Internal conductance to CO(2) diffusion and C(18)OO discrimination in C(3) leaves

(18)O discrimination in CO(2) stems from the oxygen exchange between (18)O-enriched water and CO(2) in the chloroplast, a process catalyzed by carbonic anhydrase (CA). A proportion of this (18)O-labeled CO(2) escapes back to the atmosphere, resulting in an effective discrimination against C(18)OO during photosynthesis (Delta(18)O). By constraining the delta(18)O of chloroplast water (delta(e)) by analysis of transpired water and the extent of CO(2)-H(2)O isotopic equilibrium (theta(eq)) by measurements of CA activity (theta(eq) = 0.75-1.0 for tobacco, soybean, and oak), we could apply measured Delta(18)O in a leaf cuvette attached to a mass spectrometer to derive the CO(2) concentration at the physical limit of CA activity, i.e. the chloroplast surface (c(cs)). From the CO(2) drawdown sequence between stomatal cavities from gas exchange (c(i)), from Delta(18)O (c(cs)), and at Rubisco sites from Delta(13)C (c(c)), the internal CO(2) conductance (g(i)) was partitioned into cell wall (g(w)) and chloroplast (g(ch)) components. The results indicated that g(ch) is variable (0.42-1.13 mol m(-2) s(-1)) and proportional to CA activity. We suggest that the influence of CA activity on the CO(2) assimilation rate should be important mainly in plants with low internal conductances.     

Carbonic anhydrase and its influence on carbon isotope discrimination during C4 photosynthesis. Insights from antisense RNA in Flaveria bidentis

In C4 plants, carbonic anhydrase (CA) facilitates both the chemical and isotopic equilibration of atmospheric CO2 and bicarbonate (HCO3-) in the mesophyll cytoplasm. The CA-catalyzed reaction is essential for C4 photosynthesis, and the model of carbon isotope discrimination (Delta13C) in C4 plants predicts that changes in CA activity will influence Delta13C. However, experimentally, the influence of CA on Delta13C has not been demonstrated in C4 plants. Here, we compared measurements of Delta13C during C4 photosynthesis in Flaveria bidentis wild-type plants with F. bidentis plants with reduced levels of CA due to the expression of antisense constructs targeted to a putative mesophyll cytosolic CA. Plants with reduced CA activity had greater Delta13C, which was also evident in the leaf dry matter carbon isotope composition (delta13C). Contrary to the isotope measurements, photosynthetic rates were not affected until CA activity was less than 20% of wild type. Measurements of Delta13C, delta13C of leaf dry matter, and rates of net CO2 assimilation were all dramatically altered when CA activity was less than 5% of wild type. CA activity in wild-type F. bidentis is sufficient to maintain net CO2 assimilation; however, reducing leaf CA activity has a relatively large influence on Delta13C, often without changes in net CO2 assimilation. Our data indicate that the extent of CA activity in C4 leaves needs to be taken into account when using Delta13C and/or delta13C to model the response of C4 photosynthesis to changing environmental conditions.     

A transgenic approach to understanding the influence of carbonic anhydrase on C18OO discrimination during C4 photosynthesis

The oxygen isotope composition of atmospheric CO(2) is an important signal that helps distinguish between ecosystem photosynthetic and respiratory processes. In C(4) plants the carbonic anhydrase (CA)-catalyzed interconversion of CO(2) and bicarbonate (HCO(3)(-)) is an essential first reaction for C(4) photosynthesis but also plays an important role in the CO(2)-H(2)O exchange of oxygen as it enhances the rate of isotopic equilibrium between CO(2) and water. The C(4) dicot Flaveria bidentis containing genetically reduced levels of leaf CA (CA(leaf)) has been used to test whether changing leaf CA activity influences online measurements of C(18)OO discrimination (Delta(18)O) and the proportion of CO(2) in isotopic equilibrium with leaf water at the site of oxygen exchange (theta). The Delta(18)O in wild-type F. bidentis, which contains high levels of CA relative to the rates of net CO(2) assimilation, was less than predicted by models of Delta(18)O. Additionally, Delta(18)O was sensitive to small decreases in CA(leaf). However, reduced CA activity in F. bidentis had little effect on net CO(2) assimilation, transpiration rates (E), and stomatal conductance (g(s)) until CA levels were less than 20% of wild type. The values of theta determined from measurements of Delta(18)O and the (18)O isotopic composition of leaf water at the site of evaporation (delta(e)) were low in the wild-type F. bidentis and decreased in transgenic plants with reduced levels of CA activity. Measured values of theta were always significantly lower than the values of theta predicted from in vitro CA activity and gas exchange. The data presented here indicates that CA content in a C(4) leaf may not represent the CA activity associated with the CO(2)-H(2)O oxygen exchange and therefore may not be a good predictor of theta during C(4) photosynthesis. Furthermore, uncertainties in the isotopic composition of water at the site of exchange may also limit the ability to accurately predict theta in C(4) plants.     

The role of phosphoenolpyruvate carboxylase during C4 photosynthetic isotope exchange and stomatal conductance

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) plays a key role during C(4) photosynthesis and is involved in anaplerotic metabolism, pH regulation, and stomatal opening. Heterozygous (Pp) and homozygous (pp) forms of a PEPC-deficient mutant of the C(4) dicot Amaranthus edulis were used to study the effect of reduced PEPC activity on CO(2) assimilation rates, stomatal conductance, and (13)CO(2) (Delta(13)C) and C(18)OO (Delta(18)O) isotope discrimination during leaf gas exchange. PEPC activity was reduced to 42% and 3% and the rates of CO(2) assimilation in air dropped to 78% and 10% of the wild-type values in the Pp and pp mutants, respectively. Stomatal conductance in air (531 mubar CO(2)) was similar in the wild-type and Pp mutant but the pp mutant had only 41% of the wild-type steady-state conductance under white light and the stomata opened more slowly in response to increased light or reduced CO(2) partial pressure, suggesting that the C(4) PEPC isoform plays an essential role in stomatal opening. There was little difference in Delta(13)C between the Pp mutant (3.0 per thousand +/- 0.4 per thousand) and wild type (3.3 per thousand +/- 0.4 per thousand), indicating that leakiness (), the ratio of CO(2) leak rate out of the bundle sheath to the rate of CO(2) supply by the C(4) cycle, a measure of the coordination of C(4) photosynthesis, was not affected by a 60% reduction in PEPC activity. In the pp mutant Delta(13)C was 16 per thousand +/- 3.2 per thousand, indicative of direct CO(2) fixation by Rubisco in the bundle sheath at ambient CO(2) partial pressure. Delta(18)O measurements indicated that the extent of isotopic equilibrium between leaf water and the CO(2) at the site of oxygen exchange () was low (0.6) in the wild-type and Pp mutant but increased to 0.9 in the pp mutant. We conclude that in vitro carbonic anhydrase activity overestimated as compared to values determined from Delta(18)O in wild-type plants.     

Discrimination in the dark. Resolving the interplay between metabolic and physical constraints to phosphoenolpyruvate carboxylase activity during the crassulacean acid metabolism cycle

A model defining carbon isotope discrimination (delta13C) for crassulacean acid metabolism (CAM) plants was experimentally validated using Kalanchoe daigremontiana. Simultaneous measurements of gas exchange and instantaneous CO2 discrimination (for 13C and 18O) were made from late photoperiod (phase IV of CAM), throughout the dark period (phase I), and into the light (phase II). Measurements of CO2 response curves throughout the dark period revealed changing phosphoenolpyruvate carboxylase (PEPC) capacity. These systematic changes in PEPC capacity were tracked by net CO2 uptake, stomatal conductance, and online delta13C signal; all declined at the start of the dark period, then increased to a maximum 2 h before dawn. Measurements of delta13C were higher than predicted from the ratio of intercellular to external CO2 (p(i)/p(a)) and fractionation associated with CO2 hydration and PEPC carboxylations alone, such that the dark period mesophyll conductance, g(i), was 0.044 mol m(-2) s(-1) bar(-1). A higher estimate of g(i) (0.085 mol m(-2) s(-1) bar(-1)) was needed to account for the modeled and measured delta18O discrimination throughout the dark period. The differences in estimates of g(i) from the two isotope measurements, and an offset of -5.5 per thousand between the 18O content of source and transpired water, suggest spatial variations in either CO2 diffusion path length and/or carbonic anhydrase activity, either within individual cells or across a succulent leaf. Our measurements support the model predictions to show that internal CO2 diffusion limitations within CAM leaves increase delta13C discrimination during nighttime CO2 fixation while reducing delta13C during phase IV. When evaluating the phylogenetic distribution of CAM, carbon isotope composition will reflect these diffusive limitations as well as relative contributions from C3 and C4 biochemistry.     

Carbon isotopic composition and oxygen isotopic enrichment in phloem and total leaf organic matter of European beech (Fagus sylvatica L.) along a climate gradient

This study investigated the influence of climate on the carbon isotopic composition (sigma13C) and oxygen isotopic enrichment (delta18O) above the source water of different organic matter pools in European beech. In July and September 2002, sigma13C and delta18O were determined in phloem carbohydrates and in bulk foliage of adult beech trees along a transect from central Germany to southern France, where beech reaches its southernmost distributional limit. The data were related to meteorological and physiological parameters. The climate along the transect stretches from temperate [subcontinental (SC)] to submediterranean (SM). Both sigma13Cleaf and delta18Oleaf were representative of site-specific long-term environmental conditions. sigma13C of leaves collected in September was indicative of stomatal conductance, vapour pressure deficit (VPD) and radiation availability of the current growing season. delta18O was mainly correlated to mean growing season relative humidity (RH) and VPD. In contrast to the leaves, sigma13Cphloem varied considerably between July and September and was well correlated with canopy stomatal conductance (Gs) in a 2 d integral prior to phloem sampling. The relationship between sigma13C and delta18O in both leaves and phloem sap points, however, to a combined influence of stomatal conductance and photosynthetic capacity on the variation of sigma13C along the transect. delta18Ophloem could be described by applying a model that included 18O fractionation associated with water exchange between the leaf and the atmosphere and with the production of organic matter. Hence, isotope signatures can be used as effective tools to assess the water balance of beech, and thus, help predict the effects of climatic change on one of the ecologically and economically most important tree species in Central Europe.     

C4 photosynthetic isotope exchange in NAD-ME- and NADP-ME-type grasses

Monitoring photosynthetic isotope exchange is an important tool for predicting the influence of plant communities on the global carbon cycle in response to climate change. C(4) grasses play an important role in the global carbon cycle, but their contribution to the isotopic composition of atmospheric CO(2) is not well understood. Instantaneous measurements of (13)CO(2) (Delta(13)C) and C(18)OO (Delta(18)O) isotope exchange in five NAD-ME and seven NADP-ME C(4) grasses have been conducted to investigate the difference in photosynthetic CO(2) isotopic fractionation in these subgroups. As previously reported, the isotope composition of the leaf material (delta(13)C) was depleted in (13)C in the NAD-ME compared with the NADP-ME grasses. However, Delta(13)C was not different between subtypes at high light, and, although Delta(13)C increased at low light, it did so similarly in both subtypes. This suggests that differences in leaf delta(13)C between the C(4) subtypes are not caused by photosynthetic isotope fractionation and leaf delta(13)C is not a good indicator of bundle sheath leakiness. Additionally, low carbonic anhydrase (CA) in C(4) grasses may influences Delta(13)C and should be considered when estimating the contribution of C(4) grasses to the global isotopic signature of atmospheric CO(2). It was found that measured Delta(18)O values were lower than those predicted from leaf CA activities and Delta(18)O was similar in all species measured. The Delta(18)O in these C(4) grasses is similar to low Delta(18)O previously measured in C(4) dicots which contain 2.5 times the leaf CA activity, suggesting that leaf CA activity is not a predictor of Delta(18)O in C(4) plants.     

Combining delta 13 C and delta 18 O analyses to unravel competition, CO2 and O3 effects on the physiological performance of different-aged trees

Combined delta(13)C and delta(18)O analyses of leaf material were used to infer changes in photosynthetic capacity (A(max)) and stomatal conductance (g(l)) in Fagus sylvatica and Picea abies trees growing under natural and controlled conditions. Correlation between g(l) and delta(18)O in leaf cellulose (delta(18)O(cel)) allowed us to apply a semi-quantitative model to infer g(l) from delta(18)O(cel) and also interpret variation in delta(13)C as reflecting variation in A(max). Extraction of leaf cellulose was necessary, because delta(18)O from leaf organic matter (delta(18)O(LOM)) and delta(18)O(cel) was not reliably correlated. In juvenile trees, the model predicted elevated carbon dioxide (CO(2)) to reduce A(max) in both species, whereas ozone (O(3)) only affected beech by reducing CO(2) uptake via lowered g(l). In adult trees, A(max) declined with decreasing light level as g(l) was unchanged. O(3) did not significantly affect isotopic signatures in leaves of adult trees, reflecting the higher O(3) susceptibility of juvenile trees under controlled conditions. The isotopic analysis compared favourably to the performance of leaf gas exchange, underlining that the semi-quantitative model approach provides a robust way to gather time-integrated information on photosynthetic performance of trees under multi-faced ecological scenarios, in particular when information needed for quantitative modelling is only scarcely available.     

Photosynthetic Fractionation of the Stable Isotopes of Oxygen and Carbon

Isotope discrimination during photosynthetic exchange of O2 and CO2 was measured using enzyme, thylakoid, and whole cell preparations. Evolved oxygen from isolated spinach thylakoids was isotopically identical (within analytical error) to its source water. Similar results were obtained with Anacystis nidulans Richter and Phaeodactylum tricornutum Bohlin cultures purged with helium. For consumptive reactions, discrimination ([delta], where 1 + [delta]/1000 equals the isotope effect, k16/k18 or k12/k13) was determined by analysis of residual substrate (O2 or CO2). The [delta] for the Mehler reaction, mediated by ferredoxin or methylviologen, was 15.3[per mille (thousand) sign]. Oxygen isotope discrimination during oxygenation of ribulose-1,5-bisphosphate (RuBP) catalyzed by RuBP carboxylase/oxygenase (Rubisco) was 21.3[per mille (thousand) sign] and independent of enzyme source, unlike carbon isotope discrimination: 30.3[per mille (thousand) sign] for spinach enzyme and 19.6 to 23[per mille (thousand) sign] for Rhodospirillum rubrum and A. nidulans enzymes, depending on reaction conditions. The [delta] for O2 consumption catalyzed by glycolate oxidase was 22.7[per mille (thousand) sign]. The expected overall [delta] for photorespiration is about 21.7[per mille (thousand) sign]. Consistent with this, when Asparagus sprengeri Regel mesophyll cells approached the compensation point within a sealed vessel, the [delta]18O of dissolved O2 came to a steady-state value of about 21.5[per mille (thousand) sign] relative to the source water. The results provide improved estimates of discrimination factors in several reactions prominent in the global O cycle and indicate that photorespiration plays a significant part in determining the isotopic composition of atmospheric oxygen.     

A new measurement technique reveals temporal variation in delta18O of leaf-respired CO2

The oxygen isotope composition of CO(2) respired by Ricinus communis leaves (delta(18)O(R)) was measured under non-steady-state conditions with a temporal resolution of 3 min using a tunable diode laser (TDL) absorption spectrometer coupled to a portable gas exchange system. The SD of delta(18)O measurement by the TDL was +/- 0.2 per thousand and close to that of traditional mass spectrometers. Further, delta(18)O(R) values at isotopic steady state were comparable to those obtained using traditional flask sampling and mass spectrometric techniques for R. communis grown and measured in similar environmental conditions. As well as higher temporal resolution, the online TDL method described here has a number of advantages over mass spectrometric techniques. At isotopic steady state among plants grown at high light, the "one-way flux" model was required to accurately predict delta(18)O(R). A comparison of measurements and the model suggests that plants grown under low-light conditions have either a lower proportion of chloroplast CO(2) that isotopically equilibrates with chloroplast water, or more enriched delta(18)O of CO(2) in the chloroplast that has not equilibrated with local water. The high temporal resolution of isotopic measurements allowed the first measurements of delta(18)O(R) when stomatal conductance was rapidly changing. Under non-steady-state conditions, delta(18)O(R) varied between 50 and 220 per thousand for leaves of plants grown under different light and water environments, and varied by as much as 100 per thousand within 10 min for a single leaf. Stomatal conductance ranged from 0.001 to 1.586 mol m(-2) s(-1), and had an important influence on delta(18)O(R) under non-steady-state conditions not only via effects on leaf water H(2) (18)O enrichment, but also via effects on the rate of the one-way fluxes of CO(2) into and out of the leaf.     

Discrimination Processes and Shifts in Carboxylation during the Phases of Crassulacean Acid Metabolism

The magnitude and extent of Crassulacean acid metabolism (CAM) activity in two Clusia species was manipulated to investigate the regulation of the distinct CAM phases. First, in response to leaf-air vapor pressure deficit at night, changes in leaf conductance altered on-line carbon-isotope discrimination throughout the theoretical range for dark CO2 uptake during CAM. These ranged from the limit set by phosphoenolpyruvate carboxylase (PEPc) (-6[per mille (thousand) sign], [delta]13C equivalent of -2[per mille (thousand) sign]) to that imposed by diffusion limitation (+4[per mille (thousand) sign], [delta]13C equivalent of -12[per mille (thousand) sign]), but the lowest carbon-isotope discrimination occurred when P[square root]pa was only 0.7. Second, when the availability of external or internal sources of CO2 was reduced for both field- and greenhouse-grown plants, CO2 uptake by day via PEPc during phase II largely compensated. Third, by reducing the dark period, plants accumulated low levels of acidity, and CO2 uptake occurred throughout the subsequent light period. Discrimination switched from being dominated by PEPc (phase II) to ribulose 1,5-bisphosphate carboxylase/oxygenase (phase III), with both enzymes active during phase IV. Under natural conditions, photochemical stability is maintained by extended PEPc activity in phase II, which enhances acid accumulation and delays decarboxylation until temperature and light stress are maximal at midday.     

Toward using δ13C of ecosystem respiration to monitor canopy physiology in complex terrain

In 2005 and 2006, air samples were collected at the base of a Douglas-fir watershed to monitor seasonal changes in the delta13CO2 of ecosystem respiration (delta13C(ER)). The goals of this study were to determine whether variations in delta13C(ER) correlated with environmental variables and could be used to predict expected variations in canopy-average stomatal conductance (Gs). Changes in delta13C(ER) correlated weakly with changes in vapor pressure deficit (VPD) measured 0 and 3-7 days earlier and significantly with soil matric potential (psi(m)) (P value <0.02) measured on the same day. Midday G (s) was estimated using sapflow measurements (heat-dissipation method) at four plots located at different elevations within the watershed. Values of midday Gs from 0 and 3-7 days earlier were correlated with delta13C(ER), with the 5-day lag being significant (P value <0.05). To examine direct relationships between delta13C(ER) and recent Gs, we used models relating isotope discrimination to stomatal conductance and photosynthetic capacity at the leaf level to estimate values of stomatal conductance ("Gs-I") that would be expected if respired CO2 were derived entirely from recent photosynthate. We compared these values with estimates of Gs using direct measurement of transpiration at multiple locations in the watershed. Considering that the approach based on isotopes considers only the effect of photosynthetic discrimination on delta13C(ER), the magnitude and range in the two values were surprisingly similar. We conclude that: (1) delta13C(ER) is sensitive to variations in weather, and (2) delta13C(ER) potentially could be used to directly monitor average, basin-wide variations in Gs in complex terrain if further research improves understanding of how delta13C(ER) is influenced by post-assimilation fractionation processes.     

Stomatal behavior of ozone-sensitive and -insensitive coneflowers (Rudbeckia laciniata var. digitata) in Great Smoky Mountains National Park

* Morphological and physiological attributes were assessed to elucidate the underlying mechanisms of ozone (O(3)) sensitivity in a highly sensitive species, cutleaf coneflower (Rudbeckia laciniata var. digitata). * Foliage at the same height in the canopy on paired O(3)-sensitive and -insensitive cutleaf coneflowers was assessed for level of foliar symptoms, stomatal density, stomatal responsiveness to dynamic changes in light and leaf-to-air vapor pressure deficit (VPD), steady-state responses to light and CO(2), intrinsic transpirational efficiency, and plant water balance. * There were no morphological differences between the sensitivity types that might have contributed to greater O(3) uptake in sensitive individuals. Stomata of sensitive plants were less responsive than those of insensitive plants to experimentally increased and decreased light intensities, and to increased VPD. O(3)-insensitive plants had greater intrinsic transpirational efficiencies, greater maximum assimilation rates under saturating CO(2) and light, and greater carboxylation rates. * Different physiological attributes vary independently within an individual plant, which collectively confer sensitivity or insensitivity to O(3) injury.     

Association between carbonyl sulfide uptake and (18)Δ during gas exchange in C(3) and C(4) leaves

Carbonyl sulfide (COS) and C(18)OO exchange by leaves provide potentially powerful tracers of biosphere-atmosphere CO(2) exchange, and both are assumed to depend on carbonic anhydrase (CA) activity and conductance along the diffusive pathway in leaves. We investigated these links using C(3) and C(4) plants, hypothesizing that the rates of COS and C(18)OO exchange by leaves respond in parallel to environmental and biological drivers. Using CA-deficient antisense lines of C(4) and C(3) plants, COS uptake was essentially eliminated and discrimination against C(18)OO exchange ((18)Δ) greatly reduced, demonstrating CA's key role in both processes. (18)Δ showed a positive linear correlation with leaf relative uptake (LRU; ratio of COS to CO(2) assimilation rates, A(s)/A(c), normalized to their respective ambient concentrations), which reflected the effects of stomatal conductance on both COS and C(18)OO exchange. Unexpectedly, a decoupling between A(s) and (18)Δ was observed in comparing C(4) and C(3) plants, with a large decrease in (18)Δ but no parallel reduction in A(s) in the former. This could be explained by C(4) plants having higher COS concentrations at the CA site (maintaining high A(s) with reduced CA) and a high phosphoenolpyruvate carboxylase/CA activity ratio (reducing (18)O exchange efficiency between CO(2) and water, but not A(s)). Similar A(s) but higher A(c) in C(4) versus C(3) plants resulted in lower LRU values in the former (1.16 ± 0.20 and 1.82 ± 0.18 for C(4) and C(3), respectively). LRU was, however, relatively constant in both plant types across a wide range of conditions, except low light (<191 μmol photon m(-2) s(-1)).     

Photosynthetic performance of an Arabidopsis mutant with elevated stomatal density (sdd1-1) under different light regimes

In the Arabidopsis mutant sdd1-1, a point mutation in a single gene (SDD1) causes specific alterations in stomatal density and distribution. In comparison to the wild type (C24), abaxial surfaces of sdd1-1 rosette leaves have about 2.5-fold higher stomatal densities. This mutant was used to study the consequence of stomatal density on photosynthesis under various light regimes. The increased stomatal density in the mutant had no significant influence on the leaf CO(2) assimilation rate (A) under constant light conditions. Mutant and wild-type plants contained similar amounts of carbohydrates under these conditions. However, exposure of plants to increasing photon flux densities resulted in differences in gas exchange and the carbohydrate metabolism of the wild type and mutant. Increased stomatal densities in sdd1-1 enabled low-light-adapted plants to have 30% higher CO(2) assimilation rates compared to the wild type when exposed to high light intensities. After 2 d under high light conditions leaves of sdd1-1 accumulated 30% higher levels of starch and hexoses than wild-type plants.     

Factors affecting the isotopic composition of organic matter. (1) Carbon isotopic composition of terrestrial plant materials

The stable isotope composition of the light elements (i.e., H, C, N, O and S) of organic samples varies significantly and, for C, is also unique and distinct from that of inorganic carbon. This is the result of (1) the isotope composition of reactants, (2) the nature of the reactions leading to formation and post-formational modification of the samples, (3) the environmental conditions under which the reactions took place, and (4) the relative concentration of the reactants compared to that of the products (i.e., [products]/[reactants] ratio). This article will examine the carbon isotope composition of terrestrial plant materials and its relationship with the above factors. delta13C(PDB) values of terrestrial plants range approximately from -8 to -38%, inclusive of C3-plants (-22 to -38%), C4-plants (-8 to -15%) and CAM-plants (-13 to -30%). Thus, the delta13C(PDB) values largely reflect the photosynthesis pathways of a plant as well as the genetics (i.e., species difference), delta13C(PDB) values of source CO2, relevant humidity, CO2/O2 ratios, wind and light intensity etc. Significant variations in these values also exist among different tissues, different portions of a tissue and different compounds. This is mainly a consequence of metabolic reactions. Animals mainly inherit the delta13C(PDB) values of the foods they consume; therefore, their delta13C(PDB) values are similar. The delta13C(PDB) values of plant materials, thus, contain information regarding the inner workings of the plants, the environmental conditions under which they grow, the delta13C(PDB) values of CO2 sources etc., and are unique. Furthermore, this uniqueness is passed on to their derivative matter, such as animals, humus etc. Hence, they are very powerful tools in many areas of research, including the ecological and environmental sciences.     

Prevention of stomatal closure by immunomodulation of endogenous abscisic acid and its reversion by abscisic acid treatment: physiological behaviour and morphological features of tobacco stomata

Transgenic tobacco ( Nicotiana tabacum L.) plants ubiquitously accumulating a single-chain variable-fragment (scFv) antibody against abscisic acid (ABA) to high concentrations in the endoplasmic reticulum (RA plants) show a wilty phenotype. High stomatal conductance and loss of CO(2) and light dependence of stomatal conductance are typical features of these plants. ABA was applied to these plants either via the petioles or by daily spraying over several weeks in order to normalise the phenotype. During the long-term experiments, scFv protein concentrations, total and (calculated) free ABA contents, and stomatal conductance and its dependence on CO(2) concentration and light intensity were monitored. The wilty phenotype of transgenic plants could not be normalised by short-term treatment with ABA via the petioles. Only a daily long-term treatment during plant development normalised the physiological behaviour completely. Scanning electron microscopy of stomata showed morphological changes in RA plants compared with wild-type plants that, for structural reasons, prevented regular stomatal movements. After long-term treatment with ABA this defect could be completely eliminated. Guard-cell-specific expression of the anti-ABA scFv did not cause any changes in physiological behaviour compared to the wild type. In addition, mesophyll-specific expression starting in leaves that were already fully differentiated resulted in normal phenotypes, too. We conclude that changes in distribution and availability of ABA in the cells of developing leaves of RA plants cause the development of structural features in stomata that prevent normal function.     

C4 Photosynthesis (The CO2-Concentrating Mechanism and Photorespiration)

Despite previous reports of no apparent photorespiration in C4 plants based on measurements of gas exchange under 2 versus 21% O2 at varying [CO2], photosynthesis in maize (Zea mays) shows a dual response to varying [O2]. The maximum rate of photosynthesis in maize is dependent on O2 (approximately 10%). This O2 dependence is not related to stomatal conductance, because measurements were made at constant intercellular CO2 concentration (Ci); it may be linked to respiration or pseudocyclic electron flow. At a given Ci, increasing [O2] above 10% inhibits both the rate of photosynthesis, measured under high light, and the maximum quantum yield, measured under limiting light ([phi]CO2). The dual effect of O2 is masked if measurements are made under only 2 versus 21% O2. The inhibition of both photosynthesis and [phi]CO2 by O2 (measured above 10% O2) with decreasing Ci increases in a very similar manner, characteristically of O2 inhibition due to photorespiration. There is a sharp increase in O2 inhibition when the Ci decreases below 50 [mu]bar of CO2. Also, increasing temperature, which favors photorespiration, causes a decrease in [phi]CO2 under limiting CO2 and 40% O2. By comparing the degree of inhibition of photosynthesis in maize with that in the C3 species wheat (Triticum aestivum) at varying Ci, the effectiveness of C4 photosynthesis in concentrating CO2 in the leaf was evaluated. Under high light, 30[deg]C, and atmospheric levels of CO2 (340 [mu]bar), where there is little inhibition of photosynthesis in maize by O2, the estimated level of CO2 around ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the bundle sheath compartment was 900 [mu]bar, which is about 3 times higher than the value around Rubisco in mesophyll cells of wheat. A high [CO2] is maintained in the bundle sheath compartment in maize until Ci decreases below approximately 100 [mu]bar. The results from these gas exchange measurements indicate that photorespiration occurs in maize but that the rate is low unless the intercellular [CO2] is severely limited by stress.     

Reduction of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by Antisense RNA in the C4 Plant Flaveria bidentis Leads to Reduced Assimilation Rates and Increased Carbon Isotope Discrimination

Transgenic Flaveria bidentis (a C4 species) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were used to examine the relationship between the CO2 assimilation rate, Rubisco content, and carbon isotope discrimination. Reduction in the amount of Rubisco in the transgenic plants resulted in reduced CO2 assimilation rates and increased carbon isotope discrimination of leaf dry matter. The H2O exchange was similar in transgenic and wild-type plants, resulting in higher ratios of intercellular to ambient CO2 partial pressures. Carbon isotope discrimination was measured concurrently with CO2 and H2O exchange on leaves of the control plants and T1 progeny with a 40% reduction in Rubisco. From the theory of carbon isotope discrimination in the C4 species, we conclude that the reduction in the Rubisco content in the transgenic plants has led to an increase in bundle-sheath CO2 concentration and CO2 leakage from the bundle sheath; however, some down-regulation of the C4 cycle also occurred.