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     

Wax as a Mechanism for Protection against Photoinhibition — A Study of Cotyledon orbiculata

The leaves of the Crassulacean acid metabolism plant Cotyledon orbiculata have a waxy coating which is highly reflective but can be easily removed by brushing. This provided an ideal system in which to investigate the role of epidermal wax as a possible photoprotectant. Removal of the wax, prior to exposure to natural sunlight, resulted in substantial decreases in F_v/F_m and in severe cases evidence of photoinhibitory damage, as indicated by a rise in F_o. Leaves from which wax had been removed also showed higher conversion of violaxanthin to zeaxanthin than waxed leaves. Recovery of brushed leaves over a 12 day period was correlated with an increase in the total pool of xanthophyll cycle components. This study suggests that the presence of highly reflective wax on the epidermis may confer significant photoprotection to plants exposed to high solar radiation environments.     

INORGANIC CARBON LIMITATION OF PHOTOSYNTHESIS IN ULVA ROTUNDATA (CHLOROPHYTA)1

A computerized oxygen electrode Astern was used to make rapid and accurate measurements of photosynthetic light and dissolved inorganic carbon (DIC) response cures with a macroalga. Ulva rotundata Blid. was grown in an outdoor, continuous flow system in seawater under sunlight or 9% of sunlight at Beaufort, North Carolina. The light compensation points in the shade- and sun-grown plants, measured in seawater, were at photon flux densities (PFDs) of 16 and 27 μmol. Photons·m^?2·s^?1, respectively but the quantum yield of O₂ evolution was not significantly different. Rates of photosynthesis in seawater per unit area of thallus under saturating light and rates of dark respiration were about 1.5-fold higher in sun- than in shade-grown plants. The concentration of DIC in seawater (approximately 2 mM) limited photosynthesis at absorbed PFDs above 60–70 μmol photons·m^?2·s^?1 Addition of 20 mM inorganic carbon had no effect on quantum yield but caused about a 1.5-fold increase in the light-saturated photosynthetic rate in both shade- and sun-grown Ulva. The effect of DIC supplementation was greatest in plants grown in October and least in plants grown in June. The light- and DIC-saturated rate of photosynthesis in seawater was similar to the maximum rate obtained by exposing Ulva to 10% CO₂, in the gas phase. The carbon isotope values (δ¹³C, reflecting the ¹³C/¹²C ratio compared to a standard) of Ulva grown in the same seawater supply were dependent on light and agitation. Samples from Beaufort Inlet were more negative (δ¹³C value, ?20.03‰) than those grown in bright light with agitation (δ¹³C value, ?17.78‰ outdoors; ?17.23‰ indoors), which may indicate DIC supply limited carbon uptake in seawater.     

Diffusional Contribution to Carbon Isotope Fractionation during Dark CO(2) Fixation in CAM Plants

A mathematical model is developed which can be used to predict in vivo carbon isotope fractionations associated with carbon fixation in plants in terms of diffusion, CO₂ hydration, and carboxylation components. This model also permits calculation of internal CO₂ concentration for comparison with results of gas-exchange experiments. The isotope fractionations associated with carbon fixation in Kalanchoë daigremontiana and Bryophyllum tubiflorum have been measured by isolation of malic acid following dark fixation and enzymic determination of the isotopic composition of carbon-4 of this material. Corrections are made for residual malic acid, fumarase activity, and respiration. Comparison of these data with calculations from the model indicates that the rate of carbon fixation is limited principally by diffusion, rather than by carboxylation. Processes subsequent to the initial carboxylation also contribute to the over-all isotopic composition of the plant.     

Resolution of rat renin substrates by isoelectric focusing.

Pooled plasmas from normal or binephrectomized rats and perfusates of isolated livers were used as sources of renin substrate for isoelectric focusing. After desalting, preliminary fractionation (plasma only), and concentration, the preparations were focused in a pH 3--10 gradient on 20-cm glass plates layered with Sephadex slurry. The pH 4--6 region, containing all the substrate, was scraped from this plate and refocused in a pH 4--6 gradient. Substrate content of 1-cm strips of slurry from half of the plate was determined by both radioimmunoassay and bioassay of angiotensin resulting from incubation with added renin. Corresponding strips from the other half of the plate were incubated without renin as a control for any preformed angiotensin. The asymmetry and broad distribution (pH 4--5) of substrate from different sources suggested the existence of more than one form. Higher resolution achieved by using the high substrate concentration of postnephrectomy plasma and 0.5-cm strips of slurry on 20-cm or 40-cm plates revealed peaks and shoulders of substrate activity. Our data suggest that multiple forms of substrate are synthesized by the liver and circulate in plasma. Postnephrectomy rat plasma appears to contain relatively more substrate(s) with higher isoelectric points than in normal plasma, possibly an accumulation of forms ordinarily degraded by endogenous renal renin.     

Malice's wonderland: Research funding and peer review

The players all played at once, without waiting for turns, quarreling all the while and fighting for the hedgehogs; and in a very short time the queen was in a furious passion and went stamping about, and shouting “off with his head” or “off with her head,” about once a minute. Alice began to feel very uneasy: to be sure she had not as yet had any dispute with the queen, but she knew that it might happen any minute, “and then” thought she, “what would become of me? They're dreadfully fond of beheading people here: the great wonder is, that there's any one left alive”.     

Activation and measurement of plasma prorenin in the rat.

Prorenin determination in rat plasma has been problematic from the outset. Consequently, its existence is questioned by some and its quantity by others, making it difficult for knowledge to advance as to its function relative to the renin system. The present study examines major variables in the determination of rat plasma prorenin and renin, notably different prorenin activation protocols involving blood samples obtained under various conditions from animals under different anesthetics. We found that a trypsin activation step with 5 mg/mL plasma, 60 min at 23 degrees C, followed by a PRA step of 10 min at 37 degrees C, resulted in the highest prorenin estimates, up to approximately 400 ng.mL-1.h-1 in terms of angiotensin I, as compared with published values of 0-190, based on other protocols. These estimates were obtained despite considerable destruction of angiotensinogen (renin substrate) by trypsin. Cryoactivation of prorenin was much less effective than in human plasma but, when followed by trypsin, it facilitated greater activation than with trypsin alone. Comparable fresh and fresh-frozen plasmas had similar prorenin-renin values, but lower values were observed in plasmas that had been repeatedly frozen and thawed. Conscious rats and those anesthetized with Inactin or ether had higher renins and prorenins than those anesthetized with methoxyflurane or halothane. Rats with kidneys in place during blood collection had higher renins (but not prorenins) than those whose kidneys were clamped off, suggesting that last-minute renin release during blood collection had occurred. We conclude that (i) trypsin generates increased renin, or renin-like, activity in plasma, suggesting activation of a precursor; (ii) on this basis, high prorenin levels exist in normal rat plasma; (iii) renin and prorenin levels are variously influenced by different anesthetics and blood handling procedures; (iv) variation in prorenin levels suggests that it is a dynamic (functional?) component of the renin system; (v) prorenin measurements are heavily influenced by methodological variations during the trypsin step or the subsequent PRA step; (vi) using standardized methodology, the rat can serve as a model for investigating the function of prorenin in normotension and hypertension.     

Population dynamics of "null" and Thy1lo lymphocytes in mouse bone marrow: genesis of cells with natural killer cell lineage characteristics.

The population dynamics of "null" small lymphocytes lacking B and T lineage markers in mouse bone marrow have been examined using a combination of immunolabeling and hydroxyurea (HU) deletion techniques. The binding of the B lineage-associated mAb, 14.8, and anti-Thy1.2 to bone marrow cells has been detected radioautographically. Null cells lacking 14.8 and Thy1.2 determinants (14.8- Thy1-) formed a substantial subset (12-14%) of bone marrow small lymphocytes, representing 0.5 x 10(6) cells per femur (2-3% of nucleated cells). HU treatment revealed an exceptionally rapid turnover of the null small lymphocyte population (T1/2, 7.5 hr) compared with 14.8+ cells (T1/2, 20.5 hr) and Thy1+ cells (T1/2, 53 hr). Small lymphocytes bearing low intensities of Thy1 (Thy1lo) were also rapidly renewed (T1/2, 28 hr) whereas those with high intensities of Thy1 (Thy1hi) were renewed only slowly (T1/2, 123 hr). During ontogeny, null small lymphocytes first appeared in the fetal liver by Day 11 and the fetal spleen by Day 16, but increased rapidly in the bone marrow in early postnatal life. Double immunolabeling techniques demonstrated that 10% of null small lymphocytes in the bone marrow expressed NK1.1 antigen, while larger proportions bound to tumor (YAC.1) cells in vitro and displayed Fc receptors. The NK1.1-bearing fraction of null small lymphocytes in bone marrow was depleted by HU treatment only after an initial delay. NK1.1 was also expressed on subsets of Thy1lo cells and Thy1hi cells. The results have revealed the continuous production in mouse bone marrow of null and Thy1lo small lymphocytes, totaling 1-3 x 10(7) cells/day and 1.2 x 10(6) cells/day, respectively. The findings suggest that the large-scale production of null lymphocytes in mouse bone marrow includes the genesis of NK lineage cells which express NK1.1 and Thy1lo during a period of terminal maturation.     

Measurements of the Engagement of Cyanide-Resistant Respiration in the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana with the Use of On-Line Oxygen Isotope Discrimination

Discrimination against ¹⁸O during dark respiration in tissues of Kalanchoë daigremontiana, Medicago sativa, and Glycine max was measured using an on-line system that enabled direct measurements of the oxygen fractionation of samples in a gas-phase leaf disk electrode unit. Discrimination factors for cytochrome pathway respiration were 18.6 to 19.8%_o for all tissues. However, discrimination in cyanide-resistant respiration was significantly higher in green tissues (30.4-31.2%_o) compared with nongreen tissues (25.3-25.9%_o). Using these discrimination factors, the partitioning of electron transport to these pathways was calculated from measurements of discrimination in the absence of inhibitors. Changes in flux through the alternative pathway were measured during the light and dark phases of Crassulacean acid metabolism in leaf disks of K. daigremontiana. The flux of electrons through the alternative pathway was higher during deacidification than during the other phases of Crassulacean acid metabolism. The increase in alternative pathway electron flux accounted for all of the increased respiration in the light phase. Despite this increase, simultaneous measurements of malate concentration and respiratory flux confirm that only a small proportion of the total malate decarboxylation occurs in the mitochondria.     

Relation of birth weight and childhood respiratory infection to adult lung function and death from chronic obstructive airways disease.

OBJECTIVE--To examine whether birth weight, infant weight, and childhood respiratory infection are associated with adult lung function and death from chronic obstructive airways disease. DESIGN--Follow up study of men born during 1911-30 whose birth weights, weights at 1 year, and childhood illnesses were recorded at the time by health visitors. SETTING--Hertfordshire, England. SUBJECTS--5718 men born in the county during 1911-30 and a subgroup of 825 men born in the county during 1920-30 and still living there. MAIN OUTCOME MEASURES--Death from chronic obstructive airways disease, mean forced expiratory volume in one second (FEV1) and forced vital capacity (FVC), and respiratory symptoms. RESULTS--55 men died of chronic obstructive airways disease. Death rates fell with increasing birth weight and weight at 1 year. Mean FEV1 at age 59 to 70 years, adjusted for height and age, rose by 0.06 litre (95% confidence interval 0.02 to 0.09) with each pound (450 g) increase in birth weight, independently of smoking habit and social class. Bronchitis or pneumonia in infancy was associated with a 0.17 litre (0.02 to 0.32) reduction in adult FEV1 and with an increased odds ratio of wheezing and persistent sputum production in adult life independently of birth weight, smoking habit, and social class. Whooping cough in infancy was associated with a 0.22 litre (0.02 to 0.42) reduction in adult FEV1. CONCLUSIONS--Lower birth weight was associated with worse adult lung function. Intrauterine influences which retard fetal weight gain may irrecoverably constrain the growth of the airways. Bronchitis, pneumonia, or whooping cough in infancy further reduced adult lung function. They also retarded infant weight gain. Consistent with this, death from chronic obstructive airways disease in adult life was associated with lower birth weight and weight at 1 year. Promoting lung growth in fetuses and infants and reducing the incidence of lower respiratory tract infection in infancy may reduce the incidence of chronic obstructive airways disease in the next generation.