Different responses of two contrasting wheat genotypes to abscisic acid application

Purpose of this study was to investigate different responses of two wheat genotypes (Triticum aestivum L.) from the wet and dry climate regions to exogenous abscisic acid (ABA) application under well-watered and water-stressed conditions. Exogenous ABA was applied to the leaves by spraying and changes in dry matter accumulation and allocation, endogenous ABA content and carbon isotope ratio (δ¹³C) were monitored. The ABA application significantly decreased stem height, total biomass, total leaf area, total grain mass and leaf area/mass ratio, and significantly increased root/aboveground biomass ratio, endogenous ABA content and δ¹³C under well-watered and water-stressed conditions. Compared with the wet climate genotype, the dry climate genotype was more responsive to exogenous ABA application, resulting in lower stem height, total biomass, total leaf area, total grain mass and leaf area/mass ratio, and higher root/aboveground biomass ratio, endogenous ABA content and δ¹³C under all experimental treatments.The research was supported by the Program of “100 Distinguished Young Scientists” and “ Knowledge Innovation Engineering” of the Chinese Academy of Sciences (No. KSCX2-SW-115).     

Leaf traits and photosynthetic parameters of saplings and adult trees of co-existing species in a temperate broad-leaved forest

In Central European forestry the establishment of broad-leaved mixed forests is attaining increasing importance, but little information exists about gas exchange characteristics of some of the tree species involved, which are less abundant today. In an old-growth forest in Central Germany (Hainich, Thuringia), (i) I compared morphological and chemical leaf traits that are indicative of leaf gas exchange characteristics among eight co-existing species, and (ii) analysed photosynthetic parameters of saplings and adult trees (lower and upper canopy level) in four of these species (Acer pseudoplatanus L., Carpinus betulus L., Fraxinus excelsior L. and Tilia platyphyllos Scop.).Leaves from the upper canopy in the eight species studied varied significantly in their specific leaf area (12.9–19.4 m² kg⁻¹), stomatal density (125–313 stomata mm⁻²), leaf nitrogen concentration (95–157 mmol N m⁻²) and δ¹³C content (–27.81 to –25.85‰). F. excelsior and C. betulus were largely contrasting species, which suggests that the species, which were studied in more detail, include the widest difference in leaf gas exchange among the co-existing species. The saplings of the four selected species exhibited shade acclimated leaves with net photosynthesis rates at saturating irradiance (A_max) between 5.0 and 6.4 μmol m⁻² s⁻¹. In adult trees A_max of fully sunlit leaves was more variable and ranged from 10.5 (C. betulus) to 16.3 μmol m⁻² s⁻¹ (F. excelsior). However, less negative δ¹³C values in F. excelsior sun leaves point to a strong limitation in gas exchange. In the lower canopy of adult trees A_max of F. excelsior (12.0 μmol m⁻² s⁻¹) was also greater than that of A. pseudoplatanus, C. betulus and T. platyphyllos (5.0–5.6 μmol m⁻² s⁻¹). This can be explained by the small leaf area and the absence of shade leaves in mature F. excelsior trees. Thus, a considerable variation in leaf traits and gas exchange was found among the co-existing tree species. The results suggest that species-specific characteristics increase the spatial heterogeneity of canopy gas exchange and should be taken into account in the interpretation and prediction of gas flux from mixed stands.In der Forstwirtschaft Mitteleuropas gewinnt die Begründung von Laubmischwäldern zunehmend an Bedeutung, aber über Eigenschaften im Gasaustausch einiger beteiligter Baumarten, die heute nicht so häufig sind, ist wenig bekannt. In einem Altbestand in Mitteldeutschland (Hainich, Thüringen) habe ich (i) morphologische und chemische Eigenschaften von Sonnenblättern, die Hinweise auf Charakteristika im Blattgaswechsel geben, an acht koexistierenden Baumarten untersucht, und (ii) Photosyntheseparameter von juvenilen und adulten Bäumen (unteres und oberes Kronenniveau) von vier dieser Arten (Acer pseudoplatanus L., Carpinus betulus L., Fraxinus excelsior L. and Tilia platyphyllos Scop.) erhoben.Blätter aus dem oberen Kronenraum der acht untersuchten Arten variierten signifikant in der spezifischen Blattfläche (12.9–19.4 m² kg⁻¹), der Stomatadichte (125–313 Stomata mm⁻²), dem Blattstickstoffgehalt (95–157 mmol N m⁻²) und den δ¹³C-Werten (–27.81 bis –25.85‰). In diesem Kollektiv zeigten F. excelsior und C. betulus groβe Unterschiede, was darauf hindeutet, dass die Arten, die genauer untersucht wurden, die Spannweite an Gaswechseleigenschaften unter den koexistierenden Baumarten umfassen. Die Jungpflanzen der vier ausgewählten Arten besaßen Schattenblätter, deren Netto-Photosyntheserate bei hoher Lichtintensität (A_max) zwischen 5.0 and 6.4 μmol m⁻² s⁻¹ variierte. An Sonnenblättern von Altbäumen war A_max variabler und lag zwischen 10.5 (C. betulus) und 16.3 μmol m⁻² s⁻¹ (F. excelsior). Allerdings weisen hohe δ¹³C-Werte in Sonnenblättern von F. excelsior auf eine starke Limitierung des Gasaustauschs hin. Auch in der unteren Krone der Altbäume war A_max von F. excelsior (12.0 μmol m⁻² s⁻¹) höher als A_max von A. pseudoplatanus, C. betulus und T. platyphyllos (5.0–5.6 μmol m⁻² s⁻¹). Dies kann durch die geringe Blattfläche und die Abwesenheit von Schattenblättern in der Krone adulter Bäume von F. excelsior erklärt werden. Zwischen den koexistierenden Baumarten wurde somit in Bezug auf Blatteigenschaften und Photosyntheseparameter eine erhebliche Variation festgestellt. Die Ergebnisse legen nahe, dass artspezifische Eigenschaften die räumliche Heterogenität des Gaswechsels im Kronenraum erhöhen und bei der Interpretation und Vorhersage von Gasflüssen über Mischbeständen berücksichtigt werden sollten.     

Organ-specific changes in the activity and subunit composition of glutamine-synthetase isoforms of barley (Hordeum vulgare L.) after growth on different levels of NH+ 4

One cytosolic glutamine synthetase (GS, EC 6.3.1.2) isoform (GS 1a) was active in the germinating seeds of barley (Hordeum vulgare L.). A second cytosolic GS isoform (GS 1b) was separated from the leaves as well as the roots of 10-d-old seedlings. The chloroplastic isoform (GS 2) was present and active only in the leaves. The three GS isoforms were active in N-supplied (NH⁺ ₄ or NO ₃ ^– ) as well as in N-free-grown seedlings. This indicates (i) that a supply of nitrogen to the germinating seeds was not necessary for the induction of the GS isoforms and (ii) that no nitrogen-specific isoforms appeared during growth of seedlings with different nitrogen sources. The activity of GS, however, depended on the seedlings' nitrogen source: the specific activity was much higher in the leaves and much lower in the roots of NH⁺ ₄-grown barley than in the respective organs of NO ₃ ^– -fed or N free-grown plants. With increasing concentrations of NH⁺ ₄ (supplied hydroponically during growth), the specific activity of GS 1b increased in the leaves, but decreased in the roots. The activity of GS 2 (leaf) also increased with increasing NH⁺ ₄ supply, whereas GS 1a activity (leaf and root) was not affected. The changes in the activities of GS 1b and GS 2 were correlated with changes in the subunit compositions of the active holoenzymes: growth at increased levels of external NH⁺ ₄ resulted in an increased abundance of one of the four GS subunits, and of two of the five GS 1b subunits in the leaves. In the roots, however, the abundance of these two GS 1b subunits was decreased under the same growth conditions, indicating an organ-specific difference either in the expression of the genes coding for the respective GS 1b subunits or in the assembly of the GS 1b holoenzymes. Furthermore, growth at different levels of NH⁺ ₄ resulted in changes in the substrate affinities of the isoforms GS 1b (root and leaf) and GS 2 (leaf), presumably due to the changes in the subunit compositions of the active holoenzymes.Abbreviations FPLC fast protein liquid chromatography - GHA -glutamyl hydroxamate - GS glutamine synthetase Dr. Roger Wallsgrove's (Rothamsted Experimental Station, Harpenden, UK) generous gift of GS antiserum is greatly appreciated.     

Physiological comparisons of two soybean cultivars differing in canopy photosynthesis. I. Variation in vertical 14CO2 labelling and dry weight partitioning

Differences in canopy apparent photosynthesis (CAP) among soybean [Glycine max (L.) Merr.] genotypes have been shown to be correlated to seed yields. Since the physiological basis for such differences in CAP is unknown, two cultivars known to differ in CAP, Tracy and Davis, were studied during the 1978–1980 growing seasons. The CAP and dry weights of component plant parts were determined. In 1978 and 1979, ¹⁴CO₂ uptake by vertical leaf strata was determined and specific leaf weight (SLW) and leaf area index (LAI) were determined for corresponding strata in 1979 and 1980. Measurements were taken on several dates during reproductive growth. With the exception of CAP, all measurements (¹⁴C uptake, dry weights) were made in layers within the canopy. CAP on some dates were significantly higher in Tracy than in Davis and integrated CAP values from a certain growth period, labeled as R5 to R7, averaged 16 percent higher in Tracy for the three years studied. No differences in the relative recovery of ¹⁴C from different layers of leaves in the canopy were found. This indicates that variations in canopy structure or leaf orientation did not play a major role in the CAP differences between cultivars. The differences seem related to variations in leaf dry weights. Overall, Tracy exhibited 13.5, 19.2, and 13.2 percent greater leaf dry weights than Davis during 1978, 1979, and 1980, respectively. These differences in leaf dry weight seem largely due to a differences in the SLW. Data from these experiments indicate that differences in soybean CAP values were associated with differences in SLW.Abbreviations CAP Canopy Apparent Photosynthesis - CER Carbondioxide Exchange Rates - EST Eastern Standard Time - LAI Leaf Area Index - LSD Least Significant Difference - POPOP 1,4-bis-[2(5-phenyloxazdyl)]-benzene - PPO 2,5-diphenyloxazole - SLW Specific Leaf Weight     

Purification and characterisation of the cardenolide-specificβ-glucohydrolase CGH II from Digitalis lanata leaves

A cardenolide-hydrolysing β-D-glucosidase was isolated from young leaves of Digitalis lanata. Since this enzyme differs from the cardenolide glucohydrolase (CGH) described and characterised previously, it was termed cardenolide glucohydrolase II (CGH II). CGH II was detected in various Digitalis tissue cultures as well as in young leaves of D. lanata. The latter source was used as the starting material for the isolation and purification of CGH II. The specific enzyme activity reached about 15 pkat·mg^–1 protein in buffered leaf extracts. Optimal CGH II activity was seen at around pH 6.0 and 50 °C. CGH II was purified about 600-fold by anion exchange chromatography, size exclusion chromatography and hydroxyapatite chromatography. The apparent molecular mass of CGH II was 65 kDa as determined by SDS-PAGE. CGH II exhibited a high substrate specificity towards cardenolide disaccharides, especially to those with a 1-4-β-linked glucose-digitoxose moiety such as glucoevatromonoside. The K_m- and V_max-values for this particular substrate were calculated to be 101 μM and 19.8 nkat·mg^–1 protein, respectively.     

Resource use by salmonids in riverine,lacustrine and marine environments: Evidence from stable isotope analysis

Synopsis We measured stable isotope ratios (δ¹³C and δ¹⁵N) of invertebrates, Atlantic salmon, Salmo salar, and brook trout, Salvelinus fontinalis, in three distinct freshwater environments (headwater tributary, ultra-oligotrophic lake, and main-stem river) in the Western Brook system, Newfoundland, Canada. Large differences in the stable carbon signatures of invertebrates allowed the identification of organic matter assimilation from each environment by resident parr and migrating smolts. Brook trout captured in the headwater tributary in June had a carbon signature characteristic of the tributary, while those collected in August had enriched ¹³C (maximum = −15.6‰) and ¹⁵N (maximum = 12.8‰) values. These enriched carbon and nitrogen signatures were indicative of foraging at sea. There was a low correlation between δ¹³C and δ¹⁵N (r² = 0.198) for individual fish that was likely due to the confounding influence of trout feeding in the lake and the lower main-stem of the river, where δ¹³C of food sources was high but δ¹⁵N was low. Smolts emigrating from Western Brook Pond where they had been foraging (based on lacustrine carbon signatures) were significantly larger than those emigrating from a nursery brook and the main river in the same basin, despite having the same median age. These results suggest better growth opportunities in the lake environment. Trout fork length was positively correlated with δ¹³C and δ¹⁵N, demonstrating that larger individuals had been feeding outside the brook. These results support previous studies that found increased growth potential for salmonids in lacustrine and marine environments, and further, indicate possible adaptive advantages for salmonid movement away from natal brooks.     

The importance of seed reserves for seedling performance: an integrated approach using morphological,physiological, and stable isotope techniques

To investigate how seed reserves affect early seedling performance, we conducted a factorial greenhouse experiment using Lithocarpus densiflora (Tanoak). Seedlings were grown from large (5.8±0.7 g) and small (3.2±0.4 g) seeds and, following shoot emergence, seeds were either removed or left attached. Seedlings were harvested for quantification of biomass and ¹³C at seven time periods following seed removal (2, 4, 8, 16, 32, 64, 128 days) and seedling photosynthesis was measured three separate time periods (2–4, 49–82, 95–128 days after seed removal). Biomass increased for all seedlings, but the increase was significantly larger for seedlings with attached seeds than with removed seeds. Seed removal just after shoot emergence significantly decreased seedling biomass, but seed removal 64 days after shoot emergence had no effect on seedling biomass. Seedling photosynthesis per unit leaf area varied by time and seed presence, but not by seed size. At the first period, seedlings with attached seeds had significantly higher photosynthetic rates than seedlings with removed seeds, at the second period there was no effect of seed removal, and at the third time period seedlings with attached seeds had significantly lower photosynthetic rates than seedlings with removed seeds. Despite temporal variation in photosynthesis per unit leaf area, seedlings with attached seeds always had significantly greater leaf area than seedlings with removed seeds, resulting in significantly higher total plant photosynthesis at all three time periods. The ¹³C values of both the leaves and roots were more similar to that of the seed for seedlings with attached seeds than for seedlings with removed seeds, however, seed removal and seed size strongly affected root ¹³C. This study demonstrates that seed reserves have important effects on the early growth, physiology, and ¹³C of L. densiflora seedlings.     

Organic matter rain rates, oxygen availability, and vital effects from benthic foraminiferal δC in the historic Skagerrak, North Sea

The sediment cores 225514 and 225510 were recovered from 420 and 285 m water depth, respectively. They were investigated for their benthic foraminiferal δ¹³C during the last 500 years. Both cores were recovered from the southern flank of the Skagerrak. The δ¹³C values of Uvigerina mediterranea and other shallow infaunal species in both cores indicate that organic matter rain rates to the seafloor varied around a mean value until approximately AD 1950 after which they increased. This increase might result from changes in the North Atlantic Current System and a co-occurring persistently high North Atlantic Oscillation index state in the 1980s to 1990s, rather than from anthropogenic eutrophication. Using δ¹³C mean values of multiple species, we reconstruct δ¹³C gradients of dissolved inorganic carbon (DIC) within pore waters for the time periods AD 1500 to 1950 and AD 1950 to 2000. The calculated δ¹³C_DIC ranges, interpreted as indicating total organic matter remineralization due to respiration, are generally bigger in Core 225514 than in Core 225510. Since mean δ¹³C values of U. mediterranea suggest that organic matter rain rates were similar at both locations, differences in total organic matter remineralization are attributed to differing oxygen availability. However, oxygen concentrations in the overlying bottom water masses are not likely to have differed significantly. Thus, we suggest that organic matter remineralization was controlled by oxygen availability within the sediments, reflecting strong differences in sedimentation rates at the two investigated core sites. Based on the assumptions that tests of benthic foraminiferal species inhabiting the same microhabitat depth should show equal δ¹³C values unless they are affected by vital effects and that Globobulimina turgida records pore water δ¹³C_DIC, we estimate microhabitat-corrected vital effects for several species with respect to G. turgida: > 0.7‰ for Cassidulina laevigata, > 1.3‰ for Hyalinea balthica, and > 0.7‰ for Melonis barleeanus. Melonis zaandami seems to closely record pore water δ¹³C_DIC.     

Oxygen isotope values in bone carbonate and collagen are consistently offset for New World monkeys

Stable oxygen isotopes are increasingly used in ecological research. Here, I present oxygen isotope (δ¹⁸O) values for bone carbonate and collagen from howler monkeys (Alouatta palliata), spider monkeys (Ateles geoffroyi) and capuchins (Cebus capucinus) from three localities in Costa Rica. There are apparent differences in δ¹⁸O_carbonate and δ¹⁸O_collagen among species. Monkeys from moist forest have significantly lower isotope values than those from drier localities. Because patterns are similar for both substrates, discrimination (Δ) between δ¹⁸O_carbonate and δ¹⁸O_collagen is relatively consistent among species and localities (17.6 ± 0.9‰). Although this value is larger than that previously obtained for laboratory rats, consistency among species and localities suggests it can be used to compare δ¹⁸O_carbonate and δ¹⁸O_collagen for monkeys, and potentially other medium-bodied mammals. Establishing discrimination for oxygen between these substrates for wild monkeys provides a foundation for future environmental and ecological research on modern and ancient organisms.     

Intraspecific differences in metabolic rates shape carbon stable isotope trophic discrimination factors of muscle tissue in the common teleost Eurasian perch (Perca fluviatilis)

1. Stable isotopes represent a unique approach to provide insights into the ecology of organisms. δ¹³C and δ¹⁵N have specifically been used to obtain information on the trophic ecology and food‐web interactions. Trophic discrimination factors (TDF, Δ¹³C and Δ¹⁵N) describe the isotopic fractionation occurring from diet to consumer tissue, and these factors are critical for obtaining precise estimates within any application of δ¹³C and δ¹⁵N values. It is widely acknowledged that metabolism influences TDF, being responsible for different TDF between tissues of variable metabolic activity (e.g., liver vs. muscle tissue) or species body size (small vs. large). However, the connection between the variation of metabolism occurring within a single species during its ontogeny and TDF has rarely been considered.
2. Here, we conducted a 9‐month feeding experiment to report Δ¹³C and Δ¹⁵N of muscle and liver tissues for several weight classes of Eurasian perch (Perca fluviatilis), a widespread teleost often studied using stable isotopes, but without established TDF for feeding on a natural diet. In addition, we assessed the relationship between the standard metabolic rate (SMR) and TDF by measuring the oxygen consumption of the individuals.
3. Our results showed a significant negative relationship of SMR with Δ¹³C, and a significant positive relationship of SMR with Δ¹⁵N of muscle tissue, but not with TDF of liver tissue. SMR varies inversely with size, which translated into a significantly different TDF of muscle tissue between size classes.
4. In summary, our results emphasize the role of metabolism in shaping‐specific TDF (i.e., Δ¹³C and Δ¹⁵N of muscle tissue) and especially highlight the substantial differences between individuals of different ontogenetic stages within a species. Our findings thus have direct implications for the use of stable isotope data and the applications of stable isotopes in food‐web studies.

     

Investigation of the porphyrins accumulation in barley leaves incubated with Mn cations and δ-aminolevulinic acid

In greening etiolated primary leaves of barley (Hordeum vulgare L.), Mn²⁺ ions have been shown to inhibit chlorophyll (Chl) accumulation in a dose dependent manner and to lead to an accumulation of protoporphyrin IX (Proto) and Mg-protoporphyrin IX monomethyl ester (MgPE). The amount of MgPE that accumulated, was 2 times higher than Proto. In the dark, Proto and MgPE were observed to have accumulated to high levels in seven-day old green and etiolated leaves in the presence of 5 mmol/L Mn²⁺, but only if 5 mmol/L δ-aminolevulinic acid (ALA) was present. The 24 hours of irradiation of the green barley leaves treated in this way, resulted in a photodynamic destruction of Proto and MgPE as well as of Chl and carotenoids (Car). The observed porphyrin accumulation caused by the Mn²⁺ ions was reversed in the presence of active iron (Fe²⁺). This effect was observed when the iron concentration in incubation solutions was half the Mn²⁺ concentration, most effective for porphyrin synthesis, i.e. 5 mmol/L. The action of Mn²⁺ on porphyrin accumulation is also discussed.     

不同模拟增雨下白刺比叶面积和叶干物质含量的比较

以荒漠生态系统典型植物白刺(Nitraria tangutorum)为研究对象,根据内蒙古磴口多年平均降水量和植物生长规律,设计两个增雨时段(生长季前期与生长季后期),每个增雨时段设置两个增雨梯度(72.5mm/a(50%)、145mm/a(100%)),对天然白刺灌丛进行增雨实验,研究了不同模拟增雨处理下2012年与2013年生长季白刺叶片的比叶面积(SLA)与叶干物质含量(LDMC)的变化。结果表明,增雨处理可以增加白刺叶片的SLA及LDMC,同时期增雨100%处理对SLA及LDMC的影响大于50%处理,但同时期增雨的两个处理之间无显著差异;白刺叶片SLA在生长季前期对水分响应明显,LDMC则在生长季后期对水分反应敏感;相同增雨处理,2012年白刺叶片SLA及LDMC的净增加值高于2013年;SLA与LDMC在2012年呈显著负相关,在2013年虽呈负相关,但相关性不显著。在未来降雨增加的背景下,荒漠植物白刺叶片SLA与LDMC对增雨具有较强的协调适应能力,在不同生长季节可以通过改变不同的叶片性状来适应环境变化。     

The influence of nitrogen concentration and ammonium/nitrate ratio on N-uptake,mineral composition and yield of citrus

In short-term water culture experiments with different ¹⁵N labeled ammonium or nitrate concentrations, citrus seedlings absorbed NH₄ ⁺ at a higher rate than NO₃ ^–. Maximum NO₃ ^– uptake by the whole plant occurred at 120 mg L^–1 NO₃ ^–-N, whereas NH₄ ⁺ absorption was saturated at 240 mg L^–1 NH₄ ⁺-N. ¹⁵NH₄ ⁺ accumulated in roots and to a lesser degree in both leaves and stems. However, ¹⁵NO₃ ^– was mostly partitioned between leaves and roots.Adding increasing amounts of unlabeled NH₄ ⁺ (15–60 mg L^–1 N) to nutrient solutions containing 120 mg L^–1 N as ¹⁵N labeled nitrate reduced ¹⁵NO₃ ^– uptake. Maximum inhibition of ¹⁵NO₃ ^– uptake was about 55% at 2.14 mM NH₄ ⁺ (30 mg L^–1 NH₄ ⁺-N) and it did not increase any further at higher NH₄ ⁺ proportions.In a long-term experiment, the effects of concentration and source of added N (NO₃ ^– or NH₄ ⁺) on nutrient concentrations in leaves from plants grown in sand were evaluated. Leaf concentration of N, P, Mg, Fe and Cu were increased by NH₄ ⁺ versus NO₃ ^– nutrition, whereas the reverse was true for Ca, K, Zn and Mn.The effects of different NO₃ ^–-N:NH₄ ⁺-N ratios (100:0, 75:25, 50:50, 25:75 and 0:100) at 120 mg L^–1 total N on leaf nutrient concentrations, fruit yield and fruit characteristics were investigated in another long-term experiment with plants grown in sand cultures. Nitrogen concentrations in leaves were highest when plants were provided with either NO₃ ^– or NH₄ ⁺ as a sole source of N. Lowest N concentration in leaves was found with a 75:25 NO₃ ^–-N/NH₄ ⁺-N ratio. With increasing proportions of NH₄ ⁺ in the N supply, leaf nutrients such as P, Mg, Fe and Cu increased, whereas Ca, K, Mn and Zn decreased. Yield in number of fruits per tree was increased significantly by supplying all N as NH₄ ⁺, although fruit weight was reduced. The number of fruits per tree was lowest with the 75:25 NO₃ ^–-N:NH₄ ⁺-N ratio, but in this treatment fruits reached their highest weight. Rind thickness, juice acidity, and colour index of fruits decreased with increasing NH₄ ⁺ in the N supply, whereas the % pulp and maturity index increased. Percent of juice in fruits and total soluble solids were only slightly affected by NO₃ ^–:NH₄ ⁺ ratio.     

Seasonal reversibility of acclimation to irradiance in leaves of common box (Buxus sempervirens L.) in a deciduous forest

Understorey shade plants are seasonally exposed to dramatic changes in light conditions in deciduous forests related with the dynamics of the overstorey leaf phenology. These transitions are commonly followed by changes in herb plant communities, but shade-tolerant evergreen species must be able to adapt to changing light conditions. In this work we checked the photoprotective responses of evergreen species to acclimate to the shady summer environment and reversibly de-acclimate to a more illuminated environment after leaf fall on deciduous overstoreys. For that purpose we have followed the process of light acclimation in leaves of common box (Buxus sempervirens) during the winter to spring transition, which decrease irradiance in the understorey, and conversely during the transition from summer to autumn. Four parameters indicative of the structure and degree of acclimation of the photosynthetic apparatus have been studied: chlorophyll a/b ratio which is supposed to be inversely proportional to the antenna size, α/β-carotene which increases in shade acclimated leaves and the pools of α-tocopherol and xanthophyll cycle pigments (VAZ) which are two of the main photoprotection mechanisms in plants. Among these parameters, chlorophyll a/b ratio and VAZ pool responded finely to changes in irradiance indicating that modifications in the light harvesting size and photoprotective capacity contribute to the continuous acclimation and de-acclimation of long-lived evergreen leaves.     

Analysis of N-methylhistamine by gas chromatography–mass spectrometry

A gas chromatography–electron capture mass spectrometry assay has been developed for the histamine H₃ receptor agonist, N^α-methylhistamine (N^α-MH). The assay is linear from 50 pg–10 ng, with a limit of detection of 50 pg/ml for gastric juice and plasma, and 50 pg/sample for bacteria (10⁷–10⁸ CFU) and gastric tissue (5–10 mg wet weight). The limits of quantification are 100 pg/ml for gastric juice (%RSD=1.4) and plasma (%RSD=9.4), and 100 pg/sample for bacteria (%RSD=3.9) and tissue (%RSD=5.8). N^α-MH was not present in human plasma, but low levels (1.4 ng/ml and 0.4 ng/ml) were detected in two samples of human gastric juice obtained from patients infected with Helicobacter pylori.     

Up-regulation of Brain N-Methyl- -aspartate Receptors Following Multiple Intracerebro- ventricular Injections of [ -Pen, -Pen] Enkephalin and [ -Ala, Glu]deltorphin II in Mice

Bhargava, H. N., S. Kumar and J. T. Bian. Up-regulation of brain N-methyl- -aspartate receptors following multiple intracerebroventricular injections of [ -Pen², -Pen⁵]enkephalin and [ -Ala², Glu⁴]deltorphin II in mice. Peptides 18(10) 1609–1613, 1997.—The effects of chronic administration of [ -Pen², -Pen⁵]enkephalin and [ -Ala², Glu⁴]deltorphin II, the selective agonists of the δ₁- and δ₂-opioid receptors, on the binding of [³H]MK-801, a noncompetitive antagonist of the N-methyl- -aspartate receptor, were determined in several brain regions of the mouse. Male Swiss-Webster mice were injected intracerebroventricularly (i.c.v.) with [ -Pen², -Pen⁵]enkephalin or [ -Ala², Glu⁴]deltorphin II (20 μg/mouse) twice a day for 4 days. Vehicle injected mice served as controls. Previously we have shown that the above treatment results in the development of tolerance to their analgesic activity. The binding of [³H]MK-801 was determined in brain regions (cortex, midbrain, pons and medulla, hippocampus, striatum, hypothalamus and amygdala). At 5 nM concentration, the binding of [³H]MK-801 was increased in cerebral cortex, hippocampus, and pons and medulla of [ -Pen², -Pen⁵]enkephalin treated mice. In [ -Ala², Glu⁴]deltorphin II treated mice, the binding of [³H]MK-801 was increased in cerebral cortex and hippocampus. The changes in the binding were due to increases in the B_max value of [³H]MK-801. It is concluded that tolerance to δ₁- and δ₂-opioid receptor agonists is associated with up-regulation of brain N-methyl- -aspartate receptors, however, some brain areas affected differ with the two treatments. The results are consistent with the recent observation from this laboratory that N-methyl- -aspartate receptors antagonists block tolerance to the analgesic action of δ₁- and δ₂-opioid receptor agonists.     

CFTR mediates bicarbonate-dependent activation of miR-125b in preimplantation embryo development

Although HCO₃⁻ is known to be required for early embryo development, its exact role remains elusive. Here we report that HCO₃⁻ acts as an environmental cue in regulating miR-125b expression through CFTR-mediated influx during preimplantation embryo development. The results show that the effect of HCO₃⁻ on preimplantation embryo development can be suppressed by interfering the function of a HCO₃⁻-conducting channel, CFTR, by a specific inhibitor or gene knockout. Removal of extracellular HCO₃⁻ or inhibition of CFTR reduces miR-125b expression in 2 cell-stage mouse embryos. Knockdown of miR-125b mimics the effect of HCO₃⁻ removal and CFTR inhibition, while injection of miR-125b precursor reverses it. Downregulation of miR-125b upregulates p53 cascade in both human and mouse embryos. The activation of miR-125b is shown to be mediated by sAC/PKA-dependent nuclear shuttling of NF-κB. These results have revealed a critical role of CFTR in signal transduction linking the environmental HCO₃⁻ to activation of miR-125b during preimplantation embryo development and indicated the importance of ion channels in regulation of miRNAs.     

Isolation and Ex Vivo Culture of Vδ1+CD4+γδ T Cells,an Extrathymic αβT-cell Progenitor

The thymus, the primary organ for the generation of αβ T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of αβT cells. Yet, thymic involution begins early in life leading to a drastically reduced output of naïve αβT cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic αβT cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. γδ T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a γδ T cell subset by showing that the scarce entity of CD4⁺ Vδ1⁺γδ T cells can transdifferentiate into αβT cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. Vδ1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4⁺ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4⁺ Vδ1⁺γδ T cells. This knowledge is prerequisite for studies that relate to this αβT cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.     

Effects of β-glucanase supplementation of barley diets on growth performance of broilers

A trial was performed to examine the effects of levels of barley substitution and supplementation with β-glucanase in a corn–soybean diet on growth performance and intestinal characteristics of broiler chickens. The experiment involved five levels of barley substituted for corn (0, 125, 250, 500, and 1000 g/kg) and two levels of β-glucanase supplement (with 0.5 g/kg and without) in a factorial arrangement with two replicates. Four hundred day-old commercial strain Harber broiler chickens were randomly allocated into twenty groups of ten dietary treatments for a six-week feeding trial, growing (0–3 week) and finishing period (4–6 week). Unless supplemented with β-glucanase, broilers receiving the diet with more than 250 g/kg of the barley substitute gained slower during the growth period. Conversely, supplementing β-glucanase did not improve total weight gain (0–6 weeks) with a diet of 500 g/kg barley substitution. As the level of barley substitution increased, feed intake in the growing period decreased significantly and viscosity of the intestinal contents increased. However, such an increase did not significantly influence feed conversion (P>0.05). Supplementation with β-glucanase on diet up to 250 g/kg of barley substitute not only enhanced body weight gains of growing broilers, but also improved the live-weight of six-week-old broilers (P<0.05).