Carbon isotope composition of C4 grasses is influenced by light and water supply

The carbon isotope composition of C⁴ grasses has the potential to be used as an indicator of changes in the isotopic composition and concentration of atmospheric CO2, especially for climate reconstruction. The usefulness of C₄ grasses for this purpose hinges on the assumption that their photosynthetic discrimination against ¹³C remains constant in a wide range of environmental conditions. We tested this assumption by examining the effects of light and water stress on the carbon isotope composition of C₄ grasses using different biochemical subtypes (NADP-ME, NAD-ME, PCK) in glasshouse experiments. We grew 14 different C₄ grass species in four treatments: sun-watered, sun-drought, shade-watered and shade-drought. Carbon isotope discrimination (Δ) rarely remained constant. In general, Δ values were lowest in sun-watered grasses, greater for sun-drought plants and even higher for plants of the shade-watered treatment. The highest Δ values were generally found in the most stressed grasses, the shade-drought plants. Grasses of the NADP-ME subtype were the least influenced by a change in environmental variables, followed by PCK and NAD-ME subtypes. Water availability affected the carbon isotope discrimination less than light limitation in PCK and NAD-ME subtypes, but similarly in NADP-ME subtypes. In another experiment, we studied the effect of increasing light levels (150 to 1500 μmol photons m^?2 s^?1) on the Δ values of 18 well-watered C₄ grass species. Carbon isotope discrimination remained constant until photon flux density (PFD) was less than 700 μmol photons m^?2 s^?1. Below this light level, Δ values increased with decreasing irradiance for all biochemical subtypes. The change in A was less pronounced in NADP-ME and PCK than in NAD-ME grasses. Grasses grown in the field and in the glasshouse showed a similar pattern. Thus, caution should be exercised when using C4 plants under varying environmental conditions to monitor the concentration or carbon isotopic composition of atmospheric CO2 in field/glasshouse studies or climate reconstruction.     

Carbon isotope discrimination and bundle sheath leakiness in three C(4) subtypes grown under variable nitrogen,water and atmospheric CO(2) supply

The changes in composition and productivity of semi-arid C(4) grassland, anticipated with rising atmospheric CO(2), will depend on soil water and nutrient availability. The interactive effects of soil resource limitation and elevated CO(2 )on these grasses, furthermore, may vary among C(4) biochemical subtypes (NADP-ME, NAD-ME, PCK) that differ in bundle sheath leakiness (Phi) responses to drought and nitrogen supply. To address C(4) subtype responses to soil resource gradients, the carbon isotope discrimination (Delta), bundle sheath leakiness (Phi), leaf gas exchange (A, g(s), c(i)/c(a)) and above-ground biomass accumulation were measured on three dominant grasses of semi-desert grassland in south-eastern Arizona. Bouteloua curtipendula (PCK), Aristida glabrata (NADP-ME) and the non-native Eragrostis lehmanniana (NAD-ME) were grown in controlled-environment chambers from seed under a complete, multi-factorial combination of present ambient (370 ppm) and elevated (690 ppm) CO(2) concentration and under high and low water and nitrogen supply. E. lehmanniana (NAD-ME) had the highest photosynthetic rate (A) and lowest Phi compared to the other two grasses when grown under low nitrogen and water availability. However, favourable water and nitrogen supply and elevated atmospheric CO(2) enhanced photosynthetic performance and above-ground biomass production of B. curtipendula (PCK) to a greater extent than in A. glabrata and E. lehmanniana. Contrary to pre vious studies, Phi and Delta in the NADP-ME subtype (A. glabrata) were most affected by changing environmental conditions compared to the other subtypes; deviations from the classic NADP-ME anatomy in Aristida could have accounted for this result. Overall, response of semi-arid grasslands to rising atmospheric CO(2) may depend more on species-specific responses to drought and nitrogen limitation than on general C(4) subtype responses.     

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.     

Faster Rubisco is the key to superior nitrogen-use efficiency in NADP-malic enzyme relative to NAD-malic enzyme C4 grasses

In 27 C4 grasses grown under adequate or deficient nitrogen (N) supplies, N-use efficiency at the photosynthetic (assimilation rate per unit leaf N) and whole-plant (dry mass per total leaf N) level was greater in NADP-malic enzyme (ME) than NAD-ME species. This was due to lower N content in NADP-ME than NAD-ME leaves because neither assimilation rates nor plant dry mass differed significantly between the two C4 subtypes. Relative to NAD-ME, NADP-ME leaves had greater in vivo (assimilation rate per Rubisco catalytic sites) and in vitro Rubisco turnover rates (k(cat); 3.8 versus 5.7 s(-1) at 25 degrees C). The two parameters were linearly related. In 2 NAD-ME (Panicum miliaceum and Panicum coloratum) and 2 NADP-ME (Sorghum bicolor and Cenchrus ciliaris) grasses, 30% of leaf N was allocated to thylakoids and 5% to 9% to amino acids and nitrate. Soluble protein represented a smaller fraction of leaf N in NADP-ME (41%) than in NAD-ME (53%) leaves, of which Rubisco accounted for one-seventh. Soluble protein averaged 7 and 10 g (mmol chlorophyll)(-1) in NADP-ME and NAD-ME leaves, respectively. The majority (65%) of leaf N and chlorophyll was found in the mesophyll of NADP-ME and bundle sheath of NAD-ME leaves. The mesophyll-bundle sheath distribution of functional thylakoid complexes (photosystems I and II and cytochrome f) varied among species, with a tendency to be mostly located in the mesophyll. In conclusion, superior N-use efficiency of NADP-ME relative to NAD-ME grasses was achieved with less leaf N, soluble protein, and Rubisco having a faster k(cat).     

The activities of PEP carboxylase and the C4 acid decarboxylases are little changed by drought stress in three C4 grasses of different subtypes

The C(4) photosynthetic pathway involves the assimilation of CO(2) by phosphoenolpyruvate carboxylase (PEPC) and the subsequent decarboxylation of C(4) acids. The enzymes of the CO(2) concentrating mechanism could be affected under water deficit and limit C(4) photosynthesis. Three different C(4) grasses were submitted to gradually induced drought stress conditions: Paspalum dilatatum (NADP-malic enzyme, NADP-ME), Cynodon dactylon (NAD-malic enzyme, NAD-ME) and Zoysia japonica (PEP carboxykinase, PEPCK). Moderate leaf dehydration affected the activity and regulation of PEPC in a similar manner in the three grasses but had species-specific effects on the C(4) acid decarboxylases, NADP-ME, NAD-ME and PEPCK, although changes in the C(4) enzyme activities were small. In all three species, the PEPC phosphorylation state, judged by the inhibitory effect of L: -malate on PEPC activity, increased with water deficit and could promote increased assimilation of CO(2) by the enzyme under stress conditions. Appreciable activity of PEPCK was observed in all three species suggesting that this enzyme may act as a supplementary decarboxylase to NADP-ME and NAD-ME in addition to its role in other metabolic pathways.     

Assessing environmental and physiological controls over water relations in a Scots pine (Pinus sylvestris L.) stand through analyses of stable isotope composition of water and organic matter

This study investigated the influence of meteorological, pedospheric and physiological factors on the water relations of Scots pine, as characterized by the origin of water taken up, by xylem transport as well as by carbon isotope discrimination (Delta13C) and oxygen isotope enrichment (Delta18O) of newly assimilated organic matter. For more than 1 year, we quantified delta2H and delta18O of potential water sources and xylem water as well as Delta13C and Delta18O in twig and trunk phloem organic matter biweekly, and related these values to continuously measured or modelled meteorological parameters, soil water content, stand transpiration (ST) and canopy stomatal conductance (G(s)). During the growing season, delta18O and delta2H of xylem water were generally in a range comparable to soil water from a depth of 2-20 cm. Long residence time of water in the tracheids uncoupled the isotopic signals of xylem and soil water in winter. Delta18O but not Delta13C in phloem organic matter was directly indicative of recent environmental conditions during the whole year. Delta18O could be described applying a model that included 18O fractionation associated with water exchange between leaf and atmosphere, and with the production of organic matter as well as the influence of transpiration. Phloem Delta13C was assumed to be concertedly influenced by G(s) and photosynthetically active radiation (PAR) (as a proxy for photosynthetic capacity). We conclude that isotope signatures can be used as effective tools (1) to characterize the seasonal dynamics in source and xylem water, and (2) to assess environmental effects on transpiration and G(s) of Scots pine, thus helping to understand and predict potential impacts of climate change on trees and forest ecosystems.     

Immunogold localization of photosynthetic enzymes in leaves of various C4 plants, with particular reference to pyruvate orthophosphate dikinase

Cellular localization of photosynthetic enzymes was investigated by immunogold electron microscopy for leaves of nine C4 grasses (three NADP-malic enzyme (NADP-ME)subtype species, three NAD-malic enzyme (NAD-ME) subtype species, and three phosphoenolpyruvate carboxykinase (PCK) subtype species), two C4 sedges (NADP-ME subtype species) and two C4 dicots (an NADP-ME and an NADP/NAD-ME subtype species). In leaves of all species, immunogold labelling was present for phosphoenolpyruvate carboxylase in the cytosol of the mesophyll cells (MC) and for ribulose-1,5-bisphosphate carboxylase/oxygenase in the chloroplasts of the bundle sheath cells (BSC). However, considerable specific variation was found in the intercellular patterns of labelling for pyruvate orthophosphate dikinase (PPDK). In the NADP-ME grasses, two NAD-ME grasses, and the dicots, significant labelling for PPDK was present in the both the BSC and the MC chloroplasts. In the other NAD-ME grass, the PCK grasses, and the sedges, labelling for PPDK was present almost exclusively in the chloroplasts of the MC. These patterns were observed in the leaves of both young seedlings and mature plants. These results indicate that the accumulation of PPDK in leaves of C4 plants is not necessarily restricted to the MC, although the chloroplasts of the MC accumulate more than those of the BSC.Key words: C4 plants, immunolocalization, phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, ribulose-1,5-bisphosphate carboxylase/oxygenase.      

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.     

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.     

Relationship between leaf nitrogen and photosynthetic rate for three NAD-ME and three NADP-ME C4 grasses

Theoretical considerations have suggested that there may be differences in photosynthetic nitrogen use efficiency (PNUE) among plants that use different biochemical variants of C(4) photosynthesis. To test this hypothesis we examined the leaf nitrogen content and photosynthetic rates of six grass species (three of C(4) subtype NAD-ME and three of C(4) subtype NADP-ME) grown over a wide range of nitrogen supply. While there were significant differences among the species in various traits, there were no consistent differences between the C(4) subtypes in either leaf nitrogen content at a given level of nitrogen supply or in the leaf nitrogen-photosynthesis relationship. We suggest that species-level variation in photosynthetic nitrogen use efficiency among C(4) species is large enough to mask any differences that may be due to C(4) subtype.     

Large daily variation in 13C-enrichment of leaf-respired CO2 in two Quercus forest canopies

The use of the 13C : 12C isotopic ratio (delta13C) of leaf-respired CO2 to trace carbon fluxes in plants and ecosystems is limited by little information on temporal variations in delta13C of leaf dark-respired CO2 (delta13Cr) under field conditions. Here, we explored variability in delta13Cr and its relationship to key respiratory substrates from collections of leaf dark-respired CO2, carbohydrate extractions and gas exchange measurements over 24-h periods in two Quercus canopies. Throughout both canopies, delta13Cr became progressively 13C-enriched during the photoperiod, by up to 7%, then 13C-depleted at night relative to the photoperiod. This cycle could not be reconciled with delta13C of soluble sugars (delta13Css), starch (delta13Cst), lipids (delta13Cl), cellulose (delta13Cc) or with calculated photosynthetic discrimination (Delta). However, photoperiod progressive enrichment in delta13Cr was correlated with cumulative carbon assimilation (r2 = 0.91). We concluded that there is considerable short-term variation in delta13Cr in forest canopies, that it is consistent with current hypotheses for 13C fractionation during leaf respiration, that leaf carbohydrates cannot be used as surrogates for delta13Cr, and that diel changes in leaf carbohydrate status could be used to predict changes in delta13Cr empirically.     

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.     

C4GEM, a genome-scale metabolic model to study C4 plant metabolism

Leaves of C(4) grasses (such as maize [Zea mays], sugarcane [Saccharum officinarum], and sorghum [Sorghum bicolor]) form a classical Kranz leaf anatomy. Unlike C(3) plants, where photosynthetic CO(2) fixation proceeds in the mesophyll (M), the fixation process in C(4) plants is distributed between two cell types, the M cell and the bundle sheath (BS) cell. Here, we develop a C(4) genome-scale model (C4GEM) for the investigation of flux distribution in M and BS cells during C(4) photosynthesis. C4GEM, to our knowledge, is the first large-scale metabolic model that encapsulates metabolic interactions between two different cell types. C4GEM is based on the Arabidopsis (Arabidopsis thaliana) model (AraGEM) but has been extended by adding reactions and transporters responsible to represent three different C(4) subtypes (NADP-ME [for malic enzyme], NAD-ME, and phosphoenolpyruvate carboxykinase). C4GEM has been validated for its ability to synthesize 47 biomass components and consists of 1,588 unique reactions, 1,755 metabolites, 83 interorganelle transporters, and 29 external transporters (including transport through plasmodesmata). Reactions in the common C(4) model have been associated with well-annotated C(4) species (NADP-ME subtypes): 3,557 genes in sorghum, 11,623 genes in maize, and 3,881 genes in sugarcane. The number of essential reactions not assigned to genes is 131, 135, and 156 in sorghum, maize, and sugarcane, respectively. Flux balance analysis was used to assess the metabolic activity in M and BS cells during C(4) photosynthesis. Our simulations were consistent with chloroplast proteomic studies, and C4GEM predicted the classical C(4) photosynthesis pathway and its major effect in organelle function in M and BS. The model also highlights differences in metabolic activities around photosystem I and photosystem II for three different C(4) subtypes. Effects of CO(2) leakage were also explored. C4GEM is a viable framework for in silico analysis of cell cooperation between M and BS cells during photosynthesis and can be used to explore C(4) plant metabolism.     

Non-steady state effects in diurnal 180 discrimination by Picea sitchensis branches in the field

We report diurnal variations in 18O discrimination (18 delta) during photosynthesis (18 delta A) and respiration (18 delta R) of Picea sitchensis branches measured in branch chambers in the field. These observations were compared with predicted 18 delta (18 delta pred) based on concurrent measurements of branch gas exchange to evaluate steady state and non-steady state (NSS) models of foliage water 18O enrichment for predicting the impact of this ecosystem on the Delta 18O of atmospheric CO2. The non-steady state approach substantially improved the agreement between 18 delta pred and observed 18 delta (18 delta obs) compared with the assumption of isotopic steady state (ISS) for the Delta 18O signature of foliage water. In addition, we found direct observational evidence for NSS effects: extremely high apparent 18 delta values at dusk, dawn and during nocturnal respiration. Our experiments also show the importance of bidirectional foliage gas exchange at night (isotopic equilibration in addition to the net flux). Taken together, neglecting these effects leads to an underestimation of daily net canopy isofluxes from this forest by up to 30%. We expect NSS effects to be most pronounced in species with high specific leaf water content such as conifers and when stomata are open at night or when there is high relative humidity, and we suggest modifications to ecosystem and global models of delta 18O of CO2.     

Life form-specific variations in leaf water oxygen-18 enrichment in Amazonian vegetation

Leaf water (18)O enrichment (Delta(o)) influences the isotopic composition of both gas exchange and organic matter, with Delta(o) values responding to changes in atmospheric parameters. In order to examine possible influences of plant parameters on Delta(o) dynamics, we measured oxygen isotope ratios (delta(18)O) of leaf and stem water on plant species representing different life forms in Amazonia forest and pasture ecosystems. We conducted two field experiments: one in March (wet season) and another in September (dry season) 2004. In each experiment, leaf and stem samples were collected at 2-h intervals at night and hourly during the day for 50 h from eight species including upper-canopy forest trees, upper-canopy forest lianas, and lower-canopy forest trees, a C(4) pasture grass and a C(3) pasture shrub. Significant life form-related differences were detected in (18)O leaf water values. Initial modeling efforts to explain these observations over-predicted nighttime Delta(o) values by as much as 10 per thousand. Across all species, errors associated with measured values of the delta(18)O of atmospheric water vapor (delta(v)) appeared to be largely responsible for the over-predictions of nighttime Delta(o) observations. We could not eliminate collection or storage of water vapor samples as a possible error and therefore developed an alternative, plant-based method for estimating the daily average delta(v) value in the absence of direct (reliable) measurements. This approach differs from the common assumption that isotopic equilibrium exists between water vapor and precipitation water, by including transpiration-based contributions from local vegetation through (18)O measurements of bulk leaf water. Inclusion of both modified delta(v) and non-steady state features resulted in model predictions that more reliably predicted both the magnitude and temporal patterns observed in the data. The influence of life form-specific patterns of Delta(o) was incorporated through changes in the effective path length, an important but little known parameter associated with the Péclet effect.     

Effects of intraleaf variations in carbonic anhydrase activity and gas exchange on leaf C18OO isoflux in Zea mays

Variation in the C18OO content of atmospheric CO2 (delta18Oa) can be used to distinguish photosynthesis from soil respiration, which is based on carbonic anhydrase (CA)-catalyzed 18O exchange between CO2 and 18O-enriched leaf water (delta18Ow). Here we tested the hypothesis that mean leaf delta18Ow and assimilation rates can be used to estimate whole-leaf C18OO flux (isoflux), ignoring intraleaf variations in CA activity and gas exchange parameters. We observed variations in CA activity along the leaf (> 30% decline from the leaf center toward the leaf ends), which were only partially correlated to those in delta18Ow (7 to 21 per thousand), delta18O and delta13C of leaf organic matter (25 to 30 per thousand and -12.8 to -13.2 per thousand, respectively), and substomatal CO2 concentrations (intercellular CO2 concentrations, c(i), at the leaf center were approximately 40% of those at the leaf tip). The combined effect of these variations produced a leaf-integrated isoflux that was different from that predicted based on bulk leaf values. However, because of canceling effects among the influencing parameters, isoflux overestimations were only approximately 10%. Conversely, use of measured parameters from a leaf segment could produce large errors in predicting leaf-integrated C18OO fluxes.     

Experimental evidence for diel variations of the carbon isotope composition in leaf, stem and phloem sap organic matter in Ricinus communis

Carbon isotope fractionation in metabolic processes following carboxylation of ribulose-1,5-bisphosphate (RuBP) is not as well described as the discrimination during photosynthetic CO(2) fixation. However, post-carboxylation fractionation can influence the diel variation of delta(13)C of leaf-exported organic matter and can cause inter-organ differences in delta(13)C. To obtain a more mechanistic understanding of post-carboxylation modification of the isotopic signal as governed by physiological and environmental controls, we combined the modelling approach of Tcherkez et al., which describes the isotopic fractionation in primary metabolism with the experimental determination of delta(13)C in leaf and phloem sap and root carbon pools during a full diel course. There was a strong diel variation of leaf water-soluble organic matter and phloem sap sugars with relatively (13)C depleted carbon produced and exported during the day and enriched carbon during the night. The isotopic modelling approach reproduces the experimentally determined day-night differences in delta(13)C of leaf-exported carbon in Ricinus communis. These findings support the idea that patterns of transitory starch accumulation and remobilization govern the diel rhythm of delta(13)C in organic matter exported by leaves. Integrated over the whole 24 h day, leaf-exported carbon was enriched in (13)C as compared with the primary assimilates. This may contribute to the well-known--yet poorly explained--relative (13)C depletion of autotrophic organs compared with other plant parts. We thus emphasize the need to consider post-carboxylation fractionations for studies that use delta(13)C for assessing environmental effects like water availability on ratio of mole fractions of CO(2) inside and outside the leaf (e.g. tree ring studies), or for partitioning of CO(2) fluxes at the ecosystem level.     

18O pattern and biosynthesis of natural plant products

Oxygen atoms in plant products originate from CO(2), H(2)O and O(2), precursors with quite different delta18O values. Furthermore their incorporation by different reactions implies isotope effects. On this base the resulting non-statistical 18O distributions in natural compounds are discussed. The delta18O value of cellulose is correlated to that of the leaf water, and the observed 18O enrichment (approximately +27 per thousand) is generally attributed to an equilibrium isotope effect between carbonyl groups and water. However, as soluble and heterotrophically synthesised carbohydrates show other correlations, a non-statistical 18O distribution - originating from individual biosynthetic reactions - is postulated for carbohydrates. Similarly, the delta18O values of organic acids, carbonyl compounds, alcohols and esters indicate water-correlated, but individual 18O abundances (e.g. O from acyl groups approximately +19% above water), depending upon origin and biosyntheses. Alcoholic groups introduced by monooxygenase reactions, e.g. in sterols and phenols, show delta18O values near +5 per thousand, in agreement with an assumed isotope fractionation factor of approximately 1.02 on the reaction with atmospheric oxygen (delta18O=+23.5 per thousand). Correspondingly, a "thermodynamically ordered isotope distribution" is only observed for oxygen in some functional groups correlated to an origin from CO(2) and H(2)O, not from O(2). The individual isotopic increments of functional groups permit the prediction of global delta18O values of natural compounds on the basis of their biosynthesis.     

Manganese requirement for optimum photosynthesis and growth in NAD-malic enzyme C-4 species

Despite the evidence for a critical role of Mn in malate decarboxylation and CO₂ release for carbon fixation reactions in C-4 plants, there is a lack of information on their Mn requirement. The objective of this study was to establish Mn levels needed for optimum growth and photosynthesis of four agriculturally important C-4 species, NAD-ME C-4 pearl millet and purple amaranth, and NADP-ME C-4 corn and sorghum, as compared to two C-3 species, wheat and squash. Plants were grown hydroponically in a complete nutrient solution with Mn concentrations ranging from 0 to 100 μM. We report that under these conditions, C-3 and NADP-ME C-4 plants reached their maximum biomass production with 2–5 μM Mn, the concentration commonly used in plant nutrient media. In contrast, Mn concentrations supporting maximum performance of NAD-ME C-4 plants were up to 20-fold higher and ranged between 50 and 100 μM. Although leaf tissue Mn concentrations increased in parallel with Mn nutrition in all plants, the higher leaf Mn had no effect on NADP-ME C-4 or C-3 plants, but it caused a large, up to 100%, increase in net photosynthetic rate in NAD-ME C-4 species. The highest photosynthetic rates across the spectrum of photon flux density were recorded for C-3 and NADP-ME C-4 plants receiving 2–5 μM Mn, and for NAD-ME C-4 species millet and amaranth supplied with 50 or 100 μM Mn, respectively. Squash (C-3) plants were the most sensitive to Mn and their photosynthetic rate was severely depressed with more than 10 μM Mn. The increase in photosynthetic rates of NAD-ME C-4 species occurred without an increase in stomatal conductance, eliminating CO₂ uptake as the main cause. We propose that the higher photosynthetic rates in NAD-ME C-4 species supplied with higher Mn were a result of increased activation of the Mn-dependent NAD-ME in bundle sheath cells, producing greater CO₂ supply for Calvin cycle reactions. This is, to our knowledge, the first report on a significantly higher Mn requirement for optimum photosynthesis and biomass production of NAD-ME C-4 species.