The extrafloral nectaries of cowpea (Vigna unguiculata (L.) Walp.) II. Nectar composition,origin of nectar solutes,and nectary functioning

Nectar was collected from the extrafloral nectaries of leaf stipels and inflorescence stalks, and phloem sap from cryopunctured fruits of cowpea plants. Daily sugar losses as nectar were equivalent to only 0.1–2% of the plant's current net photosynthate, and were maximal in the fourth week after anthesis. Sucrose:glucose:fructose weight ratios of nectar varied from 1.5:1:1 to 0.5:1:1, whereas over 95% of phloem-sap sugar was sucrose. [¹⁴C]Sucrose fed to leaves was translocated as such to nectaries, where it was partly inverted to [¹⁴C]glucose and [¹⁴C]fructose prior to or during nectar secretion. Invertase (EC 3.2.1.26) activity was demonstrated for inflorescence-stalk nectar but not stipel nectar. The nectar invertase was largely associated with secretory cells that are extruded into the nectar during nectary functioning, and was active only after osmotic disruption of these cells upon dilution of the nectar. The nectar invertase functioned optimally (phloem-sap sucrose as substrate) at pH 5.5, with a starting sucrose concentration of 15% (w/v). Stipel nectar was much lower in amino compounds relative to sugars (0.08–0.17 mg g^-1 total sugar) than inflorescence nectar (22–30 mg g^-1) or phloem sap (81–162 mg g^-1). The two classes of nectar and phloem sap also differed noticeably in their complements of organic acids. Xylem feeding to leaves of a range of ¹⁴C-labelled nitrogenous solutes resulted in these substrates and their metabolic products appearing in fruit-phloem sap and adjacent inflorescence-stalk nectar. ¹⁴C-labelled asparagine, valine and histidine transferred freely into phloem and appeared still largely as such in nectar. ¹⁴C-labelled glycine, serine, arginine and aspartic acid showed limited direct access to phloem and nectar, although labelled metabolic products were transferred and secreted. The ureide allantoin was present in phloem, but absent from both types of nectar. Models of nectary functioning are proposed.     

Expression of a yeast-derived invertase in companion cells results in long-distance transport of a trisaccharide in an apoplastic loader and influences sucrose transport

Companion cell-specific expression of a cytosolic invertase from yeast (Saccharomyces cerevisiae) was used as a tool to synthesise oligosaccharides in the sieve element/companion cell complex and study whether oligosaccharides could be transported in the phloem of an apoplastically loading species. Potato (Solanum tuberosum L.) plants expressing the invertase under the control of the Agrobacterium tumefaciens rolC promoter produced the trisaccharide 6-kestose in leaves, which was transported via the phloem and accumulated in tubers of transgenic plants. In graft experiments with rolC invertase plants as scion and wild-type rootstocks, 6-kestose accumulated in tubers to levels comparable to sucrose. This shows that long-distance transport of oligosaccharides is possible in apoplastically loading plants, which normally transport only sucrose. The additional transport route for assimilates neither led to elevated photosynthetic activity nor to increased tuber yield. Enhanced sucrose turnover in companion cells caused large amounts of glucose and fructose to be exuded from leaf petioles, and elevated levels of sucrose were detected in phloem exudates. While the latter indicates a higher capacity for sucrose loading into the phloem due to increased metabolic activity of companion cells, the massive release of hexoses catalysed by the invertase seemed to interfere with assimilate delivery to sink organs.Abbreviations HPAEC High-performance liquid anion-exchange chromatography - SE–CCC Sieve element/companion cell complex - WT Wild type     

Patterns of Assimilate Production and Translocation in Muskmelon (Cucumis melo L.) : II. Low Temperature Effects

Muskmelon (Cucumis melo L.) plants were exposed to a 10°C chilling treatment for 72 hours, which induced leaf chilling injury symptoms (wilting, appearance of water-soaked areas, necrosis). Chilling caused an accumulation of starch, sucrose, hexoses (glucose and fructose), and certain amino acids (glutamate, aspartate, and citrulline) in source leaf tissues, but no accumulation of stachyose or other galactosyl-oligosaccharides occurred. Chilling also caused a general increase in sugar (stachyose, raffinose, sucrose) and amino acid content of the phloem sap, although rates of phloem transport were apparently reduced. Pretreatment of the leaves with a 20-milligram per liter abscisic acid (ABA) spray before chilling prevented the appearance of chilling injury symptoms. ABA pretreatment had little or no affect on sugar accumulation in leaf tissues but greatly reduced or eliminated the chilling-induced amino acid accumulation. Higher levels of aspartate and particularly of arginine were found in phloem saps from ABA-pretreated plants. The data indicate that changes in leaf metabolism caused by environmental stresses such as chilling may change the composition of cucurbit phloem sap. This raises the possibility that some of the deleterious effects of stress on sink tissues may, in part, be due to alterations in the nature of the assimilate supply.     

Decreased sucrose content triggers starch breakdown and respiration in stored potato tubers (Solanum tuberosum)

To change the hexose-to-sucrose ratio within phloem cells, yeast-derived cytosolic invertase was expressed in transgenic potato (Solanum tuberosum cv. Desirée) plants under control of the rolC promoter. Vascular tissue specific expression of the transgene was verified by histochemical detection of invertase activity in tuber cross-sections. Vegetative growth and tuber yield of transgenic plants was unaltered as compared to wild-type plants. However, the sprout growth of stored tubers was much delayed, indicating impaired phloem-transport of sucrose towards the developing bud. Biochemical analysis of growing tubers revealed that, in contrast to sucrose levels, which rapidly declined in growing invertase-expressing tubers, hexose and starch levels remained unchanged as compared to wild-type controls. During storage, sucrose and starch content declined in wild-type tubers, whereas glucose and fructose levels remained unchanged. A similar response was found in transgenic tubers with the exception that starch degradation was accelerated and fructose levels increased slightly. Furthermore, changes in carbohydrate metabolism were accompanied by an elevated level of phosphorylated intermediates, and a stimulated rate of respiration. Considering that sucrose breakdown was restricted to phloem cells it is concluded that, in response to phloem-associated sucrose depletion or hexose elevation, starch degradation and respiration is triggered in parenchyma cells. To study further whether elevated hexose and/or hexose-phosphates or decreased sucrose levels are responsible for the metabolic changes observed, sucrose content was decreased by tuber-specific expression of a bacterial sucrose isomerase. Sucrose isomerase catalyses the reversible conversion of sucrose into palatinose, which is not further metabolizable by plant cells. Tubers harvested from these plants were found to accumulate high levels of palatinose at the expense of sucrose. In addition, starch content decreased slightly, while hexose levels remained unaltered, compared with the wild-type controls. Similar to low sucrose-containing invertase tubers, respiration and starch breakdown were found to be accelerated during storage in palatinose-accumulating potato tubers. In contrast to invertase transgenics, however, no accumulation of phosphorylated intermediates was observed. Therefore, it is concluded that sucrose depletion rather than increased hexose metabolism triggers reserve mobilization and respiration in stored potato tubers.     

Accumulation of hexoses in leaf vacuoles: Studies with transgenic tobacco plants expressing yeast-derived invertase in the cytosol,vacuole or apoplasm

The subcellular distribution of hexoses, sucrose and amino acids among the stromal, cytosolic and vacuolar compartments was analysed by a nonaqueous fractionation technique in leaves of tobacco (Nicotiana tabaccum L.) wild-type and transgenic plants expressing a yeast-derived invertase in the cytosolic, vacuolar or apoplasmic compartment. In the wild-type plants the amino acids were found to be located in the stroma and in the cytosol, sucrose mainly in the cytosol and up to 98% of the hexoses in the vacuole. In the leaves of the various transformants, where the contents of hexoses were greater than in wild-type plants, again 97–98% of these hexoses were found in the vacuoles. It is concluded that leaf vacuoles contain transporters for the active uptake of glucose and fructose against a high concentration gradient. A comparison of estimated metabolite concentrations in the subcellular compartments of wild-type and transformant plants indicated that the decreased photosynthetic capacity of the transformants is not due to an osmotic effect on photosynthesis, as was shown earlier to be the case in transformed potato leaves, but is the result of a long-term dedifferentiation of tobacco leaf cells to heterotrophic cells.Abbreviations apo-inv tobacco plant with yeast invertase in the apoplasm - Chl chlorophyll - cy-inv tobacco plant with yeast invertase in the cytosol - vac-inv tobacco plant with yeast invertase in the vacuole - WT wild-type tobacco plant The authors thank A. Großpietsch for her able technical assistance. This work has been supported by the Bundesminister für Forschung und Technologie.     

Transport of sucrose, not hexose, in the phloem

Several lines of evidence indicate that glucose and fructose are essentially absent in mobile phloem sap. However, this paradigm has been called into question, especially but not entirely, with respect to species in the Ranunculaceae and Papaveraceae. In the experiments in question, phloem sap was obtained by detaching leaves and placing the cut ends of the petioles in an EDTA solution. More hexose than sucrose was detected. In the present study, these results were confirmed for four species. However, almost identical results were obtained when the leaf blades were removed and only petiole stubs were immersed. This suggests that the sugars in the EDTA solution represent compounds extracted from the petioles, rather than sugars in transit in the phloem. In further experiments, the leaf blades were exposed to (14)CO(2) and, following a chase period, radiolabelled sugars in the petioles and EDTA exudate were identified. Almost all the radiolabel was in the form of [(14)C]sucrose, with little radiolabelled hexose. The data support the long-held contention that sucrose is a ubiquitous transport sugar, but hexoses are essentially absent in the phloem stream.     

Comparison of the contents of sucrose and amino acids in the leaves, phloem sap and taproots of high and low sugar-producing hybrids of sugar beet (Beta vulgaris L.)

The contents of sucrose and amino acids in the leaves, phloemsap and taproots have been analysed in three experimental hybridsof sugar beet and compared with earlier analysed leaf and phloemsap contents in spinach and barley. The three hybrids accumulatedsucrose and amino acids to various extents in the mature rootsas well as in the young taproots (9–12 weeks). The differencesin the sucrose-to-amino acid ratios in the taproots were reflectedin the corresponding ratios in the phloem sap. The leaf contentsof sucrose and amino acids in the three hybrids were found tobe very similar to each other and also to those in spinach andbarley. In contrast, the phloem concentration of sucrose (1.3M) was much higher, and that of amino acids much lower thanin spinach and barley. In the taproots, the overall concentrationof sucrose was about half that in the phloem sap. From thesefindings it is con cluded that the decisive factor in the highsucrose accumulation in sugar beet roots is the very efficientprocess of phloem loading in the leaves. The patterns of theamino acids in the phloem sap and in the taproots resembledthose in the leaves, indicating that there is no special transportform for a-amino nitrogen from the leaves to the roots, butall amino acids which are present in the cytosol are translocated. Key words: Amino acids, Beta vulgaris L., phloem sap, sucrose, tap roots, transport     

Changes in the amino acid composition and protein modification in phloem sap of rice

Pure phloem sap was collected from insects feeding on rice (Oryza sativa L.) leaves by a laser technique similar to the aphid stylet technique. Rapid circulation of nitrogen in the sieve tubes was demonstrated directly using ¹⁵N as a tracer. Application to the roots of the metabolic inhibitors of amino acids, aminooxyacetate and methioninesulfoximine, changed the amino acid composition in the sieve tubes. Feeding methionine to leaf tips resulted in its bulk transfer into the sieve tubes. In vitro experiments confirmed the existence of protein kinases in the pure rice phloem sap. The phosphorylation status of the sieve tube sap proteins was affected by the light regime. The possibility that changes in chemical composition or protein modification such as phosphorylation in the sieve tubes might affect plant growth are discussed.Analysis of pure phloem sap collected from rice plants by insect laser technique has shown dynamic changes in the chemical composition and the quality of proteins in the sap.     

Translocation from leaves to fruits of a legume,studied by a phloem bleeding technique: Diurnal changes and effects of continuous darkness

Summary Diurnal changes in the carbohydrates of leaf laminae and fruits and in the bleeding of sugar and amino acids from fruit phloem were followed by successive sampling from a population of Lupinus albus L. plants. Phloem sap was collected for a standard 5 min period from cut distal tips of attached fruits. Daily fluctuations in leaf dry matter resulted largely from changes in starch and sugar. Leaf sugar rose to a maximum in the afternoon, starch to a maximum at, or shortly after, dusk. Leaves lost sugar and starch from dusk to dawn. Phloem bleeding rate varied little over a daily cycle but sucrose levels fluctuated from a noon maximum of 12–13% (w/v) to a dawn minimum of 9–10%. The rhythm of phloem sugar levels matched closely those of fruit and leaf. Phloem amino acid levels fluctuated in phase with that of sucrose: the relative composition of the amino fraction did not vary significantly over the daily cycle. Pulse feeding of source leaves with ¹⁴CO₂ at different times in the photoperiod allowed study of the pattern of release of labelled photosynthate to the fruit phloem and the build up and depletion of ¹⁴C starch in leaves. Plants transferred to continuous darkness showed a rapid decline in output and concentration of phloem sap solutes, and translocated nitrogen to their fruits at only one quarter of the rate of control plants retained in natural daylight. The combined data from the experiments showed that the rate of output of sugar from cut phloem of a fruit was directly related to the current level of sugar in leaves. When leaf sugar levels were low (5–10 mg ml tissue water^-1) sugar in phloem was 10–11 times more concentrated than in source leaves, but at high leaf sugar levels (25–30 mg ml^-1) this concentration difference was only 3–4 fold.     

Amino Acid and sucrose content determined in the cytosolic, chloroplastic, and vacuolar compartments and in the Phloem sap of spinach leaves

Amino acid and sucrose contents were analyzed in the chloroplastic, cytosolic, and vacuolar compartments and in the phloem sap of illuminated spinach leaves (Spinacia oleracea L.). The determination of subcellular metabolite distribution was carried out by nonaqueous fractionation of frozen and lyophilized leaf material using a novel three-compartment calculation method. The phloem sap was collected by aphid stylets which had been severed by a laser beam. Subcellular analysis revealed that the amino acids found in leaves are located mainly in the chloroplast stroma and in the cytosol, the sum of their concentrations amounting to 151 and 121 millimolar, respectively, whereas the amino acid concentrations in the vacuole are one order of magnitude lower. The amino acid concentrations in the phloem sap are found to be not very different from the cytosolic concentrations, whereas the sieve tube concentration of sucrose is found to be one order of magnitude higher than in the cytosol. It is concluded that the phloem loading results in a preferential extraction of sucrose from the source cells.     

The changes in invertase activity and the content of sugars in the course of adaptation of potato plants to hypothermia

The pattern of changes in the activity of various forms of invertase (acid soluble, alkaline, and acid insoluble) and the content of sugars (glucose, fructose, and sucrose) in the course of plant adaptation to prolonged (6 days) hypothermia (5°C) was investigated in the leaves of potato plants (Solanum tuberosum L., cv. Desiree) produced in vitro. We used the wild-type plants as a control and transformed plants with carbohydrate metabolism modified by inserting the yeast gene for invertase (apoplastic enzyme). In the course of adaptation to hypothermia, the activity of acid invertase was shown to rise and the content of sucrose and glucose to increase in the leaves of both genotypes. The greatest activity of acid invertases by the third day of cold acclimation corresponded to the peak level of sugars; in transformed plants, these characteristics exceeded those in the control plants. The transformed plants were more cold resistant than the control plants as suggested by the lack of disturbance of ion permeability of their membranes. It was concluded that owing to accumulation of low-molecular carbohydrates in the course of cold acclimation caused by activation of acid invertase cold resistant plants better adapt to temperature drop.     

Diurnal changes in assimilate concentrations and fluxes in the phloem of castor bean (Ricinus communis L.) and tansy (Tanacetum vulgare L.)

Reports about diurnal changes of assimilates in phloem sap are controversial. We determined the diurnal changes of sucrose and amino acid concentrations and fluxes in exudates from cut aphid stylets on tansy leaves (Tanacetum vulgare), and sucrose, amino acid and K(+) concentrations and fluxes in bleeding sap of castor bean pedicel (Ricinus communis). Approximately half of the tansy sieve tubes exhibited a diurnal cycle of sucrose concentrations and fluxes in phloem sap. Data from many tansy plants indicated an increased sucrose flux in the phloem during daytime in case of low N-nutrition, not at high N-nutrition. The sucrose concentration in phloem sap of young Ricinus plants changed marginally between day and night, whereas the sucrose flux increased 1.5-fold during daytime (but not in old Ricinus plants). The amino acid concentrations and fluxes in tansy sieve tubes exhibited a similar diurnal cycle as the sucrose concentrations and fluxes, including their dependence on N-nutrition. The amino acid fluxes, but not the concentrations, in phloem sap of Ricinus were higher at daytime. The sucrose/amino acid ratio showed no diurnal cycle neither in tansy nor in Ricinus. The K(+)-concentrations in phloem sap of Ricinus, but not the K(+) fluxes, decreased slightly during daytime and the sucrose/K(+)-ratio increased. In conclusion, a diurnal cycle was observed in sucrose, amino acid and K(+) fluxes, but not necessarily in concentrations of these assimilates. Because of the large variations between different sieve tubes and different plants, the nutrient delivery to sink tissues is not homeostatic over time.     

Transgenic tobacco plants expressing yeast-derived invertase in either the cytosol, vacuole or apoplast: a powerful tool for studying sucrose metabolism and sink/source interactions

In higher plants sucrose plays a central roles with respect to both short-term storage and distribution of photoassimilates formed in the leaf. Sucrose is synthesized in the cytosol, transiently stored in the vacuole and exported via the apoplast. In order to elucidate the role of the different compartments with respect to sucrose metabolism, a yeast-derived invertase was directed into the cytosol and vacuole of transgenic tobacco plants. This was in addition to the targeting of yeast-derived invertase into the apoplast described previously. Vacuolar targeting was achieved by fusing an N-terminal portion (146 amino acids long) of the vacuolar protein patatin to the coding region of the mature invertase protein. Transgenic tobacco plants expressing the yeast-derived invertase in different subcellular compartments displayed dramatic phenotypic differences when compared to wild-type plants. All transgenic plants showed stunted growth accompanied by reduced root formation. Starch and soluble sugars accumulated in leaves indicating that the distribution of sucrose was impaired in all cases. Expression of cytosolic yeast invertase resulted in the accumulation of starch and soluble sugars in both very young (sink) and older (source) leaves. The leaves were curved, indicating a more rapid cell expansion or cell division at the upper side of the leaf. Light-green sectors with reduced photosynthetic activity were evenly distributed over the leaf surface. With the apoplastic and vacuolar invertase, the phenotypical changes induced only appear in older (source) leaves. The development of bleached and/or necrotic sectors was linked to the source state of a leaf. Bleaching followed the sink to source transition, starting at the rim of the leaf and moving to the base. The bleaching was paralleled by the inhibition of photosynthesis.     

Developmental changes in carbohydrate content and sucrose degrading enzymes in tuberising stolons of potato (Solanum tuberosum)

Tuberising stolon tips of potato ( Solanum tuberosum L. cv. Record) accumulate starch and sucrose but the hexose content, particularly fructose, declines rapidly. Similar changes occur in the region 2 cm behind the swelling apex but the decline in glucose is far more pronounced than in the developing tuber. Tuberisation is characterised by an apparent switch from an invertase-dominated sucrolytic system (both acid and alkaline invertases [EC 3.2.1.26] are present) to one dominated by sucrose synthase (EC 2.4.1.13). Sucrose synthase and fructokinase (EC 2.7.1.4) activities were, at a maximum, ca 10- and 5-fold higher, respectively in the swelling stolon tip compared with the non-tuberising region. At the highest starch contents attained, the starch level in the young developing tuber was approximately double that in the adjacent non-tuberising stolon region. Immunoblots revealed that developmental changes in sucrose synthase. fructokinase and alkaline invertase polypeptides corresponded with enzyme activities. Antibodies raised against the N-terminal amino acid sequence of a soluble invertase purified from mature tubers did not detect significant quantities of a polypeptide in stolons and young, developing tubers. Antibodies raised against an in vitro expression product of an apoplastic invertase cloned from a leaf cDNA library detected a polypeptide in developing tubers but not in mature ones. However, expression of the protein did not correlate well with acid invertase activity during early tuber formation.     

Further Studies of the Phloem Loading Process in Leaves of Barley and Spinach. The Comparison of Metabolite Concentrations in the Apoplastic Compartment with those in the Cytosolic Compartment and in the Sieve Tubes1

The concentrations of sucrose, amino acids, nitrate and malate in the apoplastic compartment of illuminated leaves of barley and spinach were determined and compared with the corresponding concentrations in the cytosolic compartment of mesophyll cells and in the phloem sap, as measured previously with plants grown under identical conditions. The concentrations of sucrose and amino acids in the apoplast are found to be much lower than in the cytosol and in the phloem sap, indicating that not only the uptake into the phloem of sucrose, but also of amino acids, requires transport against a concentration gradient. The gradient of sucrose and amino acids between the cytosol and the apoplast was maintained when phloem transport had been blocked by cold girdling. Apparently, the efflux of sucrose and amino acids from the source cells to the apoplast is regulated in such a way that it meets the requirements of phloem transport. The percentages of the single amino acids as part of the total amino acids are quite similar in the cytosol, apoplast and phloem sap. The ratio of sucrose to the total amino acids in the cytosol is similar to that in the apoplast but about five times higher in the phloem sap. It appears from these results that the preferential extraction of sucrose over amino acids from the source cells to the phloem is due to the uptake from the apoplast into the phloem.     

Control of the hexose content of potato tubers

The amounts of glucose and fructose in a range of harvested tubers of Solanum tuberosum were compared with the labelling of these hexoses by [U-¹⁴C]sucrose supplied to the tubers. Hexose content varied. Fructose was more heavily labelled than glucose. There was no correlation between the amounts of glucose and fructose in the tuber and their labelling. The maximum catalytic activities of α-glucan phosphorylase, acid invertase, alkaline invertase, sucrose synthase, α-amylase and β-amylase in tubers stored for 17 weeks at 5° and at 10° were estimated. The values showed no clear correlation with hexose content, but provided sound evidence that starch breakdown was phosphorolytic. It is suggested that the amounts of glucose and fructose in mature harvested tubers may be determined more by the partitioning of the translocated sucrose during the development of the tubers than by the metabolism of the harvested tuber.     

Variation in Eastern cottonwood (Populus deltoides Bartr.) phloem sap content caused by leaf development may affect feeding site selection behavior of the aphid, Chaitophorous populicola Thomas (Homoptera: Aphididae)

Apterous populations of Chaitophorous populicola Thomas (Homoptera: Aphididae) appear to track Eastern cottonwood (Populus deltoides Bartr.) leaf development. Few aphids occur on mature leaves. Marked individual aphids on leaves of different developmental stages were observed through a period of new leaf initiation. Nymph and adult C. populicola frequently track leaf development by moving up to younger leaves. A comparison of phloem sap constituents and leaf toughness among leaf developmental stages revealed some differences that could be used by C. populicola to determine leaf age. Phloem sap exudates, collected from P. deltoides leaves of different developmental stages, were analyzed by high-performance liquid chromatography for free amino acids and the phenolic glycoside salicin. Sucrose concentration in exudates, indicative of phloem sap exudation rate, was uniform among leaf stages. Of 20 amino acids examined, only aspartic acid and gamma-amino-n-butyric acid (GABA) concentrations differed significantly between leaf stages. Forward stepwise discriminant function analysis showed that seven of the amino acids analyzed are useful for classifying leaf maturity groupings. Aphid-infested cottonwoods had lower cystine concentrations in phloem sap than aphid-free plants. Salicin concentration was significantly higher in new leaves. Leaf toughness was assessed by lignin density and distance measurements in petiole cross-sections. Rapidly expanding leaves had significantly less lignification and new leaves had shorter distances to the vascular bundles than senescent leaves. These physiological and phytochemical differences among P. deltoides leaf developmental stages may contribute to the leaf stage selection patterns exhibited by the aphid, C. populicola.     

Phloem loading in peach: Symplastic or apoplastic?

Sorbitol and sucrose are the two main soluble carbohydrates in mature peach leaves. Both are translocated in the phloem, in peach as in other rosaceous trees. The respective role of these two soluble carbohydrates in the leaf carbon budget, and their phloem loading pathway, remain poorly documented. Though many studies have been carried out on the compartmentation and export of sucrose in sucrose-transporting species, far less is known about sorbitol in species transporting both sucrose and sorbitol. Sorbitol and sucrose concentrations were measured in several tissues and in sap, in 2-month-old peach (Prunus persica L. Batsch) seedlings, i.e. leaf blade, leaf main vein, petiole, xylem sap collected using a pressure bomb, and phloem sap collected by aphid stylets. The sorbitol to sucrose molar ratio depended on the tissue or sap, the highest value (about 7) found in the leaf main vein. Sorbitol concentration in the phloem sap was about 560 mM, whereas that of sucrose was about 140 mM. The lowest sorbitol and sucrose concentrations were observed in xylem sap collected from the shoot. The volume of the leaf apoplast, estimated by infiltration with ³H-inulin, represented about 17% of the leaf blade water content. This volume was used to calculate a global intracellular concentration for each carbohydrate in the leaf blade. Following these simplifying assumptions, the calculated concentration gradient between the leaf's intracellular compartment and phloem sap is nil for sorbitol and could thus allow for the symplastic loading of the phloem of this alditol. However, infiltration of ¹⁴C-labelled source leaves with 2 mMp-chloromercuribenzenesulfonic acid (PC-MBS), a potent inhibitor of the sucrose carrier responsible for phloem loading in sucrose-transporting plants, had a significant effect on the exudation of both labelled sucrose and sorbitol from the phloem. Therefore, in peach, which is a putative symplastic loader according to minor vein anatomy and sorbitol concentration gradients, apoplastic loading may predominate.     

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.