Phylogenetic relationships between Mediterranean and middle-Asian wild species of the genus Hordeum L. as revealed by biochemical and molecular markers.

The phylogenetic relationships of 60 accessions of the genus Hordeum (29 Mediterranean and 20 middle-Asian wild accessions, together with nine American accessions and two of unknown origin), representing together nine species, were investigated by AFLP markers. Three hundred sixty six AFLP fragments were used for studying the molecular genetic diversity among the studied species, 339 out of them were polymorphic. Forty seven protein bands were obtained from the water soluble and the water insoluble seed storage protein by SDS-PAGE electrophoresis of 18 accessions representing nine species (two accessions/species). One band was common to all species and the other 46 bands were polymorphic. The phylogenetic tree deduced from AFLP analysis is concordant to a large extent with that deduced from seed storage protein. Highly significant cophenetic correlation coefficient was obtained between both AFLP (0.96) and seed storage protein (0.89) indicating the reliability of the results. The studied taxa were clustered according to their genome type. All Mediterranean and middle-Asian wild accessions could be integrated into the existing phylogenetic scheme.

Carrier-mediated uptake and phloem systemy of a 350-Dalton chlorinated xenobiotic with an alpha-amino acid function.

In a previous paper we have shown that epsilon-(phenoxyalkanecarboxylyl)-L-Lys conjugates are potent inhibitors of amino acid transport systems and that it is possible to modulate the uptake inhibition by hydrophobic or hydrophilic additions in the 4-position of the aromatic ring (J.F. Chollet, C. Delétage, M. Faucher, L. Miginiac, J.L. Bonnemain [1997] Biochem Biophys Acta 1336: 331-341). In this report we demonstrate that epsilon-(2,4-dichlorophenoxyacetyl)-L-Lys (2,4D-Lys), one of the largest molecules of the series and one of the most potent inhibitors, is a highly permeant conjugate. Uptake of 2,4D-Lys by broad bean (Vicia faba) leaf discs is mediated by an active carrier system (Km1 = 0.2 mM; Vmax1 = 2.4 nmol x cm(-2) x h(-1) at pH 5.0) complemented by an important diffusive component. Among the compounds tested (neutral, basic, and acidic amino acids, auxin, glutathione, and sugars), only the aromatic amino acids clearly compete with 2,4D-Lys. The conjugate accumulates in the vein network, is exported toward the growing organs, and exhibits a distribution pattern different from that of the herbicide moiety. However, over time 2,4D-Lys progressively splits into 2,4D and lysine. Analyses by high-performance liquid chromatography and liquid scintillation spectrometry of the phloem sap collected from the castor bean system, used as a systemy test, indicate decreasing capacities of 2,4D, 2,4D-Lys, and glyphosate, respectively, to move from the epidermis cell wall to the sieve element. Our results show that it is possible to design synthesis of large-size xenobiotics (approximately 350 D) with a lipophilic pole, exhibiting high mobility within the vascular system.

23Na and (1)H NMR microimaging of intact plants.

(23)Na NMR microimaging is described to map, for the first time, the sodium distribution in living plants. As an example, the response of 6-day-old seedlings of Ricinus communis to exposure to sodium chloride concentrations from 5 to 300 mM was observed in vivo using (23)Na as well as (1)H NMR microimaging. Experiments were performed at 11.75 T with a double resonant (23)Na-(1)H probehead. The probehead was homebuilt and equipped with a climate chamber. T(1) and T(2) of (23)Na were measured in the cross section of the hypocotyl. Within 85 min (23)Na images with an in-plane resolution of 156 x 156 micrometer were acquired. With this spatial information, the different types of tissue in the hypocotyl can be discerned. The measurement time appears to be short compared to the time scale of sodium uptake and accumulation in the plant so that the kinetics of salt stress can be followed. In conclusion, (23)Na NMR microimaging promises great potential for physiological studies of the consequences of salt stress on the macroscopic level and thus may become a unique tool for characterizing plants with respect to salt tolerance and salt sensitivity.

Assimilate export by leaves of ricinus communis L. growing under normal and elevated carbon dioxide concentrations: the same rate during the day, a different rate at night

Castor bean (Ricinus communis L.) plants were grown for 5-7 weeks in a controlled environment at 350 &mgr;l l(-1) or 700 &mgr;l l(-1) CO(2). Carbon assimilation, assimilate deposition, dark respiration and assimilate mobilization were measured in leaves 2, 3 and 4 (counted from the base of the plant), and a balance sheet of carbon input and export was elaborated for both CO(2) concentrations. Carbon dioxide assimilation was nearly constant over the illumination period, with only a slight depression occurring at the end of the day in mature source leaves, not in young source leaves. Assimilation was ca. 40% higher at 700 &mgr;l l(-1) than at 350 &mgr;l l(-1) CO(2). The source leaves increased steadily in weight per unit area during the first 3 weeks, more at 700 &mgr;l l(-1) than at 350 &mgr;l l(-1) CO(2). On top of an irreversible weight increase, there was a large gain in dry weight during the day, which was reversed during the night. This reversible weight gain was constant over the life time of the leaf and ca. 80% higher at 700 &mgr;l l(-1) than at 350 &mgr;l l(-1). Most of it was due to carbohydrates. The carbon content (as a percentage) was not altered by the CO(2) treatment. Respiration was 25% higher in high-CO(2) plants when based on leaf area, but the same when based on dry weight. The rate of carbon export via the phloem was the same during the daytime in plants grown at 350 &mgr;l l(-1) and 700 &mgr;l l(-1) CO(2). During the night the low-CO(2) plants had only 50% of the daytime export rate, in contrast to the high-CO(2) plants which maintained the high export rate. It was concluded that the phloem loading system is saturated during the daytime in both CO(2) regimes, whereas during the night the assimilate supply is reduced in plants in the normal CO(2) concentration. Two-thirds of the carbon exported from the leaves was permanently incorporated as plant dry matter in the residual plant parts. This "assimilation efficiency" was the same for both CO(2) regimes. It is speculated that under 350 &mgr;l l(-1) CO(2) the growing Ricinus plant operates at sink limitation during the day and at source limitation during the night.

Cloning of the cDNA for glutaredoxin, an abundant sieve-tube exudate protein from Ricinus communis L. and characterisation of the glutathione-dependent thiol-reduction system in sieve tubes.

Sieve-tube exudate protein (STEP) from Ricinus communis L. seedlings consists of a characteristic set of more than 100 different polypeptides, against which a complex antiserum was raised. This antiserum cross-reacted with dominant protein species (molecular weights 10-30 kDa) present in the sieve-tube exudate and, to a lesser extent, with proteins in tissue extracts of Ricinus and a wide range of other plant species. For further elucidation of the nature of individuals STEPs in the sieve tubes the anti-STEP serum was used to screen a cDNA expression library constructed from Ricinus cotyledon mRNA. Two clones that differed in the 3' untranslated region encoded a protein of 11 kDa which showed striking homology to bacterial and eucaryotic glutaredoxin sequences. Glutaredoxin activity was confirmed for the recombinant protein after overexpression in Escherichia coli and characterised in detail in sieve-tube exudate. Michaelis Menten constants (Km) for reduced glutathione and cysteine were 2 mM and 50 microns, respectively. Besides L-cysteine, dehydroascorbate and protein disulphides were also reduced by the activity present in the sieve-tube exudate. Glutathione, which is the obligate donor of reduced thiols for glutaredoxin, was present in sieve-tube sap in millimolar concentrations (up to 3 mM) with a ratio of total to oxidised glutathione of 3:1. It is suggested that glutaredoxin and glutathione in sieve tubes prevent oxidative damage and may be involved in redox regulation of sieve-tube proteins.

Sucrose Accumulation in the Sugarcane Stem Is Regulated by the Difference between the Activities of Soluble Acid Invertase and Sucrose Phosphate Synthase.

To assess the relative importance of morphological and biochemical factors in the regulation of sucrose (Suc) accumulation in the sugarcane (Saccharum spp. hybrids) stem, we investigated morphological and biochemical correlates of Suc accumulation among parents and progeny of a family segregating for differences. In contrast to the parents, no relationship was observed between morphology and the level of Suc accumulation among the progeny. The level and timing of Suc accumulation in the whole stalk and within individual internodes was correlated with the down-regulation of soluble acid invertase (SAI) activity. High SAI activity prevented most, but not all, Suc accumulation. There was a critical threshold of SAI activity above which high concentrations of Suc did not accumulate. This low level of SAI activity was always exceeded in the internodes of the lower-Suc-storing genotypes. However, low activity of SAI was not sufficient by itself to account for the Suc accumulation in the higher-Suc-storing genotypes. Major differences in Suc accumulation among the population were attributed to the difference between activities of SAI and Suc phosphate synthase, provided SAI is below the critical threshold concentration. This result is not unexpected, since the pathway of Suc transport for storage involves Suc hydrolysis and resynthesis.

Correlation-peak imaging.

Identification and quantitation in conventional 1H spectroscopic imaging in vivo is often hampered by the small chemical-shift range. To improve the spectral resolution of spectroscopic imaging, homonuclear two-dimensional correlation spectroscopy has been combined with phase encoding of the spatial dimensions. From the theoretical description of the coherence-transfer signal in the Fourier-transform domain, a comprehensive acquisition and processing strategy is presented that includes optimization of the width and the position of the acquisition windows, matched filtering of the signal envelope, and graphical presentation of the cross peak of interest. The procedure has been applied to image the spatial distribution of the correlation peaks from specific spin systems in the hypocotyl of castor bean (Ricinus communis) seedlings. Despite the overlap of many resonances, correlation-peak imaging made it possible to observe a number of proton resonances, such as those of sucrose, beta-glucose, glutamine/glutamate, lysine, and arginine.

Phloem loading--not metaphysical, only complex: towards a unified model of phloem loading.

Phloem loading comprises the entire pathway of phloem-mobile solutes from their place of generation (or delivery) to the sieve tubes in a sequence of transport steps across or passing by several different cell types. Each of these steps can be classified as symplastic or apoplastic. The detailed anatomical-cytological work in the past ten years made clear that the symplastic continuity from mesophyll to sieve tubes may be very different for different plant species or even in different vein orders. Therefore data from one species are not transferable to another species and a well-rounded picture involving different experimental methods has to be aimed at for each species separately. The information obtained with the Ricinus seedling, where phloem loading and sieve tube sap analysis can be achieved relatively easily, is presented. The analysis of the radioactive labelling of sucrose from the sieve tubes of cotyledons, in which external and intracellular sucrose had been differently labelled, revealed that at sucrose concentrations close to the natural one, 50% of sucrose is loaded directly from the external medium. The other 50% is first taken up by mesophyll and then released for uptake into the sieve tubes. No bundle tissue works as obligate, intermediate sucrose storage. The apoplast therefore definitely serves as a transit reservoir for sucrose destined to be loaded into the sieve tubes. The sieve tube sap contains glycolytic metabolites at concentrations higher than found in the hypocotyl tissue, whereas the corresponding glycolytic enzymes are missing. It is concluded that the enzymes are sequestered in the companion cell or by parietal membrane stacks. Not only the sieve tubes but nearly all cotyledonary cells are equipped with a sucrose-H(+) symporter able to achieve sucrose accumulation and sensitive to inhibition by high salt concentrations or SH reagents. A cDNA clone coding for a sucrose carrier was isolated. It is transcribed at approximately the same level in most organs of the seedling and throughout the germination period. Leaves of adult Ricinus have significantly lower levels of this transcript. Recirculation of excess, phloem-delivered solutes from the sink back to the source is shown not only to be a common feature of long-distance transport, but the only way that an imbalance between supply to and consumption of nutrients in the sink can be adjusted in the source. It is a pathway by which sink activity regulates phloem loading. Non-invasive NMR imaging revealed the flow rates and flow speeds in phloem and xylem in the intact seedling and proved directly the existence of an internal circulating solution flow. A unified model of phloem loading is proposed, based on a pump-and-leak model, where active sucrose carriers (and other carriers) accumulate solutes in the sieve tubes with a concomitant build-up of pressure resulting in mass flow. Plasmodesmata are leaks (as are the transport carriers, too), slowing down the transport rate, but they also serve as diffusion channels for substances which are produced in the neighbouring cell. Therefore, compounds, which are not made in the sieve tubes themselves are translocated together with the bulk solution of sieve tube sap.

Plant histochemistry by correlation peak imaging.

Using a new NMR correlation-peak imaging technique, we were able to investigate noninvasively the spatial distribution of carbohydrates and amino acids in the hypocotyl of castor bean seedlings. In addition to the expected high sucrose concentration in the phloem area of the vascular bundles, we could also observe high levels of sucrose in the cortex parenchyma, but low levels in the pith parenchyma. In contrast, the glucose concentration was found to be lower in the cortex parenchyma than in the pith parenchyma. Glutamine and/or glutamate was detected in the cortex parenchyma and in the vascular bundles. Lysine and arginine were mainly visible in the vascular bundles, whereas valine was observed in the cortex parenchyma, but not in the vascular bundles. Although the physiological significance of these metabolite distribution patterns is not known, they demonstrate the potential of spectroscopic NMR imaging to study noninvasively the physiology and spatial metabolic heterogeneity of living plants.

Gradients of turgor, osmotic pressure, and water potential in the cortex of the hypocotyl of growing ricinus seedlings : effects of the supply of water from the xylem and of solutes from the Phloem.

To evaluate the possible role of solute transport during extension growth, water and solute relations of cortex cells of the growing hypocotyl of 5-day-old castor bean seedlings (Ricinus communis L.) were determined using the cell pressure probe. Because the osmotic pressure of individual cells (pi(i)) was also determined, the water potential (psi) could be evaluated as well at the cell level. In the rapidly growing part of the hypocotyl of well-watered plants, turgor increased from 0.37 megapascal in the outer to 1.04 megapascal in the inner cortex. Thus, there were steep gradients of turgor of up to 0.7 megapascal (7 bar) over a distance of only 470 micrometer. In the more basal and rather mature region, gradients were less pronounced. Because cell turgor approximately pi(i) and psi approximately 0 across the cortex, there were also no gradients of psi across the tissue. Gradients of cell turgor and pi(i) increased when the endosperm was removed from the cotyledons, allowing for a better water supply. They were reduced by increasing the osmotic pressure of the root medium or by cutting off the cotyledons or the entire hook. If the root was excised to interrupt the main source for water, effects became more pronounced. Gradients completely disappeared and turgor fell to 0.3 megapascal in all layers within 1.5 hours. When excised hypocotyls were infiltrated with 0.5 millimolar CaCl(2) solution under pressure via the cut surface, gradients in turgor could be restored or even increased. When turgor was measured in individual cortical cells while pressurizing the xylem, rapid responses were recorded and changes of turgor exceeded that of applied pressure. Gradients could also be reestablished in excised hypocotyls by abrading the cuticle, allowing for a water supply from the wet environment. The steep gradients of turgor and osmotic pressure suggest a considerable supply of osmotic solutes from the phloem to the growing tissue. On the basis of a new theoretical approach, the data are discussed in terms of a coupling between water and solute flows and of a compartmentation of water and solutes, both of which affect water status and extension growth.

The lipid-transfer protein C of Ricinus communis L.: isolation of two cDNA sequences which are strongly and exclusively expressed in cotyledons after germination.

Differential screening of cDNA libraries from Ricinus communis L. cotyledons at the day 4 of germination against those at day 2 led to the isolation of two cDNA sequences which code for type C of the "nonspecific lipid-transfer protein". The two clones are nearly identical in the coding region, which in each case starts with a sequence coding for a signal peptide. In the non-coding regions there is only 40% identity. Both clones are expressed only in the cotyledons and are strongly developmentally regulated, being exclusively expressed after the second day of germination with a maximum at days 4 and 5. No transcripts were found in the hypocotyls, roots or endosperm of seedlings and none in sink and source leaves of mature Ricinus plants.

Phloem loading in Ricinus cotyledons: sucrose pathways via the mesophyll and the apoplasm.

Ricinus communis cv. Carmencita seedlings with their cotyledons incubated in sucrose solution and their hypocotyls cut to induce exudation of phloem sap, constitute a system of sucrose fluxes into and out of the cotyledons. This system was characterized with respect to quasi-steady-state conditions of sucrose uptake and export and then used to investigate the pathways of sucrose during phloem loading. The redistribution of (14)C-labelled internal sucrose between the three "compartments", cotyledons (mesophyll), exudate (sieve tubes) and incubation medium (cell-wall space), was measured in the presence or absence of external nonlabelled sucrose. It was found that mesophyll-derived labelled and external sucrose compete at uptake sites in the apoplasm. On the basis of the specific radioactivity of sucrose which reflects the proportionate intermixture of mesophyll-derived and external sucrose in the three "compartments", it was determined that about 50% of the sucrose exported is loaded directly from the apoplasm, while the other half takes the route via the mesophyll. It was confirmed that mesophyll-derived sucrose is released into the apoplasm, so that the existence of an indirect apoplasmic loading pathway is established. Calculations depending on the concentration gradients of labelled and non-labelled sucrose in the cell-wall space are presented to quantify tentatively the proportions of direct and indirect apoplasmic as well as symplasmic loading.

Solute distribution between vacuole and cytosol of sugarcane suspension cells: Sucrose is not accumulated in the vacuole.

The compartmentation of solutes in suspension cells of Saccharum sp. during different growth phases in batch culture was determined using CuCl2 to permeabilize the plasma membrane of the cells. The efflux of cytosolic and vacuolar pools of sugars, cations and phosphate was monitored, and the efflux data for phosphate were compared and corrected using data from compartmentation analysis of phosphate as determined by (31)P-nuclear magnetic resonance spectroscopy. The results show that sucrose is not accumulated in the vacuoles at any phase of the growth cycle. On the other hand, glucose and fructose are usually accumulated in the vacuole, except at the end of the cell-culture cycle when equal distribution of glucose and fructose between the cytosol and the vacuole is found. Both Na(+) and Mg(2+) are preferentially located in the vacuoles, but follow the same tendency as glucose and fructose with almost complete location in the vacuole in the early culture phases and increasing cytosolic concentration with increasing age of the cell culture. Potassium ions are always clearly accumulated in the cytosol at a concentration of about 80 mM; only about 20% of the cellular K(+) is located inside the vacuole. Cytosolic phosphate is little changed during the cell cycle, whereas the vacuolar phosphate pool changes according to total cellular phosphate. In general there are two different modes of solute compartmentation in sugarcane cells. Some solutes, fructose, glucose, Mg(2+) and Na(+), show high vacuolar compartmentation when the total cellular content of the respective solute is low, whereas in the case of ample supply the cytosolic pools increase. For other solutes, phosphate and K(+), the cytosolic concentration tends to be kept constant, and only excess solute is stored in the vacuole and remobilized under starvation conditions. The behaviour of sucrose is somewhat intermediate and it appears to equilibrate easily between cytosol and vacuole.

Sucrose uptake into vacuoles of sugarcane suspension cells.

Uptake of sucrose into vacuoles of suspension cells of Saccharum sp. (sugarcane) was investigated using a vacuole-isolation method based on osmotic- and pH-dependent lysis of protoplasts. Vacuoles took up sucrose at high rates without the influence of tonoplast energization on sucrose transport. Neither addition of ATP or pyrophosphate nor dissipation of the membrane potential or the pH gradient by ionophores changed uptake rates appreciably. Generation of an ATP-dependent pH gradient across the tonoplast was measured in vacuoles and tonoplast vesicles by fluorescence quenching of quinacrine. No H(+) efflux could be measured by addition of sucrose to energized vacuoles or vesicles so that there was no evidence for a sucrose/H(+) antiport system. Uptake rates of glucose and other sugars were similar to those of sucrose indicating a relatively non-specific sugar uptake into the vacuoles. Sucrose uptake was concentration-dependent, but no clear saturation kinetics were found. Strict dependence on medium pH and inhibition of sucrose transport by p-chloromercuriphenylsulfonic acid (PCMBS) indicate that sucrose uptake into sugarcane vacuoles is a passive, carrier-mediated process.

Sucrose storage in cell suspension cultures of Saccharum sp. (sugarcane) is regulated by a cycle of synthesis and degradation.

We have investigated the regulation of sucrose storage in cell-suspension cultures of sugarcane. When grown in batch culture, sucrose accumulation commences after about 5 d, when the nitrogen supply is exhausted. Sucrose storage is also induced by decreasing the nitrogen supply to cells growing in a chemostat. The measured activity of sucrose-phosphate synthase is high enough to account for the rate of sucrose accumulation, provided precautions are taken to avoid the hydrolysis of UDP during the assay. The cells contained high sucrose-synthase activity but pulsing experiments with [(14)C]glucose and unlabelled fructose indicated that this enzyme did not contribute substantially to the synthesis of sucrose, because the glucosyl and fructosyl moieties of sucrose were equally labelled. Several lines of evidence demonstrate the presence of a cycle in which sucrose is synthesized and degraded simultaneously; sucrosephosphate-synthase activity doubles during the phase when the cells are actively storing sucrose but activity is also high after storage has ceased, or when the sucrose is being remobilised; pulse experiments with [(14)C]fructose also showed that sucrose synthesis occurs not only during the storage phase, but also after storage has stopped and during the rapid mobilisation of sucrose; the cells contain high activities of sucrose synthase and alkaline invertase and these are both at a maximum when sucrose storage is occurring; even during the storage phase. [(14)C]fructose pulses lead to labelling of free glucose which is evidence for rapid synthesis and degradation of sucrose. It is proposed that the rate and extent of sucrose storage is regulated by this cycle of synthesis and degradation. Measurements of enzyme activities and metabolite levels are presented, and it is discussed which factors could contribute to the regulation of these two opposing fluxes and, hence, the rate of net sucrose storage and mobilisation.

Microautoradiographic studies of the role of mesophyll and bundle tissues of the Ricinus cotyledon in sucrose uptake.

The aim of the study was to show which tissues and cell types of the cotyledon of Ricinus communis L. are responsible for uptake of sucrose by H(+)-sucrose symport. The cotyledons were incubated in labelled sucrose for up to 20 min and then the amount of radioactivity in each cell type of the cotyledon was assessed by microautoradiography. It was found that 50% of the label was present in the spongy mesophyll, and 10-15% was in the bundles, the epidermal layers and the palisade parenchyma. The sieve tubes contained only 2-3% of the label. The addition of sucrose to cotyledons depolarized the membrane of spongy-mesophyll cells by 33 mV. Therefore, it was concluded that the previously found H(+)-sucrose symport is at least partly located at the spongy mesophyll. No precursor-like behaviour of the label in mesophyll or bundle-sheath cells was observed in pulse-chase experiments, which indicates a direct uptake of sucrose by the sieve tube-companion cell complex from the apoplast.

Transfer of amino acids and nitrate from the roots into the xylem of Ricinus communis seedlings.

The loading of amino acids and nitrate into the xylem was investigated by collection and analysis of root-pressure exudate from the cut hypocotyl stumps of seedlings of Ricinus communis L. Glutamine was found to be the dominant amino acid in the exudate and also to be the amino acid which is transferred to the xylem most rapidly and accumulated to the greatest extent. The comparison between uptake and xylem loading showed significant differences in specificity between these two transport reactions, indicating a different set of transport systems. Nitrate is transferred to the xylem at a higher relative rate than any amino acid despite the great nitrate-storage capacity of the root system. Thus the supply of nitrate to Ricinus plants leads to enhanced nitrogen allocation to the shoots.

Charge and acidity compensation during proton-sugar symport in Chlorella: The H(+)-ATPase does not fully compensate for the sugar-coupled proton influx.

This study was undertaken in order to demonstrate the extent to which the activity of the plasmalemma H(+)-ATPase compensates for the charge and acidity flow caused by the sugar-proton symport in cells of chlorella vulgaris Beij.. Detailed analysis of H(+) and K(+) fluxes from and into the medium together with measurements of respiration, cytoplasmic pH, and cellular ATP-levels indicate three consecutive phases after the onset of H(+) symport. Phase 1 occurred immediately after addition of sugar, with an uptake of H(+) by the hexoseproton symport and charge compensation by K(+) loss from the cells and, to a smaller degree, by loss of another ion, probably a divalent cation. This phase coincided with strong membrane depolarization. Phase 2 started approximately 5 s after addition of sugar, when the acceleration of the H(+)-ATPase caused a slow-down of the K(+) efflux, a decrease in the cellular ATP level and an increase in respiration. The increased respiration was most probably responsible for a pronounced net acidification of the medium. This phase was inhibited in deuterium oxide. In phase 3, finally, a slow rate of net H(+) uptake and K(+) loss was established for several further minutes, together with a slight depolarization of the membrane. There was hardly any pH change in the cytoplasm, because the cytoplasmic buffering capacity was high enough to stabilize the pH for several minutes despite the net H(+) fluxes. The quantitative participation of the several phases of H(+) and K(+) flow depended on the pH of the medium, the ambient Ca(2+) concentration, and the metabolic fate of the transported sugar. The results indicate that the activity of the H(+)-ATPase never fully compensated for H(+) uptake by the sugar-symport system, because at least 10% of symport-caused charge inflow was compensated for by K(+) efflux. The restoration of pH in the cytoplasm and in the medium was probably achieved by metabolic reactions connected to increased glycolysis and respiration.

Transport of hexoses by the phloem of Ricinus communis L. seedlings.

The sieve-tube sap of Ricinus communis L. seedlings has been analysed to determine whether or not hexoses can be taken up by the phloem. Under natural conditions, i.e. with the endosperm attached to the cotyledons, glucose and fructose occurred only in trace amounts in the sieve-tube sap. Incubation of the cotyledons with hexoses in the concentration range 25-200 mM caused a rapid and substantial uptake of hexoses into the phleom, where they appeared eventually in the sieve-tube sap at the same concentration as in the incubation medium. Phloem loading of glucose, 3-O-methyl-glucose and sorbitol occurred easily, whereas fructose was less well loaded. glucose and to a larger extent fructose were also transformed to sucrose, which was loaded into the phloem. The loading of hexoses into the sieve tubes as observed in the experimental exudation system also occurred in the intact seedling, but transloction in the latter soon came to a standstill, probably because of lack of consumption by the sink tissues. These results indicate that the virtual absence of hexoses in the sievetube sap under in-vivo conditions is not because of the inability of the phloem-loading system to transport the monosaccharides but because of the absence of sufficiently high concentrations in the apoplast.

Sucrose transport into the phloem of Ricinus communis L. seedlings as measured by the analysis of sieve-tube sap.

Careful cutting of the hypocotyl of Ricinus communis L. seedlings led to the exudation of pure sieve-tube sap for 2-3 h. This offered the possibility of testing the phloem-loading system qualitatively and quantitatively by incubating the cotyledons with different solutes of various concentrations to determine whether or not these solutes were loaded into the sieve tubes. The concentration which was achieved by loading and the time course could also be documented. This study concentrated on the loading of sucrose because it is the major naturally translocated sieve-tube compound. The sucrose concentration of sieve-tube sap was approx. 300 mM when the cotyledons were buried in the endosperm. When the cotyledons were excised from the endosperm and incubated in buffer, the sucrose concentration decreased gradually to 80-100 mM. This sucrose level was maintained for several hours by starch breakdown. Incubation of the excised cotyledons in sucrose caused the sucrose concentration in the sieve tubes to rise from 80 to 400 mM, depending on the sucrose concentration in the medium. Thus the sucrose concentration in the sieve tubes could be manipulated over a wide range. The transfer of labelled sucrose to the sieve-tube sap took 10 min; full isotope equilibration was finally reached after 2 h. An increase of K(+) in the medium or in the sieve tubes did not change the sucrose concentration in the sievetube sap. Similarly the experimentally induced change of sucrose concentration in the sieve tubes did not affect the K(+) concentration in the exudate. High concentrations of K(+), however, strongly reduced the flow rate of exudation. Similar results were obtained with Na(+) (data not shown). The minimum translocation speed in the sieve tubes in vivo was calculated from the growth increment of the seedling to be 1.03 m·h(-1), a value, which on average was also obtained for the exudation system with the endosperm attached. This comparison of the in-vivo rate of phloem transport and the exudation rate from cut hypocotyls indicates that sink control of phloem transport in the seedlings of that particular age was small, if there was any at all, and that the results from the experimental exudation system were probably not falsified by removal of the sink tissues.