Planta (1992)188:35,J~361
Pl~__t~ 9 Springer-Verlag1992
Localization of galactinol, raffinose, and stachyose synthesis in Cucurbita pepo leaves Dwight U. Beebe* and Robert Turgeon Section of Plant Biology, Division of Biological Sciences, Cornell University, Ithaca NY 14853, USA Received 14 February; accepted 12 June 1992
Abstract. The biochemical pathway of stachyose synthesis was localized by immunocytochemical and 14Clabeling techniques in mature Cucurbita pepo L. leaves. Galactinol synthase (GAS; EC 2.4.1.123), the first unique enzyme in this pathway, was immunolocalized within the intermediary cells of minor veins in conventionally fixed and cryo-fixed, resin-embedded sections using potyclonal anti-GaS antibodies and protein A-gold. Intermediary cells are specialized companion ceils with extensive symplastic connections to the bundle sheath. Gold particles were not seen over the non-specialized companion cells of larger veins or over intermediary cells in young leaves prior to the sink-source transition. In another approach to localization, radiolabel was measured in isolated mesophyll tissue and whole tissue of leaves that were lyophilized following a 90-s exposure to 14CO2. Mesophyll, obtained by abrasion of the leaf surface, contained labeled sucrose, galactinol, raffinose and stachyose. However, the latter three labeled compounds constituted a smaller proportion of the neutral fraction than in whole-tissue samples, which also contained minor veins. We conclude that synthesis of galactinol, raffinose, and stachyose occurs in both mesophyll and intermediary cells, predominantly the latter. Key words: Cucurbita (stachyose synthesis) - Galactinol synthase (immunolocalization) - Phloem loading - Raffinose - Stachyose synthesis
Introduction Raffinose and stachyose are translocated, in addition to sucrose, in a large and taxonomically diverse group of plants, and stachyose is often the predominant sugar in * To whom correspondence should be addressed; FAX: 1(607)255-5407 Abbreviations: GaS=galactinol synthase; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis
phloem exudate (Zimmermann and Ziegler 1975). While it is well established that sucrose is synthesized in mesophyll cells and is then loaded into the phloem of minor veins in preparation for long-distance transport, much less is known about the synthesis and loading of raffinose and stachyose. While phloem loading is generally considered to involve an apoplastic step (for review see Geiger and Fondy 1991), some lines of evidence indicate that loading in stachyose-translocating plants is symplastic, i.e. sugar, or sugar precursors, may move from the mesophyll to the minor-vein phloem entirely within the plasmodesmataconnected cytoplasm of contiguous cells (for review, see Turgeon and Beebe 1991). One such indication of a symplastic pathway is that certain minor-vein companion cells (intermediary cells) in stachyose-translocating species are specialized in that they are connected to bundle-sheath cells by very large numbers ofplasmodesmata. Intermediary cells have not been found in species that translocate sucrose alone. We recently proposed a mechanism for symplastic phloem loading in stachyose-translocating plants (Turgeon and Gowan 1990; Turgeon 1991). An important component of this model is that raffinose and stachyose are synthesized in intermediary cells, rather than the mesophyll. At the time this model was presented, the weight of evidence was in favor of the mesophyll as the site of raffinose and stachyose synthesis (for review, see Turgeon and Gowan 1990; Turgeon and Beebe 1991). However, Holthaus and Schmitz (1991) subsequently reported immunocytochemical data indicating that stachyose synthase is present in intermediary cells, but not the mesophyll, of Cucumis melo. They suggested that galactinol, the galactose donor for raffinose and stachyose synthases, is made in the mesophyll and is transported to the phloem, where the tri- and tetrasaccharides are produced for export. Galactinol is synthesized from myoinositol and UDP-galactose by galactinol synthase (Kandler and Hopf 1982). While the immunocytochemical data of Holthaus and Schmitz (1991) are compelling, other results are difficult
D.U. Beebe and R. Turgeon: Stachyose synthesis in Cucurbita leaves to reconcile with their scheme. Raffinose a n d stachyose synthesis have been detected in isolated m e s o p h y l l tissue a n d p r o t o p l a s t s b y M a d o r e a n d her colleagues ( M a d o r e a n d W e b b 1982; M a d o r e et al. 1988) a n d H o l t h a u s a n d Schmitz (1991) also observed t h a t p r o t o p l a s t s have raffinose s y n t h a s e activity. I n this p a p e r we p r o v i d e a n e x p l a n a t i o n for these inconsistencies: o u r data, o b t a i n e d b y i m m u n o l o c a l i z a t i o n o f g a l a c t i n o l synthase (GAS), a n d 14C-labeling techniques, indicate that the complete p a t h w a y to stachyose, i n c l u d i n g g a l a c t i n o l synthesis, is p r e s e n t in b o t h mesophyU a n d i n t e r m e d i a r y cells o f Cucurbita pepo. I m m u n o c y t o c h e m i c a l results have been p u b l i s h e d in a b s t r a c t f o r m (Beebe a n d T u r g e o n 1991).
Material and methods
Plant material. Cucurbitapepo L. var. Melopepo cv. zucchini (Burpee hybrid zucchini) and cv. torticollis (early prolific straightneck squash) seeds were obtained from Burpee Seed Co. (Warminster, Penn., USA) and plants were grown in a greenhouse as described previously (Turgeon and Gowan 1990). Mature leaves were used in all studies, with the exception of a young zucchini leaf undergoing the sink-source transition.
Galactinol-synthase purification and antibody preparation. Bruce Schweiger, Phillip Kerr, and John Pierce (Crop Research Laboratory, E.I. du Pont de Nemours, Newark, Del., USA) prepared the purified enzyme and kindly provided the anti-GaS polyclonal antibodies. Galactinol synthase was purified from mature zucchini leaves essentially as described by Smith et al. (1991), with the following exceptions : 50 mM Mops (3-[N-morpholino]propanesulfonic acid-NaOH), pH 7, containing 2 mM MnC12, was used as the buffer in all steps; a Phenyl-Sepharose column (Pharmacia LKB, Piscataway, N.Y. ; USA) was used for the hydrophobic-interactionchromatography step, with ammonium sulphate (20%) substituted for NaC1; a Spectra/Gel AcA 54 column (Spectrum Medical Industries, Los Angeles, Calif., USA) was used for the size-exclusion chromatography step, and the final purification step utilized an FPLC Mono Q column (Pharmacia), eluting the sample with an NaC1 gradient. The purified protein was then run multiple times on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels with high-range protein molecular weight standards (GIBCO BRL, Buffalo, N.Y. ; USA) to determine the molecular weight. The only protein revealed by Coomassie Blue staining was a doublet band of 38 kDa molecular weight. Galactinol synthase was purified to a specific activity of 23.3 lamol-min-1.(mg protein)- 1 To prepare the antibodies, the 38-kDa doublet was cleanly excised from an SDS-PAGE gel and pulverized using a hand-held homogenizer. The resulting powder was sent to Hazelton Research Laboratories (Denver, Penn., USA) for antibody production. Prior to injecting the antigen, blood to provide preimmune serum was drawn from the rabbits. For the primary injection, 300 lag of GaS was mixed to a final volume of I ml with phosphate-buffered saline. This was then combined with 1 ml of Freund's complete adjuvant, split between two rabbits, and injected intradermally. Three weeks later, the rabbits were boosted with subcutaneous injections of 75 lag GaS in Freund's incomplete adjuvant. At the fifth week, the animals were again boosted as described, then boosted twice more at twoweek intervals. Blood was collected at two-week intervals corresponding to the boost dates. Boosts were discontinued after the fourth boost. Serum was collected at two-week intervals until the sixteenth week, when the animals were exsanguinated. Serum collected at the eighth week was used for the immunolocalization study. The specificity of the antibody was assessed by Western-blot analysis against crude extract alone and crude extract containing
355 increasing amounts (up to 50%) of the purified enzyme. Zucchini crude extract, the crude extract/purified enzyme mixtures, and prestained high-range protein molecular weight markers (GIBCO BRL) were run on an SDS-PAGE gel at 20 lal per lane. The proteins on the gel were then blotted onto nitrocellulose. The SDS-PAGE gel was stained for proteins with Amido Black and the Western blot was probed with the anti-GaS serum at 1: 500 dilution. Sensitivity of the anti-GaS serum from the various collections was tested against purified GaS on a slot-blot Western at concentrations of 0, 1, 5, and 10 ng (based on enzyme activity) of purified enzyme.
Chemical fixation. Leaf tissue, sampled from intercostal regions, was fixed in 4 % (v/v) paraformaldehyde, 0.5 % (v/v) glutaraldehyde, 2 mM CaC12, in 50 mM Pipes (1,4-piperazinediethanesulfonicacidNaOH), pH 7.2, at 4 ~ for 6-8 h. Fixed tissue was washed in buffer (four times for 10 min each), dehydrated in ethanol, and infiltrated with L. R. White resin (London Resin Co., Hampshire, UK), all at 4 ~ Infiltrated samples were embedded in gelatin capsules and polymerized for 24 h at 60 ~ Cryofixation. Leaf pieces (%16 c m 2) w e r e sampled as above and aspirated in 20 mM Mes (2-[N-morpholino]ethanesulfonic acidNaOH, 20 mM KC1, 20 mM CaC12; pH 5.5) containing 100 mM or 200 mM sucrose. These pieces were cut into smaller (1 mm 2) segments, frozen in a Balzers HPM010 high-pressure freezer (Balzers Union, Liechtenstein), freeze-substituted in 0.5 % (w/v) uranyl acetate (UA) in anhydrous acetone for 24 h at - 90 ~ transferred to a fresh solution of 0.5% (w/v) UA plus 0.1% osmium tetroxide (w/v) in acetone at - 2 0 ~ for 24 h, then to acetone for 2-3 h at 4 ~ and then brought to room temperature. Samples were rinsed in fresh acetone, transferred to anhydrous ethanol: acetone (1:1 by volume) for 15 min, then to ethanol, and embedded in L. R. White resin. Polymerization was as described above. Immunocytochemistry. Thin sections (silver to pale gold) were cut with a diamond knife and picked up on formvar/carboncoated nickel slot grids. Grids were floated on 50-lal drops of either 10 mM Tris-buffered saline (TBST: 2-amino-2-[hydroxymethyl]-1,3propanediol-NaOH, 150 mM NaC1, 0.3% [v/v] Tween 20; pH 7.2), or 10 mM phosphate-buffered saline (PBST: potassium phosphate buffer, 150 mM NaC1, 0.3% [v/v] Twcen 20, pH 7.2) for 30 min. Both buffers contained 1% bovine serum albumin, fraction V powder (BSA; Sigma Chemical Company., St. Louis, Mo., USA). Grids were then transferred to 25 gl drops of antibody (1 : 200, diluted in either TBST-BSA or PBST-BSA) for 60 min. Grids were then washed six times for 5 min each on 50-lal drops of buffer-BSA and transferred to 25-p.1 drops of 15-nm-diameter BioCell EM-grade protein A-gold (1:30, 1:40, or 1:50 diluted in buffer-BSA; Ted Pella, Redding, Calif., USA) for 60 min. Grids were then washed seven times for 5 min each on 50-lal drops: twice in buffer-BSA, twice in buffer only, and three times in double-distilled H20. Solutions were passed through 0.45-lam filters before dilution of antibody or protein A-gold, and all steps were carried out at room temperature. Preimmune serum and buffer-BSA alone were used as controls in place of anti-GaS serum. Sections, either unstained or contrasted with uranyl acetate and lead citrate, were examined with a Philips (Eindhoven, The Netherlands) EM300 transmission electron microscope at 80 kV. Morphometry and quantification of gold label. Areas of cell components were quantified using the point-counting method of Weibel (1979). Total numbers of gold particles over cell components were determined by counting at least 20 cells of each type, taken from several different tissue samples. Density of label was expressed as the number of gold particles per l.tm 2 a r e a .
Analysis of labeled photosynthase. Plants were illuminated with a water-filtered incandescent 1000-W metal-halide lamp providing approx. 800 gmol photons 9m 2 - s- 1 (photosynthetically active radiation) at plant level. One hour later a mature leaf was enclosed in a plastic bag and 14CO2 (2.0 MBq), generated in the
Fig. 1. A S D S - P A G E of zucchini leaf crude extract with and without purified GaS, and prestained high-range protein molecularweight markers. A 12.5% separating gel was used and stained with Amido Black. Molecular-weight-marker lane was loaded with 7 ~tl, while all sample lanes were loaded with 20 lal. Lanes are as follows : 1 98% (v/v) crude extract, 1.6% purified GaS; 2 96.2% crude extract, 3.2% purified GaS; 3 93.8% crude extract, 6.2% purified GaS; 4 87.6% crude extract, 12.4% purified GaS; 5 75% crude extract,
25% purified GaS; 6 molecular weight markers; 7 50% crude extract, 50% purified GaS; 8 crude extract only. B Western blot on nitrocellulose of proteins from the S D S - P A G E of A, probed with anti-GaS polyclonal antibodies, diluted 1 : 500. Lanes are identified as in A. Darts indicate the position of the 38-kDa GaS protein. Note the presence of a weakly staining band just below the heavily stained band in lane 7 of B
Fig. 2A, B. Portions of minor-vein phloem cells from squash leaf tissue prepared by high-pressure freezing and freeze substitution. Sections are unstained to better illustrate the presence of gold particles. A Intermediary cell from section incubated in preimmune serum, followed by protein A-gold. Cell is essentially unlabeled; only a single gold particle (dart) is present. Dense portion of cell in lower middle of figure is cytoplasm, an artifact resulting from
high pressure freezing. V, vacuole, x 19152; bar=0.5 lam. B Two intermediary cells (/) and a sieve-tube member (S) from a section incubated in anti-GaS serum, followed by protein A-gold. Note high density of labelling over cytosol, with virtually no label over mitochondria (M) or vacuoles (I1). A single gold particle (dart) is present over the lumen of the sieve-tube member, x 18992; bar = 0.5 ~tm
D.U. Beebe and R. Turgeon: Stachyose synthesis in Cucurbita leaves
357
barrel of a syringe by addition of excess 80% lactic acid to Na2 14CO3 (6.6 9 105 MBq 9mmol-1), was injected into the bag without delay. Thirty seconds later the bag was removed and after a further 60 s in room atmosphere the leaf was excised, quickly placed between stainless steel screens, and frozen in powdered, solid CO2. Frozen tissue was lyophilized as described by Weisberg et al. (1988), except that the tissue was not compressed. For whole-tissue sampling, discs (5.2 mm diameter) of lyophilized tissue were removed with a cork borer. To obtain samples of mesophyll, free of minor and major veins, the upper (adaxial) surface of the lyophilized tissue was gently abraded with fine sandpaper (400, wet or dry; 3M, St. Paul, Minn., USA), under a dissecting microscope, to remove the epidermis and some of the underlying mesophyll cells. More of the mesophyll was then removed with a camel-hair brush until the minor veins were just visible. Care was taken not to abrade the veins. Leaf discs and mesophyll samples were extracted in a mixture of methanol: water: chloroform (12:3:5 by volume) at 70 ~ for 1 h (Haissig and Dickson 1979). Water (3 ml per 5 ml solvent) was then added to cause phase separation and, following centrifugation in a clinical centrifuge to separate the phases, the aqueous portion was passed through ion-exchange membranes (Bio-Rex AG50W cation membrane [H + form] and AGI anion membrane [carbonate form]; Bio-Rad, Richmond, Calif., USA). Sufficient unlabeled sugars were added to the eluate to allow subsequent visualization and the sugars were resolved by two-dimensional TLC (Turgeon and Gowan 1990). Spots from vanillin-sprayed plates (Touchstone and Dobbins 1983, p. 173) were scraped into scintillation vials and the radioactivity counted.
Sink-source transition. A single mature leaf was enclosed in a plastic bag while attached to the plant and labeled with 14CO2 (0.5 MBq) as described above, for 5 min. Two hours later, samples were removed from a growing leaf that was passing through the sinksource transition. The samples were chemically fixed for immunolocalization as described above and the rest of the leaf was quickly excised, placed between stainless-steel screens, flash-frozen in solid CO2, lyophilized, and autoradiographed (Turgeon and Gowan 1992).
Fig. 3. A Portion ofa palisade-mesophyllcell from squash leaf tissue prepared by high-pressure freezing and freeze substitution. Section was incubated in anti-GaS serum, followed by protein A-gold. Section is unstained to better view gold particles. Only two gold particles (darts) are present over the chloroplast. Label is absent from vacuole (V) and other components of the cell. IS, intercellular space; SG, starch grain. • 19152; bar = 0.5 jam. B Portion of minor-
vein phloem from zucchini leaf tissue undergoing sink-to-source transition. Tissue was chemically fixed. Section was incubated in anti-GaS serum, followed by protein A-gold, and is unstained to better view gold particles. Label is absent from ail cell types. Only a single gold particle (dart) is present over the cytosol of a vascularparenchyma cell (P). CC, companion cell; L intermediary cell; S, sieve-tube member. • 19152; bar = 0.5 lam.
Results Specificity and sensitivity of the anti-GaS antibodies. W e s t e r n - b l o t a n a l y s i s i n d i c a t e d t h a t the a n t i b o d i e s recognize a 3 8 - k D a G a S d o u b l e t b a n d , t h e l a r g e r o f the t w o b a n d s s t a i n i n g m u c h m o r e i n t e n s e l y t h a n the s m a l l e r (Fig. 1 B, b e g i n n i n g in lane 2 a n d c l e a r l y b y l a n e 3). B o t h b a n d s o f the d o u b l e t h a v e G a S activity, verified b y t h e i r a b i l i t y to c a t a l y z e the synthesis o f g a l a c t i n o l f r o m myo-inositol a n d U D P - g a l a c t o s e in v i t r o (Phillip K e r r , p e r s o n a l c o m m u n i cation). These results i n d i c a t e t h a t for W e s t e r n b l o t t i n g , a p p r o x . 20-30% g r e a t e r e n z y m e a c t i v i t y t h a n t h a t p r e s e n t in the c r u d e e x t r a c t (Fig. 1 B, l a n e 8) is r e q u i r e d for a clear signal to be o b s e r v e d . T h e s e results also s h o w t h a t the a n t i b o d i e s d o n o t c r o s s - r e a c t w i t h a n y o f the o t h e r p r o t e i n s p r e s e n t in t h e c r u d e e x t r a c t (Fig. 1B, L a n e
358
D.U. Beebe and R. Turgeon: Stachyose synthesis in Cucurbita leaves
Table 1. Distribution of protein A-colloidal gold particles in transverse sections of mature squash leaves following incubation with immune serum (anti-GaS antibodies) or preimmune serum. Data Cell type
Intermediary Bundle sheath Palisade mesophyll Spongy mesophyll
Treatment
Immune Preimmune Immune Preimmune Immune Preimmune Immune Preimmune
are mean numbers of gold particles per I~m 2 of cross sectional area. SEs are in parentheses. NA = not applicable, NP = not present in sections
Cell compartment Cytosol Nucleus
Mitochondria
Chloroplasts
Vacuole
Cell wall
1.49 (0.20) 0.03 (0.01) 0.07 (0.01) 0.03 (0.01) 0.10 (0.01) 0.04 (0) 0.06 (0.01) 0.03 (0)
0.20 (0.03) 0.01 (0,01) 0.16 (0.09) 0.20 (0.06) 0.12 (0,03) 0.10 (0.02) 0.12 (0.03) 0.12 (0.04)
NA NA 0.16 (0.02) 0.10 (0.02) 0.15 (0.01) 0.08 (0.01) 0.17 (0.02) 0.11 (0.01)
0.08 (0.02) 0 (0) 0.02 (0) 0 (0) 0.01 (0) 0 (0) 0.02 (0) 0 (0)
0.24 (0.03) 0.02 (0.01) 0.67 (0.08) 0.04 (0.01) 0.35 (0.05) 0.03 (0.01) 0.70 (0.07) 0.12 (0.03)
0.96 (0.30) 0.04 (0.01) 0.03 (0.01) 0.02 (0.01) 0.18 (0.02) 0.07 (0.01) NP NP
8). Anti-GaS serum, f r o m the bleed selected for the immunolocalization study, gave a positive signal at a 1 : 10,000 dilution against 1 ng (based on enzyme activity) of purified GaS (data not shown). N o reaction product was detected over control slots. Immunolocalization o f GaS. Polyclonal antibodies to GaS labeled the cytosol o f intermediary cells much more densely than did p r e i m m u n e serum (Fig. 2). In other cell types, counts of gold particles over the cytosol were the same as, or slightly higher than, those over control sections incubated in p r e i m m u n e serum (Fig. 3A; Table 1). Since these cytosol counts in other cell types were elevated no m o r e than counts over cell walls, chloroplasts, and mitochondria, we attributed the extra signal to nonspecific binding. Cell walls were particularly "sticky", as has been noted in other immunolocalization studies (see H e r m a n 1989). N o appreciable difference in labeling patterns was found in comparisons o f the two cultivars used. Although the patterns of labeling were the same in chemically- and cryo-fixed samples, the density of label was greater in the latter. Within intermediary cells, specific labeling by gold particles was not detected over mitochondria or vacuoles (Table 1). Significant numbers o f gold particles were found over intermediary-cell nuclei, but not over the nuclei o f other cell types. Therefore, although the signal over the intermediary-cell nucleus was not as strong as that over the cytosol, it appeared to be specific for the GaS antibody. As a test of specificity for GaS, we used immature (importing) tissue f r o m leaves that were approximately half-grown; i m m a t u r e tissue does not synthesize raffinose or stachyose (Turgeon and W e b b 1975). Translocation experiments were performed on these plants immediately before the samples were removed to determine the location o f the sink-source b o u n d a r y in the sampled leaves (Turgeon 1989). Tissue that was close to the boundary, but still importing, was used for immunolocalization studies (Fig. 4). In this immature tissue, no label above control levels was detected in any cell type, including intermediary cells (Fig. 3B). In larger m i n o r veins there are two types of abaxial c o m p a n i o n cells: intermediary cells and "ordinary"
Fig. 4. Autoradiograph of zucchini leaf undergoing sink-to-source transition that was sampled to provide the section illustrated in Fig. 3B. Blackened areas indicate regions that imported radiolabeled sugars from a source leaf exposed to 14CO2. Stippled circles indicate actual areas sampled. Leaf area is reduced 40% by lyophilization. x 1; bar = 1.0 cm c o m p a n i o n cells that are similar in appearance to the minor-vein companion cells of species that do not translocate raffinose sugars (Turgeon et al. 1975). These "ordinary" companion cells, found usually in the interior of the vein and having relatively few plasmodesmatal connections to surrounding cells, were not labeled by antibodies to GaS (Fig. 5) in mature leaves. Also, there was no specific labeling of the adaxial phloem, in which the c o m p a n i o n cell is not specialized as
D.U. Beebe and R. Turgeon: Stachyose synthesis in Cucurbita leaves
359
Fig. 5. Portions of abaxial phloem of a large minor vein from squash leaf tissue prepared by high-pressure freezing and freeze substitution. Sections are unstained to better illustrate the presence of gold particles. A Section was incubated in preimmune serum, followed by protein A-gold. Label is virtually absent from all cell types. Only a single gold particle (dart) is present over the plasmalemma of the companion cell (CC). L intermediary cell; M, mitochondria; P,
vascular-parenchyma cell; S, sieve-tube member; V, vacuole. x 18992; bar=0.5 gm. B Section was incubated in anti-GaS, followed by protein A-gold. Note high density of label over the cytosol of the intermediary cell (/) and the virtual lack of labelling elsewhere. Label seen over other cell types is due to nonspecific binding. Ceils and cell components are identified as in A. x 18992; bar = 0.5 gm
an intermediary cell (not shown). It is unlikely that stachyose is synthesized in adaxial phloem since the solute content o f these cells is low (Turgeon and Hepler 1989) and, in m i c r o a u t o r a d i o g r a p h y studies with Cucumis melo leaves, they are not labeled following application of 1~CO2 (Schmitz et ah 1987).
tinol, and raft• oligosaccharides are labeled very soon after 14CO z is applied (20 s or less; not shown). To determine whether labeled sugars were present in mesophyll, samples o f this tissue were obtained by abrasion and the neutral fraction was analyzed by T L C (Table 2). Labeled galactinol, raffinose, and stachyose were present, although in very small amounts, while approx. 80 % of the label in the neutral fraction was in sucrose. In the whole-tissue samples, which contained veins as well as mesophyll, the p r o p o r t i o n o f label in
Label• of sugars with 1"C. The immunolocalization technique failed to demonstrate the presence of G a S in the mesophyll, even though gatactinol ( M a d o r e and W e b b 1982; Schmitz and Holthaus 1986; Holthaus and Schmitz 1991) and stachyose ( M a d o r e and W e b b 1982; M a d o r e et al. 1988) synthesis has been detected in this tissue in cucurbits. However, immunolocalization has limited sensitivity. Therefore, a m o r e sensitive procedure was developed that coupled radiolabeling with physical isolation of mesophyll. Leaves that were still attached to the plant were exposed to 14CO2 for 30 s and, following a further 60-s chase in r o o m atmosphere, the labeled c o m p o u n d s were immobilized by flash-freezing and the tissue was lyophilized. The relatively short 90-s total time period was employed so that there would be only limited m o v e m e n t of labeled c o m p o u n d s between cell types. Preliminary experiments indicated that sucrose, galac-
Table 2. Radiolabeled compounds in mature zucchini leaf tissue. Leaves were exposed to 14COz for 30 s followed by 60 s in room atmosphere, then flash-frozen and lyophilized. Assays were conducted on isolated mesophyll tissue and whole-tissue. Results are expressed as the percentage of radiolabel in the neutral fraction. Values are means • SE Radiolabeled compound
Mesophyll tissue
Whole-leaf tissue
Sucrose Galactinol Raft• Stachyose Other
80.6i 3.0 • 1.7• 1.9 • 12.9 •
42.8 • 6.2 • 7.4• 9.9 • 33.8 •
1.1 0.3 0.4 0.2
3.6 0.4 1.1 2.3 2.8
360 galactinol, raffinose, and stachyose was higher than in the mesophyll samples, while there was proportionately less sucrose (Table 2); these results are expected if galactinol, raffinose, and stachyose are made in the veins. In the mesophyll there was more label in galactinol than in either raffinose or stachyose, while in the whole-tissue, the label was more evenly distributed between the three compounds. Discussion
Synthesis and transport of sucrose, the sole sugar translocated in many species, has been studied extensively: it is made in the mesophyll cells of the leaf and is then loaded, against a concentration gradient, into the phloem (Lucas and Madore 1988). Less well studied is the synthesis of raffinose oligosaccharides, the predominant export sugars of many plants, especially tropical trees and vines. We demonstrate here that, in C. pepo, raffinose, stachyose, and the galactose donor for these molecules are synthesized in the transport tissue itself, i.e., in the intermediary cells of the minor-vein phloem. Evidence to date has pointed to mesophyll cells as the source of galactinol (Madore and Webb 1982; Schmitz and Holthaus 1986; Holthaus and Schmitz 1991) and it has been postulated that this precursor to raffinose and stachyose then migrates to the veins (Schmitz and Holthaus 1986; Holthaus and Schmitz 1991). However, it should be noted that experiments, cited above, demonstrating galactinol synthesis in the mesophyll, do not rule out the possibility that it is also made in the phloem. Indeed, the results presented here indicate that galactinol is probably made in both locations. The immunological evidence clearly indicates that there is GaS in intermediary cells, and short-term experiments with 14CO 2 reveal the presence of labeled galactinol in the mesophyll. Why do antibodies to GaS not label the mesophyll? The most likely reason is that the enzyme is present in too low a concentration for detection by immunocytochemistry. We found that cryofixation increased sensitivity by at least tenfold over chemical fixation but, even so, a small amount of enzyme dispersed over the entire mesophyll cytosol may be present at too low a concentration to aliow labeling at a level distinguishable from nonspecific labeling over control tissue. This may also be the reason that Holthaus and Schmitz (1991) did not detect stachyose synthase in the mesophyll by immunocytochemistry. It is possible that the absence of label in the mesophyll is due to the specificity of the antibodies for an intermediary-cell isoenzyme; indeed, the purified enzyme was resolved as a doublet protein band. However, the antibodies were polyclonal and both members of the protein doublet were used to raise them, yet specific signal was present only over the intermediary cells in tissue sections. The fact that two protein bands had GaS activity does not necessarily indicate the presence of isoenzymes: the minor, lower molecular-weight, member of the doublet could also be a slightly degraded form of the enzyme. Smith et al. (1991) found no evidence for the presence of
D.U. Beebeand R. Turgeon : Stachyosesynthesis in Cucurbita leaves galactinol synthase isoenzymes in C. pepo leaves, but they did not rule out the possibility that they are present. Another explanation for the presence of labeled compounds in the mesophyll after exposure of the leaf to 1 4 C 0 2 is that some transport from the veins to the mesophyll took place during the course of the experiment. Indeed, plasmodesmata between intermediary cells and the mesophyll are open to passage of low-molecularweight compounds (Turgeon and Hepler 1989). We have speculated that the plasmodesmata between intermediary cells and bundle sheath allow the passage of smaller molecules such as sucrose and galactinol while restricting the passage of tri- and tetrasaccharides (Turgeon 199l). If this is the case, leakage from veins to the mesophyll could have been selective. Still another explanation for the presence of labeled galactinol, raffinose, and stachyose in mesophyll samples is that they are contaminants, introduced by movement of label during lyophilization or subsequent handling, or by the accidental abrasion of veins. This seems highly unlikely, however. Our freeze-preservation procedures yield macroautoradiographs of minor veins with high resolution, indicating very little, if any, spread of label (for example, see Turgeon 1987). Furthermore, the minor veins are easily recognized in lyophilized tissue and we discarded any mesophyll samples that could have been contaminated with vein tissue. If the label detected in mesophyll samples had come from the veins, the proportions of galactinol, raffinose, and stachyose would have been the same in mesophyll and whole-tissue samples and they were not. Since galactinol (Madore and Webb 1982; Schmitz and Holthaus 1986; Madore et al. 1988; Holthaus and Schmitz 1991) and stachyose (Madore and Webb 1982; Madore et al. 1988) synthesis has been detected in isolated protoplasts and tissues from cucurbit leaves, we favor the straightforward interpretation that the entire pathway to stachyose is present in both mesophyll and intermediary cells. We came to the same conclusion in studies of sugar synthesis in Coleus blumei (Turgeon and Gowan 1992). Why is stachyose synthesized in the mesophyll as well as the phloem? Perhaps the stachyose in mesophyll cells is stored during the day and catabolized at night while stachyose that is synthesized in the phloem is used for immediate export. The discovery that galactinol, raffinose, and stachyose are made in the phloem raises several interesting questions. How is specificity for export sugars conferred? In those plants that transport sucrose alone, specificity is thought to be the result of selective release of this sugar to the apoplast and-or selective, carrier-mediated uptake from the apoplast into the sieve-element-companion-cell complex (Geiger and Fondy 1991). This explanation does not suffice for raffinose and stachyose. What is the driving force for solute accumulation in the intermediary cell? It is unlikely to be proton symport from the apoplast, as postulated for species that translocate only sucrose, since experiments on Coleus (Turgeon and Gowan 1990, 1992) and the cucurbits (Madore and Webb 1981; Schmitz et al. 1987) indicate that sucrose
D.U. Beebe and R. Turgeon: Stachyose synthesis in Cucurbita leaves and other potential precursors to stachyose p r o b a b l y do not enter the minor-vein phloem by this route. Yet solute levels are as high as in other species: the intermediary cells of C. pepo (Turgeon and Hepler 1989) and Coleus (Fisher 1986) do not plasmolyze in 1.2 M mannitol. We have postulated that the intermediary cell acts as a "molecular size-discrimination t r a p " by allowing lowmolecular-weight precursor(s) to enter the cell while preventing larger sugars such as raffinose and stachyose from diffusing away (Turgeon 1991). Solute accumulation could also be a consequence o f active transport within the intermediary cell (Turgeon 1991). Finally, how is synthesis o f sucrose in the mesophyll coordinated with raffinose and stachyose synthesis in the phloem, and how is carbon partitioning regulated in these plants? Answers to these questions will come only after we have a m u c h m o r e detailed understanding of sugar synthesis in stachyose-translocating species. We thank John Pierce, Phillip Kerr, and Bruce Schweiger for the gift of anti-GaS antibody and M.K. Kandasamy for helpful discussions. 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 Fisher, D.G. (1986) Ultrastructure, plasmodesmatal frequency, and solute concentration in green areas of variegated Coleus btumei Benth. leaves. Planta 169, 141-152 Geiger, D.R., Fondy, B.R. (199t) Regulation of carbon allocation and partitioning: status and research agenda. In: Recent advances in phloem transport and assimilate compartmentation, pp. 1-9, Bonnemain, J.-L., Delrot, S., Lucas, W.J., Dainty, J., eds. Ouest Editions, Nantes, France Haissig, B.E., Dickson, R.E. (1979) Starch measurement in plant tissue using enzymatic hydrolysis. Physiol. Plant. 47, 151-157 Herman, E.M. (1989) Colloidal gold labeling of acrylic resinembedded plant tissues. In: Colloidal gold: principles, methods, and applications, vol. 2, pp. 303-322, Hayat, M.A., ed. Academic Press, New York Holthaus, U., Schmitz, K. (1991) Distribution and immunolocalization of stachyose synthase in Cucumis melo L. Planta 185, 479-486 Kaudler, O., Hopf, H. (1982) Oligosaccharides based on sucrose (sucrosyl oligosaccharides). In: Encyclopedia of plant physiology, N.S., vol. 13A: Plant carbohydrates I, Intracellular carbohydrates, pp 348-383, Loewus, F.A., Tanner, W., eds. Springer, Berlin Heidelberg New York
361 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, Preiss, J., ed. Academic Press, New York Madore, M.A., Webb, J.A. (1981) Leaf free space analysis and vein loading in Cucurbita pepo. Can. J. Bot. 59, 225--2257 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. (1985) 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 Smith, P.T., Kuo, T.M., Crawford, C.G. (1991) Purification and characterization of galactinol synthase from mature zucchini squash leaves. Plant Physiol 96, 693-698 Touchstone, J.C., Dobbins, M.F. (1983) Practice of thin layer chromatography, 2nd edn. Wiley, New York Turgeon, R. (1987) Phloem unloading in tobacco sink leaves: insensitivity to anoxia indicates a symplastic pathway. Planta 171, 73-81 Turgeon, R. (1989) The sink-source transition in leaves. Annu. Rev. Plant Physiol Plant Mol. Biol. 40, 119-138 Turgeon, R. (199 i) Symplastic phloem loading and the sink-source transition in leaves: a model. In: Recent advances in phloem transport and assimilate compartmentation, pp. 18-22, Bonnemain, 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., Gowan, E. (1992) Sugar synthesis and phloem loading in Coleus blumei leaves. Planta 187, 388-394 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., Webb, J.A. (1975) Leaf development and phloem transport in Cucurbita pepo: carbon economy. Planta 123, 53~52 Turgeon, R., Webb, J.A., Evert, R.F. (1975) UItrastructure of minor veins of Cucurbita pepo leaves. Protoplasma 83, 217-232 Weibel, E.R. (1979) Stereological methods, vol. 1: Practical methods for biological morphometry. Academic Press, London Orlando Weisberg, L.A., Wimmers, L.E., Turgeon, R. (1988) Photoassimi!ate-transport characteristics of nonchlorophyltous and green tissue in variegated leaves of Coleus blumei Benth. Planta 175, 1-8 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
Pl~__t~ 9 Springer-Verlag1992
Localization of galactinol, raffinose, and stachyose synthesis in Cucurbita pepo leaves Dwight U. Beebe* and Robert Turgeon Section of Plant Biology, Division of Biological Sciences, Cornell University, Ithaca NY 14853, USA Received 14 February; accepted 12 June 1992
Abstract. The biochemical pathway of stachyose synthesis was localized by immunocytochemical and 14Clabeling techniques in mature Cucurbita pepo L. leaves. Galactinol synthase (GAS; EC 2.4.1.123), the first unique enzyme in this pathway, was immunolocalized within the intermediary cells of minor veins in conventionally fixed and cryo-fixed, resin-embedded sections using potyclonal anti-GaS antibodies and protein A-gold. Intermediary cells are specialized companion ceils with extensive symplastic connections to the bundle sheath. Gold particles were not seen over the non-specialized companion cells of larger veins or over intermediary cells in young leaves prior to the sink-source transition. In another approach to localization, radiolabel was measured in isolated mesophyll tissue and whole tissue of leaves that were lyophilized following a 90-s exposure to 14CO2. Mesophyll, obtained by abrasion of the leaf surface, contained labeled sucrose, galactinol, raffinose and stachyose. However, the latter three labeled compounds constituted a smaller proportion of the neutral fraction than in whole-tissue samples, which also contained minor veins. We conclude that synthesis of galactinol, raffinose, and stachyose occurs in both mesophyll and intermediary cells, predominantly the latter. Key words: Cucurbita (stachyose synthesis) - Galactinol synthase (immunolocalization) - Phloem loading - Raffinose - Stachyose synthesis
Introduction Raffinose and stachyose are translocated, in addition to sucrose, in a large and taxonomically diverse group of plants, and stachyose is often the predominant sugar in * To whom correspondence should be addressed; FAX: 1(607)255-5407 Abbreviations: GaS=galactinol synthase; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis
phloem exudate (Zimmermann and Ziegler 1975). While it is well established that sucrose is synthesized in mesophyll cells and is then loaded into the phloem of minor veins in preparation for long-distance transport, much less is known about the synthesis and loading of raffinose and stachyose. While phloem loading is generally considered to involve an apoplastic step (for review see Geiger and Fondy 1991), some lines of evidence indicate that loading in stachyose-translocating plants is symplastic, i.e. sugar, or sugar precursors, may move from the mesophyll to the minor-vein phloem entirely within the plasmodesmataconnected cytoplasm of contiguous cells (for review, see Turgeon and Beebe 1991). One such indication of a symplastic pathway is that certain minor-vein companion cells (intermediary cells) in stachyose-translocating species are specialized in that they are connected to bundle-sheath cells by very large numbers ofplasmodesmata. Intermediary cells have not been found in species that translocate sucrose alone. We recently proposed a mechanism for symplastic phloem loading in stachyose-translocating plants (Turgeon and Gowan 1990; Turgeon 1991). An important component of this model is that raffinose and stachyose are synthesized in intermediary cells, rather than the mesophyll. At the time this model was presented, the weight of evidence was in favor of the mesophyll as the site of raffinose and stachyose synthesis (for review, see Turgeon and Gowan 1990; Turgeon and Beebe 1991). However, Holthaus and Schmitz (1991) subsequently reported immunocytochemical data indicating that stachyose synthase is present in intermediary cells, but not the mesophyll, of Cucumis melo. They suggested that galactinol, the galactose donor for raffinose and stachyose synthases, is made in the mesophyll and is transported to the phloem, where the tri- and tetrasaccharides are produced for export. Galactinol is synthesized from myoinositol and UDP-galactose by galactinol synthase (Kandler and Hopf 1982). While the immunocytochemical data of Holthaus and Schmitz (1991) are compelling, other results are difficult
D.U. Beebe and R. Turgeon: Stachyose synthesis in Cucurbita leaves to reconcile with their scheme. Raffinose a n d stachyose synthesis have been detected in isolated m e s o p h y l l tissue a n d p r o t o p l a s t s b y M a d o r e a n d her colleagues ( M a d o r e a n d W e b b 1982; M a d o r e et al. 1988) a n d H o l t h a u s a n d Schmitz (1991) also observed t h a t p r o t o p l a s t s have raffinose s y n t h a s e activity. I n this p a p e r we p r o v i d e a n e x p l a n a t i o n for these inconsistencies: o u r data, o b t a i n e d b y i m m u n o l o c a l i z a t i o n o f g a l a c t i n o l synthase (GAS), a n d 14C-labeling techniques, indicate that the complete p a t h w a y to stachyose, i n c l u d i n g g a l a c t i n o l synthesis, is p r e s e n t in b o t h mesophyU a n d i n t e r m e d i a r y cells o f Cucurbita pepo. I m m u n o c y t o c h e m i c a l results have been p u b l i s h e d in a b s t r a c t f o r m (Beebe a n d T u r g e o n 1991).
Material and methods
Plant material. Cucurbitapepo L. var. Melopepo cv. zucchini (Burpee hybrid zucchini) and cv. torticollis (early prolific straightneck squash) seeds were obtained from Burpee Seed Co. (Warminster, Penn., USA) and plants were grown in a greenhouse as described previously (Turgeon and Gowan 1990). Mature leaves were used in all studies, with the exception of a young zucchini leaf undergoing the sink-source transition.
Galactinol-synthase purification and antibody preparation. Bruce Schweiger, Phillip Kerr, and John Pierce (Crop Research Laboratory, E.I. du Pont de Nemours, Newark, Del., USA) prepared the purified enzyme and kindly provided the anti-GaS polyclonal antibodies. Galactinol synthase was purified from mature zucchini leaves essentially as described by Smith et al. (1991), with the following exceptions : 50 mM Mops (3-[N-morpholino]propanesulfonic acid-NaOH), pH 7, containing 2 mM MnC12, was used as the buffer in all steps; a Phenyl-Sepharose column (Pharmacia LKB, Piscataway, N.Y. ; USA) was used for the hydrophobic-interactionchromatography step, with ammonium sulphate (20%) substituted for NaC1; a Spectra/Gel AcA 54 column (Spectrum Medical Industries, Los Angeles, Calif., USA) was used for the size-exclusion chromatography step, and the final purification step utilized an FPLC Mono Q column (Pharmacia), eluting the sample with an NaC1 gradient. The purified protein was then run multiple times on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels with high-range protein molecular weight standards (GIBCO BRL, Buffalo, N.Y. ; USA) to determine the molecular weight. The only protein revealed by Coomassie Blue staining was a doublet band of 38 kDa molecular weight. Galactinol synthase was purified to a specific activity of 23.3 lamol-min-1.(mg protein)- 1 To prepare the antibodies, the 38-kDa doublet was cleanly excised from an SDS-PAGE gel and pulverized using a hand-held homogenizer. The resulting powder was sent to Hazelton Research Laboratories (Denver, Penn., USA) for antibody production. Prior to injecting the antigen, blood to provide preimmune serum was drawn from the rabbits. For the primary injection, 300 lag of GaS was mixed to a final volume of I ml with phosphate-buffered saline. This was then combined with 1 ml of Freund's complete adjuvant, split between two rabbits, and injected intradermally. Three weeks later, the rabbits were boosted with subcutaneous injections of 75 lag GaS in Freund's incomplete adjuvant. At the fifth week, the animals were again boosted as described, then boosted twice more at twoweek intervals. Blood was collected at two-week intervals corresponding to the boost dates. Boosts were discontinued after the fourth boost. Serum was collected at two-week intervals until the sixteenth week, when the animals were exsanguinated. Serum collected at the eighth week was used for the immunolocalization study. The specificity of the antibody was assessed by Western-blot analysis against crude extract alone and crude extract containing
355 increasing amounts (up to 50%) of the purified enzyme. Zucchini crude extract, the crude extract/purified enzyme mixtures, and prestained high-range protein molecular weight markers (GIBCO BRL) were run on an SDS-PAGE gel at 20 lal per lane. The proteins on the gel were then blotted onto nitrocellulose. The SDS-PAGE gel was stained for proteins with Amido Black and the Western blot was probed with the anti-GaS serum at 1: 500 dilution. Sensitivity of the anti-GaS serum from the various collections was tested against purified GaS on a slot-blot Western at concentrations of 0, 1, 5, and 10 ng (based on enzyme activity) of purified enzyme.
Chemical fixation. Leaf tissue, sampled from intercostal regions, was fixed in 4 % (v/v) paraformaldehyde, 0.5 % (v/v) glutaraldehyde, 2 mM CaC12, in 50 mM Pipes (1,4-piperazinediethanesulfonicacidNaOH), pH 7.2, at 4 ~ for 6-8 h. Fixed tissue was washed in buffer (four times for 10 min each), dehydrated in ethanol, and infiltrated with L. R. White resin (London Resin Co., Hampshire, UK), all at 4 ~ Infiltrated samples were embedded in gelatin capsules and polymerized for 24 h at 60 ~ Cryofixation. Leaf pieces (%16 c m 2) w e r e sampled as above and aspirated in 20 mM Mes (2-[N-morpholino]ethanesulfonic acidNaOH, 20 mM KC1, 20 mM CaC12; pH 5.5) containing 100 mM or 200 mM sucrose. These pieces were cut into smaller (1 mm 2) segments, frozen in a Balzers HPM010 high-pressure freezer (Balzers Union, Liechtenstein), freeze-substituted in 0.5 % (w/v) uranyl acetate (UA) in anhydrous acetone for 24 h at - 90 ~ transferred to a fresh solution of 0.5% (w/v) UA plus 0.1% osmium tetroxide (w/v) in acetone at - 2 0 ~ for 24 h, then to acetone for 2-3 h at 4 ~ and then brought to room temperature. Samples were rinsed in fresh acetone, transferred to anhydrous ethanol: acetone (1:1 by volume) for 15 min, then to ethanol, and embedded in L. R. White resin. Polymerization was as described above. Immunocytochemistry. Thin sections (silver to pale gold) were cut with a diamond knife and picked up on formvar/carboncoated nickel slot grids. Grids were floated on 50-lal drops of either 10 mM Tris-buffered saline (TBST: 2-amino-2-[hydroxymethyl]-1,3propanediol-NaOH, 150 mM NaC1, 0.3% [v/v] Tween 20; pH 7.2), or 10 mM phosphate-buffered saline (PBST: potassium phosphate buffer, 150 mM NaC1, 0.3% [v/v] Twcen 20, pH 7.2) for 30 min. Both buffers contained 1% bovine serum albumin, fraction V powder (BSA; Sigma Chemical Company., St. Louis, Mo., USA). Grids were then transferred to 25 gl drops of antibody (1 : 200, diluted in either TBST-BSA or PBST-BSA) for 60 min. Grids were then washed six times for 5 min each on 50-lal drops of buffer-BSA and transferred to 25-p.1 drops of 15-nm-diameter BioCell EM-grade protein A-gold (1:30, 1:40, or 1:50 diluted in buffer-BSA; Ted Pella, Redding, Calif., USA) for 60 min. Grids were then washed seven times for 5 min each on 50-lal drops: twice in buffer-BSA, twice in buffer only, and three times in double-distilled H20. Solutions were passed through 0.45-lam filters before dilution of antibody or protein A-gold, and all steps were carried out at room temperature. Preimmune serum and buffer-BSA alone were used as controls in place of anti-GaS serum. Sections, either unstained or contrasted with uranyl acetate and lead citrate, were examined with a Philips (Eindhoven, The Netherlands) EM300 transmission electron microscope at 80 kV. Morphometry and quantification of gold label. Areas of cell components were quantified using the point-counting method of Weibel (1979). Total numbers of gold particles over cell components were determined by counting at least 20 cells of each type, taken from several different tissue samples. Density of label was expressed as the number of gold particles per l.tm 2 a r e a .
Analysis of labeled photosynthase. Plants were illuminated with a water-filtered incandescent 1000-W metal-halide lamp providing approx. 800 gmol photons 9m 2 - s- 1 (photosynthetically active radiation) at plant level. One hour later a mature leaf was enclosed in a plastic bag and 14CO2 (2.0 MBq), generated in the
Fig. 1. A S D S - P A G E of zucchini leaf crude extract with and without purified GaS, and prestained high-range protein molecularweight markers. A 12.5% separating gel was used and stained with Amido Black. Molecular-weight-marker lane was loaded with 7 ~tl, while all sample lanes were loaded with 20 lal. Lanes are as follows : 1 98% (v/v) crude extract, 1.6% purified GaS; 2 96.2% crude extract, 3.2% purified GaS; 3 93.8% crude extract, 6.2% purified GaS; 4 87.6% crude extract, 12.4% purified GaS; 5 75% crude extract,
25% purified GaS; 6 molecular weight markers; 7 50% crude extract, 50% purified GaS; 8 crude extract only. B Western blot on nitrocellulose of proteins from the S D S - P A G E of A, probed with anti-GaS polyclonal antibodies, diluted 1 : 500. Lanes are identified as in A. Darts indicate the position of the 38-kDa GaS protein. Note the presence of a weakly staining band just below the heavily stained band in lane 7 of B
Fig. 2A, B. Portions of minor-vein phloem cells from squash leaf tissue prepared by high-pressure freezing and freeze substitution. Sections are unstained to better illustrate the presence of gold particles. A Intermediary cell from section incubated in preimmune serum, followed by protein A-gold. Cell is essentially unlabeled; only a single gold particle (dart) is present. Dense portion of cell in lower middle of figure is cytoplasm, an artifact resulting from
high pressure freezing. V, vacuole, x 19152; bar=0.5 lam. B Two intermediary cells (/) and a sieve-tube member (S) from a section incubated in anti-GaS serum, followed by protein A-gold. Note high density of labelling over cytosol, with virtually no label over mitochondria (M) or vacuoles (I1). A single gold particle (dart) is present over the lumen of the sieve-tube member, x 18992; bar = 0.5 ~tm
D.U. Beebe and R. Turgeon: Stachyose synthesis in Cucurbita leaves
357
barrel of a syringe by addition of excess 80% lactic acid to Na2 14CO3 (6.6 9 105 MBq 9mmol-1), was injected into the bag without delay. Thirty seconds later the bag was removed and after a further 60 s in room atmosphere the leaf was excised, quickly placed between stainless steel screens, and frozen in powdered, solid CO2. Frozen tissue was lyophilized as described by Weisberg et al. (1988), except that the tissue was not compressed. For whole-tissue sampling, discs (5.2 mm diameter) of lyophilized tissue were removed with a cork borer. To obtain samples of mesophyll, free of minor and major veins, the upper (adaxial) surface of the lyophilized tissue was gently abraded with fine sandpaper (400, wet or dry; 3M, St. Paul, Minn., USA), under a dissecting microscope, to remove the epidermis and some of the underlying mesophyll cells. More of the mesophyll was then removed with a camel-hair brush until the minor veins were just visible. Care was taken not to abrade the veins. Leaf discs and mesophyll samples were extracted in a mixture of methanol: water: chloroform (12:3:5 by volume) at 70 ~ for 1 h (Haissig and Dickson 1979). Water (3 ml per 5 ml solvent) was then added to cause phase separation and, following centrifugation in a clinical centrifuge to separate the phases, the aqueous portion was passed through ion-exchange membranes (Bio-Rex AG50W cation membrane [H + form] and AGI anion membrane [carbonate form]; Bio-Rad, Richmond, Calif., USA). Sufficient unlabeled sugars were added to the eluate to allow subsequent visualization and the sugars were resolved by two-dimensional TLC (Turgeon and Gowan 1990). Spots from vanillin-sprayed plates (Touchstone and Dobbins 1983, p. 173) were scraped into scintillation vials and the radioactivity counted.
Sink-source transition. A single mature leaf was enclosed in a plastic bag while attached to the plant and labeled with 14CO2 (0.5 MBq) as described above, for 5 min. Two hours later, samples were removed from a growing leaf that was passing through the sinksource transition. The samples were chemically fixed for immunolocalization as described above and the rest of the leaf was quickly excised, placed between stainless-steel screens, flash-frozen in solid CO2, lyophilized, and autoradiographed (Turgeon and Gowan 1992).
Fig. 3. A Portion ofa palisade-mesophyllcell from squash leaf tissue prepared by high-pressure freezing and freeze substitution. Section was incubated in anti-GaS serum, followed by protein A-gold. Section is unstained to better view gold particles. Only two gold particles (darts) are present over the chloroplast. Label is absent from vacuole (V) and other components of the cell. IS, intercellular space; SG, starch grain. • 19152; bar = 0.5 jam. B Portion of minor-
vein phloem from zucchini leaf tissue undergoing sink-to-source transition. Tissue was chemically fixed. Section was incubated in anti-GaS serum, followed by protein A-gold, and is unstained to better view gold particles. Label is absent from ail cell types. Only a single gold particle (dart) is present over the cytosol of a vascularparenchyma cell (P). CC, companion cell; L intermediary cell; S, sieve-tube member. • 19152; bar = 0.5 lam.
Results Specificity and sensitivity of the anti-GaS antibodies. W e s t e r n - b l o t a n a l y s i s i n d i c a t e d t h a t the a n t i b o d i e s recognize a 3 8 - k D a G a S d o u b l e t b a n d , t h e l a r g e r o f the t w o b a n d s s t a i n i n g m u c h m o r e i n t e n s e l y t h a n the s m a l l e r (Fig. 1 B, b e g i n n i n g in lane 2 a n d c l e a r l y b y l a n e 3). B o t h b a n d s o f the d o u b l e t h a v e G a S activity, verified b y t h e i r a b i l i t y to c a t a l y z e the synthesis o f g a l a c t i n o l f r o m myo-inositol a n d U D P - g a l a c t o s e in v i t r o (Phillip K e r r , p e r s o n a l c o m m u n i cation). These results i n d i c a t e t h a t for W e s t e r n b l o t t i n g , a p p r o x . 20-30% g r e a t e r e n z y m e a c t i v i t y t h a n t h a t p r e s e n t in the c r u d e e x t r a c t (Fig. 1 B, l a n e 8) is r e q u i r e d for a clear signal to be o b s e r v e d . T h e s e results also s h o w t h a t the a n t i b o d i e s d o n o t c r o s s - r e a c t w i t h a n y o f the o t h e r p r o t e i n s p r e s e n t in t h e c r u d e e x t r a c t (Fig. 1B, L a n e
358
D.U. Beebe and R. Turgeon: Stachyose synthesis in Cucurbita leaves
Table 1. Distribution of protein A-colloidal gold particles in transverse sections of mature squash leaves following incubation with immune serum (anti-GaS antibodies) or preimmune serum. Data Cell type
Intermediary Bundle sheath Palisade mesophyll Spongy mesophyll
Treatment
Immune Preimmune Immune Preimmune Immune Preimmune Immune Preimmune
are mean numbers of gold particles per I~m 2 of cross sectional area. SEs are in parentheses. NA = not applicable, NP = not present in sections
Cell compartment Cytosol Nucleus
Mitochondria
Chloroplasts
Vacuole
Cell wall
1.49 (0.20) 0.03 (0.01) 0.07 (0.01) 0.03 (0.01) 0.10 (0.01) 0.04 (0) 0.06 (0.01) 0.03 (0)
0.20 (0.03) 0.01 (0,01) 0.16 (0.09) 0.20 (0.06) 0.12 (0,03) 0.10 (0.02) 0.12 (0.03) 0.12 (0.04)
NA NA 0.16 (0.02) 0.10 (0.02) 0.15 (0.01) 0.08 (0.01) 0.17 (0.02) 0.11 (0.01)
0.08 (0.02) 0 (0) 0.02 (0) 0 (0) 0.01 (0) 0 (0) 0.02 (0) 0 (0)
0.24 (0.03) 0.02 (0.01) 0.67 (0.08) 0.04 (0.01) 0.35 (0.05) 0.03 (0.01) 0.70 (0.07) 0.12 (0.03)
0.96 (0.30) 0.04 (0.01) 0.03 (0.01) 0.02 (0.01) 0.18 (0.02) 0.07 (0.01) NP NP
8). Anti-GaS serum, f r o m the bleed selected for the immunolocalization study, gave a positive signal at a 1 : 10,000 dilution against 1 ng (based on enzyme activity) of purified GaS (data not shown). N o reaction product was detected over control slots. Immunolocalization o f GaS. Polyclonal antibodies to GaS labeled the cytosol o f intermediary cells much more densely than did p r e i m m u n e serum (Fig. 2). In other cell types, counts of gold particles over the cytosol were the same as, or slightly higher than, those over control sections incubated in p r e i m m u n e serum (Fig. 3A; Table 1). Since these cytosol counts in other cell types were elevated no m o r e than counts over cell walls, chloroplasts, and mitochondria, we attributed the extra signal to nonspecific binding. Cell walls were particularly "sticky", as has been noted in other immunolocalization studies (see H e r m a n 1989). N o appreciable difference in labeling patterns was found in comparisons o f the two cultivars used. Although the patterns of labeling were the same in chemically- and cryo-fixed samples, the density of label was greater in the latter. Within intermediary cells, specific labeling by gold particles was not detected over mitochondria or vacuoles (Table 1). Significant numbers o f gold particles were found over intermediary-cell nuclei, but not over the nuclei o f other cell types. Therefore, although the signal over the intermediary-cell nucleus was not as strong as that over the cytosol, it appeared to be specific for the GaS antibody. As a test of specificity for GaS, we used immature (importing) tissue f r o m leaves that were approximately half-grown; i m m a t u r e tissue does not synthesize raffinose or stachyose (Turgeon and W e b b 1975). Translocation experiments were performed on these plants immediately before the samples were removed to determine the location o f the sink-source b o u n d a r y in the sampled leaves (Turgeon 1989). Tissue that was close to the boundary, but still importing, was used for immunolocalization studies (Fig. 4). In this immature tissue, no label above control levels was detected in any cell type, including intermediary cells (Fig. 3B). In larger m i n o r veins there are two types of abaxial c o m p a n i o n cells: intermediary cells and "ordinary"
Fig. 4. Autoradiograph of zucchini leaf undergoing sink-to-source transition that was sampled to provide the section illustrated in Fig. 3B. Blackened areas indicate regions that imported radiolabeled sugars from a source leaf exposed to 14CO2. Stippled circles indicate actual areas sampled. Leaf area is reduced 40% by lyophilization. x 1; bar = 1.0 cm c o m p a n i o n cells that are similar in appearance to the minor-vein companion cells of species that do not translocate raffinose sugars (Turgeon et al. 1975). These "ordinary" companion cells, found usually in the interior of the vein and having relatively few plasmodesmatal connections to surrounding cells, were not labeled by antibodies to GaS (Fig. 5) in mature leaves. Also, there was no specific labeling of the adaxial phloem, in which the c o m p a n i o n cell is not specialized as
D.U. Beebe and R. Turgeon: Stachyose synthesis in Cucurbita leaves
359
Fig. 5. Portions of abaxial phloem of a large minor vein from squash leaf tissue prepared by high-pressure freezing and freeze substitution. Sections are unstained to better illustrate the presence of gold particles. A Section was incubated in preimmune serum, followed by protein A-gold. Label is virtually absent from all cell types. Only a single gold particle (dart) is present over the plasmalemma of the companion cell (CC). L intermediary cell; M, mitochondria; P,
vascular-parenchyma cell; S, sieve-tube member; V, vacuole. x 18992; bar=0.5 gm. B Section was incubated in anti-GaS, followed by protein A-gold. Note high density of label over the cytosol of the intermediary cell (/) and the virtual lack of labelling elsewhere. Label seen over other cell types is due to nonspecific binding. Ceils and cell components are identified as in A. x 18992; bar = 0.5 gm
an intermediary cell (not shown). It is unlikely that stachyose is synthesized in adaxial phloem since the solute content o f these cells is low (Turgeon and Hepler 1989) and, in m i c r o a u t o r a d i o g r a p h y studies with Cucumis melo leaves, they are not labeled following application of 1~CO2 (Schmitz et ah 1987).
tinol, and raft• oligosaccharides are labeled very soon after 14CO z is applied (20 s or less; not shown). To determine whether labeled sugars were present in mesophyll, samples o f this tissue were obtained by abrasion and the neutral fraction was analyzed by T L C (Table 2). Labeled galactinol, raffinose, and stachyose were present, although in very small amounts, while approx. 80 % of the label in the neutral fraction was in sucrose. In the whole-tissue samples, which contained veins as well as mesophyll, the p r o p o r t i o n o f label in
Label• of sugars with 1"C. The immunolocalization technique failed to demonstrate the presence of G a S in the mesophyll, even though gatactinol ( M a d o r e and W e b b 1982; Schmitz and Holthaus 1986; Holthaus and Schmitz 1991) and stachyose ( M a d o r e and W e b b 1982; M a d o r e et al. 1988) synthesis has been detected in this tissue in cucurbits. However, immunolocalization has limited sensitivity. Therefore, a m o r e sensitive procedure was developed that coupled radiolabeling with physical isolation of mesophyll. Leaves that were still attached to the plant were exposed to 14CO2 for 30 s and, following a further 60-s chase in r o o m atmosphere, the labeled c o m p o u n d s were immobilized by flash-freezing and the tissue was lyophilized. The relatively short 90-s total time period was employed so that there would be only limited m o v e m e n t of labeled c o m p o u n d s between cell types. Preliminary experiments indicated that sucrose, galac-
Table 2. Radiolabeled compounds in mature zucchini leaf tissue. Leaves were exposed to 14COz for 30 s followed by 60 s in room atmosphere, then flash-frozen and lyophilized. Assays were conducted on isolated mesophyll tissue and whole-tissue. Results are expressed as the percentage of radiolabel in the neutral fraction. Values are means • SE Radiolabeled compound
Mesophyll tissue
Whole-leaf tissue
Sucrose Galactinol Raft• Stachyose Other
80.6i 3.0 • 1.7• 1.9 • 12.9 •
42.8 • 6.2 • 7.4• 9.9 • 33.8 •
1.1 0.3 0.4 0.2
3.6 0.4 1.1 2.3 2.8
360 galactinol, raffinose, and stachyose was higher than in the mesophyll samples, while there was proportionately less sucrose (Table 2); these results are expected if galactinol, raffinose, and stachyose are made in the veins. In the mesophyll there was more label in galactinol than in either raffinose or stachyose, while in the whole-tissue, the label was more evenly distributed between the three compounds. Discussion
Synthesis and transport of sucrose, the sole sugar translocated in many species, has been studied extensively: it is made in the mesophyll cells of the leaf and is then loaded, against a concentration gradient, into the phloem (Lucas and Madore 1988). Less well studied is the synthesis of raffinose oligosaccharides, the predominant export sugars of many plants, especially tropical trees and vines. We demonstrate here that, in C. pepo, raffinose, stachyose, and the galactose donor for these molecules are synthesized in the transport tissue itself, i.e., in the intermediary cells of the minor-vein phloem. Evidence to date has pointed to mesophyll cells as the source of galactinol (Madore and Webb 1982; Schmitz and Holthaus 1986; Holthaus and Schmitz 1991) and it has been postulated that this precursor to raffinose and stachyose then migrates to the veins (Schmitz and Holthaus 1986; Holthaus and Schmitz 1991). However, it should be noted that experiments, cited above, demonstrating galactinol synthesis in the mesophyll, do not rule out the possibility that it is also made in the phloem. Indeed, the results presented here indicate that galactinol is probably made in both locations. The immunological evidence clearly indicates that there is GaS in intermediary cells, and short-term experiments with 14CO 2 reveal the presence of labeled galactinol in the mesophyll. Why do antibodies to GaS not label the mesophyll? The most likely reason is that the enzyme is present in too low a concentration for detection by immunocytochemistry. We found that cryofixation increased sensitivity by at least tenfold over chemical fixation but, even so, a small amount of enzyme dispersed over the entire mesophyll cytosol may be present at too low a concentration to aliow labeling at a level distinguishable from nonspecific labeling over control tissue. This may also be the reason that Holthaus and Schmitz (1991) did not detect stachyose synthase in the mesophyll by immunocytochemistry. It is possible that the absence of label in the mesophyll is due to the specificity of the antibodies for an intermediary-cell isoenzyme; indeed, the purified enzyme was resolved as a doublet protein band. However, the antibodies were polyclonal and both members of the protein doublet were used to raise them, yet specific signal was present only over the intermediary cells in tissue sections. The fact that two protein bands had GaS activity does not necessarily indicate the presence of isoenzymes: the minor, lower molecular-weight, member of the doublet could also be a slightly degraded form of the enzyme. Smith et al. (1991) found no evidence for the presence of
D.U. Beebeand R. Turgeon : Stachyosesynthesis in Cucurbita leaves galactinol synthase isoenzymes in C. pepo leaves, but they did not rule out the possibility that they are present. Another explanation for the presence of labeled compounds in the mesophyll after exposure of the leaf to 1 4 C 0 2 is that some transport from the veins to the mesophyll took place during the course of the experiment. Indeed, plasmodesmata between intermediary cells and the mesophyll are open to passage of low-molecularweight compounds (Turgeon and Hepler 1989). We have speculated that the plasmodesmata between intermediary cells and bundle sheath allow the passage of smaller molecules such as sucrose and galactinol while restricting the passage of tri- and tetrasaccharides (Turgeon 199l). If this is the case, leakage from veins to the mesophyll could have been selective. Still another explanation for the presence of labeled galactinol, raffinose, and stachyose in mesophyll samples is that they are contaminants, introduced by movement of label during lyophilization or subsequent handling, or by the accidental abrasion of veins. This seems highly unlikely, however. Our freeze-preservation procedures yield macroautoradiographs of minor veins with high resolution, indicating very little, if any, spread of label (for example, see Turgeon 1987). Furthermore, the minor veins are easily recognized in lyophilized tissue and we discarded any mesophyll samples that could have been contaminated with vein tissue. If the label detected in mesophyll samples had come from the veins, the proportions of galactinol, raffinose, and stachyose would have been the same in mesophyll and whole-tissue samples and they were not. Since galactinol (Madore and Webb 1982; Schmitz and Holthaus 1986; Madore et al. 1988; Holthaus and Schmitz 1991) and stachyose (Madore and Webb 1982; Madore et al. 1988) synthesis has been detected in isolated protoplasts and tissues from cucurbit leaves, we favor the straightforward interpretation that the entire pathway to stachyose is present in both mesophyll and intermediary cells. We came to the same conclusion in studies of sugar synthesis in Coleus blumei (Turgeon and Gowan 1992). Why is stachyose synthesized in the mesophyll as well as the phloem? Perhaps the stachyose in mesophyll cells is stored during the day and catabolized at night while stachyose that is synthesized in the phloem is used for immediate export. The discovery that galactinol, raffinose, and stachyose are made in the phloem raises several interesting questions. How is specificity for export sugars conferred? In those plants that transport sucrose alone, specificity is thought to be the result of selective release of this sugar to the apoplast and-or selective, carrier-mediated uptake from the apoplast into the sieve-element-companion-cell complex (Geiger and Fondy 1991). This explanation does not suffice for raffinose and stachyose. What is the driving force for solute accumulation in the intermediary cell? It is unlikely to be proton symport from the apoplast, as postulated for species that translocate only sucrose, since experiments on Coleus (Turgeon and Gowan 1990, 1992) and the cucurbits (Madore and Webb 1981; Schmitz et al. 1987) indicate that sucrose
D.U. Beebe and R. Turgeon: Stachyose synthesis in Cucurbita leaves and other potential precursors to stachyose p r o b a b l y do not enter the minor-vein phloem by this route. Yet solute levels are as high as in other species: the intermediary cells of C. pepo (Turgeon and Hepler 1989) and Coleus (Fisher 1986) do not plasmolyze in 1.2 M mannitol. We have postulated that the intermediary cell acts as a "molecular size-discrimination t r a p " by allowing lowmolecular-weight precursor(s) to enter the cell while preventing larger sugars such as raffinose and stachyose from diffusing away (Turgeon 1991). Solute accumulation could also be a consequence o f active transport within the intermediary cell (Turgeon 1991). Finally, how is synthesis o f sucrose in the mesophyll coordinated with raffinose and stachyose synthesis in the phloem, and how is carbon partitioning regulated in these plants? Answers to these questions will come only after we have a m u c h m o r e detailed understanding of sugar synthesis in stachyose-translocating species. We thank John Pierce, Phillip Kerr, and Bruce Schweiger for the gift of anti-GaS antibody and M.K. Kandasamy for helpful discussions. 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 Fisher, D.G. (1986) Ultrastructure, plasmodesmatal frequency, and solute concentration in green areas of variegated Coleus btumei Benth. leaves. Planta 169, 141-152 Geiger, D.R., Fondy, B.R. (199t) Regulation of carbon allocation and partitioning: status and research agenda. In: Recent advances in phloem transport and assimilate compartmentation, pp. 1-9, Bonnemain, J.-L., Delrot, S., Lucas, W.J., Dainty, J., eds. Ouest Editions, Nantes, France Haissig, B.E., Dickson, R.E. (1979) Starch measurement in plant tissue using enzymatic hydrolysis. Physiol. Plant. 47, 151-157 Herman, E.M. (1989) Colloidal gold labeling of acrylic resinembedded plant tissues. In: Colloidal gold: principles, methods, and applications, vol. 2, pp. 303-322, Hayat, M.A., ed. Academic Press, New York Holthaus, U., Schmitz, K. (1991) Distribution and immunolocalization of stachyose synthase in Cucumis melo L. Planta 185, 479-486 Kaudler, O., Hopf, H. (1982) Oligosaccharides based on sucrose (sucrosyl oligosaccharides). In: Encyclopedia of plant physiology, N.S., vol. 13A: Plant carbohydrates I, Intracellular carbohydrates, pp 348-383, Loewus, F.A., Tanner, W., eds. Springer, Berlin Heidelberg New York
361 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, Preiss, J., ed. Academic Press, New York Madore, M.A., Webb, J.A. (1981) Leaf free space analysis and vein loading in Cucurbita pepo. Can. J. Bot. 59, 225--2257 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. (1985) 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 Smith, P.T., Kuo, T.M., Crawford, C.G. (1991) Purification and characterization of galactinol synthase from mature zucchini squash leaves. Plant Physiol 96, 693-698 Touchstone, J.C., Dobbins, M.F. (1983) Practice of thin layer chromatography, 2nd edn. Wiley, New York Turgeon, R. (1987) Phloem unloading in tobacco sink leaves: insensitivity to anoxia indicates a symplastic pathway. Planta 171, 73-81 Turgeon, R. (1989) The sink-source transition in leaves. Annu. Rev. Plant Physiol Plant Mol. Biol. 40, 119-138 Turgeon, R. (199 i) Symplastic phloem loading and the sink-source transition in leaves: a model. In: Recent advances in phloem transport and assimilate compartmentation, pp. 18-22, Bonnemain, 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., Gowan, E. (1992) Sugar synthesis and phloem loading in Coleus blumei leaves. Planta 187, 388-394 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., Webb, J.A. (1975) Leaf development and phloem transport in Cucurbita pepo: carbon economy. Planta 123, 53~52 Turgeon, R., Webb, J.A., Evert, R.F. (1975) UItrastructure of minor veins of Cucurbita pepo leaves. Protoplasma 83, 217-232 Weibel, E.R. (1979) Stereological methods, vol. 1: Practical methods for biological morphometry. Academic Press, London Orlando Weisberg, L.A., Wimmers, L.E., Turgeon, R. (1988) Photoassimi!ate-transport characteristics of nonchlorophyltous and green tissue in variegated leaves of Coleus blumei Benth. Planta 175, 1-8 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
