Leaf relative water content at 50% stomatal conductance measured by noninvasive NMR is linked to climate of origin in nine species of eucalypt.

Stomata are the gatekeepers of plant water use and must quickly respond to changes in plant water status to ensure plant survival under fluctuating environmental conditions. The mechanism for their closure is highly sensitive to disturbances in leaf water status, which makes isolating their response to declining water content difficult to characterise and to compare responses among species. Using a small-scale non-destructive nuclear magnetic resonance spectrometer as a leaf water content sensor, we measure the stomatal response to rapid induction of water deficit in the leaves of nine species of eucalypt from contrasting climates. We found a strong linear correlation between relative water content at 50% stomatal conductance (RWCgs50 ) and mean annual temperature at the climate of origin of each species. We also show evidence for stomata to maintain control over water loss well below turgor loss point in species adapted to warmer climates and secondary increases in stomatal conductance despite declining water content. We propose that RWCgs50 is a promising trait to guide future investigations comparing stomatal responses to water deficit. It may provide a useful phenotyping trait to delineate tolerance and adaption to hot temperatures and high leaf-to-air vapour pressure deficits.

In situ pod growth rate reveals contrasting diurnal sensitivity to water deficit in Phaseolus vulgaris.

The development of reproductive tissues determines plant fecundity and yield. Loading of resources into the developing reproductive tissue is thought to be under the co-limiting effects of source and sink strength. The dynamics of this co-limitation are unknown, largely due to an inability to measure the flux of resources into a developing sink. Here we use nuclear magnetic resonance (NMR) sensors to measure sink strength by quantifying rates of pod dry matter accumulation (pod loading) in Phaseolus vulgaris at 13-min intervals across the diel period. Rates of pod loading showed contrasting variation across light and dark periods during the onset of water deficit. In addition, rates of pod loading appeared decoupled from net photosynthetic rates when adjusted to the plant scale. Combined, these observations illustrate that the rate of pod development varies under water limitation and that continuous, non-invasive methodologies to measure sink strength provide insight into the governing processes that determine the development of reproductive tissues.

Chemical composition and reproductive functionality of contrasting faba bean genotypes in response to water deficit.

Water deficit (WD), a major contributor to yield reductions in faba bean (Vicia faba), is a complex phenomenon that varies across daily to seasonal cycles. Several studies have identified various morphological and physiological indicators of WD tolerance, which generally show limited water use during WD. Limited information is available on the impact of WD on nutrient content and reproductive biology of the faba bean. We studied carbohydrates, amino acids, mineral nutrients and the abundance of naturally occurring carbon isotopes (δ13 C) in leaf and grain tissues of faba bean genotypes grown under well-watered (WW) and WD conditions. δ13 C of leaf tissues were found to indicate changes in water use due to WD but this was not reflected in grain tissues. Nutrient concentrations with regard to amino acids and minerals were not influenced by WD. However, carbohydrate accumulation was found to be significant for WD, specifically through the presence of a higher concentration of myo-inositol in WD leaf tissues. Alternatively, sucrose concentration in grain tissues was reduced under WD treatment. WD hampered reproductive functionality by reducing pollen viability and germination with the severity and duration of stress and this reduction was less prominent in the drought-tolerant genotype (AC0805#4912) compared to the sensitive one (11NF010c-4). It was also demonstrated that WD caused developmental impairment in the stamen and pistil, where the pistil appeared more sensitive than stamen. These findings suggest that WD impairs pollen viability and pistil function reducing yield volume, but the nutrient content of the resulting yield is not significantly affected.

Biochemical and physiological impacts of zinc sulphate, potassium phosphite and hydrogen sulphide in mitigating stress conditions in soybean.

Soybean is the most widely grown oilseed in the world. It is an important source of protein and oil which are derived from its seeds. Drought stress is a major constraint to soybean yields. Finding alternative methods to mitigate the water stress for soybean is useful to maintain adequate crop yields. The aim of this study was to evaluate the morpho-physiological, biochemical and metabolic changes in soybean plants in two ontogenetic stages, under exposure to water deficit and treatment with zinc sulphate (ZS), potassium phosphite (PP) or hydrogen sulphide (HS). We carried out two independent experiments in the V4 and R1 development stages consisting of the following treatments: well-watered control (WW, 100% maximum water holding capacity, MWHC), water deficit (WD, 50% MWHC), PP + WW, PP + WD, HS + WW, HS + WD, ZS + WW and ZS + WD. The experimental design consisted of randomized blocks with eight treatments with five replicates. Morphological, physiological and metabolic analyses were performed 8 days after the start of the treatments for both experiments. We identified two tolerance mechanisms acting in response to compound application during water stress: the first involved the upregulation of antioxidant enzyme activity and the second involved the accumulation of soluble sugars, free amino acids and proline to facilitate osmotic adjustment. Both mechanisms are related to the maintenance of the photosynthetic parameters and cell membrane integrity. This report suggests the potential agricultural use of these compounds to mitigate drought effects in soybean plants.

Rate of photosynthetic induction in fluctuating light varies widely among genotypes of wheat.

Crop photosynthesis and yield are limited by slow photosynthetic induction in sunflecks. We quantified variation in induction kinetics across diverse genotypes of wheat for the first time. Following a preliminary study that hinted at wide variation in induction kinetics across 58 genotypes, we grew 10 genotypes with contrasting responses in a controlled environment and quantified induction kinetics of carboxylation capacity (Vcmax) from dynamic A versus ci curves after a shift from low to high light (from 50 µmol m-2 s-1 to 1500 µmol m-2 s-1), in five flag leaves per genotype. Within-genotype median time for 95% induction (t95) of Vcmax varied 1.8-fold, from 5.2 min to 9.5 min. Our simulations suggest that non-instantaneous induction reduces daily net carbon gain by up to 15%, and that breeding to speed up Vcmax induction in the slowest of our 10 genotypes to match that in the fastest genotype could increase daily net carbon gain by up to 3.4%, particularly for leaves in mid-canopy positions (cumulative leaf area index ≤1.5 m2 m-2), those that experience predominantly short-duration sunflecks, and those with high photosynthetic capacities.

Chemical and isotopic markers detect water deficit and its influence on nutrient allocation in Phaseolus vulgaris.

The impact of drought on plant growth and yield has been widely studied and is considered a major limitation to crops reaching yield potential. Less known is the impact of water deficit on the nutritional quality of the resulting yield. This study characterised the impact of water deficit on carbon assimilation, modelled water use efficiency from carbon isotope discrimination and analysed the concentration of mineral nutrients, amino acids and sugars in leaf, phloem and pod pools collected from Phaseolus vulgaris L. (common bean) grown in a controlled environment. Water deficit led to an isohydric response, impacting on carbon isotope abundance in all tissues though not translating to any significant treatment differences in water use efficiency or nutrient content in tissues over the course of plant development. The results obtained in this study demonstrate that nutrient content of P. vulgaris yield was not impacted by the availability of water. The absence of significant changes in the nutrient content of individual seeds highlights the plasticity of developing reproductive tissue to changes in whole plant water availability.

Water Deficit Elicits a Transcriptional Response of Genes Governing d-pinitol Biosynthesis in Soybean (Glycine max).

d-pinitol is the most commonly accumulated sugar alcohol in the Leguminosae family and has been observed to increase significantly in response to abiotic stress. While previous studies have identified genes involved in d-pinitol synthesis, no study has investigated transcript expression in planta. The present study quantified the expression of several genes involved in d-pinitol synthesis in different plant tissues and investigated the accumulation of d-pinitol, myo-inositol and other metabolites in response to a progressive soil drought in soybean (Glycine max). Expression of myo-inositol 1-phosphate synthase (INPS), the gene responsible for the conversion of glucose-6-phosphate to myo-inositol-1-phosphate, was significantly up regulated in response to a water deficit for the first two sampling weeks. Expression of myo-inositol O-methyl transferase (IMT1), the gene responsible for the conversion of myo-inositol into d-ononitol was only up regulated in stems at sampling week 3. Assessment of metabolites showed significant changes in their concentration in leaves and stems. d-Pinitol concentration was significantly higher in all organs sampled from water deficit plants for all three sampling weeks. In contrast, myo-inositol, had significantly lower concentrations in leaf samples despite up regulation of INPS suggesting the transcriptionally regulated flux of carbon through the myo-inositol pool is important during water deficit.

Medium term water deficit elicits distinct transcriptome responses in Eucalyptus species of contrasting environmental origin.

BACKGROUND: Climatic and edaphic conditions over geological timescales have generated enormous diversity of adaptive traits and high speciation within the genus Eucalyptus (L. Hér.). Eucalypt species occur from high rainfall to semi-arid zones and from the tropics to latitudes as high as 43°S. Despite several morphological and metabolomic characterizations, little is known regarding gene expression differences that underpin differences in tolerance to environmental change. Using species of contrasting taxonomy, morphology and physiology (E. globulus and E. cladocalyx), this study combines physiological characterizations with 'second-generation' sequencing to identify key genes involved in eucalypt responses to medium-term water limitation. RESULTS: One hundred twenty Million high-quality HiSeq reads were created from 14 tissue samples in plants that had been successfully subjected to a water deficit treatment or a well-watered control. Alignment to the E. grandis genome saw 23,623 genes of which 468 exhibited differential expression (FDR < 0.01) in one or both ecotypes in response to the treatment. Further analysis identified 80 genes that demonstrated a significant species-specific response of which 74 were linked to the 'dry' species E. cladocalyx where 23 of these genes were uncharacterised. The majority (approximately 80%) of these differentially expressed genes, were expressed in stem tissue. Key genes that differentiated species responses were linked to photoprotection/redox balance, phytohormone/signalling, primary photosynthesis/cellular metabolism and secondary metabolism based on plant metabolic pathway network analysis. CONCLUSION: These results highlight a more definitive response to water deficit by a 'dry' climate eucalypt, particularly in stem tissue, identifying key pathways and associated genes that are responsible for the differences between 'wet' and 'dry' climate eucalypts. This knowledge provides the opportunity to further investigate and understand the mechanisms and genetic variation linked to this important environmental response that will assist with genomic efforts in managing native populations as well as in tree improvement programs under future climate scenarios.

Carbon Isotope Composition of Carbohydrates and Polyols in Leaf and Phloem Sap of Phaseolus vulgaris L. Influences Predictions of Plant Water Use Efficiency.

The use of carbon isotope abundance (δ(13)C) to assess plant carbon acquisition and water use has significant potential for use in crop management and plant improvement programs. Utilizing Phaseolus vulgaris L. as a model system, this study demonstrates the occurrence and sensitivity of carbon isotope fractionation during the onset of abiotic stresses between leaf and phloem carbon pools. In addition to gas exchange data, compound-specific measures of carbon isotope abundance and concentrations of soluble components of phloem sap were compared with major carbohydrate and sugar alcohol pools in leaf tissue. Differences in both δ(13)C and concentration of metabolites were found in leaf and phloem tissues, the magnitude of which responded to changing environmental conditions. These changes have inplications for the modeling of leaf-level gas exchange based upon δ(13)C natural abundance. Estimates of δ(13)C of low molecular weight carbohydrates and polyols increased the precision of predictions of water use efficiency compared with those based on bulk soluble carbon. The use of this technique requires consideration of the dynamics of the δ(13)C pool under investigation. Understanding the dynamics of changes in δ(13)C during movement and incorporation into heterotrophic tissues is vital for the continued development of tools that provide information on plant physiological performance relating to water use.

Chloroplast genome analysis of Australian eucalypts--Eucalyptus, Corymbia, Angophora, Allosyncarpia and Stockwellia (Myrtaceae).

We present a phylogenetic analysis and comparison of structural features of chloroplast genomes for 39 species of the eucalypt group (genera Eucalyptus, Corymbia, Angophora, and outgroups Allosyncarpia and Stockwellia). We use 41 complete chloroplast genome sequences, adding 39 finished-quality chloroplast genomes to two previously published genomes. Maximum parsimony and Bayesian analyses, based on >7000 variable nucleotide positions, produced one fully resolved phylogenetic tree (35 supported nodes, 27 with 100% bootstrap support). Eucalyptus and its sister lineage Angophora+Corymbia show a deep divergence. Within Eucalyptus, three lineages are resolved: the 'eudesmid', 'symphyomyrt' and 'monocalypt' groups. Corymbia is paraphyletic with respect to Angophora. Gene content and order do not vary among eucalypt chloroplasts; length mutations, especially frame shifts, are uncommon in protein-coding genes. Some non-synonymous mutations are highly incongruent with the overall phylogenetic signal, notably in rbcL, and may be adaptive. Application of custom informatics pipelines (GYDLE Inc.) enabled direct chloroplast genome assembly, resolving each genome to finished-quality with no need for PCR gap-filling or contig order resolution. Analysis of whole chloroplast genomes resolved major eucalypt clades and revealed variable regions of the genome that will be useful in lower-level genetic studies (including phylogeography and geneflow).

The Metabolic Profile of Young, Watered Chickpea Plants Can Be Used as a Biomarker to Predict Seed Number under Terminal Drought.

Chickpea is the second-most-cultivated legume globally, with India and Australia being the two largest producers. In both of these locations, the crop is sown on residual summer soil moisture and left to grow on progressively depleting water content, finally maturing under terminal drought conditions. The metabolic profile of plants is commonly, correlatively associated with performance or stress responses, e.g., the accumulation of osmoprotective metabolites during cold stress. In animals and humans, metabolites are also prognostically used to predict the likelihood of an event (usually a disease) before it occurs, e.g., blood cholesterol and heart disease. We sought to discover metabolic biomarkers in chickpea that could be used to predict grain yield traits under terminal drought, from the leaf tissue of young, watered, healthy plants. The metabolic profile (GC-MS and enzyme assays) of field-grown chickpea leaves was analysed over two growing seasons, and then predictive modelling was applied to associate the most strongly correlated metabolites with the final seed number plant-1. Pinitol (negatively), sucrose (negatively) and GABA (positively) were significantly correlated with seed number in both years of study. The feature selection algorithm of the model selected a larger range of metabolites including carbohydrates, sugar alcohols and GABA. The correlation between the predicted seed number and actual seed number was R2 adj = 0.62, demonstrating that the metabolic profile could be used to predict a complex trait with a high degree of accuracy. A previously unknown association between D-pinitol and hundred-kernel weight was also discovered and may provide a single metabolic marker with which to predict large seeded chickpea varieties from new crosses. The use of metabolic biomarkers could be used by breeders to identify superior-performing genotypes before maturity is reached.

Site-specific responses to short-term environmental variation are reflected in leaf and phloem-sap carbon isotopic abundance of field grown Eucalyptus globulus.

The carbon isotopic composition (δ(13) C) of plant material has been used extensively as an indirect measure of carbon fixation per volume of water used. More recently, the δ(13) C of phloem sap (δ(13) C(phl) ) has been used as a surrogate measure of short-term, canopy scale δ(13) C. Using a combination of δ(13) C physiological, structural and chemical indices from leaves and phloem sap of Eucalyptus globulus at sites of contrasting water availability, we sought to identify short-term, canopy scale resource limitations. Results illustrate that δ(13) C(phl) offers valid reflections of short-term, canopy scale values of leaf δ(13) C and tree water status. Under conditions limited by water, leaf and phloem sap photoassimilates differ in (13) C abundance of a magnitude large enough to significantly influence predictions of water use efficiency. This pattern was not detected among trees with adequate water supply indicating fractionation into heterotrophic tissues that may be sensitive to plant water status. Trees employed a range of physiological, biochemical and structural adaptations to acclimate to resource limitation that differed among sites providing a useful context upon which to interpret patterns in δ(13) C. Our results highlight that such easily characterized properties are ideal for use as minimally invasive tools to monitor growth and resilience of plants to variations in resource availability.

Investigating Nutrient Supply Effects on Plant Growth and Seed Nutrient Content in Common Bean.

Low soil fertility commonly limits growth and yield production of common bean (Phaseolus vulgaris L.) in tropical regions. Impacts of nutrient limitations on production volume are well studied and are a major factor in reducing crop yields. This study characterised the impact of reduced nutrient supply on carbon assimilation and nutrient content of leaf, phloem sap and reproductive tissues of common bean grown in a controlled environment in order to detect chemical markers for changes in nutritional content. Leaf gas exchange measurements were undertaken over plant development to characterise changes to carbon assimilation under reduced nutrient supply. Samples of leaf, phloem sap and pod tissue of common bean were analysed for carbon isotope discrimination, mineral nutrient content, and amino acid concentration. Despite declines in nutrient availability leading to decreased carbon assimilation and reductions in yield, amino acid concentration was maintained in the pod tissue. Common bean can maintain the nutritional content of individual pods under varying nutrient availabilities demonstrating the resilience of processes determining the viability of reproductive tissues.

Carbon isotope and soluble metabolites reflect physiological status among contrasting faba bean genotypes in response to water deficit.

Identification and validation of biomarkers and bioindicators to select genotypes with superior tolerance to water deficit (WD) under field conditions are paramount to plant breeding programs. However, the co-occurrence of different abiotic stresses such as WD, heat, and radiation makes it difficult to develop generalized protocols to monitor the physiological health of the plant system. The study assessed the most abundant carbohydrates and sugar alcohols in five faba bean (Vicia faba) genotypes under field conditions and the abundance of naturally occurring carbon isotopes in bulk leaf material to predict water use efficiency (WUE). Plant water status and biomass accumulation were also assessed. Among the accumulated sugars, inter-specific variation in glucose was most prevalent and was found at a higher concentration (8.52 mg g-1 leaf) in rainfed trial. myo-Inositol concentrations followed that of glucose accumulation in that the rainfed trial had higher amounts compared to the irrigated trial. WUE calculated from carbon isotope abundance was consistently offset with measured WUE from measurements of leaf gas exchange. All genotypes demonstrated significant relationships between predicted and measured WUE (p < 0.05) apart from control variety PBA Warda. Thus, bulk leaf-level carbon isotope abundance can be used to calculate WUE and used as an effective selection criterion for improving WUE in faba bean breeding programs under field conditions.

Temporal imaging of drug dynamics in live cells using stimulated Raman scattering microscopy and a perfusion cell culture system.

Stimulated Raman scattering (SRS) microscopy is a powerful technique for visualising the cellular uptake and distribution of drugs and small molecules in live cells under biocompatible imaging conditions. The use of bio-orthogonal groups within the drug molecule, including alkynes and nitriles, has enabled the direct detection of a plethora of bioactive molecules in a minimally perturbative fashion. Limited progress has been made towards real-time detection of drug uptake and distribution into live cells under physiological conditions, despite the accordant potential it presents for preclinical drug development. SRS microscopy has been applied to the study of cellular dynamics of the drug 7RH, which is a potent inhibitor of dicoidin domain receptor 1 (DDR1) and prevents cellular adhesion, proliferation and migration in vitro. The uptake of 7RH into a variety of mammalian cell models was shown to be independent of DDR1 expression. Using a perfusion chamber, the recurrent treatment of live cancer cells was achieved, enabling 7RH uptake to be visualised in real-time using SRS microscopy, after which the viability of the same cellular population was assessed using commercially available fluorescent markers in a multimodal imaging experiment. The effect of 7RH treatment in combination with the chemotherapeutic, cisplatin was investigated using sequential perfusion and time-lapse imaging in the same live cell population, to demonstrate the application of the approach. SRS microscopy also identified potent inhibition of cellular adhesion and migration in breast cancer cell models with increasing 7RH treatment concentrations, thus representing a novel read-out methodology for phenotypic assays of this kind. The direct assessment of drug-cell interactions under physiological conditions offers significant potential for the preclinical drug development process.

Effect of Drought and Low P on Yield and Nutritional Content in Common Bean.

Common bean (Phaseolus vulgaris L.) production in the tropics typically occurs in rainfed systems on marginal lands where yields are low, primarily as a consequence of drought and low phosphorus (P) availability in soil. This study aimed to investigate the physiological and chemical responses of 12 bush bean genotypes for adaptation to individual and combined stress factors of drought and low P availability. Water stress and P deficiency, both individually and combined, decreased seed weight and aboveground biomass by ∼80%. Water deficit and P deficiency decreased photosynthesis and stomatal conductance during plant development. Maximum rates of carboxylation, electron transport, and triose phosphate utilization were superior for two common bean genotypes (SEF60 and NCB226) that are better adapted to combined stress conditions of water deficit and low P compared to the commercial check (DOR390). In response to water deficit treatment, carbon isotope fractionation in the leaf tissue decreased at all developmental stages. Within the soluble leaf fraction, combined water deficit and low P, led to significant changes in the concentration of key nutrients and amino acids, whereas no impact was detected in the seed. Our results suggest that common bean genotypes have a degree of resilience in yield development, expressed in traits such as pod harvest index, and conservation of nutritional content in the seed. Further exploration of the chemical and physiological traits identified here will enhance the resilience of common bean production systems in the tropics.

Metabolic, physiological and anatomical responses of soybean plants under water deficit and high temperature condition.

Water deficit (WD) combined with high temperature (HT) is the major factor limiting agriculture worldwide, and it is predicted to become worse according to the current climate change scenario. It is thus important to understand how current cultivated crops respond to these stress conditions. Here we investigated how four soybean cultivars respond to WD and HT isolated or in combination at metabolic, physiological, and anatomical levels. The WD + HT increased the level of stress in soybean plants when compared to plants under well-watered (WW), WD, or HT conditions. WD + HT exacerbates the increases in ascorbate peroxidase activity, which was associated with the greater photosynthetic rate in two cultivars under WD + HT. The metabolic responses to WD + HT diverge substantially from plants under WW, WD, or HT conditions. Myo-inositol and maltose were identified as WD + HT biomarkers and were connected to subnetworks composed of catalase, amino acids, and both root and leaf osmotic potentials. Correlation-based network analyses highlight that the network heterogeneity increased and a higher integration among metabolic, physiological, and morphological nodes is observed under stress conditions. Beyond unveiling biochemical and metabolic WD + HT biomarkers, our results collectively highlight that the mechanisms behind the acclimation to WD + HT cannot be understood by investigating WD or HT stress separately.

Compound-specific differences in (13)C of soluble carbohydrates in leaves and phloem of 6-month-old Eucalyptus globulus (Labill).

Movement of photoassimilates from leaves to phloem is an important step for the flux of carbon through plants. Fractionation of carbon isotopes during this process may influence their abundance in heterotrophic tissues. We subjected Eucalyptus globulus to 20, 25 and 28 °C ambient growth temperatures and measured compound-specific δ(13)C of carbohydrates obtained from leaves and bled phloem sap. We compared δ(13)C of sucrose and raffinose obtained from leaf or phloem and of total leaf soluble carbon, with modelled values predicted by leaf gas exchange. Changes in δ(13)C of sucrose and raffinose obtained from either leaves or phloem sap were more tightly coupled to changes in c(i)/c(a) than was δ(13)C of leaf soluble carbon. At 25 and 28 °C, sucrose and raffinose were enriched in (13)C compared to leaf soluble carbon and predicted values - irrespective of tissue type. Phloem sucrose was depleted and raffinose enriched in (13)C compared to leaf extracts. Intermolecular and tissue-specific δ(13)C reveal that multiple systematic factors influence (13)C composition during export to phloem. Predicting sensitivity of these factors to changes in plant physiological status will improve our ability to infer plant function at a range of temporal and spatial scales.

Phloem sap metabolites vary according to the interactive effects of nutrient supply and seasonal conditions in Eucalyptus globulus (Labill).

Improving the efficiency of fertilizer application is paramount to both the sustainability and profitability of forest plantations. Therefore, developing reliable, cost-effective tools to assess tree nutritional status is of great interest. This investigation sought to assess the use of phloem sap-derived metabolites as an indicator of nutritional status on a background of seasonal water availability of Eucalyptus globulus (Labill) trees grown under field conditions. Phloem is a central conduit for long-distance transport and signaling in plants and offers great promise in reflecting plant-scale resource limitations. Changes in the abundance of solutes and isotopes in phloem sap are sensitive to environmental cues. With a focus on both water and nutrient availability, we characterize patterns in phloem sugars, amino acids and the abundance of carbon isotopes in phloem sap obtained from E. globulus among different seasons and fertilizer treatments. Phloem-derived total amino acid concentration was found to increase with an increasing nitrogen (N) supply; however, this response was lost with the concurrent addition of phosphorus and at the highest level of N supply. Significant seasonal variation in all measured parameters was also detected, highlighting the need for caution in making quantitative relationships with growth. Broader implications of the interactive effects of both water supply and multi-nutrient additions and relationships with growth are discussed.

Relations of sugar composition and delta 13C in phloem sap to growth and physiological performance of Eucalyptus globulus (Labill).

We characterized differences in carbon isotopic content (delta(13)C) and sugar concentrations in phloem exudates from Eucalyptus globulus (Labill) plantations across a rainfall gradient in south-western Australia. Phloem sap delta(13)C and sugar concentrations varied with season and annual rainfall. Annual bole growth was negatively related to phloem sap delta(13)C during summer, suggesting a water limitation, yet was positively related in winter. We conclude that when water is abundant, variations in carboxylation rates become significant to overall growth. Concentrations of sucrose in phloem sap varied across sites by up to 600 mm, and raffinose by 300 mm. These compounds play significant roles in maintaining osmotic balance and facilitating carbon movement into the phloem, and their relative abundances contribute strongly to overall delta(13)C of phloem sap. Taken together, the delta(13)C and concentrations of specific sugars in phloem sap provide significant insights to functions supporting growth at the tree, site and landscape scale.