme of secondary metabolism, phenylalanine ammonium lyase, was identified for an ectomycorrhizal fungus by differential screening of a mycorrhizal library. The gene was highly expressed in hyphae grown at low external monosaccharide concentrations, but its expression was 30-fold reduced at elevated concentrations. Gene repression was regulated by hexokinase.

Carbon allocation in ectomycorrhizas: identification and expression analysis of an Amanita muscaria monosaccharide transporter.

Ectomycorrhizas are formed between certain soil fungi and fine roots of predominantly woody plants. An important feature of this symbiosis is the supply of plant-derived carbohydrates to the fungus. As a first step toward a better understanding of the molecular basis of this process, we cloned a monosaccharide transporter from the ectomycorrhizal fungus Amanita muscaria. Degenerate oligonucleotide primers were designed to match conserved regions from known fungal sugar transporters. A cDNA fragment of the transporter was obtained from mycorrhizal mRNA by reverse transcription-polymerase chain reaction. This fragment was used to identify a clone (AmMst1) encoding the entire monosaccharide transporter in a Picea abies/A. muscaria mycorrhizal cDNA library. The cDNA codes for an open reading frame of 520 amino acids, showing best homology to a Neurospora crassa monosaccharide transporter. The function of AmMST1 as monosaccharide transporter was confirmed by heterologous expression of the cDNA in a Schizosaccharomyces pombe mutant lacking a monosaccharide uptake system. AmMst1 was constitutively expressed in fungal hyphae under all growth conditions. Nevertheless, in mycorrhizas as well as in hyphae grown at monosaccharide concentrations above 5 mM, the amount of AmMst1 transcript increased fourfold. We therefore suggest that AmMst1 is upregulated in ectomycorrhizas by a monosaccharide-controlled mechanism.

Fusion and metabolism of plant cells as affected by microgravity.

Plant cell protoplasts derived from leaf tissue of two different tobacco species (Nicotiana tabacum., N. rustica L.) were exposed to short-term (sounding rocket experiments) and long-term (spacelab) microgravity environments in order to study both (electro) cell fusion and cell metabolism during early and later stages of tissue regeneration. The period of exposure to microgravity varied from 10 min (sounding rocket) to 10 d (space shuttle). The process of electro fusion of protoplasts was improved under conditions of microgravity: the time needed to establish close membrane contact between protoplasts (alignment time) was reduced (5 as compared to 15 s under 1 g) and numbers of fusion products between protoplasts of different specific density were increased by a factor of about 10. In addition, viability of fusion products, as shown by the ability to form callus, increased from about 60% to more than 90%. Regenerated fusion products obtained from both sounding-rocket and spacelab experiments showed a wide range of intermediate properties between the two parental plants. This was verified by isozyme analysis and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). In order to address potential metabolic responses, more general markers such as the overall energy state (ATP/ADP ratio), the redox charge of the diphosphopyridine nucleotide system (NADH/NAD ratio), and the pool size of fructose-2,6-bisphosphate (Fru 2,6 bisp), a regulator of the balance between glycolysis and gluconeogenesis, were determined. Responses of these parameters were different with regard to short-term and long-term exposure. Shortly after transition to reduced gravitation (sounding rocket) ratios of ATP/ADP exhibited strong fluctuation while the pool size of NAD decreased (indicating an increased NADH/NAD ratio) and that of Fru 2,6 bisp increased. As similar changes can be observed under stress conditions, this response is probably indicative of a metabolic stress compensation. Samples taken for up to 7 d of exposure to microgravity showed the opposite effect. Here, the ratios of ATP/ADP and of NADH/NAD, and the pool size of Fru 2,6 bisp were decreased. We take this an an indication of metabolic relaxation, i.e. decreased metabolic turnover. As rates of protoplast regeneration and cell division were obviously similar to 1-g controls, we conclude that under conditions of microgravity regenerating tobacco mesophyll protoplasts need less metabolic energy for the same effort.

Effect of fertilization on ozone-induced changes in the metabolism of birch (Betula pendula) leaves.

Cloned cuttings of Betula pendula Roth were grown in field fumigation chambers at Birmensdorf throughout one growing season in filtered air with either < 3 (control) or 90/40 nl l-1 O3 (day/night; ozone generated from pure oxygen). Each ozone regime was split into high and low soil nutrient regimes by watering plants with either a 0.05 % or a 0.005% solution of a fertilizer which contained macronutrients and micronutrients. Fertilization had a strong effect on plant growth, enzyme activities and the expression of ozone-induced effects at the biochemical level. The activities of PEPC and Rubisco were enhanced about threefold in the plants with high fertilization (HF). Significant effects of ozone were in most cases found only in the older leaves of the plants with low fertilization (LF), There, sucrose, glucose and fructose levels were enhanced. In both fertilization treatments, the number of starch granules along the minor veins was increased. These ozone effects point to a decreased or inhibited phloem loading. The increased PEPC activity and the enhanced malate levels in the ozone-exposed plants might be the result of a redirection of carbon flow from sucrose synthesis and translocation towards anapleurotic processes, which can feed detoxification and repair of ozone injury as indicated by enhanced respiration. These findings agree well with the observed effects of ozone in lowering the root: shoot biomass ratio. Although there was a marked reduction in the O3 /LF plants, O3 /HF plants showed no significant response. Inositol was decreased under ozone exposure in both fertilizer treatments, contrasting with the pattern for carbohydrates. These results demonstrate the role of fertilization as an important modifier of ozone-induced effects at the plant biochemical level. Well fertilized plants appear to cope better with the impact of ozone on metabolism.

Cyclic AMP, a possible regulator of glycolysis in the ectomycorrhizal fungus Amanita muscaria.

The amounts of cyclic AMP (cAMP), fructose-2,6-bisphosphate (F26BP), trehalose and glycogen were determined in cell suspension cultures of the ectomycorrhiza-forming fungus Amanita muscaria (L. ex Fr.) Hooker. For the assay of cAMF a protocol was developed that enabled the detection of as little as 50 fmol of this secondary messenger by an enzyme-linked immuno assay (EIA). Values varied from < 1 and up to 5 pmol cAMP mg1 d. wt according to the age of the fungal culture. Typically, a transient increase in cAMP occurred after c. 4 d of culture of the fungus on glucose-containing medium. This increase (up to 100%) was followed by the start of the logarithmic growth phase, and by a more persistent increase in F26BP. In parallel, glucose in the medium started to decrease, whilst the amounts of fungal carbohydrates, especially the disaccharide trehalose, increased, From these data we assume that a high initial rate of glucose uptake caused an increase in the fungal pools of storage carbohydrates and, via activation of an adenylate cyclase, of cAMP. According to data reported for yeast cells this should enhance the formation of F26BP by phosphorylation of relevant enzymes. In animal and yeast cells an increase in the concentration of F26BP stimulates glycolysis by activation of the ATP-dependent phosphofructokinase (PFK). A. muscaria also possesses an F26BP activated PFK and, under conditions of symbiosis, host-derived carbohydrates are supplied mainly in the form of glucose. The implications of these findings to the regulation of carbohydrate metabolism of symbiotic plant root/fungus structures (ectomycorrhiza) are discussed.

Coarse and Fine Control and Annual Changes of Sucrose-Phosphate Synthase in Norway Spruce Needles.

Annual changes of activity of sucrose-phosphate synthase (SPS) from spruce (Picea abies [L.] Karst.) needles were studied with respect to three regulatory levels: metabolic fine control, covalent modification (phosphorylation), and protein amount. Glucose-6-phosphate served as an allosteric activator of spruce SPS by shifting the Michaelis constant for the substrate fructose-6-phosphate from 4.2 to 0.59 mM, whereas inorganic phosphate competitively inhibited this activation. The affinity for the other substrate, UDP-glucose, was unaffected. Incubation of the crude extract with ATP resulted in a time- and concentration-dependent decrease of the maximal velocity of SPS. This inactivation was sensitive to staurosporine, a potent protein kinase inhibitor, indicating the participation of a protein kinase. Probing SPS protein with heterologous antibodies showed that the subunit of spruce SPS is an approximately 139-kD protein and that changes in the extractable activity during the course of a year were correlated with the amount of SPS protein. High SPS activities in winter were paralleled by increased levels of the activator glucose-6-phosphate and the substrate fructose-6-phosphate, indicating a high capacity for sucrose synthesis that may be necessary to maintain photosynthetic CO2 fixation in cold-hardened spruce needles.

Regeneration of plant cell protoplasts under microgravity: investigation of protein patterns by SDS-PAGE and immunoblotting.

As part of the D-2 Spacelab mission, tobacco (Nicotiana tabacum L.) protoplasts were cultured for 10 days in microgravity and successfully regenerated into microcalli, which, after further cultivation on the ground, gave rise to intact plants. Protein analysis was performed on samples taken during the initial microgravity period and compared to ground controls. Total protein content and protein patterns were monitored, as well as the cytoskeletal proteins tubulin and actin, a key enzyme of secondary metabolism, phenylalanine ammonia lyase, and the pathogenesis-related protein osmotin. None of the investigated proteins showed a gravity-dependent effect. Since relative changes due to culture age were detectable in the immunoblots as well as in the total protein pattern, an adaptation of the cells to microgravity without major modifications of their protein complement may be assumed.

Regeneration of plant cell protoplasts under microgravity: Investigation of protein patterns by SDS-PAGE and immunoblotting.

As part of the D-2 Spacelab mission, tobacco (Nicotiana tabacum L.) protoplasts were cultured for 10 days in microgravity and successfully regenerated into microcalli, which, after further cultivation on the ground, gave rise to intact plants. Protein analysis was performed on samples taken during the initial microgravity period and compared to ground controls. Total protein content and protein patterns were monitored, as well as the cytoskeletal proteins tubulin and actin, a key enzyme of secondary metabolism, phenylalanine ammonia lyase, and the pathogenesis-related protein osmotin. None of the investigated proteins showed a gravity-dependent effect. Since relative changes due to culture age were detectable in the immunoblots as well as in the total protein pattern, an adaptation of the cells to microgravity without major modifications of their protein complement may be assumed.

Electrofusion of plant cell protoplasts under microgravity--a D-2 spacelab experiment.

Plant cell protoplasts, derived from sexually incompatible plant species, have proved to be a good system for somatic hybridization by electrofusion. Under microgravity, an increase in fusion yield can be expected, especially if the parental cells differ markedly in size or specific density. On the D-2 spacelab mission flown in 1993, electrofusion experiments were performed with three different objects, i.e. tobacco as model system, Helianthus as an important crop, and Digitalis as a plant of pharmacological interest. The resulting fusion products were cultivated (along with parental cells) for 10 days under microgravity, and subsequently regenerated on ground for biochemical analysis. Results are presented on the observation of the fusion process during flight, heterofusion yields, ultrastructural investigation of fusion products immediately after fusion, and characterization of the resulting hybrids. The results are interpreted on the background of earlier microgravity-experiments on sounding rockets or parabolic flights.

Compartmentation of soluble carbohydrates, of starch and of malate in motor organs (pulvini) and other parts of Phaseolus coccineus L. leaves.

Quantitative histochemistry was used to investigate the tissue-specific compartmentation of soluble carbohydrates (sucrose, glucose, fructose), starch and malate in the laminar pulvinus, leaf blade and petiole of Phaselous coccineus L. at day and night positions of diurnal leaf movement. Total carbohydrate levels measured in a series of cross sections along individual pulvini of 24-d-old plants showed only small differences between the day and night positions of the respective leaf. In contrast, the level of malate changed during diurnal leaf movement, especially in the central part of a pulvinus. The levels of glucose and fructose in the pulvinus increased towards the transition zones between the pulvinus and lamina, and pulvinus and petiole, and this trend was even more pronounced for starch. By contrast, sucrose levels were highest in the pulvinus proper. The transverse compartmentation of metabolites was studied in distinct, approx. 0.5-mm-thick tissue slices from the central part of a pulvinus. These were dissected further into up to 14 distinct subsamples (bundle, bundle sheath, motor tissues, flanks). Irrespective of the position of the leaf (day or night), the central vascular core and the surrounding bundle sheath had high levels of sucrose (up to 500 mmol-(kg DW)(-1)) and low levels of glucose and fructose (below 100 mmol-(kg DW)(-1)), while in the cortex the situation was reversed. In the night position the level of sucrose decreased by approx. 30% in the bundle sheath and the central vascular core but not in the other sections. We thus suggest that because of the relatively small diurnal changes in their cortical pools, soluble sugars are not involved in the osmotic processes resulting in leaf movement. In contrast, pulvini from 14-d-old plants showed an interesting diurnal change in starch and malate pools in the outermost layer of the extensor. Here starch increased at night while the malate pool was lowered nearly stoichiometrically. Inverse pool sizes were found in the day position of the respective leaves. Although less significant, the opposite diurnal variation occurred in samples taken from the flexor region. We thus were able to locate areas of different carbohydrate activities in the laminar pulvinus of P. coccineus. The central vascular core, including the bundle sheath, is involved in temporary storage of photoassimilates, and the cortical regions are responsible for osmotically driven leaf movement. The results are discussed with respect to guard-cell physiology.

Ectomycorrhizins - symbiosis-specific or artitactual polypeptides from ectomycorrhizas?

Fungal mycelium of the fly agaric (Amanita muscaria [L. ex Fr.] Hooker), and inoculated or noninoculated seedlings of Norway spruce (Picea abies [L.] Karst.) were grown aseptically under controlled conditions. In order to detect symbiosis-specific polypeptides ('ectomycorrhizins', see Hubert and Martin, 1988, New Phytol. 110, 339-346) the protein patterns of (i) fungal mycelium, (ii) mycorrhizal, and (iii) non-mycorrhizal root tips were compared by means of one- and twodimensional electrophoresis on a microscale. Because of the sensitivity of these micromethods (50 and 200 ng of protein, respectively), single mycorrhizal root tips and even the minute quantities of extramatrical mycelium growing between the roots of inoculated plants could be analysed. Differences in the protein patterns of root tips could be shown within the root system of an individual plant (mycorrhizal as well as non-mycorrhizal). In addition, the protein pattern of fungal mycelium grown on a complex medium (malt extract and casein hydrolysate) differed from that of extramatrical mycelium collected from the mycorrhiza culture (pure mineral medium). Such differences in protein patterns are obviously due to the composition of the media and/or different developmental stages. Consequently, conventional analyses which use extracts of a large number of root tips, are not suitable for differentiating between these effects and symbiosis-specific differences in protein patterns. In order to detect ectomycorrhizins, it is suggested that roots and mycelium from individual, inoculated plants should be analysed. This approach eliminates the influence of differing media, and at the same time allows a correct discrimination between developmental and symbiosisspecific changes. In our gels we could only detect changes in spot intensity but could not detect any ectomycorrhizins or the phenomenon of polypeptide 'cleansing', which both characterize the Eucalyptus-Pisolithus symbiosis (Martin and Hubert, 1991, Experientia 47, 321-331). We thus suggest that these reported effects either are specific for the Eucalyptus-Pisolithus symbiosis or simply represent artifacts. The latter point of view is strengthened by a comparison of the experimental approaches.

Ectomycorrhizins - symbiosis-specific or artifactual polypeptides from ectomycorrhizas?

Fungal mycelium of the fly agaric (Amanita muscaria [L. ex Fr.] Hooker), and inoculated or noninoculated seedlings of Norway spruce (Picea abies [L.] Karst.) were grown aseptically under controlled conditions. In order to detect symbiosis-specific polypeptides ('ectomycorrhizins', see Hubert and Martin, 1988, New Phytol.110, 339-346) the protein patterns of (i) fungal mycelium, (ii) mycorrhizal, and (iii) non-mycorrhizal root tips were compared by means of one- and twodimensional electrophoresis on a microscale. Because of the sensitivity of these micromethods (50 and 200 ng of protein, respectively), single mycorrhizal root tips and even the minute quantities of extramatrical mycelium growing between the roots of inoculated plants could be analysed. Differences in the protein patterns of root tips could be shown within the root system of an individual plant (mycorrhizal as well as non-mycorrhizal). In addition, the protein pattern of fungal mycelium grown on a complex medium (malt extract and casein hydrolysate) differed from that of extramatrical mycelium collected from the mycorrhiza culture (pure mineral medium). Such differences in protein patterns are obviously due to the composition of the media and/or different developmental stages. Consequently, conventional analyses which use extracts of a large number of root tips, are not suitable for differentiating between these effects and symbiosis-specific differences in protein patterns. In order to detect ectomycorrhizins, it is suggested that roots and mycelium from individual, inoculated plants should be analysed. This approach eliminates the influence of differing media, and at the same time allows a correct discrimination between developmental and symbiosisspecific changes. In our gels we could only detect changes in spot intensity but could not detect any ectomycorrhizins or the phenomenon of polypeptide 'cleansing', which both characterize theEucalyptus-Pisolithus symbiosis (Martin and Hubert, 1991, Experientia47, 321-331). We thus suggest that these reported effects either are specific for theEucalyptus-Pisolithus symbiosis or simply represent artifacts. The latter point of view is strengthened by a comparison of the experimental approaches.

A dot-blot assay for quantitation of nanogram amounts of protein in the presence of carrier ampholytes and other possibly interfering substances.

A method for protein determination in one- and two-dimensional electrophoresis sample buffer is presented. Accurate quantitation of protein in two-dimensional electrophoresis sample buffer (9.5 M urea, 2% Nonidet P-40, 2% carrier ampholytes, and 5% 2-mercaptoethanol) required removal of carrier ampholytes prior to the assay. This was made possible by taking advantage of the mutual solubility/insolubility of carrier ampholytes/proteins in saturated ammonium sulfate solution. In addition, improvement of protein determination in denaturing electrophoresis sample buffer containing the anionic detergent sodium dodecyl sulfate and the reducing agent 2-mercaptoethanol was achieved. The assay covers a range of sensitivity from 40 ng to 20 micrograms of protein. The procedure is applicable to large numbers of samples.

Intercellular compartmentation of basic carbon pathways in motor organs (pulvini) of leaves of Phaseolus coccineus L.

The activities of enzymes which catalyze one step in each of the five major carbon pathways in green plants were measured in secondary pulvini and other tissues of Phaseolus coccineus L. leaves. We were able to detect activities of fumarase (EC 4.2.1.2; tricarboxylic-acid pathway), NAD-glyceraldehyde-phosphate dehydrogenase (NAD-GAPDH, EC 1.2.1.12; glycolysis), 6-phosphogluconate dehydrogenase (6-PGDH, EC 1.1.1.44; oxidative pentose-phosphate pathway), ribulose-1, 5-bisphosphate carboxylase (Rubisco, EC 4.1.1.39; photosynthetic carbon-reduction pathway), and of hydroxypyruvate reductase (HP-R, EC 1.1.1.81; photosynthetic carbon-oxidation pathway). On a protein basis the activities of Rubisco and HP-R in pulvinar regions were very low (below 1 and 2 mol · (kg protein) (--1) · h(--1), respectively), but the activities of fumarase and NAD-GAPDH were between 10- and 5-fold higher compared with the laminar tissue (up to 7 and 50 mol · (kg protein)(--1) · h(--1), respectively). Similarly, the protein specific activities of 6-PGDH were increased in the pulvinus (3-4 compared with approx. 1 mol · (kg protein)(--1) · h(--1) in the leaf blade). No differences in specific activities were detected between day and night positions of the leaves. By applying quantitative histochemical techniques we determined the longitudinal and transversal compartmentation of the activities of fumarase, NAD-GAPDH, and 6-PGDH in pulvinar tissues. Levels of activity of all three enzymes increased towards the middle part of the pulvinus. Here, expressed on a dry-weight (DW) basis, the analysis of cross sections showed highest activities in the outer parts of the extensor in the order given, approx. 0.6, 5, and 0.25 mol · (kg DW)(--1) · h(--1) for fumarase, NAD-GAPDH and 6-PGDH. When related to protein, levels of activity were comparably high within the inner parts of extensor and flexor, and partly also in the abaxial part of the bundle (fumarase, 6-PGDH). The tissue-specific compartmentation of the respective activities is discussed in relation to leaf movement and shows parallels with guard-cell function.