Planta

Planta (1989)177:336-341

9 Springer-Verlag1989

Transport of hexoses by the phloem of Ricinus communis L. seedlings Jose Kallarackal* and Ewald K o m o r * * Pflanzenphysiologie, Universit/it Bayreuth, Universit/itsstrasse 30, D-8580 Bayreuth, Federal Republic of Germany

Abstract. 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 m M caused a rapid and substantial uptake of hexoses into the phloem, where they appeared eventually in the sieve-tube sap at the same concentration as in the incubation medium. Phloem loading of glucose, 3-O-methylglucose 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 translocation 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. Key words: Hexose (phloem loading) - Phloem loading - Ricinus sink control (phloem) - Starch

mobilization

Introduction

The cotyledons of Ricinus seedlings take up sucrose, which is supplied from the medium, and ac* Present address: Plant Physiology Division, Kerala Forest Research Institute, Peechi 680 653, Trichur, Kerala, India ** To whom correspondence should be addressed

cumulate it in the phloem. Cutting the hypocotyl of the seedling leads to phloem exudation and the collected sap is sieve-tube sap according to all available criteria (Kallarackal et al. 1989). This experimental system allowed the unambiguous test for the ability of the phloem-loading system to transport monosaccharides into the sieve tubes. Analysis of sieve-tube sap from adult Ricinus, Yucca and other plants has shown that generally sucrose is the main carbohydrate translocated; mannitol, raffinose and a few other sugars are only found in some special plants (Zimmermann and Ziegler 1975). There is unanimous agreement that the hexoses glucose and fructose are not translocated in the phloem. The trace amounts, which are found, are blamed on possible contamination of the exudate, for instance by contact with cellwall-bound invertase or with wounded parenchyma cells. Consequently, the absence of appreciable amounts of hexoses in the sieve-tube exudate is even taken as a strong criterion for the purity of the sieve-tube sap and for the validity of the method of exudate collection. However, it is not known, whether the lack of glucose and fructose in the sieve-tube sap is caused by the phloem-loading system which refuses to take up these sugars at significant rates, or whether glucose and fructose just do not occur at the phloem-loading sites because, for instance, they are vigorously taken up by parenchyma and mesophyll cells or because they are instantaneously phosphorylated. Our experimental system, in which Ricinus cotyledons are incubated with solutes and in which the composition of the sieve-tube sap is monitored, allows us to answer the question of whether the sieve tubes are really unable to take up and transport hexoses. Material and methods Plant material. The seeds of Rieinus eommunis L. (cv. gibsonii)

were obtained from Walz, Stuttgart, FRG. The radioactive 14C-

J. Kallarackal and E. Komor: Hexose transport into the phloem sugars were purchased from Amersham-Buchler, Braunschweig, FRG, and Dupont-NEN, Dreieich, FRG. 3-O-Methylglucose was bought from Calbiochem, Frankfurt, F R G and sorbitol from Sigma, M/inchen, FRG.

337

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J 200-

4-= Growth and exudation. The cultivation of Ricinus seedlings was

as described in detail in Kallarackal et al. (1989). Briefly, the following experimental procedure was carried out when the seedlings were 6 d old. The endosperm was carefully removed from the cotyledons, which were then incubated for 2 h in a medium usually of the same composition as that used during the exudation period. Exudation was started by carefully cutting the hypocotyl at the hook. The exudation rate of different seedlings was variable and exudation usually lasted for at least 2h. The exuded sap was collected in 2-gl samples in microcapillaries and the sugars were analysed by enzymatic tests (glucosefructose-sucrose test from Boehringer, Mannheim, FRG). Radioactivity was measured in a dioxane-naphthalene-diphenyloxazol mixture (1000:100:5; by vol.) in a scintillation counter. Chromatography of samples was performed on cellulose thin layers in ethyl acetate-pyridine-water, 100:35:25 (by vol.). Invertase (Serva, Heidelberg, F R G ) was used to hydrolyze the sucrose in the samples.

Results

Hexoses in the sieve-tube sap of cotyledons incubated in sucrose. The contents of glucose and fructose in the sieve-tube sap of seedlings whose cotyledons were either embedded in the endosperm or were incubated in a medium containing sucrose, were very low. Endosperm-attached cotyledons exuded continuously approx. 1.8 m M glucose and approx. 0.6 m M fructose, amounts, which were negligible compared with 300 m M sucrose in the same exudate. Incubation of the cotyledons in 50 m M sucrose resulted in hexose concentrations similar to those obtained in the presence of endosperm, namely 2.5 m M glucose and 0.6 m M fructose. Though sucrose derived from the endosperm or supplied to the medium is partially hydrolyzed by invertase from the cotyledons and probably also by the endosperm, and though hexoses are taken up well by the cotyledons (Kriedemann and Beevers 1967b), there are only traces of glucose and fructose in the sieve-tube sap of Ricinus seedlings. Therefore it appears that the phloem-loading system does not load hexoses into the sieve tubes. Effect of incubation with glucose or 3-O-methylglucose on the sugar composition o f the sieve-tube sap. It is possible that glucose did not show up in the sieve-tube sap because of the levels of hexose in the vicinity of the phloem were too low, so that the phloem-loading system did not have access to glucose. Therefore, cotyledons were incubated for 2 h in a medium containing different concentra-

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Fig. 1. Concentration of sugars in the sieve-tube sap of Ricinus when the cotyledons were incubated in a medium containing different glucose concentrations. The cotyledons of seedlings from which the endosperm had been removed, were !incubated in glucose at the indicated concentrations for 2 h. The hypocotyls were then cut and the exudate was sampled and analyzed for approx. 2 h. The sugar concentrations in the steady state of exudation are shown. 9 Glucose; e, sucrose; zx, fructose

tions of glucose, and the sucrose and hexose contents of the exudate were determined. Surprisingly, under these conditions, substantial concentrations of glucose showed up continuously in the sievetube sap. Incubation in increasing concentrations of glucose resulted in a nearly linearly increasing concentration of glucose in the sieve-tube sap (Fig. 1). At 200 m M glucose in the incubation medium the glucose concentration in the sieve-tube sap became higher than the sucrose concentration. However, in no instance was the sieve-tube concentration of glucose appreciably above that in the medium. Low concentrations of glucose increased the sucrose concentration in the exudate until a plateau was reached and higher glucose concentrations then had no further effect. Appreciable concentrations of fructose were never found in these experiments (Fig. 1). Since incubation with glucose increased the level of sucrose in the exudate, a substantial rate of conversion of glucose to sucrose has to be assumed (unless it is postulated that addition of glucose stimulates the production of sucrose from starch in the cotyledons). Therefore, the time-course of hexose appearance in the sieve tubes is better followed using the non-metabolizable glucose analogue 3-0methylglucose. As was the case with glucose, labelled 3-O-methylglucose readily appeared in the sieve-tube sap at a concentration, which was slightly above that in the medium (Fig. 2). Chromato graphy of the labelled exudate gave no indication of metabolic transformation of 3-O-methylglucose. There was a slight accumulation of 3-O-methylglucose, 1.4-fold at best, but it cannot be excluded

338

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Fig. 2. Time-course of labelling of the sieve-tube sap of Ricinus

by addition of [14C]-3-O-methylglucoseto the incubation medium. The cotyledons of water-cultivated seedlings from which the endosperm had been removed, were incubated for 2 h in medium containing 50 mM 3-O-methylglucose with or without [~4C]-3-O-methylglucose. The hypocotyls were cut to start exudation and [14C]-3-O-methylglueose(4 kBq. ml- i) was simultaneously added to the unlabelled medium. The exudate was sampled in 2-pl portions and counted. The steady state of label exudation is reached at 1100 cpm-(2 pl) ~ by plants, which were permanently incubated in the labelled glucose analogue

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100

glucose

150

200

Fig. 3. Sugar composition of sieve-tube sap of Ricinus during incubation of the cotyledons in different concentrations of fructose. The procedure was the same as that described for in Fig. 1 except that the cotyledons were incubated in [14C]fructose (4 kBq. ml- 1)



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Effect of incubation with fructose or sorbitol on the composition of sieve-tube sap. Since glucose and 3O-methylglucose readily a p p e a r e d in the sieve-tube sap, the same was expected for fructose. H o w e v e r , i n c u b a t i o n o f the cotyledons with low concentrations o f fructose scarcely increased the fructose level in the sieve-tube sap, whereas the sucrose level increased dramatically (Fig. 3), in fact to nearly the same extent as could be achieved by i n c u b a t i o n in sucrose (Kallarackal et al. 1989). At higher concentrations o f fructose in the i n c u b a t i o n medium, e.g. above 50 raM, the sucrose c o n c e n t r a t i o n in the sieve-tube sap did not increase further and the fructose levels increased steadily up to 40 raM, a level, which was h o w e v e r far below the c o n c e n t r a t i o n in the medium. The glucose levels were hardly increased by i n c u b a t i o n in fructose, at m o s t up to 3 mM. A n o t h e r sugar, which we tested, was sorbitol. Sorbitol is interesting in two aspects: (i) it is often used as an o s m o t i c u m because it is supposed to p e r m e a t e weakly, and (ii) it is a natural c o m p o u n d in p h l o e m t r a n s p o r t in some Rosaceae. I n c u b a t i o n o f Ricinus cotyledons with labelled sorbitol led to the r e a d y a p p e a r a n c e o f radioactivity in the sieve-

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frucfose in fhe medium (raM)

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that slight e v a p o r a t i o n f r o m exuded sieve-tube sap (Kallarackal et al. 1989) h a d artificially increased the solute concentration.

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sorbilo[ in medium (raM) Fig. 4. Sugar composition of the sieve-tube of Ricinus sap during incubation of the cotyledons in sorbito]. The experimental procedure was the same as for Fig. I, except that the cotyledons were incubated in [i4C] sorbitol (4 kBq. ml-i)

tube sap. C h r o m a t o g r a p h i c separation o f the labelled exudate showed that at least 85% o f the label was in sorbitol (the rest could not be properly assigned because o f separation problems). Incubation o f the cotyledons in different concentrations o f sorbitol clearly showed the appearance o f sorbitel in the sieve-tube sap at a p p r o x i m a t e l y the same c o n c e n t r a t i o n as in the i n c u b a t i o n m e d i u m (Fig. 4). The sucrose c o n c e n t r a t i o n in the sievetube sap was hardly affected by sorbitol; the glucose c o n c e n t r a t i o n decreased f r o m approx. 1.5 m M to levels below 1 m M . The m o n o s a c c h a r i d e s glucose, fructose, 3 - 0 methylglucose and sorbitol are not only different in their ability to establish themselves at a high c o n c e n t r a t i o n in the sieve-tube sap, they might also use different p a t h w a y s to the sieve tube. 3-O-Methylglucose and sorbitol are not metabolised by Rici-

J. Kallarackal and E. Komor: Hexose transport into the phloem

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Fig. 5. Time-courses of exudation of radioactivity from the sieve tubes of Ricinus during incubation of the cotyledons in [14C]glucose, [14C]fructose or [t4C]sorbitol. Cotyledons of seedlings were incubated in 50 m M of the respective sugar for 2 h, then the hypocotyl was cut and exudation followed. At the time-point of cutting 14C-labelled sugar was added at 4 kBq.ml- 1

nus cotyledons, whereas fructose and to a smaller degree glucose are well incorporated into sucrose. This latter fraction, therefore, must necessarily have entered the symplast before it reached the sieve tubes, whereas sorbitol, 3-O-methylglucose and the non-metabolized fraction of glucose might have gone exclusively through the apoplast. A comparison of the time-courses of label appearance in the sieve-tube sap (Fig. 5) shows that there is hardly any difference in the time lag for radioactivity from fructose, sorbitol, glucose or 3-O-methylglucose (Fig. 2). Either the metabolic conversion of fructose to sucrose is not rate-limiting for phloem loading relative to the transport process itself or all sugars are loaded through the symplast. Sugar transIocation in the intact seedling. Feeding the intact seedling via the cotyledons with labelled sorbitol and analysing the amount of radioactivity in the hypocotyl and roots showed that sorbitol is also translocated into the basal parts of the seedling (Fig. 6). However, with sorbitol as well as with 3-O-methylglucose, the net translocation seemed to come to a standstill after 2 h, whereas sucrose translocation proceeded steadily for at least 5 h. It appeared that the non-metabolized sugars were either not sufficiently unloaded or were recycled continuously because they were not metabolically consumed. The translocation of the non-metabolic sugars sorbitol and 3-O-methylglucose, therefore, seems to be limited by the sink activity, which is relieved by cutting the hypocotyl.

Ricinus by incubation of the cotyledons in radioactive sugars. The cotyledons of seedlings, from which the endosperm had been removed, were incubated in 100 m M 14C-labelled sucrose, 3-O-methylglucose or sorbitol (3 kBq.ml-1). At 1-h intervals seedlings were harvested, washed and separated into cotyledons, hypocotyl and roots. The radioactivity of each of these organs was determined by drying the samples, powdering them, adding protosol (Roth, Karlsruhe, FRG) and, after 24 h, adding scintillation fluid. The scintillation vials were kept at least 1 d in darkness to avoid phosphorescence

Discussion It is well known that glucose and fructose are not normally found in the sieve-tube sap, so that the presence or absence of hexoses in a collected exudate serves as a strong criterion for the source of exudate, e.g. whether from the phloem or from the xylem, and for the purity of the collected sap. In Ricinus seedlings, hexoses were only detected in trace amounts under natural conditions, where the supply of sucrose was derived from the endosperm, though 40 m M hexoses have been measured at the cotyledon-endosperm interface (Kriedemann and Beevers 1967a). Since the mesophyll cells of Ricinus cotyledons vigorously take up hexoses, the probability of phloem loading of fructose and glucose will depend on the ratio between the supply of hexoses to the cells and the metabolic conversion of the hexoses on their way to the " phloemloading site". At concentrations below 50 m M the phloem-loading site obviously does not Obtain sufficient hexoses to load them into the sieve', tubes, whereas non-metabolized sugars, e.g. 3-O-methylglucose are readily loaded even at low concentrations. This clearly shows that the phloem-loading system will take up these sugars into the sieve tubes, if these sugars are supplied. In nature this obviously does not occur. Another aspect is that it is not certain which concentration of hexose is important, that from the apoplast or that from the symplast. If it is that from the apoplast, then the reasoning about competition between diffusion

340

to the loading site and metabolic consumption is valid. If it is that from the symplast then the intracellular hexose concentration should be decisive in the sense that it determines the concentration in the sieve-tube sap. In Ricinus cotyledons the concentration of intracellular hexoses in situ is 15 m M (Kriedemann and Beevers 1967b), i.e. ten-times higher than in the sieve-tube sap, so either they are not the precursors of sieve-tube-sap hexoses, or the symplasmic transport path is strongly selective due to transport catalysts in cells. The levels of glucose and fructose can even be well above those of sucrose under experimental conditions such as illuminating an excised leaf for hours or preventing assimilate export by heat-girdling (Wilson and Lucas 1987). Under these conditions the hexoses should show up in the sieve-tube sap, if their transport path is the same as for sucrose. Why has significant translocation of hexoses not been found previously during feeding experiments (Trip et al. 1965)? Previously, because feeding occurred via the xylem and the hexoses had to travel a long way, they could be filtered out from the xylem stream and probably never reached the phloem-loading site in sufficient quantity. This example emphasizes the advantages of the Ricinus cotyledon system, where the diffusion path to the phloem is relatively short (50 gm) so that really the offered compounds reach the phloem at a defined concentration, thus allowing the genuine transport properties of the phloem to be tested. In this context it is interesting that Wright and Fischer (1981) obtained depolarization of the sieve-tube membrane potential not only by sucrose, but also by hexoses and sorbitol. Furthermore, the capacity of phloem to transport hexoses is interesting because of the fact that the capacity of leaf tissues to take up exogenously supplied sugars has been assigned to a retrieval system (Maynard and Lucas 1982). So far, hexose uptake into leaf discs has seemed to be an excellent example of retrieval, because hexoses were obviously not accumulated into phloem (Fondy and Geiger 1977; Daie and Wilusz 1987). Now, however, at least in the case of Ricinus cotyledons, hexose transport into the sieve tubes can be proven, and it is no longer applicable to argue that a certain process is not directly connected with phloem loading because its kinetic also applies to hexose uptake. The objection could be raised that glucose or 3-O-methylglucose showed up in the sieve-tube sap because of diffusion, in the same way as many xenobiotics do. This argument cannot be fully refuted because the permeation coefficient of phloem for different sugars is not known. However, there are

J. Kallarackal and E. Komor: Hexose transport into the phloem

indications that diffusion is unlikely to be the sole mechanism of hexose uptake into the sieve tubes. The strong discrimination between glucose and sorbitol on the one hand and fructose on the other is unexpected, since membranes usually do not discriminate so decisively between the two hexoses. In addition, the "diffusion" of glucose or sorbitol would have to be very fast to achieve a permanent concentration equilibration in a flow-through system like sieve tubes with a flow speed of 2 cm. min-1. Also, it is not true that everything passes into the sieve tubes of Ricinus, for example 3-hydroxy-5,8,10-pyrenetrisulfonate, calcium ions, nitrate and fructose do not show up in the sieve tubes in concentration equilibrium with the medium (Kallarackal et al. 1989). It is possible that the hexoses are taken up as "false substrates" by ttie sucrose-transport system, but we could not find a clear interaction between the sucrose and the hexose concentrations in the sieve-tube sap in experiments, where glucose and sucrose were offered simultaneously to the cotyledons. Incubation of the cotyledons with solutes and analysis of the composition of the sieve-tube sap exuded from the cut hypocotyls thus provides a valuable system to test the real ability of the phloem to load monosaccharides. This loading cannot necessarily be equated with translocation in the intact plant because cutting the hypocotyl might release the process from sink control. With respect to sucrose, there is evidence that in seedlings sink control is only small (Kallarackal et al. 1989), but in the case of sorbitol and 3-O-methylglucose it is very different. It appears that their flow is halted after some time because of lack of consumption. These sugars might, therefore, be valuable experimentally to elucidate the effects of sink control on phloem transport. This work was generously funded by the Deutsche Forschungsgemeinschaft and A. v. Humboldt-Stiftung. The valuable discussions with Chr. Schobert and G. Orlich are gratefully acknowledged.

References Daie, J., Wilusz, E.J. (1987) Facilitated transport of glucose in isolated phloem segments of celery. Plant Physiol. 85, 711-715 Fondy, B.R., Geiger, D.R. (1977) Sugar selectivity and other characteristics of phloem loading in Beta vulgaris L. Plant Physiol. 59, 750-755 Kallarackal, J., Orlich, G., Schobert, C., Komor, E. (1989) Sucrose transport into the phloem of Ricinus communis L. seedlings as measured by the analysis of sieve-tube sap. Planta 177, 327-335 Kriedemann, P., Beevers, H. (1967a) Sugar uptake and translo-

J. Kallarackal and E. Komor: Hexose transport into the phloem cation in the castor bean seedling. I. Characteristics of transfer in intact and excised seedlings. Plant Physiol. 42, 161-173 Kriedemann, P,, Beevers, H. (1967b) Sugar uptake and translocation in the castor bean seedling. II. Sugar transformations during uptake. Plant Physiol. 42, 174-180 Maynard, J.W., Lucas, W.J. (1982) Sucrose and glucose uptake into Beta vulgaris leaf tissue: a case for general (apoplastic) retrieval systems. Plant Physiol. 70, 1436-1443 Trip, P., Nelson, C.D., Krotkov, G. (1965) Selective and preferential transIocation of t4C-labelled sugars in white ash and lilac. Plant Physiol. 40, 740-747 Wilson, C., Lucas, W.J. (1987) Influence of internal sugar levels

341 on apoplastic retrieval of exogenous sucrose in source leaf tissue. Plant Physiol. 84, 1088-1095 Wright, J.P., Fischer, D.B. (1981) Measurement of the sieve tube membrane potential. Plant Physiol. 67, 845-848 Zimmermann, M.H., Ziegler, H. (1975) List of sugars and sugar alcohols in sieve-tube exudates. In: Encyclopedia of plant physiology, N. S., vol.1 : Transport in plants I, pp. 480-503, Zimmermann, M.H., Milburn, J.A., eds. Springer, Berlin Heidelberg New York

Received 9 March; accepted 5 October 1988

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...
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