MMC and LD simulations of α-D-Manp-(1→2)-β-D-Glcp-OMe: comparison to long-range heteronuclear NMR coupling constants and to the crystal structure

The conformational flexibility and the dynamics of -D-Manp-(12)--D-Glcp-OMe have been investigated by Metropolis Monte Carlo (MMC) and Langevin dynamics (LD) simulations. The two simulation techniques employ different force fields, namely the HSEA force field and a CHARMm-based force field. The former shows less conformational flexibility than the latter, in which a multiple energy minima conformational space is sampled. Long-range heteronuclear nuclear magnetic resonance (NMR) coupling constants have been measured by selective excitations of the carbons at the glycosidic linkage. Calculated 3JC, H values from MMC and LD simulations show excellent agreement to those from NMR experiments. The X-ray crystal structure has a conformation within a region of the conformational space populated in both force fields.     

Photosynthesis and associated metabolism during development of a Theobroma cacao hybrid with the lethal factor Luteus-Pa

The recessive lethal character Luteus-Pa, expressed as a yellowing of leaves of young seedlings and followed by death approximately 60 d after emergence, presents a 3:1 segregation in crosses and/or selfpollinated plants. We evaluated quantitatively the fluorescence emission of chlorophyll (Chl), gas exchange, and chemical composition of normal and recessive homozygous cacao seedlings of the cross Pa 121×Pa 169. The characteristics of Chl fluorescence kinetics were studied in stages B2, B3, C, D, and E of leaf development, corresponding to plant ages of 9 to 12, 13 to 15, 16 to 20, 21 to 30, and >30 d, respectively. Gas exchanges were measured in mature leaves of both seedlings. In regular intervals of 3 d beginning at 33 d after emergence, the seedlings were separated into roots, stems, leaves, and cotyledons to determine the contents of saccharides (SAC) and free amino acids (FAA) and variation of the leaf Chl content. The Chl distribution in complexes of the photosynthetic apparatus was analysed by SDS-PAGE in mature leaves of both normal and recessive 32-d-old seedlings. There were variations in Chl fluorescence, gas exchanges and chemical composition of different parts of both types of seedlings. However, no significant differences were found in the Chl distribution through photosynthetic complexes of 32-d-old normal and recessive homozygous seedlings. After that period a decrease in the Chl concentration was observed in the recessive seedlings, and only minimum fluorescence (F0) was found. The F0 values were higher in the recessive seedlings than in the normal ones. The net photosynthetic rate of mature leaves was negative in agreement with low conductance, transpiration rate, and high internal CO2 concentration. These factors might have contributed to a depletion in SAC in different plant parts. Although F0 partially reflects the Chl concentration in leaf tissue, the increase in its value was probably due to a damage in reaction centres of photosystem 2. Therefore, the growth and development of recessive homozygous seedlings depended exclusively on cotyledon reserves, the depletion of which leads to death.     

Seasonal Variation of Photoinhibition of Photosynthesis in Bark from Populus Tremula L.

In the bark of Populus tremula L. photochemical efficiency of photosystem 2 (PS2) determined as Fv/Fm decreased during winter. The strongest reduction was found after cold periods. The degree of reduction depended on irradiance since the lowest levels of Fv/Fm were found on the sun-exposed side of the stem and below thin phellem. Therefore, photoinhibition was partly responsible for the reduction in Fv/Fm. The photochemical efficiency of PS2 recovered in late April about a month before the trees got leaves. In the laboratory, Fv/Fm recovered within about a week under low irradiance at 20 °C. Rapid recovery of photochemical efficiency of PS2 in the bark may be important to reduce respiratory loss of CO2 from the stem before the trees get leaves.     

Characterization of a three-phase response in gloved cold-stressed fingers

Seven gloves were studied worn by eight sedentary subjects (six men and two women) exposed to cold–dry, C–D, (mean dry bulb temperature −17.2^∘C; mean dew point temperature ), and cold–wet, C–W, ( 0^∘C; ) conditions. Mean endurance times were 75 min for the C–D and 162 min for the C–W conditions. A three-phase response pattern of the temperature in the fingers was characterized. Phase I comprised an initial period during which finger temperature remained close to the pre-exposed level, due to delayed vasoconstriction in the finger. Phase II involved an exponential-like decrease of finger temperature indicative of the onset of vasoconstriction in the finger. Phase III manifested periodic finger temperature changes due to cold induced vasodilatation (CIVD). Mean wave patterns for phase III indicated approximately 3.5 waves · h⁻¹ in the C–D but only about 2 waves · h⁻¹ in the C–W condition. Extension of endurance time, due to CIVD, was defined as the difference in time between the actual end of the experiment and the time the finger-tip would have reached the set temperature endurance limit as extrapolated by a continued exponential drop. Three overall response patterns of fingers in the cold were characterized: type A exhibiting all 3 phases; type B₁ or B₂ exhibiting either phases I+II or phases II+III; and type C showing only phase II. Considerable inter- and intra-subject variability was found. In both test conditions the final physiological thermal states of the subjects were between comfortable and slightly uncomfortable but acceptable and thus did not correlate with the responses in the fingers. Accepted: 5 January 1998     

Plasma progesterone profiles and fertility status of anestrus Zebu cattle treated with norgestomet-estradiol-eCG regimen

Zebu cattle are notorious for poor fertility characterized by late maturity and long intercalving intervals attributed to a variety of factors, including genetic, nutritional and climatic. The aim of the present investigation, therefore, was to induce fertile estrus in acyclic pubertal heifers and postpartum anestrous Zebu cows by hormonal intervention. Pubertal Hariana and Sahiwal anestrous heifers (n=51) and postpartum cows (n=55) were either assigned a placebo (controls, N=6 for each breed and parity) or treated with 10-d norgestomet (3 mg) subcutaneous ear implants, with an initial injection of 3 mg, im norgestomet + 5 mg estradiol valerate, followed by 500 IU eCG at implant withdrawal (NOR-treated groups). Jugular venous plasma samples were obtained from a total of 28 animals (controls : 4 heifers and 4 cows; NOR-treated : 12 heifers and 8 cows) on Days 0 (implant insertion), 3, 7, 9 and Day 10 (implant withdrawal), every 12 h on Days 11 and 12, and then once daily on Days 17, 24 and 31. All the samples were assayed for progesterone. Almost all (97%) heifers and 81% cows were induced to estrus, the majority (92% heifers and 79% cows) within 120 h of implant removal. Synchrony of the induced estrus was better in cows, but interval to estrus and estrus duration were significantly longer in heifers (P<0.05). Post-treatment fertility, based on Day 28 nonretum rate, first service, and overall conception rates, was better in heifers (78.9, 60.5 and 73.7%, respectively) than cows (77.1, 48.6 and 62.9%, respectively), but the differences were significant only for the overall pregnancy rate (71.8% for heifers and 51.2% for cows; P<0.05). Low pre-treatment plasma progesterone values (<0.5ng/mL) were consistent with ovarian inactivity, confirming the true anestrus status of experimental animals. Controls failed to exhibit estrus and maintained low progesterone concentrations throughout the study. In treated animals, high progesterone values from Day 17 onwards suggested ovulatory estrus. These early luteal phase progesterone concentrations in nonpregnant (P=0.06) and nonpregnant, nonretum (P<0.05) animals were low in comparison with those of pregnant animals. Good fertility resulting from breeding according to estrus, inspite of variable intervals to estrus and estrus duration, advocates its advantage over fixed-time insemination in norgestomet-treated anestrous Zebu cattle.     

Seasonal differences in xanthophyll cycle characteristics and antioxidants in Mahonia repens growing in different light environments

We investigated differences between summer and winter in photosynthesis, xanthophyll cycle-dependent energy dissipation, and antioxidant systems in populations of Mahonia repens (Lindley) Don growing in the eastern foothills of the Colorado Rocky Mountains in deep shade, full exposure, and under a single-layered canopy of Pinus ponderosa (partially shaded). In summer, increasing growth irradiance (from deep shade to partial shade to full exposure) was associated with increased xanthophyll cycle-dependent energy dissipation in PSII and an increased capacity to detoxify reactive reduced oxygen species, as measured by increases in the activities of ascorbate peroxidase, superoxide scavenging, glutathione reductase, and monodehydroascorbate reductase, as well as increases in leaf ascorbate and glutathione content. Leaves of exposed and partially shaded plants exhibited decreased capacities for photosynthetic O₂ evolution in winter compared to summer, while in the deeply shaded plants this parameter did not differ seasonally. Seasonal differences in the levels of antioxidants generally exhibited an inverse response to photosynthesis, being higher in winter compared to summer in the exposed and partially shaded populations, but remaining unchanged in the deeply shaded population. In addition, total pool size and conversion state of the xanthophyll cycle were higher in winter than in summer in all populations. These trends suggest that both xanthophyll cycle-dependent energy dissipation in PSII and the capacity to detoxify reactive reduced oxygen species responded to the level of excess light absorption. Received: 23 October 1997 / Accepted: 23 March 1998     

Plant-mediated interactions between insects and a fungal plant pathogen and the role of plant chemical responses to infection

Diverse organisms simultaneously exploit plants in nature, but most studies do not examine multiple types of exploiters like phytophagous insects and fungal, bacterial, and viral plant pathogens. This study examined patterns of induction of antipathogenic peroxidase enzymes and phenolics after infection by the cucurbit scab fungus, Cladosporium cucumerinum, and then determined if induction mediated ecological effects on Colletotrichum orbiculare, another fungal pathogen, and two insect herbivores, spotted cucumber beetles, and melon aphids. Peroxidase induction occurred in inoculated, `local,' symptom-bearing leaves 3 days after inoculation, and in `systemic,' symptom-free leaves on the same plants 1 day later. Phenolics were elevated in systemic but not in local leaves 3 days after inoculation. Detached systemic leaves from plants inoculated with C. cucumerinum developed significantly fewer and smaller lesions after challenge with C. orbiculare. Spotted cucumber beetles did not show consistently significant preferences for infected versus control leaf disks in comparisons using local or systemic leaves, but trends differed significantly between leaf positions. In no-choice tests, beetles removed more leaf area from local but not from systemic infected leaves compared to control leaves, and melon aphid reproduction was enhanced on local infected leaves. In the field, cucumber beetle and melon aphid densities did not differ between infected and control plants. Antipathogenic plant chemical responses did not predict reduced herbivory by insects. Other changes in metabolism may explain the positive direction and spatially dependent nature of plant-mediated interactions between pathogens and insects in this system. Received: 28 September 1997 / Accepted: 9 February 1998     

Induction of nitrate uptake and nitrate reductase activity in trembling aspen and lodgepole pine

¹³NO₃^– influx into the roots and in vivo nitrate reductase activity (NRA) in the roots and leaves have been measured in trembling aspen (Populus tremuloides Michx.) and lodgepole pine (Pinus contorta Dougl.) seedlings after exposure to either 0·1 or 1·5 mol m^–3 NO₃^– for varying periods up to 20 d. Both NO₃^– influx and NRA were inducible in these species and, in trembling aspen, peak induction of nitrate influx and NRA were achieved within 12 h, compared to 2–4 d for influx and 4–12 d for NRA in lodgepole pine. In trembling aspen, ≈ 30% of the total ¹³N absorbed during a 10 min influx period followed by 2 min of desorption was translocated to the shoot. In lodgepole pine, by contrast, translocation of ¹³N to the shoot was undetectable during the same time period. Root NRA as well as NO₃^– influx from 0·1 mol m^–3 NO₃^– were substantially higher in trembling aspen than in lodgepole pine at all stages of NO₃^– exposure, i.e. during the uninduced, the peak induction, and steady-state stages. In order to examine whether the lower rates of NO₃^– influx and NRA were related to proportionately fewer young (unsuberized) roots in lodgepole pine, we determined these parameters in young and old (suberized) roots of this species separately. Induction of influx and NRA were initially greater in young roots but at steady-state there were only minor differences between the young and the old roots. However, even the elevated initial rates in the young roots of lodgepole pine were substantially lower than those of aspen. In pine, influx at 1·5 mol m^–3 NO₃^– was ~ 6-fold higher than at 0·1 mol m^–3 NO₃^– and appeared to be mostly via a constitutive system. By contrast, in aspen, steady-state influxes at 0·1 and 1·5 mol m^–3 were not significantly different, being similar to the rate attained by pine at only the higher [NO₃^–]. In aspen, leaf NRA was ~ 2-fold higher than that of roots. In lodgepole pine NRA of the needles was below the detection limit. These results show that trembling aspen seedlings are better adapted for NO₃^– acquisition and utilization than lodgepole pine seedlings.     

The effects of light on induction, time courses, and kinetic patterns of net nitrate uptake in barley

Barley seedlings ( Hordeum vulgare L.) were grown hydroponically with (induced) or without (uninduced) nitrate in a light/dark cycle with high photon flux density to determine the effects of light on time courses, induction and kinetics of net nitrate uptake. Nitrate uptake was induced by external nitrate in both light and dark and was prevented by 1 mol m^–3 p-fluorophenylalanine. In high light, nitrate uptake was about 2-fold higher than in low light. During time course experiments the uptake rates oscillated due to daily light–dark changes. Rates of nitrate uptake also increased at about 2200 h during continuous darkness. This increase coincided approximately with the time at which the dark period started during the previous culture of the plants, indicating that it was due to a mechanism associated with an endogenous diurnal rhythm. When calculating the kinetics of nitrate uptake, a model with two saturable systems, including a high-affinity system (HATS) and a low-affinity system (LATS), gave the best fit to data in all treatments. The apparent affinity of the HATS ranged from 7·7 to 12·2 mmol m^–3 in induced plants in all light conditions. The effect of light on the HATS was mainly an increase of apparent V _max in the step from low to high light. In uninduced plants the HATS operated at a very low activity which was strongly enhanced during induction. Interpretation of the calculated kinetics of the LATS was much more difficult on the basis of net uptake data. The apparent affinity of the LATS increased from 24·3 mol m^–3 in low light up to 0·17 mol m^–3 after acceleration in high light. These extreme changes in apparent affinity of the LATS could not be explained satisfactorily, and the nature of this system is also discussed with respect to the method used.     

BRYOPSIS MAXIMA (CHLOROPHYTA) GLUTAMATE DEHYDROGENASE: MULTIPLE GENES AND ISOZYMES

Subcellular localization of glutamate dehydrogenase (GDH) was investigated in the green alga Bryopsis maxima. Both intact and pure chloroplasts and mitochondria were isolated by two methods: successive centrifugation and continuous Percoll density gradient centrifugation. The NADP-dependent GDH activities of the chloroplastic, mitochondrial, and cytosolic portions were estimated as 64.3, 9.8, and 25.9%, respectively, and NAD-dependent GDH activity was observed only in the chloroplasts. Three organelle-specific isozymes—chloroplastic NADP-GDH1, cytosolic/mitochondrial NADP-GDH2, and cytosolic/mitochondrial NADP-GDH3—were purified. The molecular masses of these isozymes were estimated to be the same (280 kDa). K_m values of NADP-GDH1, NADP-GDH2, and NADP-GDH3 for NADPH in the amination reaction were 30, 110, and 34 μM, respectively, and those for NADH were 185, 1490, and 974 μM, respectively, showing different cofactor affinities. Several NADP-GDHs and one NAD-GDH were induced in the chloroplasts during incubation of the collected thalli in either continuous light or darkness in aerated seawater for 0 to 5 days, whereas the cytosolic and mitochondrial NADP-GDHs decreased to an almost undetectable level in 5 days. Two distinct DNA fragments (BmF-1 and BmF-2) encoding B. maxima Okamura GDH were identified and sequenced. They showed 90% homology in their deduced amino acid sequences, whereas synonymous nucleotide substitution was observed in the third position of 52% of the codons. Genomic Southern analysis suggested that the two genes are located at two different loci on the B. maxima chromosome. Thus, B. maxima GDH has been confirmed to be multiple in terms of both protein and gene. The localization of other nitrogen-assimilating enzymes was also determined. Glutamine synthetase was located in the chloroplasts and the cytosol, glutamate synthase was located in the chloroplasts, and nitrate reductase was located in the cytosol.