Dan Yakir

The application and interpretation of Keeling plots in terrestrial carbon cycle research

[1] Photosynthesis and respiration impart distinct isotopic signatures to the atmosphere that are used to constrain global carbon source/sink estimates and partition ecosystem fluxes. Increasingly, the ‘‘Keeling plot’’ method is being used to determine the carbon isotope composition of ecosystem respiration (d 13 CR) in order to better understand the processes controlling ecosystem isotope discrimination. In this paper we synthesize emergent patterns in d 13 CR by analyzing 146 Keeling plots constructed at 33 sites across North and South America. In order to interpret results from disparate studies, we discuss the assumptions underlying the Keeling plot method and recommend standardized methods for determining d 13 CR. These include the use of regression calculations that account for error in the x variable, and constraining estimates of d 13 CR to nighttime periods. We then recalculate d 13 CR uniformly for all sites. We found a high degree of temporal and spatial variability in C3 ecosystems, with individual observations ranging from � 19.0 to � 32.6%. Mean C3 ecosystem discrimination was 18.3%. Precipitation was a major driver of both temporal and spatial variability of d 13 CR, suggesting (1) a large influence of recently fixed carbon on ecosystem respiration and (2) a significant effect of previous climatic effects on d 13 CR. These results illustrate the importance of water availability as a key control on atmospheric 13 CO2 and highlight the potential of d 13 CR as a useful tool for integrating environmental effects on dynamic canopy and ecosystem processes. INDEX TERMS: 0315 Atmospheric Composition and Structure: Biosphere/atmosphere interactions; 0322 Atmospheric Composition and Structure: Constituent sources and sinks; 1615 Global Change: Biogeochemical processes (4805); 1694 Global Change: Instruments and techniques; 3322 Meteorology and Atmospheric Dynamics: Land/atmosphere interactions; KEYWORDS: carbon cycle, carbon isotopes, ecosystem respiration, carbon dioxide, terrestrial ecosystems

The use of stable isotopes to study ecosystem gas exchange

Abstract Stable isotopes are a powerful research tool in environmental sciences and their use in ecosystem research is increasing. In this review we introduce and discuss the relevant details underlying the use of carbon and oxygen isotopic compositions in ecosystem gas exchange research. The current use and potential developments of stable isotope measurements together with concentration and flux measurements of CO2 and water vapor are emphasized. For these applications it is critical to know the isotopic identity of specific ecosystem components such as the isotopic composition of CO2, organic matter, liquid water, and water vapor, as well as the associated isotopic fractionations, in the soil-plant- atmosphere system. Combining stable isotopes and concentration measurements is very effective through the use of ”Keeling plots.” This approach allows the identification of the isotopic composition and the contribution of ecosystem, or ecosystem components, to the exchange fluxes with the atmosphere. It also allows the estimation of net ecosystem discrimination and soil disequilibrium effects. Recent modifications of the Keeling plot approach permit examination of CO2 recycling in ecosystems. Combining stable isotopes with dynamic flux measurements requires precision in isotopic sampling and analysis, which is currently at the limit of detection. Combined with the micrometeorological gradient approach (applicable mostly in grasslands and crop fields), stable isotope measurements allow separation of net CO2 exchange into photosynthetic and soil respiration components, and the evapotranspiration flux into soil evaporation and leaf transpiration. Similar applications in conjunction with eddy correlation techniques (applicable to forests, in addition to grasslands and crop fields) are more demanding, but can potentially be applied in combination with the Keeling plot relationship. The advance and potential in using stable isotope measurements should make their use a standard component in the limited arsenal of ecosystem-scale research tools.

Contribution of Semi-Arid Forests to the Climate System

The Long and Short of It Semi-arid forests cover close to 18% of Earths land surface. If climate change were to stimulate carbon accumulation in these areas, resulting changes in the forests could both promote climate cooling and warming: On one hand, forest growth would draw CO2 from the atmosphere, providing a cooling effect on climate; on the other, as forests grew and became more dense, their albedo would decrease, which would warm climate. Rotenberg and Yakir (p. 451; see the Perspective by Schimel) now report that a shift in peak photosynthetic activities from summer to early spring would, indeed, cause carbon accumulation by the forests, but that a suppression of reflected longwave radiation effect would complement the better-known (shortwave) albedo effect, doubling the amount of potential warming. Several decades of carbon accumulation would thus be necessary to counteract these radiative changes. Semi-arid forests could cool climate by sequestering CO2, but could also warm it by reducing Earth’s albedo. Forests both take up CO2 and enhance absorption of solar radiation, with contrasting effects on global temperature. Based on a 9-year study in the forests’ dry timberline, we show that substantial carbon sequestration (cooling effect) is maintained in the large dry transition zone (precipitation from 200 to 600 millimeters) by shifts in peak photosynthetic activities from summer to early spring, and this is counteracted by longwave radiation (L) suppression (warming effect), doubling the forestation shortwave (S) albedo effect. Several decades of carbon accumulation are required to balance the twofold S + L effect. Desertification over the past several decades, however, contributed negative forcing at Earth’s surface equivalent to ~20% of the global anthropogenic CO2 effect over the same period, moderating warming trends.

Protection by Isoprene against Singlet Oxygen in Leaves

Isoprene (2-methyl-1,3-butadiene) protection against effects of singlet oxygen was investigated in Myrtus communisand Rhamnus alaternus. In M. communis, singlet oxygen produced in the leaves by Rose Bengal (RB) led to a 65% decrease in net assimilation rates within 3 h, whereas isoprene emission rates showed either a 30% decrease at ambient CO2concentrations or a 70% increase under high CO2. In both cases, these changes led to an increase in calculated internal isoprene concentrations. The isoprene protection effect was directly demonstrated by fumigation of young (non-emitting) leaves, treated with RB or bromoxynil (simulating photoinhibition). There was 42% and 29% reduction in the damage to net assimilation compared with non-fumigated leaves for RB or bromoxynil, respectively. In R. alaternus, similar effects of RB on net assimilation were observed, and additional fluorescence measurements showed a significantly smaller decrease inF v/F m in isoprene-fumigated young leaves treated with RB (from 0.78 to 0.52), compared with non-fumigated leaves (from 0.77 to 0.27). The internal isoprene concentrations used in this study and possible rate of1O2 production in leaves indicate that the protective effects observed should be beneficial also under natural conditions.

Using stable isotopes of water in evapotranspiration studies

The use of stable isotopes to estimate evapotranspiration (ET) fluxes from vegetated areas is increasing. By complementing conventional net flux measurements (gradient or eddy correlation techniques), isotope analyses can allow partitioning ET between its gross components, soil evaporation and leaf transpiration. Isotopic analyses of atmospheric water vapour above canopies can also constrain, or provide alternatives for estimating ET. A brief discussion of the isotope approach is aimed at highlighting some of the uncertainties that require further research. We also demonstrate first, the application of combined concentration and isotopic gradient analysis of atmospheric water vapour above crop fields in order to estimate ET fluxes and its gross components (soil evaporation was estimated at 1.5-3.5% of mid-day ET flux in a mature wheat field). Second, we demonstrate the potential in monitoring δ ss -δ I. , the difference between predicted and measured leaf water δ 18 O values, as an indicator of seasonal variations in canopy-scale transpiration in a desert ecosystem (linear correlation between this indicator and conventional ET measurements was observed). Improving our analytical capabilities for high-precision isotopic analysis of very small water vapour samples was a limiting factor in the above applications and a method for pyrolysis and on-line 18 O analysis of 0.2-2 μL water samples is described.

Electron Partitioning between the Cytochrome and Alternative Pathways in Plant Mitochondria

The contribution of the cyanide-resistant, alternative pathway to plant mitochondrial electron transport has been studied using a modified aqueous phase on-line mass spectrometry-gas chromatography system. This technique permits direct measurement of the partitioning of electrons between the cytochrome and alternative pathways in the absence of added inhibitors. We demonstrate that in mitochondria isolated from soybean (Glycine max L. cv Ransom) cotyledons, the alternative pathway contributes significantly to oxygen uptake under state 4 conditions, when succinate is used as a substrate. However, when NADH is the substrate, addition of pyruvate, an allosteric activator of the alternative pathway, is required to achieve the same level of alternative pathway activity. Under state 3 conditions, when the reduction state of the ubiquinone pool is low, the addition of pyruvate allows the alternative pathway to compete with the cytochrome pathway for electrons from the ubiquinone pool when the cytochrome pathway is not saturated. These results provide direct experimental verification of the kinetics consequences of pyruvate addition on the partitioning of electron flow between the two respiratory pathways. This distribution of electrons between the two unsaturated pathways could not be measured using conventional oxygen electrode methods and illustrates a clear advantage of the mass spectrometry technique. These results have significant ramifications for studies of plant respiration using the oxygen electrode, particularly those studies involving intact tissues.

Comparisons of δ13C of photosynthetic products and ecosystem respiratory CO2 and their responses to seasonal climate variability

This study investigated the relationship between δ13C of ecosystem components, soluble plant carbohydrates and the isotopic signature of ecosystem respired CO2 (δ13CR) during seasonal changes in soil and atmospheric moisture in a beech (Fagus sylvatica L.) forest in the central Apennine mountains, Italy. Decrease in soil moisture and increase in air vapour pressure deficit during summer correlated with substantial increase in δ13C of leaf and phloem sap soluble sugars. Increases in δ13C of ecosystem respired CO2 were linearly related to increases in phloem sugar δ13C (r2=0.99, P≤0.001) and leaf sugar δ13C (r2=0.981, P≤0.01), indicating that a major proportion of ecosystem respired CO2 was derived from recent assimilates. The slopes of the best-fit lines differed significantly (P≤0.05), however, and were about 0.86 (SE=0.04) for phloem sugars and about 1.63 (SE=0.16) for leaf sugars. Hence, changes in isotopic signature in phloem sugars were transferred to ecosystem respiration in the beech forest, while leaf sugars, with relatively small seasonal changes in δ13C, must have a slower turnover rate or a significant storage component. No significant variation in δ13C was observed in bulk dry matter of various plant and ecosystem components (including leaves, bark, wood, litter and soil organics). The apparent coupling between the δ13C of soluble sugars and ecosystem respiration was associated with large apparent isotopic disequilibria. Values of δ13CR were consistently more depleted by about 4‰ relative to phloem sugars, and by about 2‰ compared to leaf sugars. Since no combination of the measured pools could produce the observed δ13CR signal over the entire season, a significant isotopic discrimination against 13C might be associated with short-term ecosystem respiration. However, these differences might also be explained by substantial contributions of other not measured carbon pools (e.g., lipids) to ecosystem respiration or contributions linked to differences in footprint area between Keeling plots and carbohydrate sampling. Linking the seasonal and inter-annual variations in carbon isotope composition of carbohydrates and respiratory CO2 should be applicable in carbon cycle models and help the understanding of inter-annual variation in biospheric sink strength.

Isotopic inhomogeneity of leaf water: Evidence and implications for the use of isotopic signals transduced by plants

Variations as large as 11%. in δ18O values and 50%. in δD values were observed among different fractions of water in leaves of ivy (Hedera helix) and sunflower (Helianthus annuus). This observation contradicts previous experimental approaches to leaf water as an isotopically uniform pool. Using ion analysis of the water fractions to identify sources within the leaf, we conclude that the isotopic composition of the water within cells, which is involved in biosynthesis and therefore recorded in the plant organic matter, differs substantially from that of total leaf water. This conclusion must be taken into account in studies in which isotope ratios of fossil plant cellulose are interpreted in paleoclimatic terms. In addition, our results have implications for attempts to explain the Dole effect and to account for the variations of 18O16O ratios in atmospheric carbon dioxide, since the isotopic composition of cell water, not of total leaf water, influences theδ18O values of O2 and CO2 released from plants into the atmosphere.

Natural Abundance Carbon Isotope Composition of Isoprene Reflects Incomplete Coupling between Isoprene Synthesis and Photosynthetic Carbon Flow

Isoprene emission from leaves is dynamically coupled to photosynthesis through the use of primary and recent photosynthate in the chloroplast. However, natural abundance carbon isotope composition (δ13C) measurements in myrtle (Myrtus communis), buckthorn (Rhamnus alaternus), and velvet bean (Mucuna pruriens) showed that only 72% to 91% of the variations in the δ13C values of fixed carbon were reflected in the δ13C values of concurrently emitted isoprene. The results indicated that 9% to 28% carbon was contributed from alternative, slow turnover, carbon source(s). This contribution increased when photosynthesis was inhibited by CO2-free air. The observed variations in the δ13C of isoprene under ambient and CO2-free air were consistent with contributions to isoprene synthesis in the chloroplast from pyruvate associated with cytosolic Glc metabolism. Irrespective of alternative carbon source(s), isoprene was depleted in 13C relative to mean photosynthetically fixed carbon by 4‰ to 11‰. Variable13C discrimination, its increase by partially inhibiting isoprene synthesis with fosmidomicin, and the associated accumulation of pyruvate suggested that the main isotopic discrimination step was the deoxyxylulose-5-phosphate synthase reaction.

Genomic dissection of drought resistance in durum wheat × wild emmer wheat recombinant inbreed line population.

Drought is the major factor limiting wheat productivity worldwide. The gene pool of wild emmer wheat, Triticum turgidum ssp. dicoccoides, harbours a rich allelic repertoire for morpho-physiological traits conferring drought resistance. The genetic and physiological bases of drought responses were studied here in a tetraploid wheat population of 152 recombinant inbreed lines (RILs), derived from a cross between durum wheat (cv. Langdon) and wild emmer (acc# G18-16), under contrasting water availabilities. Wide genetic variation was found among RILs for all studied traits. A total of 110 quantitative trait loci (QTLs) were mapped for 11 traits, with LOD score range of 3.0-35.4. Several QTLs showed environmental specificity, accounting for productivity and related traits under water-limited (20 QTLs) or well-watered conditions (15 QTLs), and in terms of drought susceptibility index (22 QTLs). Major genomic regions controlling productivity and related traits were identified on chromosomes 2B, 4A, 5A and 7B. QTLs for productivity were associated with QTLs for drought-adaptive traits, suggesting the involvement of several strategies in wheat adaptation to drought stress. Fifteen pairs of QTLs for the same trait were mapped to seemingly homoeologous positions, reflecting synteny between the A and B genomes. The identified QTLs may facilitate the use of wild alleles for improvement of drought resistance in elite wheat cultivars.