Updating the dual C and O isotope-Gas-exchange model: A concept to understand plant responses to the environment and its implications for tree rings.

The combined study of carbon (C) and oxygen (O) isotopes in plant organic matter has emerged as a powerful tool for understanding plant functional responses to environmental change. The approach relies on established relationships between leaf gas exchange and isotopic fractionation to derive a series of model scenarios that can be used to infer changes in photosynthetic assimilation and stomatal conductance driven by changes in environmental parameters (CO2 , water availability, air humidity, temperature, nutrients). We review the mechanistic basis for a conceptual model, in light of recently published research, and discuss where isotopic observations do not match our current understanding of plant physiological response to the environment. We demonstrate that (1) the model was applied successfully in many, but not all studies; (2) although originally conceived for leaf isotopes, the model has been applied extensively to tree-ring isotopes in the context of tree physiology and dendrochronology. Where isotopic observations deviate from physiologically plausible conclusions, this mismatch between gas exchange and isotope response provides valuable insights into underlying physiological processes. Overall, we found that isotope responses can be grouped into situations of increasing resource limitation versus higher resource availability. The dual-isotope model helps to interpret plant responses to a multitude of environmental factors.

Nitrate and ammonium differ in their impact on δ13C of plant metabolites and respired CO2 from tobacco leaves.

The carbon isotopic composition (δ13C) of foliage is often used as proxy for plant performance. However, the effect of N O 3 - vs. N H 4 + supply on δ13C of leaf metabolites and respired CO2 is largely unknown. We supplied tobacco plants with a gradient of N O 3 - to N H 4 + concentration ratios and determined gas exchange variables, concentrations and δ13C of tricarboxylic acid (TCA) cycle intermediates, δ13C of dark-respired CO2, and activities of key enzymes nitrate reductase, malic enzyme and phosphoenolpyruvate carboxylase. Net assimilation rate, dry biomass and concentrations of organic acids and starch decreased along the gradient. In contrast, respiration rates, concentrations of intercellular CO2, soluble sugars and amino acids increased. As N O 3 - decreased, activities of all measured enzymes decreased. δ13C of CO2 and organic acids closely co-varied and were more positive under N O 3 - supply, suggesting organic acids as potential substrates for respiration. Together with estimates of intra-molecular 13C enrichment in malate, we conclude that a change in the anaplerotic reaction of the TCA cycle possibly contributes to 13C enrichment in organic acids and respired CO2 under N O 3 - supply. Thus, the effect of N O 3 - vs. N H 4 + on δ13C is highly relevant, particularly if δ13C of leaf metabolites or respiration is used as proxy for plant performance.

Effect of Vapor Pressure Deficit on Gas Exchange in Wild-Type and Abscisic Acid-Insensitive Plants.

Stomata control the gas exchange of terrestrial plant leaves, and are therefore essential to plant growth and survival. We investigated gas exchange responses to vapor pressure deficit (VPD) in two gray poplar (Populus × canescens) lines: wild type and abscisic acid-insensitive (abi1) with functionally impaired stomata. Transpiration rate in abi1 increased linearly with VPD, up to about 2 kPa. Above this, sharply declining transpiration was followed by leaf death. In contrast, wild type showed a steady or slightly declining transpiration rate up to VPD of nearly 7 kPa, and fully recovered photosynthetic function afterward. There were marked differences in discrimination against 13CO2 (Δ13C) and C18OO (Δ18O) between abi1 and wild-type plants. The Δ13C indicated that intercellular CO2 concentrations decreased with VPD in wild-type plants, but not in abi1 plants. The Δ18O reflected progressive stomatal closure in wild type in response to increasing VPD; however, in abi1, stomata remained open and oxygen atoms of CO2 continued to exchange with 18O enriched leaf water. Coupled measurements of Δ18O and gas exchange were used to estimate intercellular vapor pressure, e i In wild-type leaves, there was no evidence of unsaturation of e i, even at VPD above 6 kPa. In abi1 leaves, e i approached 0.6 times saturation vapor pressure before the precipitous decline in transpiration rate. For wild type, a sensitive stomatal response to increasing VPD was pivotal in preventing unsaturation of e i In abi1, after taking unsaturation into account, stomatal conductance increased with increasing VPD, consistent with a disabled active response of guard cell osmotic pressure.

Plasticity in gas-exchange physiology of mature Scots pine and European larch drive short- and long-term adjustments to changes in water availability.

Adjustment mechanisms of trees to changes in soil-water availability over long periods are poorly understood, but crucial to improve estimates of forest development in a changing climate. We compared mature trees of Scots pine (Pinus sylvestris) and European larch (Larix decidua) growing along water-permeable channels (irrigated) and under natural conditions (control) at three sites in inner-Alpine dry valleys. At two sites, the irrigation had been stopped in the 1980s. We combined measurements of basal area increment (BAI), tree height and gas-exchange physiology (Δ13 C) for the period 1970-2009. At one site, the Δ13 C of irrigated pine trees was higher than that of the control in all years, while at the other sites, it differed in pine and larch only in years with dry climatic conditions. During the first decade after the sudden change in water availability, the BAI and Δ13 C of originally irrigated pine and larch trees decreased instantly, but subsequently reached higher levels than those of the control by 2009 (15 years afterwards). We found a high plasticity in the gas-exchange physiology of pine and larch and site-specific responses to changes in water availability. Our study highlights the ability of trees to adjust to new conditions, thus showing high resilience.

A dynamic leaf gas-exchange strategy is conserved in woody plants under changing ambient CO2 : evidence from carbon isotope discrimination in paleo and CO2 enrichment studies.

Rising atmospheric [CO2 ], ca , is expected to affect stomatal regulation of leaf gas-exchange of woody plants, thus influencing energy fluxes as well as carbon (C), water, and nutrient cycling of forests. Researchers have proposed various strategies for stomatal regulation of leaf gas-exchange that include maintaining a constant leaf internal [CO2 ], ci , a constant drawdown in CO2 (ca  - ci ), and a constant ci /ca . These strategies can result in drastically different consequences for leaf gas-exchange. The accuracy of Earth systems models depends in part on assumptions about generalizable patterns in leaf gas-exchange responses to varying ca . The concept of optimal stomatal behavior, exemplified by woody plants shifting along a continuum of these strategies, provides a unifying framework for understanding leaf gas-exchange responses to ca . To assess leaf gas-exchange regulation strategies, we analyzed patterns in ci inferred from studies reporting C stable isotope ratios (δ(13) C) or photosynthetic discrimination (∆) in woody angiosperms and gymnosperms that grew across a range of ca spanning at least 100 ppm. Our results suggest that much of the ca -induced changes in ci /ca occurred across ca spanning 200 to 400 ppm. These patterns imply that ca  - ci will eventually approach a constant level at high ca because assimilation rates will reach a maximum and stomatal conductance of each species should be constrained to some minimum level. These analyses are not consistent with canalization toward any single strategy, particularly maintaining a constant ci . Rather, the results are consistent with the existence of a broadly conserved pattern of stomatal optimization in woody angiosperms and gymnosperms. This results in trees being profligate water users at low ca , when additional water loss is small for each unit of C gain, and increasingly water-conservative at high ca , when photosystems are saturated and water loss is large for each unit C gain.

A novel methylation derivatization method for δ(18)O analysis of individual carbohydrates by gas chromatography/pyrolysis-isotope ratio mass spectrometry.

RATIONALE: The oxygen isotope ratio (δ(18)O) of carbohydrates derived from animals, plants, sediments, and soils provides important information about biochemical and physiological processes, past environmental conditions, and geographical origins, which are otherwise not available. Nowadays, δ(18)O analyses are often performed on carbohydrate bulk material, while compound-specific δ(18)O analyses remain challenging and methods for a wide range of individual carbohydrates are rare. METHODS: To improve the δ(18)O analysis of individual carbohydrates by gas chromatography/pyrolysis-isotope ratio mass spectrometry (GC/Pyr-IRMS) we developed a new methylation derivatization method. Carbohydrates were fully methylated within 24 h in an easy-to-handle one-pot reaction in acetonitrile, using silver oxide as proton acceptor, methyl iodide as methyl group carrier, and dimethyl sulfide as catalyst. RESULTS: The precision of the method ranged between 0.12 and 1.09‰ for the δ(18)O values of various individual carbohydrates of different classes (mono-, di-, and trisaccharides, alditols), with an accuracy of a similar order of magnitude, despite high variation in peak areas. Based on the δ(18)O values of the main isomers, important monosaccharides such as glucose and fructose could also be precisely analyzed for the first time. We tested the method on standard mixtures, honey samples, and leaf carbohydrates extracted from Pinus sylvestris, showing that the method is also applicable to different carbohydrate mixtures. CONCLUSIONS: The new methylation method shows unrivalled accuracy and precision for δ(18)O analysis of various individual carbohydrates; it is fast and easy-to-handle, and may therefore find wide-spread application.

Optimization of automated gas sample collection and isotope ratio mass spectrometric analysis of delta(13)C of CO(2) in air.

The application of (13)C/(12)C in ecosystem-scale tracer models for CO(2) in air requires accurate measurements of the mixing ratios and stable isotope ratios of CO(2). To increase measurement reliability and data intercomparability, as well as to shorten analysis times, we have improved an existing field sampling setup with portable air sampling units and developed a laboratory setup for the analysis of the delta(13)C of CO(2) in air by isotope ratio mass spectrometry (IRMS). The changes consist of (a) optimization of sample and standard gas flow paths, (b) additional software configuration, and (c) automation of liquid nitrogen refilling for the cryogenic trap. We achieved a precision better than 0.1 per thousand and an accuracy of 0.11 +/- 0.04 per thousand for the measurement of delta(13)C of CO(2) in air and unattended operation of measurement sequences up to 12 h.

A portable automated system for trace gas sampling in the field and stable isotope analysis in the laboratory.

A computer-controllable mobile system is presented which enables the automatic collection of 33 air samples in the field and the subsequent analysis for delta13C and delta18O stable isotope ratios of a carbon-containing trace gas in the laboratory, e.g. CO2, CO or CH4. The system includes a manifold gas source input for profile sampling and an infrared gas analyzer for in situ CO2 concentration measurements. Measurements of delta13C and delta18O of all 33 samples can run unattended and take less than six hours for CO2. Laboratory tests with three gases (compressed air with different pCO2 and stable isotope compositions) showed a measurement precision of 0.03 per thousand for delta13C and 0.02 per thousand for delta18O of CO2 (standard error (SE), n = 11). A field test of our system, in which 66 air samples were collected within a 24-hour period above grassland, showed a correlation of 0.99 (r2) between the inverse of pCO2 and delta13C of CO2. Storage of samples until analysis is possible for about 1 week; this can be an important factor for sampling in remote areas. A wider range of applications in the field is open with our system, since sampling and analysis of CO and CH4 for stable isotope composition is also possible. Samples of compressed air had a measurement precision (SE, n = 33) of 0.03 per thousand for delta13C and of 0.04 per thousand for delta18O on CO and of 0.07 per thousand for delta13C on CH4. Our system should therefore further facilitate research of trace gases in the context of the carbon cycle in the field, and opens many other possible applications with carbon- and possibly non-carbon-containing trace gases.