Soil microbial sensitivity to temperature remains unchanged despite community compositional shifts along geothermal gradients.

Climate warming may be exacerbated if rising temperatures stimulate losses of soil carbon to the atmosphere. The direction and magnitude of this carbon-climate feedback are uncertain, largely due to lack of knowledge of the thermal adaptation of the physiology and composition of soil microbial communities. Here, we applied the macromolecular rate theory (MMRT) to describe the temperature response of the microbial decomposition of soil organic matter (SOM) in a natural long-term warming experiment in a geothermally active area in New Zealand. Our objective was to test whether microbial communities adapt to long-term warming with a shift in their composition and their temperature response that are consistent with evolutionary theory of trade-offs between enzyme structure and function. We characterized the microbial community composition (using metabarcoding) and the temperature response of microbial decomposition of SOM (using MMRT) of soils sampled along transects of increasing distance from a geothermally active zone comprising two biomes (a shrubland and a grassland) and sampled at two depths (0-50 and 50-100 mm), such that ambient soil temperature and soil carbon concentration varied widely and independently. We found that the different environments were hosting microbial communities with distinct compositions, with thermophile and thermotolerant genera increasing in relative abundance with increasing ambient temperature. However, the ambient temperature had no detectable influence on the MMRT parameters or the relative temperature sensitivity of decomposition (Q10 ). MMRT parameters were, however, strongly correlated with soil carbon concentration and carbon:nitrogen ratio. Our findings suggest that, while long-term warming selects for warm-adapted taxa, substrate quality and quantity exert a stronger influence than temperature in selecting for distinct thermal traits. The results have major implications for our understanding of the role of soil microbial processes in the long-term effects of climate warming on soil carbon dynamics and will help increase confidence in carbon-climate feedback projections.

Could patterns of animal behaviour cause the observed differences in soil carbon between adjacent irrigated and unirrigated pastures?

Previous soil sampling from grazed pastures in New Zealand compared the changes of soil organic carbon (SOC) in adjacent irrigated and unirrigated portions of the same paddocks. It showed that irrigated portions had lower SOC stocks than unirrigated portions, with an average difference of 7.0 tC ha-1 or 0.6 tC ha-1 yr-1. These findings have formed the basis of an assessment for the net effect of conversion of New Zealand's grazed pastures to irrigation. However, since cattle could move freely between irrigated and unirrigated portions of the studied paddocks, there could have been different grazing intensities and/or excreta transfer between the irrigated and unirrigated portions of the same paddocks. Both these factors could have affected SOC stocks. In this study, we used the process-based model, CenW, to simulate the consequences of this possible carbon transfer via animal excreta and different grazing intensities. We found that the observed increase of 0.6 tC ha-1 yr-1 in SOC stock in the unirrigated portions could result from a transfer of 20% excreta from the irrigated to unirrigated portions (with an area ratio of 6:1) of a paddock and with the unirrigated portions being grazed only lightly with 2.0 tDM ha-1 in foliage biomass residuals remaining after grazing. That means that the observed higher SOC stocks in the unirrigated portions could potentially be attributable to the behaviour of grazing animals. We suggest that a realistic extent of carbon transfer and/or differences in grazing intensities could be sufficient to account for the observed differences in SOC stocks even if irrigation per se caused no differences in carbon stocks. It is therefore inappropriate to ascribe the change of SOC to irrigation effects based on experimental findings where SOC changes can be affected by the behaviour of grazing animals.

Temperature sensitivity of decomposition decreases with increasing soil organic matter stability.

Evaluation of the temperature sensitivity of soil organic matter (SOM) decomposition is critical for forecasting whether soils in a warming world will lose or gain carbon and, therefore, accelerate or mitigate climate warming. It is usually described, using Arrhenius kinetics, as increasing with the stability of the substrate in laboratory conditions, where substrate availability is non-limiting and where chemical recalcitrance, therefore, predominantly regulates stability. However, conditions of non-limiting subtrate availability are rare in the undisturbed soil, where physicochemical protection of substrates may control their stability. The aim of this study was to assess the temperature sensitivity of decomposition of SOM with contrasting stability in the field. Our conceptual approach was based on in situ measurements of soil CO2 efflux at a range of temperatures from root exclusion plots of increasing age (1 month and three decades) and, therefore, with SOM of increasing stability. From a set of short-term measurements in spring, using diurnal temperature variation, the relative temperature sensitivity of SOM decomposition decreased significantly (p < 0.0001) with increasing SOM stability, and was weak (Q10 < 1.3) in long-term root exclusion plots. This result was confirmed in a similar set of short-term measurements repeated later in the year, in summer, as well as from an analysis perfomed at the seasonal timscale. We provide direct field evidence that the temperature sensitivity of SOM decomposition decreases with increasing stability, in direct contrast with Arrhenius kinetics prediction, and therefore show that stability of SOM in the field cannot be the sole result of chemical recalcitrance. We conclude that the physicochemical protection of SOM, which controls SOM stability in the field, constrains the temperature sensitivity of SOM decomposition under field conditions.

Irrigation increases forage production of newly established lucerne but enhances net ecosystem carbon losses.

In New Zealand, dairy farming faces increasing scrutiny for its environmental impacts, including those on soil carbon (C) stocks; hence, alternative management practices are required. One such practice is usage of deep-rooting forage, such as lucerne (Medicago sativa L.). We measured the C and water exchange of two neighbouring lucerne fields on stony, well-drained soil for 3 years, following conversion from grassland. One field received irrigation and effluent; the other received neither. Net CO2 exchange and evaporation were measured by eddy covariance, drainage and leaching with lysimeters, and water inputs with rain gauges. Biomass removal from harvesting and grazing was recorded by direct sampling. In the conversion year, irrigated lucerne was C-neutral despite two harvests and losses from the conversion process. In the 2nd and 3rd years combined, the biomass-C removal exceeded net CO2 uptake, causing net losses of 450 g C m-2 and 210 g C m-2 for irrigated and non-irrigated lucerne, respectively. Leaching losses accounted for 1 to 9 % of annual net C uptake from the atmosphere. The ratio of ecosystem respiration to gross photosynthetic productivity (GPP) increased from <0.7 in spring to ≈ 1 in autumn. Consequently, the net C balance for both lucerne crops showed gains in the first two growth periods of each year and losses in the subsequent two to four growth periods. Irrigation made no difference to the photosynthetic water-use efficiency at field scale (GPP/evaporation), but enhanced production water-use efficiency (biomass/water input). Irrigation increased both the absolute amount of drainage and the fraction of water inputs lost by drainage. In one year, significant summer drainage occurred for the irrigated lucerne. To prevent that, soil-water content should be kept well below field capacity but above the crop's water-stress level. Such practice would likely also help retain soil carbon.

Nitrous Oxide Fluxes, Soil Oxygen, and Denitrification Potential of Urine- and Non-Urine-Treated Soil under Different Irrigation Frequencies.

Despite increased use of irrigation to improve forage quality and quantity for grazing cattle ( Linnaeus), there is a lack of data that assess how irrigation practices influence nitrous oxide (NO) emissions from urine-affected soils. Irrigation effects on soil oxygen (O) availability, a primary controller of NO fluxes, is poorly understood. It was hypothesized that increased irrigation frequency would result in lower NO emissions by increasing soil moisture and decreasing soil O concentrations. This would favor more NO reduction to dinitrogen (N). We examined effects of high (3-d) versus low (6-d) irrigation frequency with and without bovine urine addition to pasture. Nitrous oxide fluxes were measured daily for 35 d. Soil O, temperature, and water content were continuously measured at multiple depths. Inorganic nitrogen, organic carbon, and soil pH were measured at 6-d intervals. Measurements of denitrification enzyme activity with and without acetylene inhibition were used to infer the NO/(NO + N) ratio. The NO/(NO + N) ratio was lower under high- compared with low-frequency irrigation, suggesting greater potential for NO reduction to N with more frequent irrigation. Although NO fluxes were increased by urine addition, they were not affected by irrigation frequency. Soil O decreased temporarily after urine deposition, but O dynamics did not explain NO dynamics. Relative soil gas diffusivity (/) was a better predictor of NO fluxes than O concentration. On a free-draining soil, increasing irrigation frequency while providing the same total water volume did not enhance NO emissions under ruminant urine patches in a grazed pasture.

Effects of soil warming and nitrogen addition on soil respiration in a New Zealand tussock grassland.

Soil respiration (RS) represents a large terrestrial source of CO2 to the atmosphere. Global change drivers such as climate warming and nitrogen deposition are expected to alter the terrestrial carbon cycle with likely consequences for RS and its components, autotrophic (RA) and heterotrophic respiration (RH). Here we investigate the impacts of a 3°C soil warming treatment and a 50 kg ha(-1) y(-1) nitrogen addition treatment on RS, RH and their respective seasonal temperature responses in an experimental tussock grassland. Average respiration in untreated soils was 0.96±0.09 µmol m(-2) s(-1) over the course of the experiment. Soil warming and nitrogen addition increased RS by 41% and 12% respectively. These treatment effects were additive under combined warming and nitrogen addition. Warming increased RH by 37% while nitrogen addition had no effect. Warming and nitrogen addition affected the seasonal temperature response of RS by increasing the basal rate of respiration (R10) by 14% and 20% respectively. There was no significant interaction between treatments for R10. The treatments had no impact on activation energy (E0). The seasonal temperature response of RH was not affected by either warming or nitrogen addition. These results suggest that the additional CO2 emissions from New Zealand tussock grassland soils as a result of warming-enhanced RS constitute a potential positive feedback to rising atmospheric CO2 concentration.

Roots affect the response of heterotrophic soil respiration to temperature in tussock grass microcosms.

AIMS AND BACKGROUND: While the temperature response of soil respiration (R(S)) has been well studied, the partitioning of heterotrophic respiration (R(H)) by soil microbes from autotrophic respiration (R(A)) by roots, known to have distinct temperature sensitivities, has been problematic. Further complexity stems from the presence of roots affecting R(H), the rhizosphere priming effect. In this study the short-term temperature responses of R(A) and R(H) in relation to rhizosphere priming are investigated. METHODS: Temperature responses of R(A), R(H) and rhizosphere priming were assessed in microcosms of Poa cita using a natural abundance δ(13)C discrimination approach. RESULTS: The temperature response of R(S) was found to be regulated primarily by R(A), which accounted for 70 % of total soil respiration. Heterotrophic respiration was less sensitive to temperature in the presence of plant roots, resulting in negative priming effects with increasing temperature. CONCLUSIONS: The results emphasize the importance of roots in regulating the temperature response of R(S), and a framework is presented for further investigation into temperature effects on heterotrophic respiration and rhizosphere priming, which could be applied to other soil and vegetation types to improve models of soil carbon turnover.

Rapid changes in δ¹³C of ecosystem-respired CO2 after sunset are consistent with transient ¹³C enrichment of leaf respired CO2.

The CO2 respired by darkened, light-adapted, leaves is enriched in ¹³C during the first minutes, and this effect may be related to rapid changes in leaf respiratory biochemistry upon darkening. We hypothesized that this effect would be evident at the ecosystem scale. High temporal resolution measurements of the carbon isotope composition of ecosystem respiration were made over 28 diel periods in an abandoned temperate pasture, and were compared with leaf-level measurements at differing levels of pre-illumination. At the leaf level, CO2 respired by darkened leaves that had been preadapted to high light was strongly enriched in ¹³C, but such a ¹³C-enrichment rapidly declined over 60-100 min. The ¹³C-enrichment was less pronounced when leaves were preadapted to low light. These leaf-level responses were mirrored at the ecosystem scale; after sunset following clear, sunny days respired CO2 was first ¹³C enriched, but the ¹³C-enrichment rapidly declined over 60-100 min. Further, this response was less pronounced following cloudy days. We conclude that the dynamics of leaf respiratory isotopic signal caused variations in ecosystem-scale ¹²CO2/¹³) CO2 exchange. Such rapid isotope kinetics should be considered when applying ¹³C-based techniques to elucidate ecosystem carbon cycling.

A dynamic soil chamber system coupled with a tunable diode laser for online measurements of delta13C, delta18O, and efflux rate of soil-respired CO2.

High frequency observations of the stable isotopic composition of CO(2) effluxes from soil have been sparse due in part to measurement challenges. We have developed an open-system method that utilizes a flow-through chamber coupled to a tunable diode laser (TDL) to quantify the rate of soil CO(2) efflux and its delta(13)C and delta(18)O values (delta(13)C(R) and delta(18)O(R), respectively). We tested the method first in the laboratory using an artificial soil test column and then in a semi-arid woodland. We found that the CO(2) efflux rates of 1.2 to 7.3 micromol m(-2) s(-1) measured by the chamber-TDL system were similar to measurements made using the chamber and an infrared gas analyzer (IRGA) (R(2) = 0.99) and compared well with efflux rates generated from the soil test column (R(2) = 0.94). Measured delta(13)C and delta(18)O values of CO(2) efflux using the chamber-TDL system at 2 min intervals were not significantly different from source air values across all efflux rates after accounting for diffusive enrichment. Field measurements during drought demonstrated a strong dependency of CO(2) efflux and isotopic composition on soil water content. Addition of water to the soil beneath the chamber resulted in average changes of +6.9 micromol m(-2) s(-1), -5.0 per thousand, and -55.0 per thousand for soil CO(2) efflux, delta(13)C(R) and delta(18)O(R), respectively. All three variables initiated responses within 2 min of water addition, with peak responses observed within 10 min for isotopes and 20 min for efflux. The observed delta(18)O(R) was more enriched than predicted from temperature-dependent H(2)O-CO(2) equilibration theory, similar to other recent observations of delta(18)O(R) from dry soils (Wingate L, Seibt U, Maseyk K, Ogee J, Almeida P, Yakir D, Pereira JS, Mencuccini M. Global Change Biol. 2008; 14: 2178). The soil chamber coupled with the TDL was found to be an effective method for capturing soil CO(2) efflux and its stable isotope composition at high temporal frequency.

Alternate mRNA splicing in multiple human tryptase genes is predicted to regulate tetramer formation.

Tryptases are serine proteases that are thought to be uniquely and proteolytically active as tetramers. Crystallographic studies reveal that the active tetramer is a flat ring structure composed of four monomers, with their active sites arranged around a narrow central pore. This model explains why many of the preferred substrates of tryptase are short peptides; however, it does not explain how tryptase cleaves large protein substrates such as fibronectin, although a number of studies have reported in vitro mechanisms for generating active monomers that could digest larger substrates. Here we suggest that alternate mRNA splicing of human tryptase genes generates active tryptase monomers (or dimers). We have identified a conserved pattern of alternate splicing in four tryptase alleles (alphaII, betaI, betaIII, and deltaI), representing three distinct tryptase gene loci. When compared with their full-length counterparts, the splice variants use an alternate acceptor site within exon 4. This results in the deletion of 27 nucleotides within the central coding sequence and 9 amino acids from the translated protein product. Although modeling suggests that the deletion can be easily accommodated by the enzymes structurally, it is predicted to alter the specificity by enlarging the S1' or S2' binding pocket and results in the complete loss of the "47 loop," reported to be critical for the formation of tetramers. Although active monomers can be generated in vitro using a range of artificial conditions, we suggest that alternate splicing is the in vivo mechanism used to generate active tryptase that can cleave large protein substrates.

Spatial and temporal scaling of intercellular CO2 concentration in a temperate rain forest dominated by Dacrydium cupressinum in New Zealand.

Seven methods, including measurements of photosynthesis (A) and stomatal conductance (g(s)), carbon isotope discrimination, ecosystem CO2 and water vapour exchange using eddy covariance and the use of a multilayer canopy model and ecosystem Keeling plots, were employed to derive estimates of intercellular CO2 concentration (Ci) across a range of spatial and temporal scales in a low productivity rain forest ecosystem dominated by the conifer Dacrydium cupressinum Lamb. in New Zealand. Estimates of shoot and canopy Ci across temporal scales ranging from minutes to years were remarkably similar (range of 274-294 micromol mol(-1)). The gradual increase in shoot Ci with depth in the canopy was more likely attributable to decreases in A resulting from lower irradiance (Q) than to increases in g, due to changes in air saturation deficit (D). The lack of marked vertical gradients in A and g(s) at saturating Q through the canopy and the low seasonal variability in environmental conditions contributed to the efficacy of scaling Ci. However, the canopy Ci estimate calculated from the carbon isotope composition of respired ecosystem CO2 (delta13CR; 236 micromol mol(-1)) was much lower than other estimates of canopy Ci. Partitioning delta13CR into four components (soil, roots, litter and foliage) indicated root respiration as the dominant (> 50%) contributor to delta13CR. Variable time lags and differences in isotopic composition during photosynthesis and respiration make the direct estimation of canopy Ci from delta 13CR problematic.

Collection and storage of CO2 for 13C analysis: An application to separate soil CO2 efflux into root- and soil-derived components.

Soil surface CO2 efflux is comprised of CO2 from (i) root respiration and rhizosphere microbes and (ii) heterotrophic respiration from the breakdown of soil organic matter (SOM). This efflux may be partitioned between these sources using delta13C measurements. To achieve this, continuous flow isotope ratio mass spectrometry can be used and, in conjunction with 10 mL septum-capped vials, large numbers of samples may be analysed using a Finnigan MAT Delta(plus)XP interfaced to a Gas Bench II. Here we describe a number of advances to facilitate such work, including: (i) a technique for monitoring mass spectrometer performance, (ii) improvements to sample storage, and (iii) a gas-handling system for incubating and sampling the CO2 derived from roots and soils. Mass spectrometer performance was monitored using an automated refillable vial. Compressed air analysed with this system had mean delta13C of -9.61 +/- 0.16 per thousand (+/- 1sigma, n = 28) collected over four runs. Heating the butyl rubber septa used to seal the vials at 105 degrees C for 12 h improved the sample storage. After air transportation over 12 days, the isotope composition of the CO2 at ambient concentrations was unchanged (before: -35.2 +/- 0.10 per thousand, n = 4; after: -35.3 +/- 0.10 per thousand, n = 15); without heat treatment of the septa the CO2 became slightly enriched (-35.0 +/- 0.14 per thousand, n = 15). The linearity of the Gas Bench II was found to decline above 8000 micromol CO2 mol(-1). To stay within a linear range and to allow the incubation of soil and root material we describe a gas-handling system based around a peristaltic pump. Finally, we demonstrate these methods by growing a C-4 grass (Guinea grass, Panicum maximum Jacq.) in a C-3 soil. Root respiration was found to contribute between 5 and 22% to the soil surface CO2 efflux. These methodologies will facilitate experiments aimed at measuring the isotopic composition of soil-derived CO2 across a range of ecological applications.

Effects of cigarette smoke on degranulation and NO production by mast cells and epithelial cells.

Exhaled nitric oxide (eNO) is decreased by cigarette smoking. The hypothesis that oxides of nitrogen (NOX) in cigarette smoke solution (CSS) may exert a negative feedback mechanism upon NO release from epithelial (AEC, A549, and NHTBE) and basophilic cells (RBL-2H3) was tested in vitro. CSS inhibited both NO production and degranulation (measured as release of beta-hexosaminidase) in a dose-dependent manner from RBL-2H3 cells. Inhibition of NO production by CSS in AEC, A549, and NHTBE cells was also dose-dependent. In addition, CSS decreased expression of NOS mRNA and protein expression. The addition of NO inhibitors and scavengers did not, however, reverse the effects of CSS, nor did a NO donor (SNP) or nicotine mimic CSS. N-acetyl-cysteine, partially reversed the inhibition of beta-hexosaminidase release suggesting CSS may act via oxidative free radicals. Thus, some of the inhibitory effects of CSS appear to be via oxidative free radicals rather than a NOX-related negative feedback.

IL-15 induces mast cell migration via a pertussis toxin-sensitive receptor.

IL-15 induces proliferation, inhibits apoptosis and increases IL-4 production in murine mast cells. There is evidence that these activities are mediated via the uncharacterised receptor, IL-15R-X, rather than the classical three-chain IL-15 receptor. Effects of IL-15 on important aspects of mast cell biology, such as migration and degranulation, are unknown. We report that IL-15 induces migration of murine and human mast cells in a dose-dependent and biphasic manner, with peaks of migration occurring at approximately 10(-15) and approximately 10(-9) M. The potency of the response was similar to that induced by other well-established mast cell chemoattractants. Competition assays performed with murine and human mast cells indicate that both peaks of migration are due to chemotaxis. Pre-treatment of cells with pertussis toxin (PTX), a guanine nucleotide-binding regulatory protein (G-protein) inhibitor, resulted in complete inhibition of murine mast cell migration at approximately 10(-15) M IL-15, and human mast cell migration at approximately 10(-15) and approximately 10(-9) M. This demonstrates that murine and human mast cells express a PTX-sensitive receptor, activated in response to IL-15. Additionally, IL-15 did not induce degranulation in murine mast cells. Locally-produced IL-15 may contribute to mast cell recruitment during inflammatory responses, thereby acting as a linking cytokine between innate and adaptive arms of the immune system.

Photosynthesis and reflectance indices for rainforest species in ecosystems undergoing progression and retrogression along a soil fertility chronosequence in New Zealand.

Measurements of photosynthesis at saturating irradiance and CO2 partial pressure, Amax, "adjusted" normalised difference vegetation index, RaNDVI, and photochemical reflectance index, RPRI, were made on trees sampled along a soil chronosequence to investigate the relationship between carbon uptake and ecosystem development in relation to nutrient availability. Measurements were made on the three most dominant species at six sites along the sequence in South Westland, New Zealand with soil age ranging from < 6 to 120,000 years resulting from the retreat of the Franz Josef glacier. The decrease in soil phosphorus availability with increasing soil age and high soil nitrogen availability at the two youngest sites, due to the presence of a nitrogen-fixing species, provided marked differences in nutrient availability. Mean Amax was high at the two youngest sites, then decreased markedly with increasing site age. Analysis of the data for individual species within sites revealed separation of groups of species in the response of Amax to Nm and Pm, suggesting complex interactions between the two nutrients. There were strong linear relationships for leaf-level RaNDVI and RPRI with Amax, at high irradiance, showing that measurements of reflectance indices can be used to estimate Amax for foliage with a range in morphology and nutrient concentrations. Notwithstanding the change in species composition from angiosperms to conifers with increasing site age, the presence of nitrogen-fixing species, the variability in foliage morphology from flat leaves to imbricate scales and a wide range in foliar nitrogen and phosphorus concentrations, there were strong positive linear relationships between site average Amax and foliage nitrogen, Nm, and phosphorus, Pm, concentrations on a foliage mass basis. The results provide insights to interpret the regulation of photosynthesis across natural ecosystems with marked gradients in nitrogen and phosphorus availability.

Mast cell accumulation and cytokine expression in the tight skin mouse model of scleroderma.

The tight skin (Tsk) mouse develops many pathological changes seen in human scleroderma, such as increased collagen content and mast cell density. Although associations between mast cell expansion and skin fibrosis have been reported, the mechanisms underlying mast cell accumulation remain unclear. In this study, we have measured the density of skin mast cells in Tsk mice and their normal littermates (pa/pa) of 4-36 weeks of age, and in the skin heterografted between Tsk and pa/pa mice. Cytokines related to mast cell differentiation, proliferation and migration were examined by using RNase protection assays. Skin mast cell density in Tsk mice was significantly increased from 12 weeks of age, compared to that in pa/pa mice. The expression of transforming growth factor-beta1 (TGF-beta1), and to a lesser extent, stem cell factor (SCF) and interleukin-15 (IL-15) mRNA was higher in Tsk mice, compared to that in control mice. Mast cell density was unchanged in Tsk skin grafted onto pa/pa hosts, but dramatically increased in pa/pa skin grafted onto Tsk hosts. This latter mast cell hyperplasia was associated with the increases in mRNA levels of TGF-beta1, SCF and IL-15, whereas little change in cytokine levels was seen in heterografted Tsk skin. These results suggest that locally produced cytokines in Tsk skin influence mast cell accumulation in this animal model of human scleroderma.

Variation in the degree of coupling between delta13C of phloem sap and ecosystem respiration in two mature Nothofagus forests.

Day-to-day variability in the carbon isotope composition of phloem sap (delta13Chd) and ecosystem respiratory CO2 (delta13CR) were measured to assess the tightness of coupling between canopy photosynthesis (delta13Chd) and ecosystem respiration (delta13CR) in two mature Nothofagus solandri (Hook. f.) forests in New Zealand. Abundant phloem-tapping scale insects allowed repeated, nondestructive access to stem phloem sap 1-2 m above ground. delta13Chd was compared with delta13C predicted by an environmentally driven, process-based canopy photosynthesis model. Keeling plots of within-canopy CO2 were used to estimate delta13CR. By including a lag of 3 d, there was good agreement in the timing and direction of variation in delta13Chd and predictions by the canopy photosynthesis model, suggesting that delta13Chd represents a photosynthesis-weighted, integrative record of canopy photosynthesis and conductance. Significant day-to-day variability in delta13CR was recorded at one of the two forests. At this site, delta13CR reflected variability in delta13Chd only on days with <2 mm rain. We conclude that the degree of coupling between canopy photosynthesis and ecosystem respiration varies between sites, and with environmental conditions at a single site.

Involvement of chymase-mediated angiotensin II generation in blood pressure regulation.

Angiotensin I-converting enzyme (ACE) inhibitors are thought to lower blood pressure in hypertensive patients, mainly by decreasing angiotensin II (Ang II) formation. Chymase, a human mast cell protease, has recently been proposed to play a role in blood pressure regulation because of its Ang II-forming activity. Here we show that the predominant chymase mRNA species in the mouse aorta are those for types 4 and 5 isoforms, and that both are efficient Ang II-forming enzymes. Evaluation of ACE-dependent and ACE-independent Ang II-forming pathways in mast cell-deficient (Kit(w)/Kit(w-v)) mice and their mast cell-sufficient littermate (MC(+/+)) controls revealed that, in contrast to the latter, Kit(w)/Kit(w-v) mice fail to express chymase mRNAs in the vasculature and have almost no ACE-independent Ang II-forming activity in either isolated blood vessels or homogenates. Moreover, in MC(+/+) but not in Kit(w)/Kit(w-v) mice, a contribution of ACE-independent Ang II generation to blood pressure regulation was evident by a 1.6-fold greater maximal reduction in mean arterial pressure with acute ACE inhibition plus AT(1) receptor blockade than with ACE inhibition alone. Thus, mast cells are the source of the vascular ACE-independent pathway, and the antihypertensive benefit of combining ACE inhibitor therapy with AT(1) receptor antagonist therapy is most likely due to negation of chymase-catalyzed Ang II generation.

Delta tryptase is expressed in multiple human tissues, and a recombinant form has proteolytic activity.

Tryptases are neutral serine proteases selectively expressed in mast cells and have been implicated in the development of a number of inflammatory diseases including asthma. It has recently been established that the number of genes encoding human mast cell tryptases is much larger than originally believed, but it is not clear how many of these genes are expressed. A recent report suggested that the transcript for at least one of these genes, originally named mMCP-7-like tryptase, is not expressed. To further address this question, we screened tissue-specific RNA samples by RT-PCR, using primers designed to match the putative exonic sequence of this gene. We successfully generated and cloned the correctly sized RT-PCR product from mRNA isolated from the human mast cell-I cell line. Two distinct clones were identified whose nucleotide sequence matched the published sequence of the mMCP-7-like I and mMCP-7-like II genes. Transcripts were detected in a wide variety of human tissues including lung, heart, stomach, spleen, skin, and colon. A polyclonal antipeptide Ab that specifically recognizes the translated product of this transcript was used to demonstrate its expression in mast cells that reside in the colon, lung, and inflamed synovium. A recombinant form of this protein expressed in bacterial cells was able to cleave a synthetic trypsin-sensitive substrate, D-Ile-Phe-Lys pNA. These results suggest that the range of functional tryptases is larger than previously recognized. For simplicity, we suggest that the gene, transcripts, and corresponding protein product be named delta tryptase.