Role of allatostatin‐like factors from the brain of Tenebrio molitor females

The effect of brain extract from females of freshly emerged Tenebrio molitor on ovary, oocyte development, total protein content of hemolymph, and ovary was studied in 4‐day‐old adult mealworm females. Injections of extracts of 2‐brain equivalents into intact (unligatured) Tenebrio females did not affect ovarian and oocyte development. Injections of ligated females, however, with 2‐brain equivalents on day 1 and 2 after adult emergence strongly inhibited ovarian growth and oocyte development. At day 4, ligated and injected females did not develop their ovaries and pre‐vitellogenic oocytes were not found. The changes in ovarian development correlated with an increase in the concentration of soluble proteins in the hemolymph as compared with the saline‐injected controls. Additionally, a strong reduction of total protein content in ovarian tissue was observed. Reverse phase HPLC separation of a methanolic brain extract of T. molitor females showed that fraction 5 has a similar retention time to synthetic cockroach allatostatin. Fraction 5 was eluted at 12.88 min, which was closest to the internal standard Dippu‐AST I, which eluted at 12.77 min. An ELISA of fraction 5 from the methanolic brain extract using antibodies against allatostatins Grybi‐AST A1 and Grybi‐AST B1 from cricket Gryllus bimaculatus showed that fraction 5 cross‐reacted with Grybi‐AST A1 antibodies. The cross‐reactivity was similar to the synthetic allatostatin from D. punctata, which was used as a positive control. These observations demonstrate a possible role for allatostatin‐like brain factor(s) in regulating the reproductive cycle of Tenebrio molitor. © 2009 Wiley Periodicals, Inc.     

Soil 13C–15N dynamics in an N2-fixing clover system under long-term exposure to elevated atmospheric CO2

Reduced soil N availability under elevated CO₂ may limit the plant's capacity to increase photosynthesis and thus the potential for increased soil C input. Plant productivity and soil C input should be less constrained by available soil N in an N₂‐fixing system. We studied the effects of Trifolium repens (an N₂‐fixing legume) and Lolium perenne on soil N and C sequestration in response to 9 years of elevated CO₂ under FACE conditions. ¹⁵N‐labeled fertilizer was applied at a rate of 140 and 560 kg N ha^?1 yr^?1 and the CO₂ concentration was increased to 60 Pa pCO₂ using ¹³C‐depleted CO₂. The total soil C content was unaffected by elevated CO₂, species and rate of ¹⁵N fertilization. However, under elevated CO₂, the total amount of newly sequestered soil C was significantly higher under T. repens than under L. perenne. The fraction of fertilizer‐N (f_N) of the total soil N pool was significantly lower under T. repens than under L. perenne. The rate of N fertilization, but not elevated CO₂, had a significant effect on f_N values of the total soil N pool. The fractions of newly sequestered C (f_C) differed strongly among intra‐aggregate soil organic matter fractions, but were unaffected by plant species and the rate of N fertilization. Under elevated CO₂, the ratio of fertilizer‐N per unit of new C decreased under T. repens compared with L. perenne. The L. perenne system sequestered more ¹⁵N fertilizer than T. repens: 179 vs. 101 kg N ha^?1 for the low rate of N fertilization and 393 vs. 319 kg N ha^?1 for the high N‐fertilization rate. As the loss of fertilizer‐¹⁵N contributed to the ¹⁵N‐isotope dilution under T. repens, the input of fixed N into the soil could not be estimated. Although N₂ fixation was an important source of N in the T. repens system, there was no significant increase in total soil C compared with a non‐N₂‐fixing L. perenne system. This suggests that N₂ fixation and the availability of N are not the main factors controlling soil C sequestration in a T. repens system.     

Linking sequestration of 13C and 15N in aggregates in a pasture soil following 8 years of elevated atmospheric CO2

The influence of N availability on C sequestration under prolonged elevated CO₂ in terrestrial ecosystems remains unclear. We studied the relationships between C and N dynamics in a pasture seeded to Lolium perenne after 8 years of elevated atmospheric CO₂ concentration (FACE) conditions. Fertilizer‐¹⁵N was applied at a rate of 140 and 560 kg N ha^2?1 y^2?1 and depleted ¹³C‐CO₂ was used to increase the CO₂ concentration to 60 Pa pCO₂. The ¹³C–¹⁵N dual isotopic tracer enabled us to study the dynamics of newly sequestered C and N in the soil by aggregate size and fractions of particulate organic matter (POM), made up by intra‐aggregate POM (iPOM) and free light fraction (LF). Eight years of elevated CO₂ did not increase total C content in any of the aggregate classes or POM fractions at both rates of N application. The fraction of new C in the POM fractions also remained largely unaffected by N fertilization. Changes in the fractions of new C and new N (fertilizer‐N) under elevated CO₂ were more pronounced between POM classes than between aggregate size classes. Hence, changes in the dynamics of soil C and N cycling are easier to detect in the POM fractions than in the whole aggregates. Within N treatments, fractions of new C and N in POM classes were highly correlated with more new C and N in large POM fractions and less in the smaller POM fractions. Isotopic data show that the microaggregates were derived from the macro‐aggregates and that the C and N associated with the microaggregates turned over slower than the C and N associated with the macroaggregates. There was also isotopic evidence that N immobilized by soil microorganisms was an important source of N in the iPOM fractions. Under low N availability, 3.04 units of new C per unit of fertilizer N were sequestered in the POM fractions. Under high N availability, the ratio of new C sequestered per unit of fertilizer N was reduced to 1.47. Elevated and ambient CO₂ concentrations lead to similar ¹⁵N enrichments in the iPOM fractions under both low and high N additions, clearly showing that the SOM‐N dynamics were unaffected by prolonged elevated CO₂ concentrations.     

Above- and below-ground responses of C3–C4 species mixtures to elevated CO2 and soil water availability

We evaluated the influences of CO₂[Control, ～ 370 µ mol mol ^{? 1}; 200 µ mol mol ^{? 1} above ambient applied by free‐air CO₂ enrichment (FACE)] and soil water (Wet, Dry) on above‐ and below‐ground responses of C₃ (cotton, Gossypium hirsutum) and C₄ (sorghum, Sorghum bicolor) plants in monocultures and two density mixtures. In monocultures, CO₂ enrichment increased height, leaf area, above‐ground biomass and reproductive output of cotton, but not sorghum, and was independent of soil water treatment. In mixtures, cotton, but not sorghum, above‐ground biomass and height were generally reduced compared to monocultures, across both CO₂ and soil water treatments. Density did not affect individual plant responses of either cotton or sorghum across the other treatments. Total (cotton + sorghum) leaf area and above‐ground biomass in low‐density mixtures were similar between CO₂ treatments, but increased by 17–21% with FACE in high‐density mixtures, due to a 121% enhancement of cotton leaf area and a 276% increase in biomass under the FACE treatment. Total root biomass in the upper 1.2 m of the soil was not influenced by CO₂ or by soil water in monoculture or mixtures; however, under dry conditions we observed significantly more roots at lower soil depths ( > 45 cm). Sorghum roots comprised 81–85% of the total roots in the low‐density mixture and 58–73% in the high‐density mixture. CO₂‐enrichment partly offset negative effects of interspecific competition on cotton in both low‐ and high‐density mixtures by increasing above‐ground biomass, with a greater relative increase in the high‐density mixture. As a consequence, CO₂‐enrichment increased total above‐ground yield of the mixture at high density. Individual plant responses to CO₂ enrichment in global change models that evaluate mixed plant communities should be adjusted to incorporate feedbacks for interspecific competition. Future field studies in natural ecosystems should address the role that a CO₂‐mediated increase in C₃ growth may have on subsequent vegetation change.     

Activation of pre-synaptic M-type K+ channels inhibits [3H]d-aspartate release by reducing Ca2+ entry through P/Q-type voltage-gated Ca2+channels

In this study, the functional consequences of the pharmacological modulation of the M‐current (I_KM) on cytoplasmic Ca²⁺ intracellular Ca²⁺concentration ([Ca²⁺]_i) changes and excitatory neurotransmitter release triggered by various stimuli from isolated rat cortical synaptosomes have been investigated. K_v7.2 immunoreactivity was identified in pre‐synaptic elements in cortical slices and isolated glutamatergic cortical synaptosomes. In cerebrocortical synaptosomes exposed to 20 mM [K⁺]_e, the I_KM activator retigabine (RT, 10 μM) inhibited [³H]d ‐aspartate ([³H]d ‐Asp) release and caused membrane hyperpolarization; both these effects were prevented by the I_KM blocker XE‐991 (20 μM). The I_KM activators RT (0.1–30 μM), flupirtine (10 μM) and BMS‐204352 (10 μM) inhibited 20 mM [K⁺]_e‐induced synaptosomal [Ca²⁺]_i increases; XE‐991 (20 μM) abolished RT‐induced inhibition of depolarization‐triggered [Ca²⁺]_i transients. The P/Q‐type voltage‐sensitive Ca²⁺channel (VSCC) blocker ω‐agatoxin IVA prevented RT‐induced inhibition of depolarization‐induced [Ca²⁺]_i increase and [³H]d ‐Asp release, whereas the N‐type blocker ω‐conotoxin GVIA failed to do so. Finally, 10 μM RT did not modify the increase of [Ca²⁺]_i and the resulting enhancement of [³H]d ‐Asp release induced by [Ca²⁺]_i mobilization from intracellular stores, or by store‐operated Ca²⁺channel activation. Collectively, the present data reveal that the pharmacological activation of I_KM regulates depolarization‐induced [³H]d ‐Asp release from cerebrocortical synaptosomes by selectively controlling the changes of [Ca²⁺]_i occurring through P/Q‐type VSCCs.     

Stomatal acclimation to increased CO2 concentration in a Florida scrub oak species Quercus myrtifolia Willd

Native scrub‐oak communities in Florida were exposed for three seasons in open top chambers to present atmospheric [CO₂] (approx. 350 μmol mol^?1) and to high [CO₂] (increased by 350 μmol mol^?1). Stomatal and photosynthetic acclimation to high [CO₂] of the dominant species Quercus myrtifolia was examined by leaf gas exchange of excised shoots. Stomatal conductance (g_s) was approximately 40% lower in the high‐ compared to low‐[CO₂]‐grown plants when measured at their respective growth concentrations. Reciprocal measurements of g_s in both high‐ and low‐[CO₂]‐grown plants showed that there was negative acclimation in the high‐[CO₂]‐grown plants (9–16% reduction in g_s when measured at 700 μmol mol^?1), but these were small compared to those for net CO₂ assimilation rate (A, 21–36%). Stomatal acclimation was more clearly evident in the curve of stomatal response to intercellular [CO₂] (c_i) which showed a reduction in stomatal sensitivity at low c_i in the high‐[CO₂]‐grown plants. Stomatal density showed no change in response to growth in high growth [CO₂]. Long‐term stomatal and photosynthetic acclimation to growth in high [CO₂] did not markedly change the 2·5‐ to 3‐fold increase in gas‐exchange‐derived water use efficiency caused by high [CO₂].     

Different Tolerance to Light Stress in NO3−- and NH4+-Grown Phaseolus vulgaris L.

Abstract: NH₄⁺‐grown plants are more sensitive to light stress than NO₃^?‐grown plants, as indicated by reduced growth and intervenal chlorosis of French bean (Phaseolus vulgaris L.). Measuring the time course of F_v/F_m ratios under photoinhibitory light regimes did not reveal any difference in PS II damage between NO₃^?‐ and NH₄⁺‐grown plants, in spite of some indications of higher energy quenching in NO₃^?‐grown plants. Also, a direct action of NH₄⁺ as an uncoupler at the thylakoid membrane could be excluded. Instead, biochemical analysis revealed enhanced lipid peroxidation and higher activity of scavenging enzymes in NH₄⁺‐grown plants indicating that these plants make use of metabolic pathways with stronger radical formation. Evidence for higher rates of photorespiration in NH₄⁺‐grown plants came from experiments showing that electron flux and O₂ evolution were decreased by SHAM in NH₄⁺‐grown plants, and by antimycin A in NO₃^?‐grown plants. Further, the comparison of electron flux and of photoacoustic measurements of O₂ evolution suggested that in NH₄⁺‐grown plants the Mehler reaction was also increased, at least in the induction phase. However, the major cause of N form‐dependent stress sensitivity is assumed to be in the coupling between photosynthesis and respiration, i.e., NO₃^?‐grown plants can utilize the TCA cycle for the generation of C skeletons for amino acid synthesis, thus improving the ATP: reductant balance, whereas NH₄⁺‐grown plants have enhanced rates of photorespiration.     

Growth and Photosynthetic Carbon Metabolism in Tobacco Plants under an Oscillating CO2 Concentration in the Atmosphere

Abstract: The hypothesis for the present work was that photosynthetic acclimation to increased atmospheric CO₂ in Nicotiana tabacum could be prevented by an oscillating supply of CO₂. This was tested by growing half of the plants (for the 20 day period after sowing) at 700 μmol mol^‐1 CO₂ (S+ plants) and half at 350 μmol mol^‐1 CO₂ (S‐ plants) and thereafter switching them every 48 h from high to low CO₂ and vice versa. These plants were compared with plants continuously kept (from sowing onwards) at 350 μmol mol^‐1 CO₂ (C‐ plants) and 700 μmol mol^‐1 CO₂ (C+ plants). Switching plants from high to low CO₂ and vice versa (S+ and S‐) did not improve plant growth efficiency, as hypothesized. The extra carbon fixed by the leaves under increased CO₂ in the atmosphere, supplied either continuously or intermittently, was mostly stored as starch and not used to build additional structural biomass. The differences in final plant biomass, observed between S+ and S‐ plants, are explained by the CO₂ concentration in the atmosphere during the first 20 days after sowing, the oscillation in CO₂ supply thereafter is playing a smaller role in this response. Switching plants from high to low CO₂ and vice versa, also did not prevent down‐regulation of photosynthesis, despite lower leaf sugar concentrations than in C+ plants. Nitrate concentration decreased dramatically in C+, S+ and S‐ plants. The leaf C/N ratio was highest in C+ plants (ranging from 8 to 13), intermediate in S+ and S‐ plants (from 7 to 11) and lowest in C‐ plants (from 6 to 8). This supports the view that the balance between carbohydrates and nitrogen may have a triggering role in plant response under elevated CO₂. Carbon export rates by the leaves seem to be independent of total carbon assimilation, suggesting a sink limiting effect on tobacco growth and phototsynthesis under elevated CO₂.     

Interactions of tropospheric CO2 and O3 enrichments and moisture variations on microbial biomass and respiration in soil

Soil microbial biomass C (C_mic) is a sensitive indicator of trends in organic matter dynamics in terrestrial ecosystems. This study was conducted to determine the effects of tropospheric CO₂ or O₃ enrichments and moisture variations on total soil organic C (C_org), mineralizable C fraction (C_Min), C_mic, maintenance respiratory (qCO₂) or C_mic death (qD) quotients, and their relationship with basal respiration (BR) rates and field respiration (FR) fluxes in wheat‐soybean agroecosystems. Wheat (Triticum aestivum L.) and soybean (Glycine max. L. Merr) plants were grown to maturity in 3‐m dia open‐top field chambers and exposed to charcoal‐filtered (CF) air at 350 μL CO₂ L^?1; CF air + 150 μL CO₂ L^?1; nonfiltered (NF) air + 35 nL O₃ L^?1; and NF air + 35 nL O₃ L^?1 + 150 μL CO₂ L^?1 at optimum (? 0.05 MPa) and restricted soil moisture (? 1.0 ± 0.05 MPa) regimes. The + 150 μL CO₂ L^?1 additions were 18 h d^?1 and the + 35 nL O₃ L^?1 treatments were 7 h d^?1 from April until late October. While C_org did not vary consistently, C_Min, C_mic and C_mic fractions increased in soils under tropospheric CO₂ enrichment (500 μL CO₂ L^?1) and decreased under high O₃ exposures (55 ± 6 nL O₃ L^?1 for wheat; 60 ± 5 nL O₃ L^?1 for soybean) compared to the CF treatments (25 ± 5 nL O₃ L^?1). The qCO₂ or qD quotients of C_mic were also significantly decreased in soils under high CO₂ but increased under high O₃ exposures compared to the CF control. The BR rates did not vary consistently but they were higher in well‐watered soils. The FR fluxes were lower under high O₃ exposures compared to soils under the CF control. An increase in C_mic or C_mic fractions and decrease in qCO₂ or qD observed under high CO₂ treatment suggest that these soils were acting as C sinks whereas, reductions in C_mic or C_mic fractions and increase in qCO₂ or qD in soils under elevated tropospheric O₃ exposures suggest the soils were serving as a source of CO₂.     

Prostaglandin E2 modulates Na+,K+-ATPase activity in rat hippocampus: implications for neurological diseases

Prostaglandin E₂ (PGE₂) is quantitatively one of the major prostaglandins synthesized in mammalian brain, and there is evidence that it facilitates seizures and neuronal death. However, little is known about the molecular mechanisms involved in such excitatory effects. Na⁺,K⁺‐ATPase is a membrane protein which plays a key role in electrolyte homeostasis maintenance and, therefore, regulates neuronal excitability. In this study, we tested the hypothesis that PGE₂ decreases Na⁺,K⁺‐ATPase activity, in order to shed some light on the mechanisms underlying the excitatory action of PGE₂. Na⁺,K⁺‐ATPase activity was determined by assessing ouabain‐sensitive ATP hydrolysis. We found that incubation of adult rat hippocampal slices with PGE₂ (0.1–10 μM) for 30 min decreased Na⁺,K⁺‐ATPase activity in a concentration‐dependent manner. However, PGE₂ did not alter Na⁺,K⁺‐ATPase activity if added to hippocampal homogenates. The inhibitory effect of PGE₂ on Na⁺,K⁺‐ATPase activity was not related to a decrease in the total or plasma membrane immunocontent of the catalytic α subunit of Na⁺,K⁺‐ATPase. We found that the inhibitory effect of PGE₂ (1 μM) on Na⁺,K⁺‐ATPase activity was receptor‐mediated, as incubation with selective antagonists for EP1 (SC‐19220, 10 μM), EP3 (L‐826266, 1 μM) or EP4 (L‐161982, 1 μM) receptors prevented the PGE₂‐induced decrease of Na⁺,K⁺‐ATPase activity. On the other hand, incubation with the selective EP2 agonist (butaprost, 0.1–10 μM) increased enzyme activity per se in a concentration‐dependent manner, but did not prevent the inhibitory effect of PGE₂. Incubation with a protein kinase A (PKA) inhibitor (H‐89, 1 μM) and a protein kinase C (PKC) inhibitor (GF‐109203X, 300 nM) also prevented PGE₂‐induced decrease of Na⁺,K⁺‐ATPase activity. Accordingly, PGE₂ increased phosphorylation of Ser943 at the α subunit, a critical residue for regulation of enzyme activity. Importantly, we also found that PGE₂ decreases Na⁺,K⁺‐ATPase activity in vivo. The results presented here imply Na⁺,K⁺‐ATPase as a target for PGE₂‐mediated signaling, which may underlie PGE₂‐induced increase of brain excitability.     

[D‐(p‐Benzoylphenylalanine)13, tyrosine19]‐melanin‐concentrating hormone,a potent analogue for MCH receptor crosslinking

A photoreactive analogue of human melanin‐concentrating hormone was designed, [d‐ Bpa¹³,Tyr¹⁹]‐MCH, containing the d‐ enantiomer of photolabile p‐benzoylphenylalanine (Bpa) in position 13 and tyrosine for radioiodination in position 19. The linear peptide was synthesized by the continuous‐flow solid‐ phase methodology using Fmoc‐strategy and PEG‐PS resins, purified to homogeneity and cyclized by iodine oxidation. Radioiodination of [d ‐Bpa¹³,Tyr¹⁹]‐MCH at its Tyr¹⁹ residue was carried out enzymatically using solid‐ phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed‐ phase mini‐column and HPLC. Saturation binding analysis of [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH with G4F‐7 mouse melanoma cells gave a K_D of 2.2±0.2×10⁻¹⁰ mol/l and a B_max of 1047±50 receptors/cell. Competition binding analysis showed that MCH and rANF(1–28) displace [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH from the MCH binding sites on G4F‐7 cells whereas α‐MSH has no effect. Receptor crosslinking by UV‐irradiation of G4F‐7 cells in the presence of [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH followed by SDS‐polyacrylamide gel electrophoresis and autoradiography yielded a band of 45–50 kDa. Identical crosslinked bands were also detected in B16‐F1 and G4F mouse melanoma cells, in RE and D10 human melanoma cells as well as in COS‐7 cells. Weak staining was found in rat PC12 phaeochromocytoma and Chinese hamster ovary cells. No crosslinking was detected in human MP fibroblasts. These data demonstrate that [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH is a versatile photocrosslinking analogue of MCH suitable to identify MCH receptors in different cells and tissues; the MCH receptor in these cells appears to have the size of a G protein‐coupled receptor, most likely with a varying degree of glycosylation. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

N2 fixation by Acacia species increases under elevated atmospheric CO2

In the present study the effect of elevated CO₂ on growth and nitrogen fixation of seven Australian Acacia species was investigated. Two species from semi‐arid environments in central Australia (Acacia aneura and A. tetragonophylla) and five species from temperate south‐eastern Australia (Acacia irrorata, A. mearnsii, A. dealbata, A. implexa and A. melanoxylon) were grown for up to 148 d in controlled greenhouse conditions at either ambient (350 µmol mol^?1) or elevated (700 µmol mol^?1) CO₂ concentrations. After establishment of nodules, the plants were completely dependent on symbiotic nitrogen fixation. Six out of seven species had greater relative growth rates and lower whole plant nitrogen concentrations under elevated versus normal CO₂. Enhanced growth resulted in an increase in the amount of nitrogen fixed symbiotically for five of the species. In general, this was the consequence of lower whole‐plant nitrogen concentrations, which equate to a larger plant and greater nodule mass for a given amount of nitrogen. Since the average amount of nitrogen fixed per unit nodule mass was unaltered by atmospheric CO₂, more nitrogen could be fixed for a given amount of plant nitrogen. For three of the species, elevated CO₂ increased the rate of nitrogen fixation per unit nodule mass and time, but this was completely offset by a reduction in nodule mass per unit plant mass.     

Net grassland carbon flux over a subambient to superambient CO2 gradient

Increasing atmospheric CO₂ concentrations may have a profound effect on the structure and function of plant communities. A previously grazed, central Texas grassland was exposed to a 200‐µmol mol^?1 to 550 µmol mol^?1 CO₂ gradient from March to mid‐December in 1998 and 1999 using two, 60‐m long, polyethylene‐ covered chambers built directly onto the site. One chamber was operated at subambient CO₂ concentrations (200–360 µmol mol^?1 daytime) and the other was regulated at superambient concentrations (360–550 µmol mol^?1). Continuous CO₂ gradients were maintained in each chamber by photosynthesis during the day and respiration at night. Net ecosystem CO₂ flux and end‐of‐year biomass were measured in each of 10, 5‐m long sections in each chamber. Net CO₂ fluxes were maximal in late May (c. day 150) in 1998 and in late August in 1999 (c. day 240). In both years, fluxes were near zero and similar in both chambers at the beginning and end of the growing season. Average daily CO₂ flux in 1998 was 13 g CO₂ m^?2 day^?1 in the subambient chamber and 20 g CO₂ m^?2 day^?1 in the superambient chamber; comparable averages were 15 and 26 g CO₂ m^?2 day^?1 in 1999. Flux was positively and linearly correlated with end‐of‐year above‐ground biomass but flux was not linearly correlated with CO₂ concentration; a finding likely to be explained by inherent differences in vegetation. Because C₃ plants were the dominant functional group, we adjusted average daily flux in each section by dividing the flux by the average percentage C₃ cover. Adjusted fluxes were better correlated with CO₂ concentration, although scatter remained. Our results indicate that after accounting for vegetation differences, CO₂ flux increased linearly with CO₂ concentration. This trend was more evident at subambient than superambient CO₂ concentrations.     

Effect of potassium deficiency on the plasma membrane H+-ATPase of the wood ray parenchyma in poplar

The effect of K⁺ deficiency on the plasma membrane (PM) H⁺‐ATPase was studied in young stems of poplar plants (Populus tremula × tremuloides) grown with low or full‐strength K⁺ supply. Immunological assays using different antibodies were applied to test if K⁺ deficiency affects the amount of immunodetectable PM H⁺‐ATPases in the stem tissue. The monoclonal antibody clone 46 E5 B11 revealed an increased abundance of PM H⁺‐ATPases under conditions of low K⁺ supply, and immunolabelling experiments showed that this increase was restricted to vessel‐associated cells (VACs) of the wood ray parenchyma. Replacement of the monoclonal antibody by a polyclonal antibody against PM H⁺‐ATPase gave a specific immunoreactivity on blots as well as tissue sections too, but the labelling intensity showed no difference between plants with low or full‐strength K⁺ supply. Measurements of extracellular H⁺ concentrations using non‐invasive, H⁺‐selective microelectrodes revealed a lowering of the pH at the surface of VACs and an enhancement of net efflux of H⁺ in plants grown with low K⁺ supply. The present results indicate an up‐regulation of specific isoforms of the PM H⁺‐ATPase in VACs under K⁺‐deficient conditions and suggest a key role for these PM H⁺‐ATPases in unloading K⁺ from the xylem stream.     

Root responses along a subambient to elevated CO2 gradient in a C3–C4 grassland

Atmospheric CO₂ (C_a) concentration has increased significantly during the last 20 000 years, and is projected to double this century. Despite the importance of belowground processes in the global carbon cycle, community‐level and single species root responses to rising C_a are not well understood. We measured net community root biomass over 3 years using ingrowth cores in a natural C₃–C₄ grassland exposed to a gradient of C_a from preglacial to future levels (230–550 μmol mol^?1). Root windows and minirhizotron tubes were installed below naturally occurring stands of the C₄ perennial grass Bothriochloa ischaemum and its roots were measured for respiration, carbohydrate concentration, specific root length (SRL), production, and lifespan over 2 years. Community root biomass increased significantly (P<0.05) with C_a over initial conditions, with linear or curvilinear responses depending on sample date. In contrast, B. ischaemum produced significantly more roots at subambient than elevated C_a in minirhizotrons. The lifespan of roots with five or more neighboring roots in minirhizotron windows decreased significantly at high C_a, suggesting that after dense root growth depletes soil resource patches, plants with carbon surpluses readily shed these roots. Root respiration in B. ischaemum showed a curvilinear response to C_a under moist conditions in June 2000, with the lowest rates at C_a<300 μmol mol^?1 and peak activity at 450 μmol mol^?1 in a quadratic model. B. ischaemum roots at subambient C_a had higher SRLs and slightly higher carbohydrate concentrations than those at higher C_a, which may be related to drier soils at low C_a. Our data emphasize that belowground responses of plant communities to C_a can be quite different from those of the individual species, and suggest that complex interactions between and among roots and their immediate soil environment influence the responses of root physiology and lifespan to changing C_a.     

The central nervous system of Bulla gouldiana: Peptide localization

In the present study, we describe the structure of the central nervous system (CNS) of the marine gastropod Bulla gouldiana, and compare it with the structure of the CNS of the related mollusc, Aplysia californica. In addition, we performed an immunohistochemical analysis of a series of peptides, and the synaptic vesicle protein, synapsin I, in the central nervous system of B. gouldiana. The most common peptide in the B. gouldiana nervous system is the molluscan cardioexcitatory peptide (FMRFamide), which is present in a significant proportion of B. gouldiana neurons. A smaller number of neurons exhibit immunoreactivity to antisera raised against the calcitonin gene related peptide, vasopressin, vasoactive intestinal peptide, cholecystokinin, galanin and enkephalin. In some instances there is colocalization of two or more peptides. Very few neurons or axons exhibit synapsin I-like immunoreactivity. The patterns of immunoreactivity to these antisera is quite similar to the patterns that have been described in other gastropods, including Lymnaea stagnalis and Aplysia californica. These observations emphasize the importance of FMRFamide-like compounds in phylogenetically old nervous systems and indicate that compounds similar to mammalian peptides are present in the gastropod. Thus, the production of a wide variety of peptide molecules and their use in neuronal function appears to be a highly conserved phylogenetic process.     

The brain-specific protein, p42IP4 (ADAP 1) is localized in mitochondria and involved in regulation of mitochondrial Ca2+

In brain, p42^IP4 (centaurin‐α1; recently named ADAP 1, which signifies ADP ribosylation factor GTPase activating protein with dual PH domains 1, within the large family of Arf‐GTPase activating proteins) is mainly expressed in neurons. p42^IP4 operates as a dual receptor recognising two second messengers, the soluble inositol(1,3,4,5)tetrakisphosphate and the lipid phosphatidylinositol(3,4,5)trisphosphate. We show here for the first time that p42^IP4 is localized in mitochondria, isolated from rat brain and from cells transfected with p42^IP4. In rat brain mitochondria we additionally found interaction of p42^IP4 with 2′, 3′‐cyclic nucleotide 3′‐phosphodiesterase and α‐tubulin by pull‐down binding assay and by immunoprecipitation. In mitochondria from Chinese hamster ovary cells, p42^IP4 is predominantly associated with the intermembrane space and the inner membrane. This localization of p42^IP4 indicates that p42^IP4 might have a still unknown mitochondrial function. We studied whether p42^IP4 is involved in Ca²⁺‐induced permeability transition pore opening, which is important in mitochondrial events leading to programmed cell death. We used mouse neuroblastoma cells as a model for the functional studies of p42^IP4 in mitochondria. In mitochondria isolated from p42^IP4‐transfected mouse neuroblastoma cells, over‐expression of p42^IP4 significantly decreased Ca²⁺ capacity and lag time for Ca²⁺ retention. Thus, we suggest that p42^IP4 is involved in the regulation of Ca²⁺ transport in mitochondria. We propose that p42^IP4 promotes Ca²⁺‐induced permeability transition pore opening and thus destabilizes mitochondria.     

Selectivity and competitive interactions between two benthic invertebrate grazers (Asellus aquaticus and Potamopyrgus antipodarum): an experimental study using 13C- and 15N-labelled diatoms

1. Tracer experiments with two diatoms labelled with ¹³C (Nitzschia palea) and ¹⁵N (Fragilaria crotonensis), were conducted to investigate feeding selectivity and interspecific competition between the grazers Asellus aquaticus (Isopoda, Crustacea) and Potamopyrgus antipodarum (Hydrobiidae, Gastropoda). Conventional methods, such as cell counts and estimated biovolume, were used first to detect feeding preferences within the different grazer treatments. 2. The results revealed a significant decline in algal biovolume in all grazer treatments and no indications of active selectivity were observed. In contrast to conventional methods, measurements based on isotope signatures showed strong differences in tracer uptake, thus indicating different degrees of assimilation and digestion by the two grazers. 3. The selectivity index Q, which provides information on the uptake ratio of ¹³C to ¹⁵N, showed a significant time effect for both grazer species and a significant difference between single‐ and mixed‐grazer treatments for P. antipodarum. Thus, this technique enabled the direct quantification of the uptake by grazers and, therefore, served as an ideal tool for the detection of passive selectivity. 4. Our results indicate a shift in feeding preferences related to between‐species competition and a potential divergence of trophic niches when species coexist.     

Natural abundance of 13C and 15N in C3 and C4 vegetation of southern Africa: patterns and implications

The mean annual rainfall in southern Africa is found to explain over half of the observed variance in the stable nitrogen (N) isotopic signatures of C₃ vegetation in southern Africa (r²=0.54, P<0.01). The inverse relationship between the stable N isotopic signatures of foliar samples from C₃ vegetation and long‐term southern African rainfall is found on a scale larger than previously observed. A modest relationship is found between stable carbon (C) isotopic signatures of C₃ vegetation and rainfall across the region (r²=0.20, P<0.01). No such relationship is found between stable C and N isotopic signatures of C₄ vegetation and rainfall. The explanation of the relationship between ¹⁵N in C₃ vegetation and the mean annual rainfall presented here is that nutrient availability varies inversely with water availability. This suggests that water‐limited systems in southern Africa are more open in terms of nutrient cycling and therefore the resulting natural abundance of foliar ¹⁵N in these systems is enriched. The use of this relationship may be of value to those researchers modeling both the dynamics of vegetation and biogeochemistry across this region. The use of the isotopic enrichment in C₃ vegetation as a function of rainfall may provide an insight into nutrient cycling across the semi‐arid and arid regions of southern Africa. This finding has implications for the study of global change, especially as it relates to vegetation responses to changing regional rainfall regimes over time.