Gene expression during CAM induction under salt stress in Mesembryanthemum: cDNA library and increased levels of mRNA for phosphoenolpyruvate carboxylase and pyruvate orthosphosphate dikinase

Mesembryanthemum crystallinum responds to high salinity in the soil by shifting the mode of carbon assimilation from the C3 mode to Crassulacean acid metabolism (CAM). Several enzymes of carbon metabolism have increased apparent activities in the CAM mode, including phosphoenolpyruvate carboxylase (PEPcase) and pyruvate orthophosphate dikinase (PPDK). We have identified cDNA clones for PEPcase and PPDK by immunological screening of a cDNA library constructed in the protein expression vector lambda gt11. The clones were characterized by immunoblotting and RNA blotting techniques. RNA blotting showed that during CAM induction the steady-state level of mRNAs for both PEP case and PPDK increased.Abbreviations IPTG isopropyl thiogalactoside - PEP phosphoenolpyruvate - PEPcase phosphoenolpyruvate carboxylase - PPDK pyruvate orthophosphate dikinase - Xgal-5 bromo-4-chloro-3-indolyl-beta-D-galactopyranoside     

Influence of the Malate-Aspartate Shuttle on Oxidative Metabolism in Synaptosomes

beta-Methyleneaspartate, a specific inhibitor of aspartate aminotransferase (EC 2.6.1.1.), was used to investigate the role of the malate-aspartate shuttle in rat brain synaptosomes. Incubation of rat brain cytosol, "free" mitochondria, synaptosol, and synaptic mitochondria, with 2 mM beta-methyleneaspartate resulted in inhibition of aspartate aminotransferase by 69%, 67%, 49%, and 76%, respectively. The reconstituted malate-aspartate shuttle of "free" brain mitochondria was inhibited by a similar degree (53%). As a consequence of the inhibition of the aspartate aminotransferase, and hence the malate-aspartate shuttle, the following changes were observed in synaptosomes: decreased glucose oxidation via the pyruvate dehydrogenase reaction and the tricarboxylic acid cycle; decreased acetylcholine synthesis; and an increase in the cytosolic redox state, as measured by the lactate/pyruvate ratio. The main reason for these changes can be attributed to decreased carbon flow through the tricarboxylic acid cycle (i.e., decreased formation of oxaloacetate), rather than as a direct consequence of changes in the NAD+/NADH ratio. Malate/glutamate oxidation in "free" mitochondria was also decreased in the presence of 2 mM beta-methyleneaspartate. This is probably a result of decreased glutamate transport into mitochondria as a result of low levels of aspartate, which are needed for the exchange with glutamate by the energy-dependent glutamate-aspartate translocator.     

Intermediary metabolism and shell growth in the brachiopod Terebratalia transversa

Juvenile Terebratalia transversa (Brachiopoda) metabolize carbohydrates in the anterior-most marginal mantle at a rate of 0.46 μM glucose/g/hr (in vitro incubation of mantle in C¹⁴-glucose in a carrying medium of 10^-3 M non-radioactive glucose). The rate declines to 0.18μM glucose/g/hr in full-grown specimens. Carbohydrate metabolism in the marginal (anterior-most) mantle averages approximately 3.7 times greater than metabolism in (a portion of the ‘posterior’) mantle situated between the coelomic canals and the marginal mantle. This ratio remains constant in specimens of all sizes (i.e. an ontogenetic trend in the ratio is absent at p≤ 0.05). Organic acids are not detectable within the mantle (HPLC techniques) even after simulated anoxia (N₂ bubbling during mantle incubation). Glucose metabolism in vitro declines in both the marginal and ‘posterior’ mantles during anoxia and the metabolic ratio between marginal/‘posterior’ mantles becomes 1/1. We found no difference (at p≤ 0.05) in mean metabolic activity or in sue-related metabolic trends among populations from depths ranging between mean sea level and 70 m. However, the activity within the ‘posterior’ mantle was more variable in specimens from 70 m than in those from shallower habitats (10 m - mean sea level). The size of the specimens analyzed was most variable in the groups obtained from the shallowest habitats and least variable at 70 m depth. Our results may help define the energetics of fossil as well as living brachiopod shell growth. Brachiopod shell growth is known to be very slow relative to that of bivalves and our results indicate that this is a result of the animals' slow metabolism. The inflation of the valves in T. transversa is, in part, a function of the high ratio of intermediary metabolism in the marginal vs‘posterior’ mantle (i.e. parallels the relative growth rates at the shell margin vs‘posterior’ areas). We found that the bivalve, Chlamys hastata, which is commonly associated with T. transversa, has a lower ratio of metabolic activities in the ventral/dorsal mantle areas than the brachiopod has in the anterior/posterior. The difference produces a flatter shell in the bivalve in accord with allometric principles. The higher metabolic rate in the marginal vs‘posterior’ brachiopod mantle and its more pronounced decline with anaerobiosis is reflected in the greater definition of growth increments in the outer shell layer. Our results do not support recent generalizations that correlate shell thickness of a wide variety of invertebrates inversely with metabolic rate. Growth rate as determined from width of shell growth increments is a better index of metabolic rate. Although the genetic basis of glucose metabolism is unknown, the observed metabolic variability is consistent with suggestions that populations of marine organisms living in stable offshore environments are genetically more variable but morphologically more uniform than populations from shallow water. Furthermore, our results support suggestions that bivalved molluscs and brachiopods are very different metabolically, but the data are neutral with respect to theories of competitive exclusion of the two taxa throughout geologic history.     

Respiration and soluble sugar metabolism in sugar pine embryos

Embroys excised from dormant seeds of sugar pine ( Pinus lambertiana Dougl.) incubated at 25°C (non-dormancy-breaking) or stratified at 5°C (dormancy-breaking) were analyzed to determine temperature effects on the relative activities of respiration and fermentative metabolism, the levels of soluble sugers and the activities of the hydrolytic enzymes, invertase and sucrose synthase, as related to the release of dormancy and germinatio. At 25°C, despite a sharp drop in embryo oxygen uptake after 48 h, a simultaneous decline in acetaldehyde and ethanol concentrations indicated that there was not a shift to fermentative metabolism. The concentrations of soluble sugars showed no treatment effects. Embryo invertase (EC 3.2.1.26) activity changed only slightly at either temperature, while stratification was accompanied by a 4-fold increase in sucrose synthase (EC 2.4.1.13) activity (cleavage direction). Upon transfer of stratified seeds to 25°C, embryo sucrose synthase activity rapidly increased almost 10-fold, with the increase beginning prior to germination, while mvertase activity increased 20-fold, concomitant with germination.     

Uptake and metabolism of benzyladenine in the early stage of flower bud development in vitro in tobacco

Benzyladenine (BA) was found to regulate the number of flower buds regenerated in vitro from pedicel tissue of tobacco. Flower bud induction was particularly sensitive to BA levels in the range of 0.45 to 1.0 μ M , where a two-fold increase in concentration caused a threefold rise in the number of buds. When tissues were fed radioactive BA for 24h, only 9–12% of the counts were recovered in the original compound. The rest was present in metabolites, tentatively identified as the mono-, di- and triribotides, 7- and 9-glucosides and 9-riboside of BA. The amount of growth regulator taken up and the quantities of BA and its metabolites in the explants were all linearly related to the concentration of the medium. The internal BA concentration was ca 60% of the level in the medium after 24 h. When the concentration in the medium was raised, relatively more BA remained in the non-conjugated form. However, this change in the equilibrium between BA and the conjugates is too small to account for the steep rise in the curve representing concentration vs effect between 0.45 and 1.0 μ M .     

Heterotrophic metabolism and diazotrophic growth ofNostoc sp. fromCycas circinalis

The cyanobiont ofCycas circinalis (identified asNostoc sp.) was isolated and its heterotrophic metabolism was studied in free culture under nitrogen-fixing conditions. Morphology, growth rate, nitrogenase activity, biochemical composition, efficiency of assimilation of organic carbon and molecular nitrogen were determined under different conditions of energy and carbon supply. The study has revealed the high potential of the heterotrophic metabolism in this symbiotic cyanobacterium. Although low rates of metabolic activities were attained under heterotrophic conditions, the efficiencies of organic carbon utilization (0.48 g cell-carbon per g glucose-carbon in chemoheterotrophy, from 0.65 to 0.74 under photoheterotrophy) and of N₂ assimilation (35.0 mg N₂ fixed per g glucose used in chemoheterotrophy, from 58.3 to 61.9 under photoheterotrophy) displayed by this organism were among the highest ever found in diazotrophically grown microorganisms. The isolate fromC. circinalis was able to grow indefinitely in the dark under nitrogen-fixing conditions, maintaining a well balanced biosynthetic activity and the capacity to resume photosynthetic metabolism quickly. The significance of the heterotrophic potential of this symbioticNostoc is discussed.     

Temperature effects on malic-acid efflux from the vacuoles and on the carboxylation pathways in crassulacean-acid-metabolism plants

The studies described in the paper were conducted with tissue slices of Crassulacean acid metabolism (CAM) plants floating in isotonic buffer. In a first series of experiments, temperature effects on the efflux of [¹⁴C]malate and¹⁴CO₂ were studied. An increase of temperature increased the efflux from the tissue in a non-linear manner. The efflux was markedly influenced also by the temperatures applied during the pretreatment. The rates of label export in response to the temperature and the relative contributions of¹⁴CO₂ and [¹⁴C]malate to the label export were different in the two studied CAM plants (Kalanchoë daigremontiana, Sempervivum montanum). In further experiments, temperature response of the labelling patterns produced by¹⁴CO₂ fixation and light and darkness were studied. In tissue which had accumulated malate (acidified state) an increase of temperature decreased the rates of dark CO₂ fixation whilst the rates of CO₂ fixation in light remained largely unaffected. An increase of temperature shifted the labelling patterns from a C₄-type (malate being the mainly labelled compound) into a C₃-type (label in carbohydrates). No such shift in the labelling patterns could be observed in the tissue which had depleted the previously stored malate (deacidified state). The results indicate that in the acidified tissue the increase of temperature increases the efflux of malate from the vacuole by changing the properties of the tonoplast. It is assumed that the increased export of malic acid lowers the in-vivo activity of phosphoenol pyruvate carboxylase by feedback inhibition.Abbreviations CAM Crassulacean acid metabolism - FW fresh weight - PEPCase phosphoenolpyruvate carboxylase Dedicated to Professor O.L. Lange, Würzburg, on the occasion of his 60th birthday     

The effect of auxin concentration on cytokinin stability and metabolism

The stability of [³H]zeatin riboside supplied to freshly excised tobacco pith explants was found to be inversely related to -naphthaleneacetic acid concentration in the incubation medium. At higher concentrations of -naphthaleneacetic acid greater breakdown of [³H]zeatin riboside was indicated by higher levels of degradative metabolites (adenine, adenosine and adenosine nucleotides) formed. This auxin effect on cytokinin metabolism appears to be mediated, at least in part, through cytokinin oxidase. The results of in-vitro assays carried out with partially purified enzyme from corn kernels substantiale this conclusion. These findings are discussed in relation to recent observations of auxin and cytokinin levels in crown-gall tumours with altered morphology.Abbreviations FPLC fast protein liquid chromatography - HPLC high-performance liquid chromatography - IP isopentenyladenine - NAA naphthaleneacetic acid - ZR zeatin riboside     

Phosphate sequestration by glycerol and its effects on photosynthetic carbon assimilation by leaves

Glycerol induced a limitation on photosynthetic carbon assimilation by phosphate when supplied to leaves of barley (Hordeum vulgare L.) and spinach (Spinacia oleracea L.). This limitation by phosphate was evidenced by (i) reversibility of the inhibition of photosynthesis by glycerol by feeding orthophosphate (ii) a decrease in light-saturated rates of photosynthesis and saturation at a lower irradiance, (iii) the promotion of oscillations in photosynthetic CO₂ assimilation and in chlorophyll fluorescence, (iv) decreases in the pools of hexose monophosphates and triose phosphates and increases in the ratio of glycerate-3-phosphate to triose phosphate, (v) decreased photochemical quenching of chlorophyll fluorescence, and increased non-photochemical quenching, specifically of the component which relaxed rapidly, indicating that thylakoid energisation had increased. In barley there was a massive accumulation of glycerol-3-phosphate and an increase in the period of the oscillations, but in spinach the accumulation of glycerol-3-phosphate was comparatively slight. The mechanism(s) by which glycerol feeding affects photosynthetic carbon assimilation are discussed in the light of these results.Abbreviations Chl chlorophyll - C _i intercellular concentration of CO₂ - P phosphate - PGA glycerate-3-phosphate - Pi orthophosphate - triose-P sum of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate     

Regulation of malic-acid metabolism in Crassulacean-acid-metabolism plants in the dark and light: In-vivo evidence from 13C-labeling patterns after 13CO2 fixation

The labeling patterns in malic acid from dark ¹³CO₂ fixation in seven species of succulent plants with Crassulacean acid metabolism were analysed by gas chromatography-mass spectrometry and ¹³C-nuclear magnetic resonance spectrometry. Only singly labeled malic-acid molecules were detected and on the average, after 12–14 h dark ¹³CO₂ fixation the ratio of [4-¹³C] to [1-¹³C] label was 2:1. However the 4-C carboxyl contained from 72 to 50% of the label depending on species and temperature. The ¹³C enrichment of malate and fumarate was similar. These data confirm those of W. Cockburn and A. McAuley (1975, Plant Physiol. 55, 87–89) and indicate fumarase randomization is responsible for movement of label to 1-C malic acid following carboxylation of phosphoenolpyruvate. The extent of randomization may depend on time and on the balance of malic-acid fluxes between mitochondria and vacuoles. The ratio of labeling in 4-C to 1-C of malic acid which accumulated following ¹³CO₂ fixation in the dark did not change during deacidification in the light and no doubly-labeled molecules of malic acid were detected. These results indicate that further fumarase randomization does not occur in the light, and futile cycling of decarboxylation products of [¹³C] malic acid (¹³CO₂ or [1-¹³C]pyruvate) through phosphoenolpyruvate carboxylase does not occur, presumably because malic acid inhibits this enzyme in the light in vivo. Short-term exposure to ¹³CO₂ in the light after deacidification leads to the synthesis of singly and multiply labeled malic acid in these species, as observed by E.W. Ritz et al. (1986, Planta 167, 284–291). In the shortest times, only singly-labeled [4-¹³C]malate was detected but this may be a consequence of the higher intensity and better detection statistics of this ion cluster during mass spectrometry. We conclude that both phosphoenolpyruvate carboxylase (EC 4.1.1.32) and ribulose-1,5-biphosphate carboxylase (EC 4.1.1.39) are active at this time.Abbreviations CAM Crassulacean acid metabolism - GCMS gas chromatography-mass spectrometry - MS mass spectrometry - NMR nuclear magnetic resonance spectrometry - PEP phosphoenolpyruvate - RuBP ribulose 1,5-bisphosphate