Stable isotope composition of organic compounds transported in the phloem of European beech--evaluation of different methods of phloem sap collection and assessment of gradients in carbon isotope composition during leaf-to-stem transport

The analysis of stable isotope composition (delta13C, delta15N, delta18O) of phloem-transported organic matter is a useful tool for assessing short-term carbon and water balance of trees. A major constraint of the general application of this method to trees at natural field sites is that the collection of phloem sap with the "phloem bleeding" technique is restricted to particular species and plant parts. To overcome this restriction, we compared the contents (amino compounds and sugars) and isotope signatures (delta13C, delta15N, delta18O) of phloem sap directly obtained from incisions in the bark (bleeding technique) with phloem exudates where bark pieces were incubated in aqueous solutions (phloem exudation technique with and without chelating agents [EDTA, polyphosphate] in the initial sampling solution, which prevent blocking of sieve tubes). A comparable spectrum of amino compounds and sugars was detected using the different techniques. O, C, or N compounds in the initial sampling solution originating from the chelating agents always decreased precision of determination of the respective isotopic signatures, as indicated by higher standard deviation, and/or led to a significant difference of mean delta as compared to the phloem bleeding technique. Hence, depending on the element from which the ratio of heavy to light isotope is determined, compounds lacking C, N, and/or O should be used as chelating agents in the exudation solution. In applying the different techniques, delta13C of organic compounds transported in the phloem of the twig (exudation technique with polyphosphate as chelating agent) were compared with those in the phloem of the main stem (phloem bleeding technique) in order to assess possible differences in carbon isotope composition of phloem carbohydrates along the tree axis. In July, organic compounds in the stem phloem were significantly enriched in 13C by > 1.3 per thousand as compared to the twig phloem, whereas this effect was not observed in September. Correlation analysis between delta13C and stomatal conductance (Gs) revealed the gradient from the twigs to the stem observed in July may be attributed to temporal differences rather than to spatial differences in carbon isotope composition of sugars. As various authors have produced conflicting results regarding the enrichment/depletion of 13C in organic compounds in the leaf-to-stem transition, the different techniques presented in this paper can be used to provide further insight into fractionation processes associated with transport of C compounds from leaves to branches and down the main stem.     

Water shortage affects the water and nitrogen balance in Central European beech forests

Whilst forest policy promotes cultivation and regeneration of beech dominated forest ecosystems, beech itself is a highly drought sensitive tree species likely to suffer from the climatic conditions prognosticated for the current century. Taking advantage of model ecosystems with cool-moist and warm-dry local climate, the latter assumed to be representative for future climatic conditions, the effects of climate and silvicultural treatment (different thinning regimes) on water status, nitrogen balance and growth parameters of adult beech trees and beech regeneration in the understorey were assessed. In addition, validation experiments with beech seedlings were carried out under controlled conditions, mainly in order to assess the effect of drought on the competitive abilities of beech. As measures of water availability xylem flow, shoot water potential, stomatal conductance as well as delta (13)C and delta (18)O in different tissues (leaves, phloem, wood) were analysed. For the assessment of nitrogen balance we determined the uptake of inorganic nitrogen by the roots as well as total N content and soluble N compounds in different tissues of adult and young trees. Retrospective and current analysis of delta (13)C, growth and meteorological parameters revealed that beech growing under warm-dry climatic conditions were impaired in growth and water balance during periods with low rain-fall. Thinning affected water, N balance and growth mostly of young beech, but in a different way under different local climatic conditions. Under cool, moist conditions, representative for the current climatic and edaphic conditions in beech forests of Central Europe, thinning improves nutrient and water status consistent to published literature and long-term experience of forest practitioners. However, beech regeneration was impaired as a result of thinning at higher temperatures and under reduced water availability, as expected in future climate.     

Comparison of leaf gas exchange and stable isotope signature of water-soluble compounds along canopy gradients of co-occurring Douglas-fir and European beech

Combined δ(13) C and δ(18) O analyses of water-soluble leaf and twig phloem material were used to determine intrinsic water-use efficiency (iWUE) and variability of stomatal conductance at different crown positions in adult European beech (Fagus sylvatica) and Douglas-fir (Pseudotsuga menziesii) trees. Simultaneous gas exchange measurements allowed evaluation of the differences in calculating iWUE from leaf or phloem water-soluble compounds, and comparison with a semi-quantitative dual isotope model to infer variability of net photosynthesis (A(n) ) between the investigated crown positions. Estimates of iWUE from δ(13) C of leaf water-soluble organic matter (WSOM) outperformed the estimates from phloem compounds. In the beech crown, δ(13) C of leaf WSOM coincided clearly with gas exchange measurements. The relationship was not as reliable in the Douglas-fir. The differences in δ(18) O between leaf and phloem material were found to correlate with stomatal conductance. The semi-quantitative model approach was applicable for comparisons of daily average A(n) between different crown positions and trees. Intracanopy gradients were more pronounced in the beech than in the Douglas-fir, which reached higher values of iWUE at the respective positions, particularly under dry air conditions.     

Photosynthetic Gas Exchange and Discrimination against 13CO2 and C18O16O in Tobacco Plants Modified by an Antisense Construct to Have Low Chloroplastic Carbonic Anhydrase

The physiological role of chloroplastic carbonic anhydrase (CA) was examined by antisense suppression of chloroplastic CA (on average 8% of wild type) in Nicotiana tabacum. Photosynthetic gas-exchange characteristics of low-CA and wild-type plants were measured concurrently with short-term, on-line stable isotope discrimination at varying vapor pressure deficit (VPD) and light intensity. Low-CA and wild-type plants were indistinguishable in the responses of assimilation, transpiration, stomatal conductance, and intercellular CO2 concentration to changing VPD or light intensity. At saturating light intensity, low-CA plants had lower discrimination against 13CO2 than wild-type plants by 1.2 to 1.8[per mille (thousand) sign]. Consequently, tissue of the low-CA plants was higher in 13C than the control plants. It was calculated that low-CA plants had chloroplast CO2 concentrations 13 to 22 [mu]mol mol-1 lower than wild-type plants. Discrimination against C18O16O in low-CA plants was 20% of that of the wild type, confirming a role of chloroplastic CA in the mechanism of discrimination against C18O16O ([delta]C18O16O). As VPD increased, stomatal closure caused a reduction in chloroplastic C02 concentration, and since VPD and chloroplastic CO2 concentration act in opposing directions on [delta]C18O16O, no effect of VPD was seen on [delta]C18O16O.     

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.     

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.     

Physiological performance of beech (Fagus sylvatica L.) at its southeastern distribution limit in Europe: seasonal changes in nitrogen, carbon and water balance

To assess the physiological performance of drought-sensitive European beech ( Fagus sylvatica L.) under the dry Mediterranean climate prevailing at its southeastern distribution limit in Europe, we analyzed seasonal changes in carbon, nitrogen and water balance of naturally grown adult trees. We determined the foliar C and N contents, delta13C and delta18O signatures, total soluble non-protein nitrogen compounds (TSNN) in xylem, leaves, and phloem, as well as leaf water potential and photosynthetic quantum yield in northern Greece during 2003. Tissue sampling was performed in May, July, and September, while field measurements were conducted regularly. Climatic conditions for the 2003 growing season fall within the typical range of the studied area. The N- and C-related parameters displayed distinct seasonal courses. TSNN was highest in May in all tissues, and asparagine (Asn) was then the most abundant compound. Thereafter, TSNN decreased significantly in all tissues and both its concentration and composition remained constant in July and September. In both months, glutamate (Glu) prevailed in leaves, gamma-aminobutyric acid (GABA) in phloem exudates from twigs and trunks, and arginine (Arg) in the xylem sap, where loading with amino acids was rather low during that period, amounting to only 0.8 micromol N ml-1 in September. Highest total foliar N and C contents were detected in May, and the elevated abundance of nutrients as well as an increased foliar delta13C signature at the beginning of the growing season is attributed to remobilization processes. The signatures of delta18O, quantum yield and leaf water potentials varied only slightly throughout the growing season. Although summer precipitation at the study site was considerably lower compared to what is usual for typical central European beech forests, no intensive drought responses of the physiological apparatus were detected in the studied beech trees. This suggests efficient internal regulation mechanisms, constantly ensuring a favourable physiological status under the relatively dry Mediterranean climate.     

Carbon and oxygen isotope composition of organic compounds in the phloem sap provides a short-term measure for stomatal conductance of European beech (Fagus sylvatica L.)

At eight different dates during the 2000 growing season, δ¹³C and δ¹⁸O were determined in the phloem of adult beech trees growing in natural beech stands in south‐west Germany differing in stand density and local climate. In addition, stand transpiration, precipitation, photosynthetic active radiation, relative air humidity, water pressure deficit of the air, air and soil temperature, soil water potential, and sugar concentration of the phloem sap were determined directly and evaporation and canopy stomatal conductance were modelled. All parameters were related to δ¹³C. The study aimed to identify the time integral within which the δ¹³C of organic compounds transported in the phloem is an indicative measure of these environmental influences. δ¹³C of soluble carbon transported in the phloem was well correlated with mean stomatal conductance in a two‐day integral prior to phloem sampling but did not depend on either light intensity or soil water availability. A strong positive relationship between δ¹³C and δ¹⁸O pointed to observed variation in δ¹³C of phloem sap being a result of variation in stomatal conductance. Bulk leaf δ¹³C was a poor indicator of changes in environmental conditions during the growing season. From these results we conclude that the analysis of δ¹³C in soluble carbon transported in the phloem is a reliable indicator of short‐term changes in C_i/C_a. In contrast, the δ¹³C of structural carbon in beech foliage represents an integration of a range of factors that mask short‐term influences responsible for C_i/C_a.     

Seasonal courses of key parameters of nitrogen,carbon and water balance in European beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.) grown on four different study sites along a European North–South climate gradient during the 2003 drought

During the growing season of the exceptionally dry and warm year 2003, we assessed seasonal changes in nitrogen, carbon and water balance related parameters of mature naturally grown European beech (Fagus sylvatica L.) along a North–South transect in Europe that included a beech forest stand in central Germany, two in southern Germany and one in southern France. Indicators for N balance assessed at all four sites were foliar N contents and total soluble non-protein nitrogen compounds (TSNN) in xylem sap, leaves and phloem exudates; C and water balance related parameters determined were foliar C contents, δ¹³C and δ¹⁸O signatures. Tissue sampling was performed in May, July and September. The N related parameters displayed seasonal courses with highest concentrations during N remobilization in May. Decreased total foliar N contents as well as higher C/N ratios in the stands in central Germany and southern France compared to the other study sites point to an impaired N nutrition status due to lower soil N contents and precipitation perception. TSNN concentrations in leaves and phloem exudates of all study sites were in ranges previously reported, but xylem sap content of amino compounds in July was lower at all study sites when compared to literature data (c. 1 μmol N mL⁻¹). In September, TSNN concentrations increased again at the two study sites in southern Germany after a rain event, whereas they remained constant at sites in central Germany and southern France which hardly perceived precipitation during that time. Thus, TSNN concentrations in the xylem sap might be indicative for water balance related N supply in the beech trees. TSNN profiles at all study sites, however, did not indicate drought stress. Foliar δ¹³C, but not foliar C and δ¹⁸O followed a seasonal trend at all study sites with highest values in May. Differences in foliar δ¹³C and δ¹⁸O did not reflect climatic differences between the sites, and are attributed to differences in altitude, photosynthesis and δ¹⁸O signatures of the water sources. Except of low TSNN concentrations in the xylem sap, no physiological indications of drought stress were detected in the trees analysed. We suppose that the other parameters assessed might not have been sensitive to the drought events because of efficient regulation mechanisms that provide a suitable physiological setting even under conditions of prolonged water limitation. The uniform performance of the trees from southern France and central Germany under comparably dry climate conditions denotes that the metabolic plasticity of mature beech from the different sites studied might be similar.     

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.     

Isotopes reveal contrasting water use strategies among coexisting plant species in a Mediterranean ecosystem

Variation in the stable carbon and oxygen isotope composition (δ(13) C, Δ(18) O) of co-occurring plant species may reflect the functional diversity of water use strategies present in natural plant communities. We investigated the patterns of water use among 10 coexisting plant species representing diverse taxonomic groups and life forms in semiarid southeast Spain by measuring their leaf δ(13) C and Δ(18) O, the oxygen isotope ratio of stem water and leaf gas exchange rates. Across species, Δ(18) O was tightly negatively correlated with stomatal conductance (g(s) ), whereas δ(13) C was positively correlated with intrinsic water use efficiency (WUE(i) ). Broad interspecific variation in Δ(18) O, δ(13) C and WUE(i) was largely determined by differences in g(s) , as indicated by a strong positive correlation between leaf δ(13) C and Δ(18) O across species The 10 co-occurring species segregated along a continuous ecophysiological gradient defined by their leaf δ(13) C and Δ(18) O, thus revealing a wide spectrum of stomatal regulation intensity and contrasting water use strategies ranging from 'profligate/opportunistic' (high g(s) , low WUE(i) ) to 'conservative' (low g(s) , high WUE(i) ). Coexisting species maintained their relative isotopic rankings in 2?yr with contrasting rainfall, suggesting the existence of species-specific 'isotopic niches' that reflect ecophysiological niche segregation in dryland plant communities.     

Water sources and water-use efficiency in mediterranean coastal dune vegetation

In coastal environments plants have to cope with various water sources: rainwater, water table, seawater, and mixtures. These are usually characterized by different isotopic signatures ( (18)O/ (16)O and D/H ratios). Xylem water reflects the isotopic compositions of the water sources. Additionally, water-use efficiency (WUE) can be assessed with carbon isotope discrimination (Delta) analyses. Gas exchange, Delta of leaf dry matter, and isotopic composition (delta (18)O) of xylem water were measured from June to August 2001 in herbaceous perennials of mobile dunes (Ammophila littoralis, Elymus farctus) and sclerophyllous shrubs and climbers (Arbutus unedo, Pistacia lentiscus, Phillyrea angustifolia, Qercus ilex, Juniperus oxycedrus, Smilax aspera) of consolidated dunes. Assimilation rates were rather low and did not show clear seasonal patterns, possibly due to limited precipitation and generally low values of stomatal conductance. The lowest values were shown in S. aspera. Different physiological patterns were found, on the basis of delta (18)O and Delta analyses. Values of delta (18)O of xylem water of phanerophytes were remarkably constant and matched those of the water table, indicating dependence on a reliable water source; values of Delta were relatively high, indicating low intrinsic WUE, with the exception of J. oxycedrus. Surprisingly, very high delta (18)O values were found for the xylem water from S. aspera in August. This suggests retrodiffusion of leaf water to xylem sap in the stem or direct uptake of water by leaves or stems, owing to dew or fog occurrence. Low Delta values indicated high WUE in S. aspera. Contrasting strategies were shown by the species of mobile dunes: E. farctus relied on superficial water and exhibited low WUE, accordingly to its therophyte-like vegetative cycle; on the contrary, A. littoralis used deeper water sources, showing higher WUE in relation to its long-lasting vegetative habit.     

Leaf water 18O and 2H enrichment along vertical canopy profiles in a broadleaved and a conifer forest tree

Distinguishing meteorological and plant‐mediated drivers of leaf water isotopic enrichment is prerequisite for ecological interpretations of stable hydrogen and oxygen isotopes in plant tissue. We measured input and leaf water δ²H and δ¹⁸O as well as micrometeorological and leaf morpho‐physiological variables along a vertical gradient in a mature angiosperm (European beech) and gymnosperm (Douglas fir) tree. We used these variables and different enrichment models to quantify the influence of Péclet and non‐steady state effects and of the biophysical drivers on leaf water enrichment. The two‐pool model accurately described the diurnal variation of leaf water enrichment. The estimated unenriched water fraction was linked to leaf dry matter content across the canopy heights. Non‐steady state effects and reduced stomatal conductance caused a higher enrichment of Douglas fir compared to beech leaf water. A dynamic effect analyses revealed that the light‐induced vertical gradients of stomatal conductance and leaf temperature outbalanced each other in their effects on evaporative enrichment. We conclude that neither vertical canopy gradients nor the Péclet effect is important for estimates and interpretation of isotopic leaf water enrichment in hypostomatous trees. Contrarily, species‐specific non‐steady state effects and leaf temperatures as well as the water vapour isotope composition need careful consideration.     

Short-term variation in the isotopic composition of organic matter allocated from the leaves to the stem of Pinus sylvestris: effects of photosynthetic and postphotosynthetic carbon isotope fractionation

We aimed to quantify the separate effects of photosynthetic and postphotosynthetic carbon isotope discrimination on δ¹³C of the fast‐turn‐over carbon pool (water soluble organic carbon and CO₂ emitted from heterotrophic tissues), including their diel variation, along the pathway of carbon transport from the foliage to the base of the stem. For that purpose, we determined δ¹³C in total and water‐soluble organic matter of the foliage plus δ¹³C and δ¹⁸O in phloem organic matter of twigs and at three heights along the stem of Pinus sylvestris over a nine‐day period, including four measurements per day. These data were related to meteorological and photosynthesis parameters and to the δ¹³C of stem‐emitted CO₂. In the canopy (foliage and twigs), the δ¹³C of soluble organic matter varied diurnally with amplitudes of up to 1.9‰. The greatest ¹³C enrichment was recorded during the night/early morning, indicating a strong influence of starch storage and remobilization on the carbon isotope signatures of sugars exported from the leaves. ¹³C enrichment of soluble organic matter from the leaves to the twig phloem and further on to the phloem of the stem was supposed to be a result of carbon isotope fractionation associated with metabolic processes in the source and sink tissues. CO₂ emitted from the stem was enriched by 2.3–5.2‰ compared with phloem organic matter. When day‐to‐day variation was addressed, water‐soluble leaf δ¹³C and twig phloem δ¹⁸O were strongly influenced by c_i/c_a and stomatal conductance (G_s), respectively. These results show that both photosynthetic and postphotosynthetic carbon isotope fractionation influence δ¹³C of organic matter over time, and over the length of the basipetal transport pathway. Clearly, these influences on the δ¹³C of respired CO₂ must be considered when using the latter for partitioning of ecosystem CO₂ fluxes or when the assessment of δ¹³C in organic matter is applied to estimate environmental effects in c_i/c_a.     

冬小麦叶片气孔导度模型水分响应函数的参数化

植物气孔导度模型的水分响应函数用来模拟水分胁迫对气孔导度的影响过程, 是模拟缺水环境下植物与大气间水、碳交换过程的关键算法。水分响应函数包括空气湿度响应函数和土壤湿度(或植物水势)响应函数, 该研究基于田间实验观测, 分析了冬小麦(Triticum aestivum)叶片气孔导度对不同空气饱和差和不同土壤体积含水量或叶水势的响应规律。一个土壤水分梯度的田间处理在中国科学院禹城综合试验站实施, 不同水分胁迫下的冬小麦叶片气体交换过程和气孔导度以及其他的温湿度数据被观测, 同时观测了土壤含水量和叶水势。实验数据表明, 冬小麦叶片气孔导度对空气饱和差的响应呈现双曲线规律, 变化趋势显示大约1 kPa空气饱和差是一个有用的阈值, 在小于1 kPa时, 冬小麦气孔导度对空气饱和差变化反应敏感, 而大于1 kPa后则反应缓慢; 分析土壤体积含水量与中午叶片气孔导度的关系发现, 中午叶片气孔导度随土壤含水量增加大致呈现线性增加趋势, 但在平均土壤体积含水量大于大约25%以后, 气孔导度不再明显增加, 而是维持在较高导度值上下波动; 冬小麦中午叶片水势与相应的气孔导度之间, 随着叶水势的增加, 气孔导度呈现增加趋势。根据冬小麦气孔导度对空气湿度、土壤湿度和叶水势的响应规律, 研究分别采用双曲线和幂指数形式拟合了水汽响应函数, 用三段线性方程拟合了土壤湿度响应函数和植物水势响应函数, 得到的参数可以为模型模拟冬小麦的各类水、热、碳交换过程采用。     

Carbon stable isotopic composition of soluble sugars in Tillandsia epiphytes varies in response to shifts in habitat

We studied C stable isotopic composition (δ¹³C) of bulk leaf tissue and extracted sugars of four epiphytic Tillandsia species to investigate flexibility in the use of crassulacean acid metabolism (CAM) and C₃ photosynthetic pathways. Plants growing in two seasonally dry tropical forest reserves in Mexico that differ in annual precipitation were measured during wet and dry seasons, and among secondary, mature, and wetland forest types within each site. Dry season sugars were more enriched in ¹³C than wet season sugars, but there was no seasonal difference in bulk tissues. Bulk tissue δ¹³C differed by species and by forest type, with values from open-canopied wetlands more enriched in ¹³C than mature or secondary forest types. The shifts within forest habitat were related to temporal and spatial changes in vapor pressure deficits (VPD). Modeling results estimate a possible 4% increase in the proportional contribution of the C₃ pathway during the wet season, emphasizing that any seasonal or habitat-mediated variation in photosynthetic pathway appears to be quite moderate and within the range of isotopic effects caused by variation in stomatal conductance during assimilation through the C₃ pathway and environmental variation in VPD. C isotopic analysis of sugars together with bulk leaf tissue offers a useful approach for incorporating short- and long-term measurements of C isotope discrimination during photosynthesis.     

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.     

Oxygen isotope enrichment (delta18O) as a measure of time-averaged transpiration rate

Experimental evidence is presented to show that the 18O enrichment in the leaf biomass and the mean (time-averaged) transpiration rate are positively correlated in groundnut and rice genotypes. The relationship between oxygen isotope enrichment and stomatal conductance (g(s)) was determined by altering g(s) through ABA and subsequently using contrasting genotypes of cowpea and groundnut. The Peclet model for the 18O enrichment of leaf water relative to the source water is able to predict the mean observed values well, while it cannot reproduce the full range of measured isotopic values. Further, it fails to explain the observed positive correlation between transpiration rate and 18O enrichment in leaf biomass. Transpiration rate is influenced by the prevailing environmental conditions besides the intrinsic genetic variability. As all the genotypes of both species experienced similar environmental conditions, the differences in transpiration rate could mostly be dependent on intrinsic g(s). Therefore, it appears that the delta18O of leaf biomass can be used as an effective surrogate for mean transpiration rate. Further, at a given vapour pressure difference, delta18O can serve as a measure of stomatal conductance as well.     

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 responses to long-term high vapor pressure deficits mediated most limitation of photosynthesis in tomatoes

Plants grown at high vapor pressure deficit (VPD) usually present decreased photosynthesis, but stomatal and mesophyll limitation to photosynthesis remain poorly quantified. To better understand the regulation of high VPD on photosynthesis and plant growth in tomatoes, we investigated the limitation of stomatal conductance and mesophyll conductance to photosynthesis and relative importance of stomatal morphology and function in stomatal conductance. Both the net photosynthesis rate and total biomass were significantly limited by high VPD. Meanwhile, stomatal conductance and mesophyll conductance were decreased under high VPD. The stomatal conductance limitation was responsible for 60% of the total photosynthetic limitation. Moreover, a reduction in stomatal density and stomatal size occurred under high VPD, which was significantly correlated with the down-regulation of stomatal conductance. The stomatal morphology contributed to more than half the change in stomatal conductance. Nevertheless, stomatal movement was also an important factor in regulating stomatal conductance. The decrease of hydraulic conductance and transpiration rate with no significant difference in relative water content, leaf water potential, and/or osmotic potential suggested passive hydraulic regulation in the feedforward responses of stomata to high VPD.