Developing Phloem δ13C and Sugar Composition as Indicators of Water Deficit in Lupinus angustifolius

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  • 1 Faculty of Agriculture, Food and Natural Resources, The University of Sydney, Australian Technology Park, Sydney NSW, Australia 2006

The use of naturally occurring carbon isotope ratios (expressed as δ13C) obtained from phloem sap offers considerable promise for evaluations of short-term physiological status of plants. We investigate the suitability of δ13C among carbon pools isolated from both leaves and phloem sap as surrogate measures of plant gas exchange in Lupinus angustifolius L. We also investigate the use of phloem osmotic potential (ψS), and metabolite concentrations as surrogate measures of δ13C. Phloem sap carbon bled from the distal tip of the fruit offers considerably improved correlations between δ13C and plant gas exchange compared with δ13C of carbon pools obtained from extractions of excised phloem tissue and leaf soluble carbon. The concentration of phloem sugars and sugar alcohols correlates strongly with measures of phloem δ13C but not with phloem ψS. We discuss these results in the context of developing fast, reliable, in-field tools for the assessment of plant physiological status.

Abstract

The use of naturally occurring carbon isotope ratios (expressed as δ13C) obtained from phloem sap offers considerable promise for evaluations of short-term physiological status of plants. We investigate the suitability of δ13C among carbon pools isolated from both leaves and phloem sap as surrogate measures of plant gas exchange in Lupinus angustifolius L. We also investigate the use of phloem osmotic potential (ψS), and metabolite concentrations as surrogate measures of δ13C. Phloem sap carbon bled from the distal tip of the fruit offers considerably improved correlations between δ13C and plant gas exchange compared with δ13C of carbon pools obtained from extractions of excised phloem tissue and leaf soluble carbon. The concentration of phloem sugars and sugar alcohols correlates strongly with measures of phloem δ13C but not with phloem ψS. We discuss these results in the context of developing fast, reliable, in-field tools for the assessment of plant physiological status.

Plant physiological processes change in response to short-term variation in resource availability. Developing non-invasive tools for the rapid and reliable evaluation of short-term variation in resource availability is of critical importance to land managers and agriculturalists across a range of managed and natural systems.

The use of natural abundance carbon isotopes to monitor plant gas exchange is well established at the leaf level. More recently, significant attention has been devoted to the use of δ13C obtained from phloem sap (δ13Cphl) as a surrogate measure of whole canopy leaf δ13C (Cernusak et al., 2003; Keitel et al., 2006; Merchant et al., 2010c; Rascher et al., 2010; Salmon et al., 2011). Transport of metabolites through the phloem is the central pathway for long-distance transport of carbon thus has potential for spatially and temporally integrated measures of canopy-level δ13C. Carbon obtained from the phloem sap is also “filtered” from a background of leaf metabolism potentially offering a clearer reflection of 13C abundance obtained by recent photoassimilation (Merchant et al., 2010b). Improved predictions of canopy-scale gas exchange (Cernusak et al., 2005; Keitel et al., 2003) and growth (Merchant et al., 2010c) across a range of plant species support these hypotheses suggesting that techniques based on the δ13C of phloem sap may be further developed across a range of plant species.

Harvesting of phloem sap remains a major impediment to adaptations of phloem sap-based diagnostic assessments of plant function. A range of phloem bleeding and extraction techniques have been developed including stem bleeding (Pate and Arthur, 1998), phloem tissue removal and extraction (Gessler et al., 2004), and bleeding from the distal tip of the fruit (Pate et al., 1974). Whereas each technique offers advantages, their deployment is reliant on the characteristics of the plant species under investigation. Gessler et al. (2004) investigated the correlation of δ13C obtained through different phloem sap collection methods applicable to tree species; however, no study has compared such methods with phloem collected from fruit tips (e.g., Pate et al., 1974) or aligned isotope abundance with that predicted by leaf-level models of isotope discrimination and measured gas exchange.

Development of tools based on measures of isotope abundance for use in the field is also restricted by the expense and time taken to measure δ13C. Pate et al. (1998) identified that the ψS (π) of phloem sap obtained from Eucalyptus globulus trees correlates strongly with δ13C. Using a refractometer, calibrated measures of π can be used as a surrogate measure of δ13C (e.g., Tausz et al., 2008) enabling in-field detection and mitigation of water availability. More recently, significant and systematic changes in phloem sugar composition have also been detected (Merchant et al., 2010a, 2010b, 2011) to levels that would have a significant influence on π.

Transport of carbon in the phloem sap of plants requires “packaging” into highly reduced compounds. For L. angustifolius, phloem is thought to consist largely of sucrose; however, the contribution of sugar alcohols is yet to be investigated. The sugar alcohol D-pinitol is common to all legumes reaching molar concentrations equivalent to that of sucrose at the leaf level (Kuo et al., 1997; Streeter, 2001; Wanek and Richter, 1997) increasing in concentration according to plant stress (Adams et al., 1992; Ford, 1982; McManus et al., 2000; Richter and Popp, 1992) and ecological gradients (Streeter et al., 2001). To date, no study has characterized the breakdown and metabolism of D-pinitol in plant tissues. In addition, the contribution of pinitol to phloem ψS is largely unknown.

In light of recent developments in the use of phloem sap as a rapid and reliable integrated measure of leaf δ13C and the potential for use of phloem π as a cheap and reliable surrogate for δ13C, we sought to determine the effects of water deficit on the concentration of sugars and δ13C in both leaves and phloem sap of L. angustifolius. We sought to determine the suitability of phloem sap carbon δ13C, obtained by different collection techniques, to reflect measures of leaf gas exchange. We also sought to evaluate the use of changes in phloem metabolite composition, in particular changes in the ratio of sugars to sugar alcohols as both a surrogate measure of plant water status and a potential confounding effect in overall measures of phloem sap δ13C and ψS.

MATERIALS AND METHODS

Experimental design.

Seeds of Lupinus angustifolius were germinated in a mixture of peat and coarse river sand (50/50 v/v) and raised under greenhouse conditions in Creswick, Australia (lat. 31°57′ S, long. 115°52′ E). In spring (Nov. 2010), ≈2 months before the start of treatments, seedlings were transplanted into 4-L pots and grown under glasshouse conditions with a light supplement to maintain a 14-h photoperiod (0700–2100 hr) of greater than 800 μmol·m−2·s−1 photosynthetic photon flux density for a 14-h photoperiod with ambient air temperature between 28 °C (day) and 15 °C (night) ramped within 1 h at the start and conclusion of the photoperiod. Seedlings were again planted into a 50:50 mix of peat and coarse river sand with 4 g·L−1 slow-release fertiliser added (Nutricote-100 13N–13P–13 with oligo elements). On each day, plants were watered to field capacity of the soil by drip irrigation.

Eight plants were allocated to each of a control treatment (watered to field capacity daily), mild water deficit [75% of water used by control plants (determined gravimetrically)], and severe water deficit (25% of water used by control plants). Treatments started 8 weeks after germination and were applied for 6 weeks. By the time of sampling, individual plants were 3 months old, ≈20 cm in height, and the stems were ≈0.8 cm in diameter.

Leaf gas exchange measurements.

After 6 weeks, treatment conditions, measures of leaf net carbon assimilation (A, μmol CO2/m−2·s−1) and stomatal conductance (gS) (mol H2O/m−2·s−1) were determined on each plant using a LI-COR 6400 infrared gas analyzer (LICOR, Lincoln, NE). Three independent measurements were taken on each plant six times throughout the day before the collection of phloem sap (see subsequently). Gas exchange results were scaled according to leaf area within the chamber. Light conditions within the LICOR chamber were set to tracking mode to approximate the growth chamber conditions. CO2 mole fraction in the reference air stream was set to 400 μmol·mol−1 and temperature and relative humidity in the measuring chamber was maintained within the LICOR chamber to approximate ambient conditions. Net CO2 A (μmol·m−2·s−1) as well as gS to water vapor (gS, mmol·m−2·s−1) were recorded and used to compute the ratio of intercellular to atmospheric CO2 concentration ci/ca. The daily integral of assimilation-weighted ci was calculated using the approach of Cernusak et al. (2005).

Leaf tissue and phloem sap collection.

At 1400 hr on the day of LICOR measurements, one blade from each of six leaflets was taken, pooled for each plant, and immediately frozen at –86 °C. Leaves used for gas exchange measurements were not sampled to avoid collection of carbon diluted by CO2 from the LICOR reference gas canister.

Phloem sap was collected through two methods, one as an extract of excised phloem tissue and the other using a phloem bleeding technique from the distal tip of the fruit (Pate et al., 1974). Each method was conducted following gas exchange measurements between 11 and 14 h after illumination. Phloem sap collection was made late in the photoperiod to avoid the effects on δ13C of transitory starch remobilization during the night identified by Gessler et al. (2007).

Collections of phloem material for phloem sap extraction were made using a single-sided razor blade. A section of phloem material ≈0.3 cm in area was excised, weighed, placed in 1.5 mL deionized water, and incubated at 4 °C for 90 min. Phloem material was then removed and the remaining solution transferred to –86 °C. Phloem tissue was then dried at 60 °C for 12 h and reweighed. For the bleeding technique, phloem sap formed a droplet on the surface of the cut that was collected into a glass disposable pipette and bulked for each tree. Samples were transferred to –4 °C during the sampling period Before freezing at –86 °C, 3 μL of phloem sap was placed into a separate microtube for measures of osmolality (see subsequently).

Analysis of phloem sap and leaf extracts for soluble carbohydrates.

Leaf samples for metabolite analysis were extracted using a solution of methanol, chloroform, and water (MCW) according to the protocol outlined Merchant et al. (2006). For determination of δ13C, the MCW extract was substituted with a hot water extract whereby 1 mL of hot (80 °C) water was used to extract 20 mg of dried leaf material. Extracts of leaf material, phloem sap, and the bled phloem sap were dried down and resuspended into 1 mL of deionized water.

Samples were then deionized over a mixed bed resin (see Merchant et al., 2006) consisting of one part Dowex 1 × 8 (anion exchange Cl form) and one part Dowex 50W (cation exchange, formate form; Dow Chemical Company, MI) and frozen awaiting analysis. For bled phloem sap, 50 μL was diluted with deionized water to a total volume of 1 mL, deionized, dried, resuspended into 50 μL of deionized water, and then frozen awaiting analysis.

Sixty microliters of deionized MCW extracts were dried and resuspended in 400 μL anhydrous pyridine to which 50 μL of trimethylchlorosilane/bis-trimethylsilyl-trifluoroacetamide mix (1:10; Sigma Aldrich, St. Louis, MO) was added. Samples were incubated for 1 h at 75 °C and analyzed by gas chromatography (GC) within 24 h. Analysis of soluble carbohydrates was performed by gas chromatography as per Merchant et al. (2006) using a Shimadzu 17A series gas chromatograph (Shimadzu Corporation Limited, Columbia, MD) with a DB1 column (0.25 mm i.d., 30 m, 0.25-μm film thickness). Split injection was made at 300 °C with an initial oven temperature program of 60 °C for 2 min ramping to 300 °C at 10 °C·min−1 and maintained for 10 min. Column flow rate was maintained at 1.5 mL·min−1. Peak integration was made using Class VP analysis software (Shimadzu Corporation Limited). δ13C was recorded with a continuous-flow isotope ratio mass spectrometer (Delta S; Thermo Electron, Bremen, Germany).

Osmolality of phloem bled from the distal tip of the fruit was determined using a freeze point depression osmometry using an OSMOMAT 030 cryoscopic osmometer (Gonotec, Berlin, Germany). Using solute concentrations obtained from GC analysis, molar concentrations of solutes in phloem sap were converted to ψSs) using the van’t Hoff equation:
DE1
where R is the universal gas constant (8.32 J·mol−1·K−1), T is the temperature in Kelvin (set at 293), and cs is the concentration of solutes in phloem sap (mol·L−1)

Isotope analysis and modeling of δ13C.

For the analysis of carbon isotope composition of the soluble extract in leaves (δ13Csol), 500 μL of extract was progressively transferred into tin cups and dried at 45 °C for 12 h and then kept over desiccant awaiting analysis. For δ13Cphlext and δ13Cphlsap, 500 μL and 5 μL, respectively, were placed into tin cups and dried at 45 °C for 12 h and then kept over desiccant until analyzed.

Isotope ratio mass spectrometry (IRMS) was used to determine the δ13C in samples. Samples were analyzed on an Isochrom mass spectrometer (Micromass, Manchester, U.K.) coupled to a Carlo Erba elemental analyser (CE Instruments, Milan, Italy). Samples were dropped from an AS200 autosampler and combusted by Dumas-combustion in a furnace kept at 1060 °C. Carbon isotope ratios are expressed in delta-notation, where δ13C = Rsample/Rstandard – 1 and R is the ratio of 13C to 12C in a sample and standard, respectively.

Predicted isotope values were calculated using the following equation originally devised by Farquhar and Richards (1984):
DE2
where ab is the fractionation caused by boundary layer (2.9%), a is the fractionation caused by gaseous diffusion (4.4%), and b is the effective fractionation caused by carboxylating enzymes (Rubisco and PEPC, ≈29%). bs is the 13C fractionation as CO2 enters solution (1.1%) and al is the 13C fractionation resulting from diffusion of CO2 in water (0.7%; Oleary, 1984). f is the fractionation resulting from photorespiration (≈11%; Lanigan et al., 2008) and Γ* is the CO2 partial pressure at which CO2 assimilation compensates the production of photorespiratory CO2 calculated according to Brooks and Farquhar (1985) by:
DE3
where T is the leaf temperature (°C). Ca was set at 400 μmol·mol−1 and Ci calculated using the LICOR 6400 software (LICOR, 2008). Mesophyll conductance (gi) at each growth temperature was estimated using the log normal calculation outlined by Warren (2008). Cc was then calculated as:
DE4

Fractionation of 13C resulting from day respiration (Rd, e′) has been ignored in modeling for our experiment (i.e., e′ = 0). Similarly, transfer resistance of the boundary layer was set to 0 (i.e., Cs = Ca).

For calculations of 13C discrimination by plants, the isotopic composition of the source (i.e., the atmosphere in the growth chamber), was assumed to be –7.8% (see Farquhar et al., 1982). Discrimination (Δ) from air was calculated using the formula:
DE5

Statistical analysis.

Effects of water deficit treatments were analyzed by analysis of variance using Statistica analytical software (Version 6; StatSoft, Tulsa, OK). P values were calculated using Tukey’s honestly significant difference post hoc test mean values. Linear regressions were calculated using a general linear model.

RESULTS

The influence of water deficit treatments over the photoperiod was observed on net photosynthetic rate with 100% and 75% treatments approximately three- and twofold higher than the 25% treatment, respectively (Fig. 1A). Similarly, gS was higher in both 100% and 75% treatments than those detected in 25% treatment with the 100% treatment sustaining higher gS rates throughout a longer proportion of the photoperiod (Fig. 1B). Delineation between treatments was also reflected in ci/ca values throughout the photoperiod (Fig. 1C).

Fig. 1.
Fig. 1.

Leaf photosynthesis, gas exchange, and ci/ca across a daily time course for 3-month-old Lupinus angustifolius subjected to 25%, 75%, and 100% of water used by control plants (determined gravimetrcally) for a period of 6 weeks. Bars represent the sem values of eight replicate plants. Gas exchange was measured on a randomly selected fully expanded leaf. Plants were grown under conditions of 25/15 °C (day/night) and between 500 and 1200 mmol·m−2·s−1 photosynthetic photon flux density (PPFD) for a photoperiod of 14 h per day.

Citation: HortScience horts 47, 6; 10.21273/HORTSCI.47.6.691

Leaf, phloem sap, and phloem extract δ13C versus modeled values.

δ13Csol, δ13Cphlext, and δ13Cphlsap were significantly enriched in 13C relative to δ13Cmodeled (P < 0.001). δ13Cphlsap was more closely aligned with δ13Cmodeled with a consistent offset of ≈4% across the range of assimilation weighted ci/ca values obtained in this study (Fig. 2; Table 1). In contrast, both the slope and intercepts of δ13Cphlext and δ13Csol significantly differed from that of δ13Cmodeled across the same range of ci/ca values (Fig. 2; Table 1).

Table 1.

Slope, intercept and regression coefficients of the relationships between leaf, phloem, and modeled δ13C values plotted against measures of ci/ca.z

Table 1.
Fig. 2.
Fig. 2.

Modeled (predicted) δ13C calculated from Eq. 2 and δ13C phloem bled δ13C extracted phloem and δ13C leaf soluble neutral fraction obtained from 3-month-old Lupinus angustifolius. Bars represent the sem values of eight replicate plants. Gas exchange was measured on a randomly selected fully expanded leaf. Plants were grown under conditions of 25/15 °C (day/night) and between 500 and 1200 mmol·m−2·s−1 photosynthetic photon flux density (PPFD) for a photoperiod of 14 h per day.

Citation: HortScience horts 47, 6; 10.21273/HORTSCI.47.6.691

Phloem sap and leaf sugar contents.

Phloem sap sucrose significantly increased by ≈80 mm in response to both the 75% and 25% treatments (Table 2). Similarly, pinitol concentrations increased more than twofold for the 75% treatment and threefold for the 25% treatment. Common reducing sugars, glucose, and fructose were both detected at less than 1 mm concentrations and, although mean values increased according to the severity of water deficit, did not significantly differ among treatments.

Table 2.

Concentrations of sucrose, pinitol, fructose, and glucose in phloem and leaves of Lupinus angustifolius plants subjected to 25%, 75%, and 100% of water used by control plants (determined gravimetrcally) for a period of 6 weeks (n = 24).z

Table 2.

In contrast, leaf tissues showed a decreasing trend in metabolite concentrations in response to water deficit treatments (Table 2). Leaf sucrose and pinitol concentrations were similar ranging between 40 and 70 μmol·g−1 dry weight.

Variation in phloem osmotic potential.

Comparison of δ13Cphlsap with that of phloem ψS calculated from analyzed phloem contents yielded a positive significant relationship (Fig. 3). Osmotic potential calculated from measured metabolites was consistently less than that measured by freeze point depression osmometry (Fig. 4). This relationship showed an increasing deviation from 1:1 with increasing ψS.

Fig. 3.
Fig. 3.

δ13C of carbon contained in phloem sap plotted against the osmotic potential (ψS) of phloem sap calculated through the additive effects of sucrose, pinitol glucose, and fructose concentrations. Phloem sap was obtained from distal fruit tip of 3-month-old Lupinus angustifolius subjected to water deficit treatments while grown under conditions of 25/15 °C (day/night) and between 500 and 1200 mmol·m−2·s−1 photosynthetic photon flux density (PPFD) for a photoperiod of 14 h per day. Water deficit treatments of 100%, 75% and 25% are proportions of daily water use by control plants.

Citation: HortScience horts 47, 6; 10.21273/HORTSCI.47.6.691

Fig. 4.
Fig. 4.

Osmotic potential in phloem sap calculated from measured metabolites plotted against osmotic potential (ψS) in phloem sap calculated from measured osmolality. Phloem sap was obtained from distal fruit tip of 3-month-old Lupinus angustifolius subjected to water deficit treatments whilst grown under conditions of 25/15 °C (day/night) and between 500 and 1200 mmol·m−2·s−1 photosynthetic photon flux density (PPFD) for a photoperiod of 14 h per day. Water deficit treatments of 100%, 75%, and 25% are proportions of daily water use by control plants.

Citation: HortScience horts 47, 6; 10.21273/HORTSCI.47.6.691

DISCUSSION

The present study confirms that δ13C of phloem sap carbon obtained through bleeding from the distal tip of the fruit represents the best reflection of plant gas exchange when compared with carbon sources either extracted from phloem or leaf tissues. We also show that D-pinitol is present in phloem sap but only constitutes a small proportion (2% to 3%) of the overall phloem sap carbon content despite representing a significant proportion of the leaf-level soluble sugars. Osmotic potential of phloem sap holds great promise as a surrogate measure of phloem sap δ13C; however, we highlight here an additional osmotic component(s) of phloem sap that may significantly influence this relationship. The influence of this additional osmotic component may have important implications for the use of refractometry (e.g., Tausz et al., 2008) as a surrogate measure of phloem sap δ13C.

Patterns in δ13C.

δ13Cphlbled best reflected those modeled values as predicted by Eq. 1 and offers valid predictions of leaf gas exchange. In contrast, carbon obtained through the phloem extraction technique and purified metabolites from leaf tissues showed poor relationships with modeled δ13C predicted by measured ci/ca. For leaf tissues, this is likely a result of a background of leaf metabolism present in the neutral fraction carbon pool. For phloem extracted from excised phloem tissue, poor relationships are likely attributable to the extraction of living material from the stem pieces that are not part of the transported carbon pool. A similar comparison between extraction techniques was made by Gessler et al. (2004) using a chelating reagent (EDTA polyphosphate) in the extraction solution. For our study, a chelating reagent was not required because phloem pieces yielded enough carbon that, when purified, remained within the detection limits of the IRMS.

Despite a superior fit for the relationship between δ13Cphlbled and modeled values, a significant offset in δ13C from leaf to phloem was observed suggesting a level of heterotrophic enrichment of ≈4%. This supports the majority of previous studies including substantial reviews detailing enrichment of phloem sap compared with leaf tissues across a range of plant genera (e.g., Bowling et al., 2008). Despite this observation, the processes underpinning this phenomenon are yet to be fully understood (see Cernusak et al., 2009). Whereas transport fractionation, respiration, and the cycling of carbon through starch biosynthesis are, among many things, likely to play an important role (see Cernusak et al., 2009), changes in phloem composition, combined with differences in fractionation events attributable to synthesis, may account for at least part of this heterotrophic enrichment (Merchant et al., 2011).

Phloem and leaf metabolites.

In response to water deficit, phloem metabolite concentrations significantly increased in agreement with previous studies across a range of plant genera (Cernusak et al., 2002; Gessler et al., 2008; Merchant et al., 2010b; Pate et al., 1998). In contrast, leaf metabolites showed significant decreases in concentration indicating reductions in photoassimilate production, increased export of photoassimilates from leaf tissues, increased respiration, increased allocation of carbon to alternative pools (e.g., starch accumulation), or a combination of effects. This pattern suggests that export of metabolites to phloem transport was not a significantly limiting factor to carbon movement during the onset of water deficit. An absence of carbohydrate accumulation in leaf tissues may have a significant influence on sugar-mediated repression of photosynthesis (e.g., Halford et al., 2003; Stitt et al., 2010) and osmotic adjustment hence impedes a plant’s responsiveness to short-term changes in resource availability and environmental conditions.

Preservation of phloem structure and function is critical for plant health. Among a range of mechanisms for preserving and protecting the phloem structure, plants “package” carbon into non-reducing forms for long-distance transport. Sucrose is the most common form in which plants transport carbon in the phloem (see Turgeon, 1996), often accompanied by raffinose oligosaccharides and/or polyols such as alditols (e.g., sorbitol, mannitol) or cyclic polyols such as D-pinitol (see Bieleski, 1982; Loescher, 1987; Noiraud et al., 2001b; Reidel et al., 2009). D-Pinitol, a methylated inositol, is of particular interest as a result of its distribution among a number of plant taxa (Angyal and Anderson, 1959; Loewus and Dickinson, 1982; Posternak, 1965), most notably the Leguminoasae. D-Pinitol has been shown to accumulate up to 28 mg·g−1 in seed tissues of leguminous species (Kuo et al., 1997) indicating the importance of phloem-mediated movement of this class of compounds. To date, no study has characterized the metabolism of methylated cyclitols in plant tissues despite their accumulation to significant concentrations. In this study, the substantial accumulation of D-pinitol in leaf tissues supports previous hypotheses for roles in osmotic adjustment (Paul and Cockburn, 1989; Streeter et al., 2001) and the sequestration of excess photochemical energy (Hare et al., 1998).

Transport of carbon in the phloem is a major sink for leaf-level carbon balance. In recognition of their important role in carbon transport, several transmembrane proteins for the movement of polyols such as mannitol and sorbitol have been characterized (Gao et al., 2003; Noiraud et al., 2001a). Despite the extensive distribution of cyclitols such as D-pinitol in leaf tissues, less is known regarding their role in the transport of carbon among plant tissues. In this study, significant concentrations of D-pinitol in the leaf, coupled with relatively minor concentrations in the phloem sap, suggest that movement may be attributable to diffusion with no phloem-loading mechanism in operation. It is important to recognize that the low concentrations of D-pinitol obtained from phloem sap in this study are not directly indicative of flux and highlight the need for further exploration of this carbon transport mechanism across a range of environmental conditions and developmental stages.

The concentration of metabolites in phloem sap increased in response to water deficit; however, no significant change in the ratio of sucrose to D-pinitol was detected. Changes in the composition of the phloem sap may have important consequences for measures of δ13C as has been shown in previous studies in Eucalyptus (Merchant et al., 2011). Whereas the significant concentrations of D-pinitol obtained here from the soluble fraction of leaves are likely to have important consequences for leaf-level measures of δ13C, it is unlikely to have significant implications for phloem sap under conditions experienced in this study.

Osmotic potential as a surrogate measure of δ13C and water status.

The concentration of phloem sugars and sugar alcohols is a viable predictor of δ13C for L. angustifolius. Previous studies investigating this relationship have yielded similar results across a range of plant species (Cernusak et al., 2002, 2003; Gessler et al., 2007; Keitel et al., 2003; Merchant et al., 2010c; Pate and Arthur, 1998). Our results also indicate the significant influence of additional solutes in phloem sap on ψS. The residual ψS unaccounted for in this study is likely to be attributed to a range of solutes, most likely organic acids and inorganic ions. Potassium has been isolated from phloem sap (Pate et al., 1974; Pritchard, 2007) and is likely to have significant influence over osmotic relations attributable to its concentration and low molecular weight. Similarly, free amino acids are often isolated from phloem sap in concentrations large enough to have a discernible influence over osmotic relations (Pritchard, 2007). The use of refractometry as a surrogate measure of phloem carbohydrate concentration requires standardization against measured sugar and sugar alcohol concentrations (e.g., Cernusak et al., 2005; Tausz et al., 2008) and assumes an insignificant or at least constant contribution of additional substances to the refraction index. Substantial differences in phloem composition have been detected across a diversity of plants grown under a broad range of conditions. For our study, a proportionally larger contribution to total phloem sap ψS was made by unknown solutes as total ψS increased. Our study suggests that the use of ψS as a surrogate measure of δ13C is valid; however, further understanding of the composition of phloem sap in L. angustifolius is required for the use of refractometry methods to be used at least under those conditions outlined in this study.

Literature Cited

  • Adams, P., Thomas, J.C., Vernon, D.M., Bohnert, H.J. & Jensen, R.G. 1992 Distinct cellular and organismic responses to salt stress Plant Cell Physiol. 33 1215 1223

    • Search Google Scholar
    • Export Citation
  • Angyal, S.J. & Anderson, L. 1959 The cyclitols Adv. Carbohydr. Chem. 14 135 212

  • Bieleski, R.L. 1982 Sugar alcohols. In: Loewus, F.A. and W. Tanner (eds.). Encyclopedia of plant physiology. Vol. 13A. Springer-Verlag, New York, NY

  • Bowling, D.R., Pataki, D.E. & Randerson, J.T. 2008 Carbon isotopes in terrestrial ecosystem pools and CO2 fluxes New Phytol. 178 24 40

  • Brooks, A. & Farquhar, G.D. 1985 Effect of temperature on the CO2/O2 specificity of ribulose-1,5-biphosphate carboxylase oxygenase and the rate of respiration in the light—Estimates from gas exchange measurements on spinach Planta 165 397 406

    • Search Google Scholar
    • Export Citation
  • Cernusak, L.A., Arthur, D.J., Pate, J.S. & Farquhar, G.D. 2003 Water relations link carbon and oxygen isotope discrimination to phloem sap sugar concentration in Eucalyptus globulus Plant Physiol. 131 1544 1554

    • Search Google Scholar
    • Export Citation
  • Cernusak, L.A., Farquhar, G.D. & Pate, J.S. 2005 Environmental and physiological controls over oxygen and carbon isotope composition of Tasmanian blue gum, Eucalyptus globulus Tree Physiol. 25 129 146

    • Search Google Scholar
    • Export Citation
  • Cernusak, L.A., Pate, J.S. & Farquhar, G.D. 2002 Diurnal variation in the stable isotope composition of water and dry matter in fruiting Lupinus angustifolius under field conditions Plant Cell Environ. 25 893 907

    • Search Google Scholar
    • Export Citation
  • Cernusak, L.A., Tcherkez, G., Keitel, C., Cornwell, W.K., Santiago, L.S., Knohl, A., Barbour, M.M., Williams, D.G., Reich, P.B., Ellsworth, D.S., Dawson, T.E., Griffiths, H.G., Farquhar, G.D. & Wright, I.J. 2009 Why are non-photosynthetic tissues generally 13C enriched compared with leaves in C3 plants? Review and synthesis of current hypotheses Funct. Plant Biol. 36 199 213

    • Search Google Scholar
    • Export Citation
  • Farquhar, G.D., O'Leary, M.H. & Berry, J.A. 1982 On the relationship between carbon isotope discrimination and the intercellular carbon dioxide concentration in leaves Aust. J. Plant Physiol. 9 121 137

    • Search Google Scholar
    • Export Citation
  • Farquhar, G.D. & Richards, R.A. 1984 Isotopic composition of plant carbon correlates with water use efficiency of wheat genotypes Aust. J. Plant Physiol. 11 539 552

    • Search Google Scholar
    • Export Citation
  • Ford, C.W. 1982 Accumulation of O-methyl-inositols in water stressed Vigna species Phytochemistry 21 1149 1151

  • Gao, Z.F., Maurousset, L., Lemoine, R., Yoo, S.D., van Nocker, S. & Loescher, W. 2003 Cloning, expression, and characterization of sorbitol transporters from developing sour cherry fruit and leaf sink tissues Plant Physiol. 131 1566 1575

    • Search Google Scholar
    • Export Citation
  • Gessler, A., Keitel, C., Kodama, N., Weston, C., Winters, A.J., Keith, H., Grice, K., Leuning, R. & Farquhar, G.D. 2007 δ13C of organic matter transported from the leaves to the roots in Eucalyptus delegatensis: Short-term variations and relation to respired CO2 Funct. Plant Biol. 34 692 706

    • Search Google Scholar
    • Export Citation
  • Gessler, A., Rennenberg, H. & Keitel, C. 2004 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 Plant Biol. 6 721 729

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    • Export Citation
  • Gessler, A., Tcherkez, G., Peuke, A.D., Ghashghaie, J. & Farquhar, G.D. 2008 Experimental evidence for diel variations of the carbon isotope composition in leaf, stem and phloem sap organic matter in Ricinus communis Plant Cell Environ. 31 941 953

    • Search Google Scholar
    • Export Citation
  • Halford, N.G., Hey, S., Jhurreea, D., Laurie, S., McKibbin, R.S., Zhang, Y. & Paul, M.J. 2003 Dissection and manipulation of metabolic signalling pathways Ann. Appl. Biol. 142 25 31

    • Search Google Scholar
    • Export Citation
  • Hare, P.D., Cress, W.A. & Van Staden, J. 1998 Dissecting the roles of osmolyte accumulation during stress Plant Cell Environ. 21 535 553

  • Keitel, C., Adams, M.A., Holst, T., Matzarakis, A., Mayer, H., Rennenberg, H. & Gessler, A. 2003 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.) Plant Cell Environ. 26 1157 1168

    • Search Google Scholar
    • Export Citation
  • Keitel, C., Matzarakis, A., Rennenberg, H. & Gessler, A. 2006 Carbon isotopic composition and oxygen isotopic enrichment in phloem and total leaf organic matter of European beech (Fagus sylvatica L.) along a climate gradient Plant Cell Environ. 29 1492 1507

    • Search Google Scholar
    • Export Citation
  • Kuo, T.M., Lowell, C.A. & Nelsen, T.C. 1997 Occurrence of pinitol in developing soybean seed tissues Phytochemistry 45 29 35

  • Lanigan, G.J., Betson, N., Griffiths, H. & Seibt, U. 2008 Carbon isotope fractionation during photorespiration and carboxylation in Senecio Plant Physiol. 148 2013 2020

    • Search Google Scholar
    • Export Citation
  • LICOR 2008 Using the LI-6400/LI-6400XT portable photosynthesis system. LI-COR Biosciences, Inc., Lincoln, NE

  • Loescher, W.H. 1987 Physiology and metabolism of sugar alcohols in higher-plants Physiol. Plant. 70 553 557

  • Loewus, F.A. & Dickinson, D.B. 1982 Cyclitols, p. 193–216. In: Loewus, F.A. and W. Tanner (eds.). Encyclopedia of plant physiology. Vol. 13A. Springer-Verlag, New York, NY

  • McManus, M.T., Bieleski, R.L., Caradus, J.R. & Barker, D.J. 2000 Pinitol accumulation in mature leaves of white clover in response to a water deficit Environ. Exp. Bot. 43 11 18

    • Search Google Scholar
    • Export Citation
  • Merchant, A., Adams, M.A., Richter, A. & Popp, M. 2006 Targeted metabolite profiling provides a functional link among eucalypt taxonomy, physiology and evolution Phytochemistry 67 402 408

    • Search Google Scholar
    • Export Citation
  • Merchant, A., Arndt, S.K., Rowell, D.M., Posch, S., Callister, A., Tausz, M. & Adams, M.A. 2010a Seasonal changes in carbohydrates, cyclitols, and water relations of 3 field grown Eucalyptus species from contrasting taxonomy on a common site Ann. For. Sci. 67

    • Search Google Scholar
    • Export Citation
  • Merchant, A., Peuke, A.D., Keitel, C., Macfarlane, C., Warren, C. & Adams, M.A. 2010b Phloem sap and leaf δ13C, carbohydrates and amino acid concentrations in Eucalyptus globulus change systematically according to flooding and water deficit treatment J. Expt. Bot. 61 1785 1793

    • Search Google Scholar
    • Export Citation
  • Merchant, A., Tausz, M., Keitel, C. & Adams, M.A. 2010c Relations of sugar composition and δ13C in phloem sap to growth and physiological performance of Eucalyptus globulus (Labill) Plant Cell Environ. 33 1361 1368

    • Search Google Scholar
    • Export Citation
  • Merchant, A., Wild, B., Richter, A., Bellot, S., Adams, M.A. & Dreyer, E. 2011 Compound-specific differences in 13C of soluble carbohydrates in leaves and phloem of 6-month-old Eucalyptus globulus (Labill) Plant Cell and Environment 34 1599 1608

    • Search Google Scholar
    • Export Citation
  • Noiraud, N., Maurousset, L. & Lemoine, R. 2001a Identification of a mannitol transporter, AgMaT1, in celery phloem Plant Cell 13 695 705

  • Noiraud, N., Maurousset, L. & Lemoine, R. 2001b Transport of polyols in higher plants Plant Physiol. Biochem. 39 717 728

  • Oleary, M.H. 1984 Measurement of the isotope fractionation associated with diffusion of carbon dioxide in aqueous solution J. Phys. Chem. 88 823 825

    • Search Google Scholar
    • Export Citation
  • Pate, J. & Arthur, D. 1998 Delta 13C analysis of phloem sap carbon: Novel means of evaluating seasonal water stress and interpreting carbon isotope signatures of foliage and trunk wood of Eucalyptus globulus Oecologia 117 301 311

    • Search Google Scholar
    • Export Citation
  • Pate, J., Shedley, E., Arthur, D. & Adams, M. 1998 Spatial and temporal variations in phloem sap composition of plantation-grown Eucalyptus globulus Oecologia 117 312 322

    • Search Google Scholar
    • Export Citation
  • Pate, J.S., Sharkey, P.J. & Lewis, O.A.M. 1974 Phloem bleeding form legume fruits—Technique for study of fruit nutrition Planta 120 229 243

  • Paul, M.J. & Cockburn, W. 1989 Pinitol, a compatible solute in Mesembryanthemum crystallinum L? J. Expt. Bot. 40 1093 1098

  • Posternak, T. 1965 The cyclitols. Hermann, Paris, France

  • Pritchard, J. 2007 Solute transport in the phloem. In: Yeo, A.R. and T.J. Flowers (eds.). Plant solute transport. Blackwell Publishing, Oxford, UK

  • Rascher, K.G., Maguas, C. & Werner, C. 2010 On the use of phloem sap δ13C as an indicator of canopy carbon discrimination Tree Physiol. 30 1499 1514

  • Reidel, E.J., Rennie, E.A., Amiard, V., Cheng, L. & Turgeon, R. 2009 Phloem loading strategies in three plant species that transport sugar alcohols Plant Physiol. 149 1601 1608

    • Search Google Scholar
    • Export Citation
  • Richter, A. & Popp, M. 1992 The physiological importance of accumulation of cyclitols in Viscum album L New Phytol. 121 431 438

  • Salmon, Y., Barnard, R.L. & Buchmann, N. 2011 Ontogeny and leaf gas exchange mediate the carbon isotopic signature of herbaceous plants Plant Cell Environ. 34 465 479

    • Search Google Scholar
    • Export Citation
  • Stitt, M., Lunn, J. & Usadel, B. 2010 Arabidopsis and primary photosynthetic metabolism—More than the icing on the cake Plant J. 61 1067 1091

  • Streeter, J.G. 2001 Simple partial purification of D-pinitol from soybean leaves Crop Sci. 41 1985 1987

  • Streeter, J.G., Lohnes, D.G. & Fioritto, R.J. 2001 Patterns of pinitol accumulation in soybean plants and relationships to drought tolerance Plant Cell Environ. 24 429 438

    • Search Google Scholar
    • Export Citation
  • Tausz, M., Merchant, A., Kruse, J., Samsa, G. & Adams, M.A. 2008 Estimation of drought-related limitations to mid-rotation aged plantation grown Eucalyptus globulus by phloem sap analysis For. Ecol. Mgt. 256 844 848

    • Search Google Scholar
    • Export Citation
  • Turgeon, R. 1996 Phloem loading and plasmodesmata Trends Plant Sci. 1 418 423

  • Wanek, W. & Richter, A. 1997 Biosynthesis and accumulation of D-ononitol in Vigna umbellata in response to drought stress Physiol. Plant. 101 416 424

    • Search Google Scholar
    • Export Citation
  • Warren, C.R. 2008 Does growth temperature affect the temperature responses of photosynthesis and internal conductance to CO2? A test with Eucalyptus regnans Tree Physiol. 28 11 19

    • Search Google Scholar
    • Export Citation

Contributor Notes

This paper was part of the colloquium, “Emerging Techniques to Evaluate and Mitigate Crop Environmental Stress in a Changing Climate” held 28 Sept. 2011 at the ASHS Conference, Waikoloa, HI, and sponsored by the Environmental Stress Physiology (STRS) Working Group.

Dr. Merchant is supported by an Australian Research Council Postdoctoral Fellowship (DP0988731).

Thank you to Dr. Antanas Spokevicius and Dr. Liubov Volkova for their assistance with the experimental setup. Thank you also to Dr. Claudia Keitel for isotopic analysis.

To whom reprint requests should be addressed; e-mail andrew.merchant@sydney.edu.au.

  • View in gallery

    Leaf photosynthesis, gas exchange, and ci/ca across a daily time course for 3-month-old Lupinus angustifolius subjected to 25%, 75%, and 100% of water used by control plants (determined gravimetrcally) for a period of 6 weeks. Bars represent the sem values of eight replicate plants. Gas exchange was measured on a randomly selected fully expanded leaf. Plants were grown under conditions of 25/15 °C (day/night) and between 500 and 1200 mmol·m−2·s−1 photosynthetic photon flux density (PPFD) for a photoperiod of 14 h per day.

  • View in gallery

    Modeled (predicted) δ13C calculated from Eq. 2 and δ13C phloem bled δ13C extracted phloem and δ13C leaf soluble neutral fraction obtained from 3-month-old Lupinus angustifolius. Bars represent the sem values of eight replicate plants. Gas exchange was measured on a randomly selected fully expanded leaf. Plants were grown under conditions of 25/15 °C (day/night) and between 500 and 1200 mmol·m−2·s−1 photosynthetic photon flux density (PPFD) for a photoperiod of 14 h per day.

  • View in gallery

    δ13C of carbon contained in phloem sap plotted against the osmotic potential (ψS) of phloem sap calculated through the additive effects of sucrose, pinitol glucose, and fructose concentrations. Phloem sap was obtained from distal fruit tip of 3-month-old Lupinus angustifolius subjected to water deficit treatments while grown under conditions of 25/15 °C (day/night) and between 500 and 1200 mmol·m−2·s−1 photosynthetic photon flux density (PPFD) for a photoperiod of 14 h per day. Water deficit treatments of 100%, 75% and 25% are proportions of daily water use by control plants.

  • View in gallery

    Osmotic potential in phloem sap calculated from measured metabolites plotted against osmotic potential (ψS) in phloem sap calculated from measured osmolality. Phloem sap was obtained from distal fruit tip of 3-month-old Lupinus angustifolius subjected to water deficit treatments whilst grown under conditions of 25/15 °C (day/night) and between 500 and 1200 mmol·m−2·s−1 photosynthetic photon flux density (PPFD) for a photoperiod of 14 h per day. Water deficit treatments of 100%, 75%, and 25% are proportions of daily water use by control plants.

  • Adams, P., Thomas, J.C., Vernon, D.M., Bohnert, H.J. & Jensen, R.G. 1992 Distinct cellular and organismic responses to salt stress Plant Cell Physiol. 33 1215 1223

    • Search Google Scholar
    • Export Citation
  • Angyal, S.J. & Anderson, L. 1959 The cyclitols Adv. Carbohydr. Chem. 14 135 212

  • Bieleski, R.L. 1982 Sugar alcohols. In: Loewus, F.A. and W. Tanner (eds.). Encyclopedia of plant physiology. Vol. 13A. Springer-Verlag, New York, NY

  • Bowling, D.R., Pataki, D.E. & Randerson, J.T. 2008 Carbon isotopes in terrestrial ecosystem pools and CO2 fluxes New Phytol. 178 24 40

  • Brooks, A. & Farquhar, G.D. 1985 Effect of temperature on the CO2/O2 specificity of ribulose-1,5-biphosphate carboxylase oxygenase and the rate of respiration in the light—Estimates from gas exchange measurements on spinach Planta 165 397 406

    • Search Google Scholar
    • Export Citation
  • Cernusak, L.A., Arthur, D.J., Pate, J.S. & Farquhar, G.D. 2003 Water relations link carbon and oxygen isotope discrimination to phloem sap sugar concentration in Eucalyptus globulus Plant Physiol. 131 1544 1554

    • Search Google Scholar
    • Export Citation
  • Cernusak, L.A., Farquhar, G.D. & Pate, J.S. 2005 Environmental and physiological controls over oxygen and carbon isotope composition of Tasmanian blue gum, Eucalyptus globulus Tree Physiol. 25 129 146

    • Search Google Scholar
    • Export Citation
  • Cernusak, L.A., Pate, J.S. & Farquhar, G.D. 2002 Diurnal variation in the stable isotope composition of water and dry matter in fruiting Lupinus angustifolius under field conditions Plant Cell Environ. 25 893 907

    • Search Google Scholar
    • Export Citation
  • Cernusak, L.A., Tcherkez, G., Keitel, C., Cornwell, W.K., Santiago, L.S., Knohl, A., Barbour, M.M., Williams, D.G., Reich, P.B., Ellsworth, D.S., Dawson, T.E., Griffiths, H.G., Farquhar, G.D. & Wright, I.J. 2009 Why are non-photosynthetic tissues generally 13C enriched compared with leaves in C3 plants? Review and synthesis of current hypotheses Funct. Plant Biol. 36 199 213

    • Search Google Scholar
    • Export Citation
  • Farquhar, G.D., O'Leary, M.H. & Berry, J.A. 1982 On the relationship between carbon isotope discrimination and the intercellular carbon dioxide concentration in leaves Aust. J. Plant Physiol. 9 121 137

    • Search Google Scholar
    • Export Citation
  • Farquhar, G.D. & Richards, R.A. 1984 Isotopic composition of plant carbon correlates with water use efficiency of wheat genotypes Aust. J. Plant Physiol. 11 539 552

    • Search Google Scholar
    • Export Citation
  • Ford, C.W. 1982 Accumulation of O-methyl-inositols in water stressed Vigna species Phytochemistry 21 1149 1151

  • Gao, Z.F., Maurousset, L., Lemoine, R., Yoo, S.D., van Nocker, S. & Loescher, W. 2003 Cloning, expression, and characterization of sorbitol transporters from developing sour cherry fruit and leaf sink tissues Plant Physiol. 131 1566 1575

    • Search Google Scholar
    • Export Citation
  • Gessler, A., Keitel, C., Kodama, N., Weston, C., Winters, A.J., Keith, H., Grice, K., Leuning, R. & Farquhar, G.D. 2007 δ13C of organic matter transported from the leaves to the roots in Eucalyptus delegatensis: Short-term variations and relation to respired CO2 Funct. Plant Biol. 34 692 706

    • Search Google Scholar
    • Export Citation
  • Gessler, A., Rennenberg, H. & Keitel, C. 2004 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 Plant Biol. 6 721 729

    • Search Google Scholar
    • Export Citation
  • Gessler, A., Tcherkez, G., Peuke, A.D., Ghashghaie, J. & Farquhar, G.D. 2008 Experimental evidence for diel variations of the carbon isotope composition in leaf, stem and phloem sap organic matter in Ricinus communis Plant Cell Environ. 31 941 953

    • Search Google Scholar
    • Export Citation
  • Halford, N.G., Hey, S., Jhurreea, D., Laurie, S., McKibbin, R.S., Zhang, Y. & Paul, M.J. 2003 Dissection and manipulation of metabolic signalling pathways Ann. Appl. Biol. 142 25 31

    • Search Google Scholar
    • Export Citation
  • Hare, P.D., Cress, W.A. & Van Staden, J. 1998 Dissecting the roles of osmolyte accumulation during stress Plant Cell Environ. 21 535 553

  • Keitel, C., Adams, M.A., Holst, T., Matzarakis, A., Mayer, H., Rennenberg, H. & Gessler, A. 2003 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.) Plant Cell Environ. 26 1157 1168

    • Search Google Scholar
    • Export Citation
  • Keitel, C., Matzarakis, A., Rennenberg, H. & Gessler, A. 2006 Carbon isotopic composition and oxygen isotopic enrichment in phloem and total leaf organic matter of European beech (Fagus sylvatica L.) along a climate gradient Plant Cell Environ. 29 1492 1507

    • Search Google Scholar
    • Export Citation
  • Kuo, T.M., Lowell, C.A. & Nelsen, T.C. 1997 Occurrence of pinitol in developing soybean seed tissues Phytochemistry 45 29 35

  • Lanigan, G.J., Betson, N., Griffiths, H. & Seibt, U. 2008 Carbon isotope fractionation during photorespiration and carboxylation in Senecio Plant Physiol. 148 2013 2020

    • Search Google Scholar
    • Export Citation
  • LICOR 2008 Using the LI-6400/LI-6400XT portable photosynthesis system. LI-COR Biosciences, Inc., Lincoln, NE

  • Loescher, W.H. 1987 Physiology and metabolism of sugar alcohols in higher-plants Physiol. Plant. 70 553 557

  • Loewus, F.A. & Dickinson, D.B. 1982 Cyclitols, p. 193–216. In: Loewus, F.A. and W. Tanner (eds.). Encyclopedia of plant physiology. Vol. 13A. Springer-Verlag, New York, NY

  • McManus, M.T., Bieleski, R.L., Caradus, J.R. & Barker, D.J. 2000 Pinitol accumulation in mature leaves of white clover in response to a water deficit Environ. Exp. Bot. 43 11 18

    • Search Google Scholar
    • Export Citation
  • Merchant, A., Adams, M.A., Richter, A. & Popp, M. 2006 Targeted metabolite profiling provides a functional link among eucalypt taxonomy, physiology and evolution Phytochemistry 67 402 408

    • Search Google Scholar
    • Export Citation
  • Merchant, A., Arndt, S.K., Rowell, D.M., Posch, S., Callister, A., Tausz, M. & Adams, M.A. 2010a Seasonal changes in carbohydrates, cyclitols, and water relations of 3 field grown Eucalyptus species from contrasting taxonomy on a common site Ann. For. Sci. 67

    • Search Google Scholar
    • Export Citation
  • Merchant, A., Peuke, A.D., Keitel, C., Macfarlane, C., Warren, C. & Adams, M.A. 2010b Phloem sap and leaf δ13C, carbohydrates and amino acid concentrations in Eucalyptus globulus change systematically according to flooding and water deficit treatment J. Expt. Bot. 61 1785 1793

    • Search Google Scholar
    • Export Citation
  • Merchant, A., Tausz, M., Keitel, C. & Adams, M.A. 2010c Relations of sugar composition and δ13C in phloem sap to growth and physiological performance of Eucalyptus globulus (Labill) Plant Cell Environ. 33 1361 1368

    • Search Google Scholar
    • Export Citation
  • Merchant, A., Wild, B., Richter, A., Bellot, S., Adams, M.A. & Dreyer, E. 2011 Compound-specific differences in 13C of soluble carbohydrates in leaves and phloem of 6-month-old Eucalyptus globulus (Labill) Plant Cell and Environment 34 1599 1608

    • Search Google Scholar
    • Export Citation
  • Noiraud, N., Maurousset, L. & Lemoine, R. 2001a Identification of a mannitol transporter, AgMaT1, in celery phloem Plant Cell 13 695 705

  • Noiraud, N., Maurousset, L. & Lemoine, R. 2001b Transport of polyols in higher plants Plant Physiol. Biochem. 39 717 728

  • Oleary, M.H. 1984 Measurement of the isotope fractionation associated with diffusion of carbon dioxide in aqueous solution J. Phys. Chem. 88 823 825

    • Search Google Scholar
    • Export Citation
  • Pate, J. & Arthur, D. 1998 Delta 13C analysis of phloem sap carbon: Novel means of evaluating seasonal water stress and interpreting carbon isotope signatures of foliage and trunk wood of Eucalyptus globulus Oecologia 117 301 311

    • Search Google Scholar
    • Export Citation
  • Pate, J., Shedley, E., Arthur, D. & Adams, M. 1998 Spatial and temporal variations in phloem sap composition of plantation-grown Eucalyptus globulus Oecologia 117 312 322

    • Search Google Scholar
    • Export Citation
  • Pate, J.S., Sharkey, P.J. & Lewis, O.A.M. 1974 Phloem bleeding form legume fruits—Technique for study of fruit nutrition Planta 120 229 243

  • Paul, M.J. & Cockburn, W. 1989 Pinitol, a compatible solute in Mesembryanthemum crystallinum L? J. Expt. Bot. 40 1093 1098

  • Posternak, T. 1965 The cyclitols. Hermann, Paris, France

  • Pritchard, J. 2007 Solute transport in the phloem. In: Yeo, A.R. and T.J. Flowers (eds.). Plant solute transport. Blackwell Publishing, Oxford, UK

  • Rascher, K.G., Maguas, C. & Werner, C. 2010 On the use of phloem sap δ13C as an indicator of canopy carbon discrimination Tree Physiol. 30 1499 1514

  • Reidel, E.J., Rennie, E.A., Amiard, V., Cheng, L. & Turgeon, R. 2009 Phloem loading strategies in three plant species that transport sugar alcohols Plant Physiol. 149 1601 1608

    • Search Google Scholar
    • Export Citation
  • Richter, A. & Popp, M. 1992 The physiological importance of accumulation of cyclitols in Viscum album L New Phytol. 121 431 438

  • Salmon, Y., Barnard, R.L. & Buchmann, N. 2011 Ontogeny and leaf gas exchange mediate the carbon isotopic signature of herbaceous plants Plant Cell Environ. 34 465 479

    • Search Google Scholar
    • Export Citation
  • Stitt, M., Lunn, J. & Usadel, B. 2010 Arabidopsis and primary photosynthetic metabolism—More than the icing on the cake Plant J. 61 1067 1091

  • Streeter, J.G. 2001 Simple partial purification of D-pinitol from soybean leaves Crop Sci. 41 1985 1987

  • Streeter, J.G., Lohnes, D.G. & Fioritto, R.J. 2001 Patterns of pinitol accumulation in soybean plants and relationships to drought tolerance Plant Cell Environ. 24 429 438

    • Search Google Scholar
    • Export Citation
  • Tausz, M., Merchant, A., Kruse, J., Samsa, G. & Adams, M.A. 2008 Estimation of drought-related limitations to mid-rotation aged plantation grown Eucalyptus globulus by phloem sap analysis For. Ecol. Mgt. 256 844 848

    • Search Google Scholar
    • Export Citation
  • Turgeon, R. 1996 Phloem loading and plasmodesmata Trends Plant Sci. 1 418 423

  • Wanek, W. & Richter, A. 1997 Biosynthesis and accumulation of D-ononitol in Vigna umbellata in response to drought stress Physiol. Plant. 101 416 424

    • Search Google Scholar
    • Export Citation
  • Warren, C.R. 2008 Does growth temperature affect the temperature responses of photosynthesis and internal conductance to CO2? A test with Eucalyptus regnans Tree Physiol. 28 11 19

    • Search Google Scholar
    • Export Citation
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