Planta (1992)187:388-394
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Sugar synthesis and phloem loading in Coleus blumei leaves Robert Turgeon* and Esther Gowan Section of Plant Biology, Division of Biological Sciences, Cornell University, Ithaca, NY 14853, USA Received 4 November 1991; accepted 5 February 1992 Abstract. Sugar-synthesis and -transport patterns were analyzed in Coleus blumei Benth. leaves to determine where galactinol, raffinose, and stachyose are made and whether phloem loading includes an apoplastic (extracellular) step or occurs entirely within the symplast (plasmodesmata-connected cytoplasm). To clarify the sequence of steps leading to stachyose synthesis, a pulse (15 s) of 14CO2 was given to attached leaves followed by a 5-s to 20-min chase: sucrose was rapidly labeled while galactinol, raffinose and stachyose were labeled more slowly and, within the first few minutes, to approximately the same degree. Leaf tissue was exposed to either 14CO2 or [14C]glucose to identify the sites of synthesis of the different sugars. A 2-min exposure of peeled leaf tissue to [~4C]glucose resulted in preferential labeling of the minor veins, as opposed to the mesophyll; galactinol, raffinose and stachyose were more heavily labeled than sucrose in these preparations. In contrast, when leaf tissue was exposed to 14CO2 for 2 min for preferential labeling of the mesophyll, sucrose was more heavily labeled than galactinol, raffinose or stachyose. We conclude that sucrose is synthesized in mesophyll cells while galactinol, raffinose and stachyose are made in the minorvein phloem. Competition experiments were performed to test the possibility that phloem loading involves monosaccharide uptake from the apoplast. Two saturable monosaccharide carriers were identified, one for glucose, galactose and 3-O-methyl glucose, and the other for fructose. Washing the apoplast of peeled leaf pieces with buffer or saturating levels of 3-O-methyl glucose, after providing a pulse of ~4CO2, did not inhibit vein loading or change the composition of labeled sugars, and less than 0.5% of the assimilated label was recovered in the incubation medium. These and previous results (Turgeon and Gowan, 1991, Plant Physiol. 94, 1244-1249) * To whom correspondence should be addressed Abbreviations: 3-OMG=3-O-methyl glucose; PCMBS=p-chloromercuribenzenesulfonic acid; SE-CCC = sieve-element-companion-cell complex
indicate that the phloem loading pathway in Coleus is probably symplastic. Key words: Coleus - Galactinol- 3-O-methyl glucosePhloem loading - Plasmodesma (transport) - Raffinose - Stachyose
Introduction Sugars of the raffinose series, primarily stachyose, are translocated in a large and taxonomically diverse group of plants (Zimmermann and Ziegler 1975). It now appears that the species that translocate substantial amounts of stachyose, at least those that have been examined closely, have a specialized type of companion cell in the minor veins (Turgeon et al. 1975; Fisher 1986; Gamalei 1989). These specialized cells, first described in Cucurbitapepo (Fischer 1885; Turgeon et al. 1975), differ from other minor-vein companion cells in that they always abut the bundle sheath and are connected to it by very large numbers of plasmodesmata (Turgeon et al. 1975; Turgeon and Beebe 1991). They are referred to as "intermediary cells", following Fischer's term for this cell type "fJbergangszellen" (1885; for discussion, see Turgeon 1989). Plants with intermediary cells include the cucurbits (Turgeon et al. 1975; Schmitz et al. 1987), Coleus blumei (Fisher 1986), and many tropical vines and trees (Gamalei 1989). The most commonly accepted hypothesis of phloem loading is that export sugar is transferred from symplast to apoplast somewhere along the loading route and that this sugar subsequently enters the sieve-element-companion-cell complex (SE-CCC) by cotransport with protons (for review, see Giaquinta 1983). However, the presence of especially numerous plasmodesmata between intermediary cells and the bundle sheath has led to speculation that the loading route is entirely symplastic in species that translocate stachyose (for review, see Turgeon and Beebe 1991). We have postulated that the
R. Turgeon and E. Gowan: Sugar synthesis in Coleus blumei leaves
raffinose and stachyose destined for export from leaves are synthesized in intermediary cells, rather than the mesophyll, and have presented a model in which sugar synthesis is mechanistically linked to phloem loading in these plants (Turgeon and Gowan 1990; Turgeon 1991). Raffinose and stachyose are synthesized by the addition of galactose to sucrose and raffinose, respectively (Kandler 1967). The galactose donor for the synthesis of both oligosaccharides is galactinol, which is synthesized from myoinositol and UDP-galactose by galactinol synthase (Kandler and Hopf 1984), the first committed enzyme in the pathway. Available data indicate that galactinol is made in rnesophyll cells (Madore and Webb 1982; Schmitz and Holthaus 1986; Holthaus and Schmitz 1991). Raffinose and stachyose synthesis has also been detected in the mesophyll of cucurbits (Beitler and Hendrix 1974; Hendrix 1977; Madore and Webb 1982; Madore et al. 1988). However, in a recent study, Holthaus and Schmitz (1991) used antibodies to purified stachyose synthase and by immunolocalization detected the enzyme in intermediary cells. Schmitz and coworkers (Holthaus and Schmitz 1991; Schmitz and Holthaus 1986; Schmitz et al. 1987) postulate that sucrose and galactinol are made in the mesophyll and are the precursors of raffinose and stachyose in intermediary cells. In this report we present evidence that the entire pathway to stachyose, including galactinol synthesis, is located in the minor-vein phloem of Coleus blumei. Our evidence also indicates that the pathway of phloem loading does not include the leaf apoplast. We postulate that sucrose, and possibly other compounds, diffuse into the phloem along an entirely symplastic route from the mesophyll and are used to make galactinol, raffinose and stachyose in intermediary ceils. Material and methods Plant material Coleus blumei Benth. cv. Candidum and tobacco (Nicotiana tabacum L. cv. Maryland Mammoth) plants were grown in a greenhouse in artificial soil as described (Weisberg et al. 1988). Seeds were from our stock and may be obtained from the authors. Green tissue from mature leaves was used in all experiments. Labelin9 with ~4C02. Plants were brought into a laboratory fumehood illuminated by a water-filtered incandescent 1000-W metalhalide lamp providing approx. 800 Ixmol p h o t o n s ' m -2 "s -~ (photosynthetically active radiation) at plant level. One hour later a mature leaf was enclosed in a plastic bag and 14CO2 (1.5 MBq), generated in the barrel of a syringe by addition of excess 80% lactic acid to Na2 14CO3 (6.6" l0 s MBq 9mmol-1), was injected into the bag without delay. After a 15-s exposure to ~4CO2 the bag was removed and, after a chase period (5 s-20 min) in room atmosphere, the leaf was quickly removed and plunged into liquid N2. Analysis of l"~C-labeled sugars. ~4CO2-1abeled, frozen leaves were ground in liquid N2 in a mortar and pestle and extracted at 60~ C in 80% ethanol (v/v). Extracts were centrifuged in a clinical centrifuge to remove leaf solids, taken to dryness under vacuum in a flash-evaporator at 40~ C, redissolved in water, and passed through ion-exchange membranes (Bio-Rex AG50W cation membrane [H § form] and AG1 anion membrane [carbonate form]; Bio-Rad, Richmond, Cal., USA). The eluant was concentrated by flash-evaporation, then by heating to 700 C under a stream of N 2. Excess CaCO 3 was added during extraction, immediately after ion-exchange chro-
389 matography, and during flash-evaporation; this prevented acidification of the extract and hydrolysis of sugars, as demonstrated in control experiments in which chromatographically pure labeled sugars were added to unlabeled extracts. Radioactive compounds were identified by two-dimensional thin-layer chromatography on silica gel GHL plates (Analteeh, Newark, Del., USA), pretreated by dipping in a solution of 0.03 M boric acid in 60% ethanol (v/v), and activated at 95-110~ C for 1 h. Solvent systems and autoradiographic procedures have been described (Turgeon and Gowan 1990). Compounds were identified by co-chromatography with standard sugars (Turgeon and Gowan 1990) and visualized by spraying the plates with vanillin solution (3 g vanillin plus 0.5 ml concentrated H2SO4 in 100 ml absolute ethanol) and heating for 15-30 min at 95-110 ~ C (Touchstone and Dobbins 1983, p. 174), or by comparison with known radi01abeled compounds.
Uptake kinetics. Leaf tissue was abraded with carborundum and discs (5.6 mm diameter) were cut under the surface of 20 mM Mes buffer (2-[N-morpholino]ethanesulfonic acid-NaOH, pH 5.5, plus 2 mM CaC12). Incubations in Mes-buffered, 14C-labeled sugar solutions (0.05-20 mM; 5--40kBq" ml- 1; 2 ml volume) were carried out in 3.5-cm diameter plastic Petri dishes for 1 h. The label in buffer-washed (30 min; 4~ C) leaf discs was measured by scintillation counting (Sun et al. 1988). Autoradioyraphy. In experiments with unpeeled leaf tissue, the upper surface of the leaf was gently abraded with carborundum and discs (5.6 mm in diameter) were cut under the surface of 20-mM Mes buffer. Discs were floated for 30 min, abraded side down, on labeled sugar solution (1 mM in Mes buffer, 30 kBq" ml- a), washed in ice-cold buffer for 30 rain, and flash-frozen in powdered, solid COz. Frozen tissue was lyophilized, pressed flat, and autoradiographed using X-ray film (Hyperfilm-[3max; Amersham Corp., Arlington, III., USA) (Weisberg et al. 1988). In other experiments, the lower epidermis was peeled from small (5-20 mm 2) pieces of leaf tissue; these pieces were incubated with 0.2 ml of labeled sugar solution (0.1 mM in Mes buffer, 60 kBq" m1-1) for varying periods of time, washed in buffer for 15 min, and flash-frozen for autoradiography. In some cases the buffer was ice-cold to prevent redistribution of label (Turgeon and Wimmers 1988) while in others the washes were at room temperature. Peeled leaf tissue was also labeled with t4COz; it was floated, peeled side down, on 2 ml of Mes buffer. The tissue was then exposed to 14CO2 (1.0 MBq) for 5 min and left on roomtemperature buffer, with agitation on a reciprocal shaker (92 trips per min), for a total of 20 or 45 rain (chase period) before flash freezing and autoradiography. In some experiments the buffer contained either mannitol or 3~O-methyl glucose (3-OMG) (100 mM) during the labeling and chase periods.
Results
Sugar synthesis and phloem loading, If raffinose and stachyose are synthesized in intermediary cells by precursors that diffuse from the mesophyll, they should be labeled relatively slowly following administration of 14CO2. Their precursors, on the other hand, should be labeled more quickly. At the earliest time periods following exposure to 14CO2 (20 s - l . 5 m i n , i n c l u d i n g the 15-s exposure), sucrose was heavily labeled (Fig. 1). T h e p r o p o r t i o n o f label in sucrose increased to approx, the 3 - m i n p o i n t a n d t h e n decreased slightly over the next 17 m i n . It was n o t possible to q u a n t i f y galactose precisely because there was some overlap with a n u n k n o w n in o u r solvent systems; however, it is clear t h a t there was little, if any, labeled galactose u n t i l the 2 - m i n time p o i n t a n d it always c o n s t i t u t e d a small p r o p o r t i o n ( < 10%) o f the
390
R. Turgeon and E. Gowan: Sugar synthesis in Coleus blumei leaves
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label at later times as well. Labeled galactinol, raffinose and stachyose were all detected at the 20-s time period but only in trace amounts. The proportion of label in these c o m p o u n d s increased substantially after 1 min and with increasing time stachyose became the predominantly labeled c o m p o u n d o f the three. To determine the time required for phloem loading to begin, leaves were labeled for 15 s as above and frozen at 1-min intervals (including the labeling period). Vein labeling was detected in m a c r o a u t o r a d i o g r a p h s at 3 min and later, but not at the 2-min time point (data not shown). Uptake o f exogenous sugars. With abraded, unpeeled leaf discs and long (30-min) uptake periods, minor veins were distinctly labeled, to approximately the same degree, following application of 14C-labeled-glucose, -fructose, -galactose, -galactinol, -sucrose, -raffinose or -stachyose (data not shown). Little, if any minor-vein labeling occurred when either mannitol or 3 - O M G were applied although there was some labeling of larger veins with these c o m p o u n d s (data not shown). O f course, long uptake periods allow extensive metabolic interconversion (Fondy and Geiger 1977; M a d o r e 1990) and spatial redistribution (Turgeon and Wimmers 1988) of labeled compounds. We therefore conducted very short uptake (15 s-3 min) experiments with 14Clabeled-glucose, - 3 - O M G , -galactinol, and -mannitol using peeled tissue to allow rapid penetration of label (Fig. 2, Table 1). When glucose was applied, label appeared m o s t readily in the veins: no vein label could be seen after 15 s of uptake, but after 30 s it was clear and distinct (Fig. 2A). Vein labeling was also apparent with 3 - O M G but t o o k longer than with glucose: no vein loading could be seen in the autoradiographs at 30 s, but after 1 min a small a m o u n t o f label in some minor veins was apparent (Fig. 2C). Rapid vein labeling was not seen with galactinol or mannitol although the veins were clearly visible when an initial 3-min incubation in these c o m p o u n d s was followed by a 15-min chase at r o o m temperature (Table 1, Fig. 2D). When tobacco, which does not synthesize raffinose oligosaccharides in the
Fig. 2A-DI Autoradiographs of C. blumei leaf tissue. The lower
epidermis was removed and tissue floated on 14C-labeled sugars for different periods of time followed by a wash in ice-cold buffer prior to freezing. White regions are radiolabeled. A Glucose, 30 s. B Glucose, 2 min. C 3-O-methyl glucose, 2 min. D Galactinol, 3 min followed by 15 min in buffer at room temperature. Bars=A 2 mm, x 5.8; B-D 1.5 mm, • 8.2 Table 1. Minor-vein loading in peeled C. blumei leaf tissue as determined by macroautoradiography. Tissue was incubated with x4C-labeled compounds for times indicated and washed for 15 min in ice-cold buffer. For the 18-min period, tissue was incubated for 3 min in labeled compound and for 15 min in buffer at room temperature. 0 =no loading, 1=week but visible loading, 2 = obvious loading but considerable label in the mesophyll, 3 = substantial loading, 4 = almost complete loading with very little label in the mesophyll, ND = not determined Time (min) 0.25 0.5
Glucose Galactose 3-OMG Galactinol
Mannitol
0 2
ND ND
0 0
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0 0
1
3
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leaves, was used in these experiments, [14C]galactinol did not label the minor veins of peeled leaf tissue, even after 15 min at r o o m temperature (data not shown). I f minor veins are adapted for apoplastic phloem loading by having an abundance of carriers for one or more sugars on the plasma m e m b r a n e of the S E - C C C , the uptake of these sugars from solution might be more pronounced in green than in white tissue since the former, but not the latter, loads photosynthate. C o m parisons were made of net influx of radiolabeled sugars into abraded discs f r o m green and white Coleus leaf tissue and green tobacco leaves. A low concentration of sugar (100 pM) was used in all cases to ensure that influx occurred predominately by carrier-mediated, rather than linear, transport (Lucas and Madore 1988). In Coleus there was generally more net influx in white than in green tissue, per unit leaf area, perhaps because it is more compact and has smaller air spaces. Proportionate to the other sugars, slightly more glucose and galactose were taken up by white tissue, otherwise there was little if any difference in the net influx of individual sugars when green and white tissues were compared (Fig. 3). In comparing uptake into tobacco and Coleus leaf tissue, net
391
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Fig. 4. Double-reciprocal plot of [14C]3_O.methy1glucose transport in C. bIumei leaf discs in the presence of unlabeled glucose ([]--[]) or galactose ( e - - e ) at 0.5 mM or in the absence of competing sugar ( 9169
composed of saturable and linear c o m p o n e n t s (Lucas and M a d o r e 1988) (Table 2). N e t influx o f these four c o m p o u n d s was partially sensitive to p-chloromercuribenzenesulfonic acid (PCMBS) to variable degrees (30-60%) in replicate experiments (data not shown), in contrast to sucrose, raffinose and stachyose uptake that is almost completely inhibited by P C M B S (Turgeon and G o w a n 1990). The linear c o m p o n e n t of uptake was inhibited to approximately the same degree as the saturable component. To determine whether these monosaccharides use the same carriers, we analyzed reciprocal plots o f saturable (carrier-mediated) uptake using all combinations of sugars as primary and competetive substrates. The uptake of glucose and 3 - O M G was mutually competetive, as is well known, and galactose competed as well (Fig. 4). Fructose did not compete with any of the other three sugars (data not shown). Metabolism o f exogenous glucose. Rapid and preferential uptake of glucose into the minor veins, as demonstrated above, provided an opportunity to test whether galactinol, raffinose and stachyose are made in the mesophyll or the veins. Peeled leaf tissue was exposed to high-specific-activity [14C]glucose (100 ~tM; 0.7 M B q 9m l - 1 ) for 2 min, washed in ice-cold buffer for 15 min, and frozen in liquid Nz. Label was found in sucrose, galactinol, raffinose, stachyose and glucose. To c o m p a r e sucrose synthesis with that of the raffinose sugars, the amount of label in four compounds (sucrose, galactinol, raffinose and stachyose) was totaled and the p r o p o r t i o n of each expressed as a percentage of this sum (Fig. 5). Most of the label was in galactinol, raffinose and stachyose, rather than sucrose. In contrast, when the same calculations were made on data from l~CO2-exposed leaves at the 2-min time point, the proportion of label in the raffinose sugars was m u c h lower, labeled sucrose being m u c h more in evidence (Fig. 5; data from Fig. 1),
Table 2. Characteristics of net 14C-sugar influx in discs of Coleus blumei leaves. Km and Vm~xvalues are for the saturable component (total minus linear component) and k values are for the linear component Sugar
Glucose Galactose 3M)MG Fructose
Km Vmax (raM) (nmol" cm -2 .h -1)
k (nmol - cm- z -[mM sugar] -1 "h -1)
0.46 0.46 0.29 0.71
1.77 1.28 0.27 1.12
10.1 11.8 6,0 11.0
7O 60 50
40 30 20 21o a.
influx of sucrose was found to be proportionately higher in the former while that of galactinol, raffinose, and stachyose was proportionately lower (Fig. 3). Kinetics ofmonosaccharide uptake. Net influx profiles of glucose, galactose, 3 - O M G and fructose in the concentration range 0.1-20 m M were all biphasic in nature,
o Stach
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Fig. 5. Relative proportions of labeled stachyose, raffinose, galactinol, and sucrose in C. blumei leaf tissue after 2 min of exposure to either [14C]glucose (open bars) or 14CO2 (shadedbars). For each treatment the label in the four compounds was totaled and the amount in each calculated as a percentage. [14C]Glucose preferentially labeled galactinol and stachyose whereas sucrose was labeled predominately by a4CO2
392
Fig. 6A, B. Autoradiography of C. blumei leaf tissue. The lower epidermis was peeled away and the tissue exposed to 14CO2 for 5 min. Following a subsequent 40-min chase period in unlabeled air, the tissue was frozen. White regions are radiolabeled. During the labeling and chase periods the tissue was floated on either 100 mM mannitol (A) or 100 mM 3-O-methyl glucose (B). There is no evident inhibition of vein loading by 3-O-methyl glucose. Bar= 1.5 mm; • 8.8 Washing the apoplast during loading. Precursors to the raffinose sugars could be delivered to the phloem from the mesophyll by an entirely symplastic pathway or by a route that includes transport through the apoplast. To distinguish between these possibilities, vein loading was analyzed in experiments where the apoplast was continually washed with buffer. Peeled or unpeeled leaf tissuc was floated on buffer, with agitation, and the tissue was labeled with ~4CO2, frozen after 20 or 45 min, and subjected to autoradiography (Fig. 6). Although unpeeled tissue was labeled more intensely with ~CO2, the autoradiographic images were the same: vein loading was as apparent with peeled tissue, in which the apoplast was washed with buffer, as with unpeeled tissue. There were no appreciable differences between images obtained at the 20- and 45-min time periods. Vein loading was not inhibited by the addition of 3 - O M G (100 raM) to the buffer (Fig. 6). We considered the possibility that, in these experiments, one or more labeled precursors of the raffinose sugars (for example, glucose) could be eliminated from the apoplast by washing while others (for example, sucrose) were able to reach the phloem via the symplast (Turgeon and Gowan 1990). If this were the case, an autoradiographic image of loading might be obtained even though the washing procedure was effective. However, such a selective loss of precursor(s) would probably lead to a change in the labeling pattern of sugars and this was not found: washing with or without mannitol or 3 - O M G did not change the proportionate composition of labeled sugars in the extract (data not shown). Very little label was lost from the apoplast of peeled tissue during the 45-min wash period: label in the buffer amounted to less than 0.5% of the soluble radioactivity in the tissue, whether the tissue was peeled or unpeeled, or whether mannitol or 3 - O M G were, or were not, included in the buffer (data not shown). Discussion
There are some indications that the pathway of phloem loading in species that translocate raffinose and stachyose is entirely symplastic (Turgeon and Beebe 1991). We have shown that phloem loading in Coleus leaves is insensitive to PCMBS, a widely used inhibitor that
R. Turgeon and E. Gowan: Sugar synthesis in Coleus blumei leaves blocks carrier-mediated uptake of sucrose, raffinose and stachyose from the apoplast (Weisberg et al. 1988; Turgeon and Gowan 1990), and that plasmodesmata joining bundle-sheath and intermediary cells in Cueurbita pepo are open to passage of fluorescent dyes (Turgeon and Hepler 1989). To provide a theoretical basis for further studies we proposed a model of symplastic phloem loading based on the compartmentation of sugar synthesis (Turgeon and Gowan 1990; Turgeon 1991). There are two essential features of the model. The first is that the raffinose and stachyose destined for export are made in the intermediary cell. The second is that the numerous plasmodesmata between the bundle sheath and intermediary cell are slightly smaller than normal so that, while sucrose can diffuse into the intermediary cell, the larger sugars are unable to leak back into the bundle sheath. The intermediary cell would therefore act as a "molecular sizediscrimination trap" for sugar to be exported in the phloem. Several lines of evidence presented here are consistent with this model in that they indicate that raffinose and stachyose destined for export are made in the phloem of Coleus leaf minor veins. First, the labeling of galactinol, raffinose and stachyose is considerably slower than that of sucrose following exposure of the leaf to 14CO2, as would be expected if precursors must migrate to the site of sugar synthesis in another compartment. The label in UDP-glucose, used in the synthesis of sucrose, can be readily transferred to UDP-galactose and galactinol, yet the time course of galactinol labeling is markedly different from that of sucrose labeling. Also, similarities in the labeling patterns of galactinol, raffinose and stachyose are consistent with the hypothesis that these compounds are all made at a site distant from the location of sucrose synthesis. Although labeled galactinol, raffinose and stachyose were detected within 20 s of exposure to 14CO2, vein loading was not apparent in autoradiographs until the 3-min time point. However, autoradiography is relatively insensitive; it will not detect vein loading until the amount of label in the veins exceeds that of the surrounding mesophyll. Since symplastie transport takes place within seconds (Tucker et al. 1989), it is reasonable to expect that a highly labeled precursor could move symplastically from nearby mesophyll cells, or bundle-sheath cells, to the intermediary cells to label galactinol, raffinose and stachyose within the 20-s time frame. It should be noted that, in contrast to our results, Webb and Gorham (1964) and Beitler and Hendrix (1974) reported that stachyose was more readily labeled than sucrose in C. pepo. Our preliminary data from C. pepo (not shown) are similar to those presented here for Coleus. We have no explanation for the discrepancies in the results of these studies. Second, exogenous [14C]glucose, which preferentially enters the minor veins, also labels galactinol, raffinose and stachyose, more strongly than sucrose. This preferential labeling of galactinol, raffinose and stachyose is in marked contrast to the preferential labeling of sucrose by 14CO2 over the same time period, presumably in the mesophyll.
393
R. Turgeon and E. Gowan: Sugar synthesis in Coleus blumei leaves Third, recent immunolocalization studies, conducted at the electron-microscope level, indicate that the first enzyme in the pathway to the raffinose sugars, galactinol synthase, is in the intermediary cells of C. pepo minor veins (Beebe and Turgeon 1991). Unfortunately, this antibody does not recognize galactinol synthase in Coleus sections (our unpublished data). It is also possible that a small amount of galactinol, raffinose and stachyose are made in the mesophyll, as reported by Madore and Webb (1982) and Madore et al. (1988) for cucurbits. However, the results presented here indicate that most galactinol is synthesized in the veins in Coleus leaves. The obvious site of synthesis within the minor veins is the intermediary cells since they are symplastically connected to the sieve elements and, judging from the number of mitochondria, very active metabolically. Further, in larger veins of the leaf, which are probably not loading photoassimilate, intermediary cells are not present. In previous experiments it was demonstrated that sucrose does not enter the phloem of Coleus leaves from the apoplast (Weisberg et al. 1988; Turgeon and Gowan 1990). Is it possible that there are other precursor(s) to galactinol, raffinose and stachyose that pass through the apoplast on their way to the phloem? The evidence here indicates not. When peeled leaf tissue is placed on buffer and agitated after exposure to 14CO2, there is no substantial loss of label to the buffer during phloem loading as would be expected if labeled compounds pass through the free space. Free-space studies such as ours have provided conflicting results in the past because, in unpeeled leaf tissue, access of the washing medium to the apoplast is inefficient (for review, see Turgeon and Beebe 1991). However, in our study, the lower epidermis was removed, allowing rapid equilibration of the apoplast and external medium. Also, in these experiments the tissue was peeled and exposed to the buffer during the entire labeling and loading periods. We therefore conclude that suffusion of the apoplast with buffer was efficient and that a measurable amount of labeled material would probably have been washed from the free space if the loading pathway was even partially apoplastic. Another approach to studying the route of loading is to inhibit uptake from the apoplast. Unfortunately, it was not possible to use PCMBS to block monosaccharide uptake since both the glucose and fructose carriers are relatively insensitive to this inhibitor, as has been shown, for example, by Daie and Wilusz (1987) and Xia and Saglio (1988). However, characterization of the glucose carrier demonstrated that it is competitively inhibited by 3-OMG, as shown often by others in different species. In subsequent experiments we found that a high concentration of 3-OMG (100 m M ; over 300 times the Kin) did not inhibit phloem loading or lead to trapping of labeled glucose in the buffer as would be expected if glucose follows an apoplastic pathway to the phloem. It is interesting that exogenous [x4C]glucose rapidly enters the minor veins in Coleus. Although this finding seems to provide support for an apoplastic loading route, it is important to remember that the behavior of an
exogenous sugar does not necessarily reflect a normal transport pathway of the endogenous compound (for discussion, see Turgeon and Beebe 1991). It is possible that these experiments demonstrate the presence of an active carrier in the veins that functions in the retrieval of glucose leaking into the free space from the phloem. On the other hand, the slow uptake of 3 - O M G into the veins argues against this interpretation: an active carrier should load glucose and 3 - O M G indiscriminately. Another possibility is that there is a passive glucosegalactose-3-OMG carrier on the intermediary cell plasma membrane and that rapid uptake of exogenous glucose into the vein is driven metabolically as it is quickly used in the synthesis of galactinol. The active versus passive nature of the glucose carrier in plants is debated (e.g. Daie and Wilusz 1987). In summary, our experiments indicate that galactinol, raffinose and stachyose in C. blumei are synthesized in the intermediary cells of the phloem from precursors that diffuse to them in the symplast. These data are consistent with the hypothesis that synthesis of export sugars is mechanistically linked to phloem loading in those species that translocate raffinose and stachyose. This research was supported by National Science Foundation Grant DCB-9104159, U.S. Department of Agriculture Competetive Grant 90000854, and Hatch funds. References
Beebe, D.U., Turgeon, R. (1991) Galactinol synthase is sequestered in intermediary cells(companion cells) of Cucurbita leaf phloem. (Abstr.) Plant Physiol. 96, Suppl., 100 Beitler, G.A., Hendrix, J.E. (1974) Stachyose: an early product of photosynthesis in squash leaves. Plant Physiol. 53, 674-676 Daie, D., Wilusz, E.J. (1987) Facilitated transport of glucose in isolated phloem segments of celery. Plant Physiol. 85, 711-715 Fischer, A. (1885) Studien fiber die Siebr6hren der Dicotylenblfitter. Ber. Verh. Sfichs. Akad. Wiss. Leipzig, Math.-Phys. KI. 37, 245-290 Fisher, D.G. (1986) Ultrastructure, plasmodesmatal frequency, and solute concentration in green areas of variegated Coleus blumei Benth. leaves. Planta 169, 141-152 Fondy, B.R., Geiger, D.R. (1977) Sugar selectivity and other characteristics of phloem loading in Beta vulgaris L. Plant Physiol. 59, 953-960 Gamalei, Y. (1989) Structure and function of leaf minor veins in trees and herbs. A taxonomic review. Trees 3, 96-110 Giaquinta, R.T. (1983) Phloem loading of sucrose. Annu. Rev. Plant Physiol. 34, 347-387 Hendrix, J.E. (1977) Phloem loading in squash. Plant Physiol. 60, 567-569 Holthaus, U., Schmitz, K. (1991) Distribution and immunolocalization of stachyose synthase in Cucumis melo L. Planta 185, 479486 Kandler, O. (1967) Biosynthesis of poly- and oligosaccharides during photosynthesis in green plants. In: Harvesting the sun, pp 131-152, San Pietro, A., Greer, F.A., Army, T.J., eds. Academic Press, New York Kandler, O., Hopf, H. (1984) Biosynthesis of oligosaccharides in vascular plants. In: Storage carbohydrates in vascular plants. Distribution, physiology and metabolism, pp 115-131, Lewis, D.H., ed. Cambridge Univ. Press, New York Lucas, W.J., Madore, M.A. (1988) Recent advances in sugar transport. In: The biochemistry of plants. A comprehensive treatise, vol. 14: Carbohydrates, pp 35 84, J. Preiss ed. Academic Press, New York
394 Madore, M.A. (1990) Carbohydrate metabolism in photosynthetic and nonphotosynthetic tissues of variegated leaves of Coleus blumei Benth. Plant Physiol. 93, 617-622 Madore, M.A., Webb, J.A. (1982) Stachyose synthesis in isolated mesophyll cells of Cucurbita pepo. Can. J. Bot. 60, 126-130 Madore, M.A., Mitchell, D.E., Boyd, C.M. (1988) Stachyose synthesis in source leaf tissue of the CAM plant Xerosicyos danguyi H Humb. Plant Physiol. 87, 588-591 Schmitz, K., Holthaus, V. (1986) Are sucrosyl-oligosaccharides synthesized in mesophyll protoplasts of mature leaves of Cucumis melo? Planta 169, 529-535 Schmitz, K., Cuypers, B., Moll, M. (1987) Pathway of assimilate transfer between mesophyll cells and minor veins in leaves of Cucumis melo L. Planta 171, 19-29 Sun, D., Wimmers, L.E., Turgeon, R. (1988) Scintillation counting of l'~C-labeled soluble and insoluble compounds in plant tissue. Anal. Biochem. 169, 424-427 Touchstone, J. C., Dobbins, M. F. (1983) Practice of thin layer chromatography, 2nd edn. Wiley, New York Tucker, J.E., Mauzerall, D., Tucker, E.B. (1989) Symplastic transport of carboxyfluorescein in staminal hairs of Setcreasea purpurea is diffusive and includes loss to the vacuole. Plant Physiol. 90, 1143-1147 Turgeon, R. (1989) The sink-source transition in leaves. Annu. Rev. Plant Physiol Plant Mol. Biol. 40, 119-138 Turgeon, R. (1991) Symplastic phloem loading and the sink-source transition in leaves: a model. In: Recent advances in phloem transport and assimilate compartmentation, pp 18-22, Bon-
R. Turgeon and E. Gowan: Sugar synthesis in Coleus blumei leaves nemain, J.-L., Delrot, S., Lucas, W. J., Dainty, J., eds. Ouest Editions, Nantes, France Turgeon, R., Beebe, D. U. (1991) The evidence for symplastic phloem loading. Plant Physiol. 96, 349-354 Turgeon, R., Gowan, E. (1990) Phloem loading in Coleus blumei in the absence of carrier-mediated uptake of export sugar from the apoplast. Plant Physiol. 94, 1244-1249 Turgeon, R., Hepler, P.K. (1989) Symplastic continuity between mesophyll and companion cells in minor veins of mature Cucurbita pepo L. leaves. Planta 179, 24-31 Turgeon, R., Wimmers, L.E. (1988) Different patterns of vein loading of exogenous [14C]sucrose in leaves of Pisum sativum and Coleus blumei. Plant Physiol. 87, 179-182 Turgeon, R., Webb, J.A., Evert, R.F. (1975) Ultrastructure of minor veins of Cucurbita pepo leaves. Protoplasma 83, 217-232 Webb, J.A., Gorham, P.R. (1964) Translocation of photosynthetically assimilated C x4 in straight-necked squash. Plant Physiol. 39, 663-672 Weisberg, L.A., Wimmers, L.E., Turgeon, R. (1988) Photoassimilate-transport characteristics of nonchlorophyUous and green tissue in variegated leaves of Coleus blumei Benth. Planta 175, 1-8 Xia, J.-H., Saglio P.H. (1988) Characterization of the hexose transport system in maize root tips. Plant Physiol. 88, 1015-1020 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 1 : Phloem transport, pp 480-503, Zimmermann, M.H., Milburn, J.A., eds. Springer, Berlin Heidelberg New York
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Sugar synthesis and phloem loading in Coleus blumei leaves Robert Turgeon* and Esther Gowan Section of Plant Biology, Division of Biological Sciences, Cornell University, Ithaca, NY 14853, USA Received 4 November 1991; accepted 5 February 1992 Abstract. Sugar-synthesis and -transport patterns were analyzed in Coleus blumei Benth. leaves to determine where galactinol, raffinose, and stachyose are made and whether phloem loading includes an apoplastic (extracellular) step or occurs entirely within the symplast (plasmodesmata-connected cytoplasm). To clarify the sequence of steps leading to stachyose synthesis, a pulse (15 s) of 14CO2 was given to attached leaves followed by a 5-s to 20-min chase: sucrose was rapidly labeled while galactinol, raffinose and stachyose were labeled more slowly and, within the first few minutes, to approximately the same degree. Leaf tissue was exposed to either 14CO2 or [14C]glucose to identify the sites of synthesis of the different sugars. A 2-min exposure of peeled leaf tissue to [~4C]glucose resulted in preferential labeling of the minor veins, as opposed to the mesophyll; galactinol, raffinose and stachyose were more heavily labeled than sucrose in these preparations. In contrast, when leaf tissue was exposed to 14CO2 for 2 min for preferential labeling of the mesophyll, sucrose was more heavily labeled than galactinol, raffinose or stachyose. We conclude that sucrose is synthesized in mesophyll cells while galactinol, raffinose and stachyose are made in the minorvein phloem. Competition experiments were performed to test the possibility that phloem loading involves monosaccharide uptake from the apoplast. Two saturable monosaccharide carriers were identified, one for glucose, galactose and 3-O-methyl glucose, and the other for fructose. Washing the apoplast of peeled leaf pieces with buffer or saturating levels of 3-O-methyl glucose, after providing a pulse of ~4CO2, did not inhibit vein loading or change the composition of labeled sugars, and less than 0.5% of the assimilated label was recovered in the incubation medium. These and previous results (Turgeon and Gowan, 1991, Plant Physiol. 94, 1244-1249) * To whom correspondence should be addressed Abbreviations: 3-OMG=3-O-methyl glucose; PCMBS=p-chloromercuribenzenesulfonic acid; SE-CCC = sieve-element-companion-cell complex
indicate that the phloem loading pathway in Coleus is probably symplastic. Key words: Coleus - Galactinol- 3-O-methyl glucosePhloem loading - Plasmodesma (transport) - Raffinose - Stachyose
Introduction Sugars of the raffinose series, primarily stachyose, are translocated in a large and taxonomically diverse group of plants (Zimmermann and Ziegler 1975). It now appears that the species that translocate substantial amounts of stachyose, at least those that have been examined closely, have a specialized type of companion cell in the minor veins (Turgeon et al. 1975; Fisher 1986; Gamalei 1989). These specialized cells, first described in Cucurbitapepo (Fischer 1885; Turgeon et al. 1975), differ from other minor-vein companion cells in that they always abut the bundle sheath and are connected to it by very large numbers of plasmodesmata (Turgeon et al. 1975; Turgeon and Beebe 1991). They are referred to as "intermediary cells", following Fischer's term for this cell type "fJbergangszellen" (1885; for discussion, see Turgeon 1989). Plants with intermediary cells include the cucurbits (Turgeon et al. 1975; Schmitz et al. 1987), Coleus blumei (Fisher 1986), and many tropical vines and trees (Gamalei 1989). The most commonly accepted hypothesis of phloem loading is that export sugar is transferred from symplast to apoplast somewhere along the loading route and that this sugar subsequently enters the sieve-element-companion-cell complex (SE-CCC) by cotransport with protons (for review, see Giaquinta 1983). However, the presence of especially numerous plasmodesmata between intermediary cells and the bundle sheath has led to speculation that the loading route is entirely symplastic in species that translocate stachyose (for review, see Turgeon and Beebe 1991). We have postulated that the
R. Turgeon and E. Gowan: Sugar synthesis in Coleus blumei leaves
raffinose and stachyose destined for export from leaves are synthesized in intermediary cells, rather than the mesophyll, and have presented a model in which sugar synthesis is mechanistically linked to phloem loading in these plants (Turgeon and Gowan 1990; Turgeon 1991). Raffinose and stachyose are synthesized by the addition of galactose to sucrose and raffinose, respectively (Kandler 1967). The galactose donor for the synthesis of both oligosaccharides is galactinol, which is synthesized from myoinositol and UDP-galactose by galactinol synthase (Kandler and Hopf 1984), the first committed enzyme in the pathway. Available data indicate that galactinol is made in rnesophyll cells (Madore and Webb 1982; Schmitz and Holthaus 1986; Holthaus and Schmitz 1991). Raffinose and stachyose synthesis has also been detected in the mesophyll of cucurbits (Beitler and Hendrix 1974; Hendrix 1977; Madore and Webb 1982; Madore et al. 1988). However, in a recent study, Holthaus and Schmitz (1991) used antibodies to purified stachyose synthase and by immunolocalization detected the enzyme in intermediary cells. Schmitz and coworkers (Holthaus and Schmitz 1991; Schmitz and Holthaus 1986; Schmitz et al. 1987) postulate that sucrose and galactinol are made in the mesophyll and are the precursors of raffinose and stachyose in intermediary cells. In this report we present evidence that the entire pathway to stachyose, including galactinol synthesis, is located in the minor-vein phloem of Coleus blumei. Our evidence also indicates that the pathway of phloem loading does not include the leaf apoplast. We postulate that sucrose, and possibly other compounds, diffuse into the phloem along an entirely symplastic route from the mesophyll and are used to make galactinol, raffinose and stachyose in intermediary ceils. Material and methods Plant material Coleus blumei Benth. cv. Candidum and tobacco (Nicotiana tabacum L. cv. Maryland Mammoth) plants were grown in a greenhouse in artificial soil as described (Weisberg et al. 1988). Seeds were from our stock and may be obtained from the authors. Green tissue from mature leaves was used in all experiments. Labelin9 with ~4C02. Plants were brought into a laboratory fumehood illuminated by a water-filtered incandescent 1000-W metalhalide lamp providing approx. 800 Ixmol p h o t o n s ' m -2 "s -~ (photosynthetically active radiation) at plant level. One hour later a mature leaf was enclosed in a plastic bag and 14CO2 (1.5 MBq), generated in the barrel of a syringe by addition of excess 80% lactic acid to Na2 14CO3 (6.6" l0 s MBq 9mmol-1), was injected into the bag without delay. After a 15-s exposure to ~4CO2 the bag was removed and, after a chase period (5 s-20 min) in room atmosphere, the leaf was quickly removed and plunged into liquid N2. Analysis of l"~C-labeled sugars. ~4CO2-1abeled, frozen leaves were ground in liquid N2 in a mortar and pestle and extracted at 60~ C in 80% ethanol (v/v). Extracts were centrifuged in a clinical centrifuge to remove leaf solids, taken to dryness under vacuum in a flash-evaporator at 40~ C, redissolved in water, and passed through ion-exchange membranes (Bio-Rex AG50W cation membrane [H § form] and AG1 anion membrane [carbonate form]; Bio-Rad, Richmond, Cal., USA). The eluant was concentrated by flash-evaporation, then by heating to 700 C under a stream of N 2. Excess CaCO 3 was added during extraction, immediately after ion-exchange chro-
389 matography, and during flash-evaporation; this prevented acidification of the extract and hydrolysis of sugars, as demonstrated in control experiments in which chromatographically pure labeled sugars were added to unlabeled extracts. Radioactive compounds were identified by two-dimensional thin-layer chromatography on silica gel GHL plates (Analteeh, Newark, Del., USA), pretreated by dipping in a solution of 0.03 M boric acid in 60% ethanol (v/v), and activated at 95-110~ C for 1 h. Solvent systems and autoradiographic procedures have been described (Turgeon and Gowan 1990). Compounds were identified by co-chromatography with standard sugars (Turgeon and Gowan 1990) and visualized by spraying the plates with vanillin solution (3 g vanillin plus 0.5 ml concentrated H2SO4 in 100 ml absolute ethanol) and heating for 15-30 min at 95-110 ~ C (Touchstone and Dobbins 1983, p. 174), or by comparison with known radi01abeled compounds.
Uptake kinetics. Leaf tissue was abraded with carborundum and discs (5.6 mm diameter) were cut under the surface of 20 mM Mes buffer (2-[N-morpholino]ethanesulfonic acid-NaOH, pH 5.5, plus 2 mM CaC12). Incubations in Mes-buffered, 14C-labeled sugar solutions (0.05-20 mM; 5--40kBq" ml- 1; 2 ml volume) were carried out in 3.5-cm diameter plastic Petri dishes for 1 h. The label in buffer-washed (30 min; 4~ C) leaf discs was measured by scintillation counting (Sun et al. 1988). Autoradioyraphy. In experiments with unpeeled leaf tissue, the upper surface of the leaf was gently abraded with carborundum and discs (5.6 mm in diameter) were cut under the surface of 20-mM Mes buffer. Discs were floated for 30 min, abraded side down, on labeled sugar solution (1 mM in Mes buffer, 30 kBq" ml- a), washed in ice-cold buffer for 30 rain, and flash-frozen in powdered, solid COz. Frozen tissue was lyophilized, pressed flat, and autoradiographed using X-ray film (Hyperfilm-[3max; Amersham Corp., Arlington, III., USA) (Weisberg et al. 1988). In other experiments, the lower epidermis was peeled from small (5-20 mm 2) pieces of leaf tissue; these pieces were incubated with 0.2 ml of labeled sugar solution (0.1 mM in Mes buffer, 60 kBq" m1-1) for varying periods of time, washed in buffer for 15 min, and flash-frozen for autoradiography. In some cases the buffer was ice-cold to prevent redistribution of label (Turgeon and Wimmers 1988) while in others the washes were at room temperature. Peeled leaf tissue was also labeled with t4COz; it was floated, peeled side down, on 2 ml of Mes buffer. The tissue was then exposed to 14CO2 (1.0 MBq) for 5 min and left on roomtemperature buffer, with agitation on a reciprocal shaker (92 trips per min), for a total of 20 or 45 rain (chase period) before flash freezing and autoradiography. In some experiments the buffer contained either mannitol or 3~O-methyl glucose (3-OMG) (100 mM) during the labeling and chase periods.
Results
Sugar synthesis and phloem loading, If raffinose and stachyose are synthesized in intermediary cells by precursors that diffuse from the mesophyll, they should be labeled relatively slowly following administration of 14CO2. Their precursors, on the other hand, should be labeled more quickly. At the earliest time periods following exposure to 14CO2 (20 s - l . 5 m i n , i n c l u d i n g the 15-s exposure), sucrose was heavily labeled (Fig. 1). T h e p r o p o r t i o n o f label in sucrose increased to approx, the 3 - m i n p o i n t a n d t h e n decreased slightly over the next 17 m i n . It was n o t possible to q u a n t i f y galactose precisely because there was some overlap with a n u n k n o w n in o u r solvent systems; however, it is clear t h a t there was little, if any, labeled galactose u n t i l the 2 - m i n time p o i n t a n d it always c o n s t i t u t e d a small p r o p o r t i o n ( < 10%) o f the
390
R. Turgeon and E. Gowan: Sugar synthesis in Coleus blumei leaves
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label at later times as well. Labeled galactinol, raffinose and stachyose were all detected at the 20-s time period but only in trace amounts. The proportion of label in these c o m p o u n d s increased substantially after 1 min and with increasing time stachyose became the predominantly labeled c o m p o u n d o f the three. To determine the time required for phloem loading to begin, leaves were labeled for 15 s as above and frozen at 1-min intervals (including the labeling period). Vein labeling was detected in m a c r o a u t o r a d i o g r a p h s at 3 min and later, but not at the 2-min time point (data not shown). Uptake o f exogenous sugars. With abraded, unpeeled leaf discs and long (30-min) uptake periods, minor veins were distinctly labeled, to approximately the same degree, following application of 14C-labeled-glucose, -fructose, -galactose, -galactinol, -sucrose, -raffinose or -stachyose (data not shown). Little, if any minor-vein labeling occurred when either mannitol or 3 - O M G were applied although there was some labeling of larger veins with these c o m p o u n d s (data not shown). O f course, long uptake periods allow extensive metabolic interconversion (Fondy and Geiger 1977; M a d o r e 1990) and spatial redistribution (Turgeon and Wimmers 1988) of labeled compounds. We therefore conducted very short uptake (15 s-3 min) experiments with 14Clabeled-glucose, - 3 - O M G , -galactinol, and -mannitol using peeled tissue to allow rapid penetration of label (Fig. 2, Table 1). When glucose was applied, label appeared m o s t readily in the veins: no vein label could be seen after 15 s of uptake, but after 30 s it was clear and distinct (Fig. 2A). Vein labeling was also apparent with 3 - O M G but t o o k longer than with glucose: no vein loading could be seen in the autoradiographs at 30 s, but after 1 min a small a m o u n t o f label in some minor veins was apparent (Fig. 2C). Rapid vein labeling was not seen with galactinol or mannitol although the veins were clearly visible when an initial 3-min incubation in these c o m p o u n d s was followed by a 15-min chase at r o o m temperature (Table 1, Fig. 2D). When tobacco, which does not synthesize raffinose oligosaccharides in the
Fig. 2A-DI Autoradiographs of C. blumei leaf tissue. The lower
epidermis was removed and tissue floated on 14C-labeled sugars for different periods of time followed by a wash in ice-cold buffer prior to freezing. White regions are radiolabeled. A Glucose, 30 s. B Glucose, 2 min. C 3-O-methyl glucose, 2 min. D Galactinol, 3 min followed by 15 min in buffer at room temperature. Bars=A 2 mm, x 5.8; B-D 1.5 mm, • 8.2 Table 1. Minor-vein loading in peeled C. blumei leaf tissue as determined by macroautoradiography. Tissue was incubated with x4C-labeled compounds for times indicated and washed for 15 min in ice-cold buffer. For the 18-min period, tissue was incubated for 3 min in labeled compound and for 15 min in buffer at room temperature. 0 =no loading, 1=week but visible loading, 2 = obvious loading but considerable label in the mesophyll, 3 = substantial loading, 4 = almost complete loading with very little label in the mesophyll, ND = not determined Time (min) 0.25 0.5
Glucose Galactose 3-OMG Galactinol
Mannitol
0 2
ND ND
0 0
ND ND
0 0
1
3
ND
1
0
0
2 3 18
4 4 4
4 4 4
2 3 4
ND ND 4
0 0 3
leaves, was used in these experiments, [14C]galactinol did not label the minor veins of peeled leaf tissue, even after 15 min at r o o m temperature (data not shown). I f minor veins are adapted for apoplastic phloem loading by having an abundance of carriers for one or more sugars on the plasma m e m b r a n e of the S E - C C C , the uptake of these sugars from solution might be more pronounced in green than in white tissue since the former, but not the latter, loads photosynthate. C o m parisons were made of net influx of radiolabeled sugars into abraded discs f r o m green and white Coleus leaf tissue and green tobacco leaves. A low concentration of sugar (100 pM) was used in all cases to ensure that influx occurred predominately by carrier-mediated, rather than linear, transport (Lucas and Madore 1988). In Coleus there was generally more net influx in white than in green tissue, per unit leaf area, perhaps because it is more compact and has smaller air spaces. Proportionate to the other sugars, slightly more glucose and galactose were taken up by white tissue, otherwise there was little if any difference in the net influx of individual sugars when green and white tissues were compared (Fig. 3). In comparing uptake into tobacco and Coleus leaf tissue, net
391
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3.0
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Fig. 4. Double-reciprocal plot of [14C]3_O.methy1glucose transport in C. bIumei leaf discs in the presence of unlabeled glucose ([]--[]) or galactose ( e - - e ) at 0.5 mM or in the absence of competing sugar ( 9169
composed of saturable and linear c o m p o n e n t s (Lucas and M a d o r e 1988) (Table 2). N e t influx o f these four c o m p o u n d s was partially sensitive to p-chloromercuribenzenesulfonic acid (PCMBS) to variable degrees (30-60%) in replicate experiments (data not shown), in contrast to sucrose, raffinose and stachyose uptake that is almost completely inhibited by P C M B S (Turgeon and G o w a n 1990). The linear c o m p o n e n t of uptake was inhibited to approximately the same degree as the saturable component. To determine whether these monosaccharides use the same carriers, we analyzed reciprocal plots o f saturable (carrier-mediated) uptake using all combinations of sugars as primary and competetive substrates. The uptake of glucose and 3 - O M G was mutually competetive, as is well known, and galactose competed as well (Fig. 4). Fructose did not compete with any of the other three sugars (data not shown). Metabolism o f exogenous glucose. Rapid and preferential uptake of glucose into the minor veins, as demonstrated above, provided an opportunity to test whether galactinol, raffinose and stachyose are made in the mesophyll or the veins. Peeled leaf tissue was exposed to high-specific-activity [14C]glucose (100 ~tM; 0.7 M B q 9m l - 1 ) for 2 min, washed in ice-cold buffer for 15 min, and frozen in liquid Nz. Label was found in sucrose, galactinol, raffinose, stachyose and glucose. To c o m p a r e sucrose synthesis with that of the raffinose sugars, the amount of label in four compounds (sucrose, galactinol, raffinose and stachyose) was totaled and the p r o p o r t i o n of each expressed as a percentage of this sum (Fig. 5). Most of the label was in galactinol, raffinose and stachyose, rather than sucrose. In contrast, when the same calculations were made on data from l~CO2-exposed leaves at the 2-min time point, the proportion of label in the raffinose sugars was m u c h lower, labeled sucrose being m u c h more in evidence (Fig. 5; data from Fig. 1),
Table 2. Characteristics of net 14C-sugar influx in discs of Coleus blumei leaves. Km and Vm~xvalues are for the saturable component (total minus linear component) and k values are for the linear component Sugar
Glucose Galactose 3M)MG Fructose
Km Vmax (raM) (nmol" cm -2 .h -1)
k (nmol - cm- z -[mM sugar] -1 "h -1)
0.46 0.46 0.29 0.71
1.77 1.28 0.27 1.12
10.1 11.8 6,0 11.0
7O 60 50
40 30 20 21o a.
influx of sucrose was found to be proportionately higher in the former while that of galactinol, raffinose, and stachyose was proportionately lower (Fig. 3). Kinetics ofmonosaccharide uptake. Net influx profiles of glucose, galactose, 3 - O M G and fructose in the concentration range 0.1-20 m M were all biphasic in nature,
o Stach
Ratf
Galol
Suc
Fig. 5. Relative proportions of labeled stachyose, raffinose, galactinol, and sucrose in C. blumei leaf tissue after 2 min of exposure to either [14C]glucose (open bars) or 14CO2 (shadedbars). For each treatment the label in the four compounds was totaled and the amount in each calculated as a percentage. [14C]Glucose preferentially labeled galactinol and stachyose whereas sucrose was labeled predominately by a4CO2
392
Fig. 6A, B. Autoradiography of C. blumei leaf tissue. The lower epidermis was peeled away and the tissue exposed to 14CO2 for 5 min. Following a subsequent 40-min chase period in unlabeled air, the tissue was frozen. White regions are radiolabeled. During the labeling and chase periods the tissue was floated on either 100 mM mannitol (A) or 100 mM 3-O-methyl glucose (B). There is no evident inhibition of vein loading by 3-O-methyl glucose. Bar= 1.5 mm; • 8.8 Washing the apoplast during loading. Precursors to the raffinose sugars could be delivered to the phloem from the mesophyll by an entirely symplastic pathway or by a route that includes transport through the apoplast. To distinguish between these possibilities, vein loading was analyzed in experiments where the apoplast was continually washed with buffer. Peeled or unpeeled leaf tissuc was floated on buffer, with agitation, and the tissue was labeled with ~4CO2, frozen after 20 or 45 min, and subjected to autoradiography (Fig. 6). Although unpeeled tissue was labeled more intensely with ~CO2, the autoradiographic images were the same: vein loading was as apparent with peeled tissue, in which the apoplast was washed with buffer, as with unpeeled tissue. There were no appreciable differences between images obtained at the 20- and 45-min time periods. Vein loading was not inhibited by the addition of 3 - O M G (100 raM) to the buffer (Fig. 6). We considered the possibility that, in these experiments, one or more labeled precursors of the raffinose sugars (for example, glucose) could be eliminated from the apoplast by washing while others (for example, sucrose) were able to reach the phloem via the symplast (Turgeon and Gowan 1990). If this were the case, an autoradiographic image of loading might be obtained even though the washing procedure was effective. However, such a selective loss of precursor(s) would probably lead to a change in the labeling pattern of sugars and this was not found: washing with or without mannitol or 3 - O M G did not change the proportionate composition of labeled sugars in the extract (data not shown). Very little label was lost from the apoplast of peeled tissue during the 45-min wash period: label in the buffer amounted to less than 0.5% of the soluble radioactivity in the tissue, whether the tissue was peeled or unpeeled, or whether mannitol or 3 - O M G were, or were not, included in the buffer (data not shown). Discussion
There are some indications that the pathway of phloem loading in species that translocate raffinose and stachyose is entirely symplastic (Turgeon and Beebe 1991). We have shown that phloem loading in Coleus leaves is insensitive to PCMBS, a widely used inhibitor that
R. Turgeon and E. Gowan: Sugar synthesis in Coleus blumei leaves blocks carrier-mediated uptake of sucrose, raffinose and stachyose from the apoplast (Weisberg et al. 1988; Turgeon and Gowan 1990), and that plasmodesmata joining bundle-sheath and intermediary cells in Cueurbita pepo are open to passage of fluorescent dyes (Turgeon and Hepler 1989). To provide a theoretical basis for further studies we proposed a model of symplastic phloem loading based on the compartmentation of sugar synthesis (Turgeon and Gowan 1990; Turgeon 1991). There are two essential features of the model. The first is that the raffinose and stachyose destined for export are made in the intermediary cell. The second is that the numerous plasmodesmata between the bundle sheath and intermediary cell are slightly smaller than normal so that, while sucrose can diffuse into the intermediary cell, the larger sugars are unable to leak back into the bundle sheath. The intermediary cell would therefore act as a "molecular sizediscrimination trap" for sugar to be exported in the phloem. Several lines of evidence presented here are consistent with this model in that they indicate that raffinose and stachyose destined for export are made in the phloem of Coleus leaf minor veins. First, the labeling of galactinol, raffinose and stachyose is considerably slower than that of sucrose following exposure of the leaf to 14CO2, as would be expected if precursors must migrate to the site of sugar synthesis in another compartment. The label in UDP-glucose, used in the synthesis of sucrose, can be readily transferred to UDP-galactose and galactinol, yet the time course of galactinol labeling is markedly different from that of sucrose labeling. Also, similarities in the labeling patterns of galactinol, raffinose and stachyose are consistent with the hypothesis that these compounds are all made at a site distant from the location of sucrose synthesis. Although labeled galactinol, raffinose and stachyose were detected within 20 s of exposure to 14CO2, vein loading was not apparent in autoradiographs until the 3-min time point. However, autoradiography is relatively insensitive; it will not detect vein loading until the amount of label in the veins exceeds that of the surrounding mesophyll. Since symplastie transport takes place within seconds (Tucker et al. 1989), it is reasonable to expect that a highly labeled precursor could move symplastically from nearby mesophyll cells, or bundle-sheath cells, to the intermediary cells to label galactinol, raffinose and stachyose within the 20-s time frame. It should be noted that, in contrast to our results, Webb and Gorham (1964) and Beitler and Hendrix (1974) reported that stachyose was more readily labeled than sucrose in C. pepo. Our preliminary data from C. pepo (not shown) are similar to those presented here for Coleus. We have no explanation for the discrepancies in the results of these studies. Second, exogenous [14C]glucose, which preferentially enters the minor veins, also labels galactinol, raffinose and stachyose, more strongly than sucrose. This preferential labeling of galactinol, raffinose and stachyose is in marked contrast to the preferential labeling of sucrose by 14CO2 over the same time period, presumably in the mesophyll.
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R. Turgeon and E. Gowan: Sugar synthesis in Coleus blumei leaves Third, recent immunolocalization studies, conducted at the electron-microscope level, indicate that the first enzyme in the pathway to the raffinose sugars, galactinol synthase, is in the intermediary cells of C. pepo minor veins (Beebe and Turgeon 1991). Unfortunately, this antibody does not recognize galactinol synthase in Coleus sections (our unpublished data). It is also possible that a small amount of galactinol, raffinose and stachyose are made in the mesophyll, as reported by Madore and Webb (1982) and Madore et al. (1988) for cucurbits. However, the results presented here indicate that most galactinol is synthesized in the veins in Coleus leaves. The obvious site of synthesis within the minor veins is the intermediary cells since they are symplastically connected to the sieve elements and, judging from the number of mitochondria, very active metabolically. Further, in larger veins of the leaf, which are probably not loading photoassimilate, intermediary cells are not present. In previous experiments it was demonstrated that sucrose does not enter the phloem of Coleus leaves from the apoplast (Weisberg et al. 1988; Turgeon and Gowan 1990). Is it possible that there are other precursor(s) to galactinol, raffinose and stachyose that pass through the apoplast on their way to the phloem? The evidence here indicates not. When peeled leaf tissue is placed on buffer and agitated after exposure to 14CO2, there is no substantial loss of label to the buffer during phloem loading as would be expected if labeled compounds pass through the free space. Free-space studies such as ours have provided conflicting results in the past because, in unpeeled leaf tissue, access of the washing medium to the apoplast is inefficient (for review, see Turgeon and Beebe 1991). However, in our study, the lower epidermis was removed, allowing rapid equilibration of the apoplast and external medium. Also, in these experiments the tissue was peeled and exposed to the buffer during the entire labeling and loading periods. We therefore conclude that suffusion of the apoplast with buffer was efficient and that a measurable amount of labeled material would probably have been washed from the free space if the loading pathway was even partially apoplastic. Another approach to studying the route of loading is to inhibit uptake from the apoplast. Unfortunately, it was not possible to use PCMBS to block monosaccharide uptake since both the glucose and fructose carriers are relatively insensitive to this inhibitor, as has been shown, for example, by Daie and Wilusz (1987) and Xia and Saglio (1988). However, characterization of the glucose carrier demonstrated that it is competitively inhibited by 3-OMG, as shown often by others in different species. In subsequent experiments we found that a high concentration of 3-OMG (100 m M ; over 300 times the Kin) did not inhibit phloem loading or lead to trapping of labeled glucose in the buffer as would be expected if glucose follows an apoplastic pathway to the phloem. It is interesting that exogenous [x4C]glucose rapidly enters the minor veins in Coleus. Although this finding seems to provide support for an apoplastic loading route, it is important to remember that the behavior of an
exogenous sugar does not necessarily reflect a normal transport pathway of the endogenous compound (for discussion, see Turgeon and Beebe 1991). It is possible that these experiments demonstrate the presence of an active carrier in the veins that functions in the retrieval of glucose leaking into the free space from the phloem. On the other hand, the slow uptake of 3 - O M G into the veins argues against this interpretation: an active carrier should load glucose and 3 - O M G indiscriminately. Another possibility is that there is a passive glucosegalactose-3-OMG carrier on the intermediary cell plasma membrane and that rapid uptake of exogenous glucose into the vein is driven metabolically as it is quickly used in the synthesis of galactinol. The active versus passive nature of the glucose carrier in plants is debated (e.g. Daie and Wilusz 1987). In summary, our experiments indicate that galactinol, raffinose and stachyose in C. blumei are synthesized in the intermediary cells of the phloem from precursors that diffuse to them in the symplast. These data are consistent with the hypothesis that synthesis of export sugars is mechanistically linked to phloem loading in those species that translocate raffinose and stachyose. This research was supported by National Science Foundation Grant DCB-9104159, U.S. Department of Agriculture Competetive Grant 90000854, and Hatch funds. References
Beebe, D.U., Turgeon, R. (1991) Galactinol synthase is sequestered in intermediary cells(companion cells) of Cucurbita leaf phloem. (Abstr.) Plant Physiol. 96, Suppl., 100 Beitler, G.A., Hendrix, J.E. (1974) Stachyose: an early product of photosynthesis in squash leaves. Plant Physiol. 53, 674-676 Daie, D., Wilusz, E.J. (1987) Facilitated transport of glucose in isolated phloem segments of celery. Plant Physiol. 85, 711-715 Fischer, A. (1885) Studien fiber die Siebr6hren der Dicotylenblfitter. Ber. Verh. Sfichs. Akad. Wiss. Leipzig, Math.-Phys. KI. 37, 245-290 Fisher, D.G. (1986) Ultrastructure, plasmodesmatal frequency, and solute concentration in green areas of variegated Coleus blumei Benth. leaves. Planta 169, 141-152 Fondy, B.R., Geiger, D.R. (1977) Sugar selectivity and other characteristics of phloem loading in Beta vulgaris L. Plant Physiol. 59, 953-960 Gamalei, Y. (1989) Structure and function of leaf minor veins in trees and herbs. A taxonomic review. Trees 3, 96-110 Giaquinta, R.T. (1983) Phloem loading of sucrose. Annu. Rev. Plant Physiol. 34, 347-387 Hendrix, J.E. (1977) Phloem loading in squash. Plant Physiol. 60, 567-569 Holthaus, U., Schmitz, K. (1991) Distribution and immunolocalization of stachyose synthase in Cucumis melo L. Planta 185, 479486 Kandler, O. (1967) Biosynthesis of poly- and oligosaccharides during photosynthesis in green plants. In: Harvesting the sun, pp 131-152, San Pietro, A., Greer, F.A., Army, T.J., eds. Academic Press, New York Kandler, O., Hopf, H. (1984) Biosynthesis of oligosaccharides in vascular plants. In: Storage carbohydrates in vascular plants. Distribution, physiology and metabolism, pp 115-131, Lewis, D.H., ed. Cambridge Univ. Press, New York Lucas, W.J., Madore, M.A. (1988) Recent advances in sugar transport. In: The biochemistry of plants. A comprehensive treatise, vol. 14: Carbohydrates, pp 35 84, J. Preiss ed. Academic Press, New York
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R. Turgeon and E. Gowan: Sugar synthesis in Coleus blumei leaves nemain, J.-L., Delrot, S., Lucas, W. J., Dainty, J., eds. Ouest Editions, Nantes, France Turgeon, R., Beebe, D. U. (1991) The evidence for symplastic phloem loading. Plant Physiol. 96, 349-354 Turgeon, R., Gowan, E. (1990) Phloem loading in Coleus blumei in the absence of carrier-mediated uptake of export sugar from the apoplast. Plant Physiol. 94, 1244-1249 Turgeon, R., Hepler, P.K. (1989) Symplastic continuity between mesophyll and companion cells in minor veins of mature Cucurbita pepo L. leaves. Planta 179, 24-31 Turgeon, R., Wimmers, L.E. (1988) Different patterns of vein loading of exogenous [14C]sucrose in leaves of Pisum sativum and Coleus blumei. Plant Physiol. 87, 179-182 Turgeon, R., Webb, J.A., Evert, R.F. (1975) Ultrastructure of minor veins of Cucurbita pepo leaves. Protoplasma 83, 217-232 Webb, J.A., Gorham, P.R. (1964) Translocation of photosynthetically assimilated C x4 in straight-necked squash. Plant Physiol. 39, 663-672 Weisberg, L.A., Wimmers, L.E., Turgeon, R. (1988) Photoassimilate-transport characteristics of nonchlorophyUous and green tissue in variegated leaves of Coleus blumei Benth. Planta 175, 1-8 Xia, J.-H., Saglio P.H. (1988) Characterization of the hexose transport system in maize root tips. Plant Physiol. 88, 1015-1020 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 1 : Phloem transport, pp 480-503, Zimmermann, M.H., Milburn, J.A., eds. Springer, Berlin Heidelberg New York
