- Email: [email protected]
Uptake of [U-14C]glucose into subcellular particles of wheat seedling roots
ARCHIVES
OF
Uptake
BIOCHEMISTRY
AND
BIOPHYSICS
of [U-14C]GI ucose
163, 702-711 (1972)
into
Subcellular
Seedling R. JILKA, Kansas
State University,
AND
P. NORDIN
of Biochemistry,
Received
of Wheat
Roots’
0. BROWN, Department
Particles
May
Manhattan,
Kansas
66606
1, 1972
Etiolated seedling roots from Triticum vulgarae were pulsed 2 hr with [U-W]glucase. Root tips were homogenized in a glass-Teflon homogenizer. After preliminary purification, the subcellular particles were separated on a sucrose density gradient. Particulate bands were obtained corresponding to densities of 1.137(T), 1.167(M), 1206(B), and a pellet >1.22(P). Subcellular particles that could be identified by electron microscopy in the respective fractions were as follows: T-golgi dictyosomes, M-microsomes and perhaps lysosomes, B-mitochondria and vesicles or lysosomes, P-mitochondria and lysosomes or vesicles. Each fraction contained base-soluble, radioactive, high molecular weight material, which was more heavily concentrated in the bottom band and pellet and was only slightly degraded by pronase. More than 50% of the pronase-resistant material from the combined fractions could be hydrolyzed to radioactive xylose, arabinose, and galactose by acid or hemicellulase. It was concluded that a hemicellulose-like polysaccharide is widely distributed throughout the subcellular particles of wheat seedling roots.
Current concepts of plant cell wall heteropolysaccharide biosynthesis invoke the Golgi apparatus and Golgi-derived secretory vesicles (14). Wheat roots rapidly incorporated tritiated glucose into Golgi dictyosomes, and electron microscope autoradiographic studies demonstrated that these organelles were among the first to be labeled (5). The labeled material was subsequently transported to the slime layer of the plasmalemma via Golgi-derived secretory vesicles. The same process has been observed for other plant tissues (6-S). Thus it has been proposed that the Golgi apparatus carries out the biosynthesis and packaging of hemicellulose and pectin into secretory vesicles which are transported to the cell wall (3, 4). Histochemical studies also support that concept (9, 10). Cellulose, however, is probably synthesized by an entirely different route (2).
MATERIALS
1 Contribution No. 137, Department of Biochemistry, Kansas Agricultural Experiment Station, Kansas State University, Manhattan, KS 66506; taken in part from a Master’s thesis (R.J.) and a Ph.D. dissertation (O.B.).
@ 1972 by Academic Press, of reproduction in any form
Inc. reserved.
AND
METHODS
Radioactive material. [U-Wlglucose, specific activity 240 mCi/mM was obtained from Schwarz Bioresearch, Orangeburg, NY. Enzymes. Hemicellulase, control number 7196, 702
Copyright All rights
It is difficult to correlate biochemical experiments with histochemical and autoradiographic studies. Thus, while particulate synthetase systems which catalyze the transfer of sugars from sugar nucleotides to a growing polysaccharide chain have been isolated from a variety of plant sources (ll-17), identity of the synthetase particles is not clear (IS-20), primarily because it is so difficult to break plant cell walls without damaging the organelles. Crude Golgi preparations from wheat roottip cells contain hemicellulose (21). Direct evidence is presented here that hemicellulose molecules are present in Golgi dictyosomes as well as in faster sedimenting particles. A method based on that of Morre’ and Mollenhauer (22) was applicable for isolating relatively pure organelles from wheat root tissue.
POLYSACCHARIDES
OF PLANT
and pectinase, control number 6417, were obtained from Nutritional Biochemicals Co., Cleveland, OH. Both enzymes were precipitated twice from saturated ammonium sulfate at 0°C and dialyzed overnight against 3 liters of 1 mnn sodium acetate buffer (pH 5.0) before use. Pronase, B grade, 45,000 Pu/g, lot number 45046, was obtained from Calbiochem, Los Angeles, CA. Preparation and incuhtion of seedlings. Wheat seeds (Kansas varieties), previously treated 30 min with 0.5% formaldehyde were germinated in the dark at room temperature 60 hr on rust-proof screens sandwiched between moist filter paper. Only the roots grew through the screen. For radiometabolite incubation experiments, the intact seedlings were then pulsed 2 hr with 200 pCi [U-‘%]glucose in an incubation medium of 10 mM glucose and 1 mu potassium phosphate buffer (pH 6.5) applied to a filter paper with which the roots were in contact. The roots were recovered by “shaving” the screen wit,h a razor blade. Approximately 25-30 g of wheat roots were used for each experiment. Isolation of organelles. The isolation procedure is a modification of that of Morre’ and Mollenhauer (22). The etiolated root tips were ground in approximately 25 ml of a homogenization medium composed of 1% dextran, 1 mu calcium chloride, 75-100 mu glutaraldehyde, 0.5 M sucrose dissolved in a stock buffer, which consisted of 0.6001 M manganese chloride and 0.1 M sodium phosphate buffer (pH 7.2). Glutaraldehyde was omitted from the homogenizat,ion medium when the preparation was used to identify enzyme activities present in the isolated particles. The roots were ground in a glass-Teflon homogenizer machined to a clearance of 0.026 in. The homogenate was squeezed through several layers of cheesecloth. All operations of the isolation procedure were performed at 4°C and all sucrose solutions used were made with stock buffer. The filtrate was transferred to cellulose nitrate centrifuge tubes and centrifuged 1 hr at 48009 in a Beckman SW-41 rotor. The supernatant solution was transferred to another cellulose nitrate tube, and a 0.5 ml 1.8 M sucrose pad was layered underneath the supernatant. The tube was centrifuged 1 hr at 43,000g and a band of particulate material was collected on top of the sucrose pad. This material was removed and resuspended in 2 ml of stock buffer. Approximately 10 ml of 0.5 M sucrose solution was layered underneath the resuspended material and another 0.5 ml 1.8 M sucrose pad was layered underneath the entire solution. The tube was centrifuged 1 hr at 43,500g and the particulate material sedimenting on top of the 1.8 M sucrose pad was designated as “washed crude Golgi.” Washed crude Golgi was resuspended in 1.5-2.0
SUBCELLULAR
703
PARTICLES
ml of stock buffer and layered on top of a discontinuous sucrose gradient in a 6-ml cellulose nitrate centrifuge tube. The sucrose gradient consisted of 0.5 ml of 1.5 M sucrose, 1.0 ml of 1.25 M sucrose, 1.0 ml of 1.0 M sucrose, and 1.0 ml of 0.5 M sucrose. The tube was centrifuged in a SW-50 L rotor 3 hr at 102,OOOg. Isopyknic conditions developed, as evidenced by the formation of a continuous gradient, after centrifugation. The four particulate bands resulting on the density gradient which were clearly evident were designated as top, middle, bottom, and pellet. The particulate material was pipetted off the gradient, resuspended in stock buffer, and pelleted. Enzymatic determinations, prdtein assays (23), or chemical extractions were performed on the pelleted material. Alternatively, to determine the density at which each band sedimented or to determine the radioactivity incorporated into each band, the bottom of the density gradient tube was pierced and O.l-ml fractions were collected for refractive index measurements (from which densities were calculated) or for liquid scintillation counting. Electron microscopy. Pelleted materials in the centrifuge tubes were drained and wiped dry with tissue paper. The pellets were fixed with 1% osmium tetroxide in 0.05 M sodium phosphate buffer (pH 7.2) for 24 hr at 4°C. The materials were aspirated of the osmium tetroxide and dehydrated with a series of ethanol solutions. The dehydrated materials were infiltrated and embedded by the
IO
20
30
ml
FIG. 1. Sucrose density gradient radioactivity scan. Wheat seedlings were incubated with [U-‘4C]glucose, and subcellular fractions were isolated on a density gradient. After piercing the bottom of the density gradient centrifuge tube, O.l-ml fractions were collected on glass fiber filters, dried, and counted. T = top band M = middle band B = bottom band
d = 1.137 g cm-3 d = 1.167 g cmp3 d = 1.206 g cm-”
704
JILKA,
BROWN,
method of Kushida (24) at 50°C for 46 hr: the blocks were sectioned on the LKB Ultrotome with glass knives, picked up on copper grids, and double stained with uranyl acetate and lead citrate. The stained sections were rinsed and viewed in a modified RCA EMU 2-D electron microscope. Chemical extraction of particulate fractions. The pelleted particulate material was extracted at 4°C with methanol-chloroform-0.2 M formic acid (12:5:3 v/v/v) (25) % hr. After low-speed centrifugation and removal of the supernatant, the remaining insoluble material was extracted with
FIG. 2. Positive stained trifugation. Magnification:
electron micrograph X 47,300.
AND
NORDIN
0.05 M KOH x hr at 100°C. The lipid layer was extracted once with distilled water and assayed for radioactivity. The KOH hydrolyzate was filtered and neutralized with concentrated KH~POI Resulting material was then treated with 200 fig of pronase 50 hr at 37°C with 4y0 ethanol added as preservative (26). The hydrolyzate was subjected to Sephadex G-75 chromatography with 5 mu sodium phosphate buffer (pH 8). One-milliliter fractions were collected and counted as described. Enzymatic hydrolysis of labeled pronase-resistant KOH-soluble material. Hemicellulase was checked
of top band from
density
gradient
cen-
POLYSACCHARIDES
OF PLANT
for hydrolytic activity against hemicellulose, polygalacturonic acid, and denatured bovine serum albumin. The hemicellulsse released various hexoses and pentoses from hemicellulose and a very small amount of galacturonic acid from polygalacturonic acid. Less than 5% of the amino acids (by weight) was released from the denatured BSA. The pronase-resistant KOH-soluble material of the washed crude Golgi (fraction I) was treated at room temperature at pH 5.0 with 50 ~1 each of the pectinase and hemicellulase enzyme solutions for 2 hr. The reaction was stopped by gentle heating and the hydrolyzate was subjected to Sephadex
FIG. 3. Positive stained trifugation. Magnification:
electron micrograph X 41,170.
SUBCELLULAR
705
PARTICLES
G-75 column chromatography. Another sample of fraction I was either treated with the enzymes 24 hr at pH 5.0 at room temperature or hydrolyzed with 1 N HCl 2 hr at 100°C. Products were examined by silica gel thin layer chromatography. Acetone-water (9:l v/v) was used to separate monosaccharides on sodium acetate-impregnated plates, and butanol-acetic acid-water (2: 1: 1 v/v) was used to separate uranic acids (27). The hydrolyzate was cochromatographed with 1% sugar standards. After drying, the area above the hydrolyzate origin was divided into horizontal l-cm strips and scraped into scintillation vials for
of middle
band from density
gradient
cen-
706
JILKA,
BROWN,
counting. The remainder of the plate was sprayed with diphenylamine-aniline (27)) and position of the authentic sugars noted with respect to the l-cm strips. Confirmatory tests for sugars were obtained by paper chromatography [butanol-pyridine-water; 10:3:3 v/v with silver nitrate dip (21)]. Liquid scintillation counting. A fluor of 0.5% PPO in toluene was used for counting in a Beckman LS-200B Scintillation Counter. Fractions from column chromatography experiments were prepared for counting by lyophilization in a counting vial containing a glass fiber filter. Silica gel
FIG. 4. Positive stained trifugation. Magnification:
electron micrograph x 47,150.
AND
NORDIN
scrapings from thin plates were counted directly in the vial containing the fluor. As the quenching level was essentially constant, no quenching corrections were made. RESULTS
Uptake of 14CActivity into Bands Figure 1 shows a density gradient radioactivity scan of the particulate bands from wheat seedlings incubated in [UJ4C]glucc.se. Incorporation of 14Cactivity into each band is evident. The pellet was labeled to approxi-
of bottom
band from density
gradient
cen-
POLYSACCHARIDES
OF PLANT
mately the same extent as the bottom band. Electron micrographs corresponding to radioactive peaks are shown in Figs. 2-5. Electron Microscopic Identijcation of the Bands Bands isolated on the density gradient were identifiable morphologically. Figure 2 shows an electron micrograph of the top band, which contains characteristic (28) stacks of Golgi cisternae with no other recog-
FIG. 5. Positive tion. Magnification:
stained electron X 34,600.
micrograph
SUBCELLULAR
707
PARTICLES
nizable cell organelles as contaminants, but with the characteristic transition and fr’agmentation forms of the Golgi, which result from isolation procedures, and which have been described by Morre’ and Mollenhauer (22). The top band sedimented at a density of 1.137 g/gm3, which is comparable to the density of Golgi apparatus isolated by other workers (19, 29). The middle band sedimented at a density of 1.167 g/cm3, which is characteristic of that
of pellet
from density
gradient
centrifuga-
708
JILKA,
BROWN,
of isolated plant lysosomes (30, 31), but its appearance (Fig. 3) is predominantly that of microsomes (29, 32, 33) with some dictyosomal and lysosomal contamination. The bottom band, with a density of 1.206 g/cm”, correlates with the density of plant mitochondria isolated by other workers (19). and the electron micrograph shown in Fig. 4 has a high proportion of the easily recognized mitochondrial structures (34). Several small dense vesicles bound by a single membrane also appear in this band. The pellet material (Fig. 5) contains some mitochondria in addition to dense single membrane-bound particles and some very dense fragmentation products. Ident$cation
of Biosynthetic Enzymes in Bands
AND
NORDIN
Polysaccharide was identified by its elution at the void volume on Bio-gel P-2 and by observing that it yielded radioactive glucose upon hydrolysis with acid. Radioactive sucrose was identified by its Rf and by its susceptibility to invertase. UDPG hydrolase was identified by the formation of radioactive glucose during incubation. Comparisons on the basis of protein content showed that glucan synthetase was more concentrated in the pellet and bottom band, while UDPG hydrolase was so active in the top Golgi band that synthetase activities were minimal. Sucrose synthetase was most heavily concentrated in the bottom band. Significant transferase activities toward n-xylose and N-acetylglucosamine were not demonstrated.
Several enzymes exhibiting activity toward UDP-glucose could be demonstrated by incubating aliquots of the bands and pellet separately with UDP-[14C]glucose and a mixture of monosaccharide acceptors (D-ghcase, n-fructose, D-xylose and N-acetylglucosamine) . For this purpose, glutaraldehyde was omitted in the homogenization medium, Quantitative assays were not possible because of competing reactions. However, the following enzymes could be identified by their products: glucan synthetase, UDPglucose hydrolase, and sucrose synthetase. TABLE DISTRIBUTION
Band
Top Middle Bottom Pellet
I
OF ACTIVITY IN SUBCELLULAR PARTICLES" 70 Lipid soluble 56.4 51.1 54.6 35.5
% KOH insoluble 20.8 25.8 20.8 34.1
22.6 22.8 24.6 30.5
0 Distribution of ‘4C activity in bands as percentages of total band activity. Aliquots of the lipid extract and the KOH-soluble material were counted on glass fiber filters. The KOH-insoluble material was counted direct in the counting solution. All figures represent an average of three determinations, except that top band figures represent an average of two det,erminations.
600
P
300 0
10
20
30
40
FIG. 6. Fractionation of pronaae hydrolyzate of KOH-soluble fraction on Sephadex G-75. The KOH-soluble fraction of each band was treated 50 hr with pronase and subjected to Sephadex G-75 chromatography on a 1 X 30 cm column eluted with 5 mu sodium phosphate buffer (pH 8). Onemilliliter fractions were collected, lyophilized, and counted.
POLYSACCHARIDES
OF PLANT
Chemical Distribution of Radioactivity @Bands Chemical distribution of the radioactivity of each band was investigated by fractionating each into three solubility classes: lipid soluble, KOH soluble, and KOH insoluble (Table I). Data in Table I indicate that the KOH-soluble labeled material was somewhat more concentrated in the pellet, although the other three bands contained significant quantities also. No other significant trends were evident. Effect of Pronase 012KOH-Soluble Materials
Labeled
Results of Sephadex G-75 chromatography of the pronase-treated KOH-soluble material of each band show that all bands have pronase-resistant high molecular weight material (Fig. 6). Pronase is a broad specificity proteolytic enzyme, so formation of low molecular weight products indicates the presence of protein. Pronase-resistant material is presumably all polysaccharide. Chemical Nature of Fraction I The high molecular weight KOH-soluble fraction resistant to pronase was designated
I
SUBCELLULAR
709
fraction I. For a study of its chemical nature it was isolated from washed crude Golgi, since “total activities incorporated” were low. Washed crude Golgi contain all the subcellular particles. Fraction I was treated with a hemicellulase-pectinase enzyme preparation and subsequently chromatographed on Sephadex G-75 (Fig. 7). Release of low molecular weight material was demonstrated. A silica gel, thin layer chromatogram scan of the enzyme hydrolyzate is shown in Fig. 8. Labeled substances were present migrating as galactose, arabinose, xylose, and an unidentified fast-running compound, and perhaps glucose. The labeled material present at the origin was immobile in a solvent for uranic acids (1-butanol-acetic acid-water 2 : 1: 2 v . v). Acid hydrolysis of fraction I and subsequent thin layer chromatography gave the results shown in Fig. 8. The only difference observed was that more xylose and less origin material were present in the acid hydrolyzate scan. No uranic acids could be demonstrated in either one. Confirmatory tests for xylose, arabinose, galactose and glucose were obtained by paper chromatography. The sugars reacted toward silver ni-
“0 J.
IO
PARTICLES
“G I
20
30
40
ml
FIG. 7. Fractionation of Fraction I hemicellulase-pectinase hydrolyzate on Sephadex G-75. Fraction I was treated 2 hr with the hemicellulase-pectinase enzyme system. The hydrolyzate was eluted on a 1 X 33 cm Sephadex G-75 column with 1 mu potassium phosphate buffer (pH 8). One-milliliter fractions were collected, lyophilized, and countedVo = void volume; VG = glucose elution volume.
JILKA,
;I
abed
a
b
e
c
d
BROWN,
f
e
f
cm
FIG. 8. Thin layer chromatography hydrolyzate tinase, lower (c) mannose, curonolactone.
of fraction I and pec(upper = hemicellulase = HCl). (a) galactose, (b) glucose, (d) arabinose, (e) xylose, (f) glu-
dip in the typical sugars.
trate
manner of reducing
DISCUSSION
The separation of subcellular particles reported here is an application of Morre and Mollenhauer’s (22) method to wheat root tips. It. appears that one can obtain morphologically distinct organelles from wheat root tips in sufficient quantity to perform biochemical studies. A polysaccharide is present in the Golgi fraction but ,clearly it is more heavily concentrated in the faster sedimenting bands. As the Golgi dictyosomes are labeled more rapidly than other organelles, 2 hr of incubation in labeled glucose is presumably suffi-
AND
NORDIN
cient for complete polysaccharide molecules to be synthesized (5). Thus, secretory vesicles, judged to separate at densities corresponding to the bottom and pellet fractions (lo), would be labeled also. This work, however, clearly demonstrated the presence of polysaccharide in all subcelMar fractions isolated from wheat root tips. The principal monosaccharides are xylose, arabinose and galactose which are characteristic of hemicellulose. Golgi preparations from pea seedlings (35) also have been reported to contain monosaccharides characteristic of hemicellulose. The fact that we find polysaccharide in all bands does not appear to be due to incomplete resolution or complexing by glutaraldehyde. Preparations without glutaraldehyde also contained a high molecular weight fraction in each band. Furthermore, the high molecular weight material is not easily removed by prolonged washing or sonication w . The enzyme studies demonstrated the localization of glucan synthetase in the bottom and pellet material. Other workers have also reported that these enzymes are found in particles migrating much faster than Golgi bodies during centrifugation (18). The only enzyme of significance that we found in the Golgi band was UDPG hydrolase, which agrees with previous studies (36). As plasmalemma fragments probably were present in our pellet fraction, it is tempting to conclude that heteropolysaccharide synthesis occurs primarily in the plasmalemma. However, secretory vesicles from the Golgi may also sediment. at densities corresponding to the pellet, and bottom fractions on the density gradient. Thus, the polysaccharide in the faster-sedimenting particles might reflect secretory granules. Currently available methods do not, permit their complete separation from other particles in wheat root tips. The “hemicellulose” found is, in any case, a precursory substance. When incubation in labeled glucose was followed by a pulse of “cold” glucose (21), radioactivity of the hemicellulose was greatly reduced. Our findings agree with earlier autoradiographic and histochemical studies (5, 6, 9, lo), showing cell wall precursors in plant cells.
POLYSACCHARIVES
OF PLANT
SUBCELLULAR
REFERENCES 1. NORTHCOTE, D. H. (1971) Endeavour 30,26-33. F., AND MCGINNIS, G. D. (1971) 2. SHAFIZADEH, Advan. Cnrbohyd. Chem. Biochem. 26, 297349. D. H. (1969) Essays Biochem. 6, 3. NORTHCOTE, 9@138. K. (1967) Annu. Rev. Plant 4. MUHLETHALER, Physiol. 18, l-24. J. 1). 5. NORTHCOTX, D. H., AND PICKETT-HR.IPS, (1966) Biochem. J. 98, 159-167. J. D. (1967) J. Ultrastruct. 6. PICKETT-HEAPS, Res. 18, 287-303. J. D. (1966) Planta 71, 1-14. 7. PICKETT-HEAPS, 8. WOODING, F. B. P. (1968) J. Cell Sci. 3, 71-80. J. D. (1968) J. Cell Sci. 3, 9. PICKETT-HICAPS, 5564. W. J., MORRE’, D. J., AND 10. VANDERWOUDE, BRACKER, C. E. (1971) J. Cell Sci. 8,331-351. 11. ELBICIN, A. D. (1969) J. Biol. Chem. 244, 16081616. J. B., AND HASSID, W. Z. (1966) 12. PRIDHAM, Biochem. J. 100, 21P. 13. KAUSS, H. (1967) Biochim. Biophys. Acta 148, 572-574. 14. BAILEY, R. W., AND HASSID, W. Z. (1966) Proc. Nat. Acad. Sci. USA 66, 158G-1593. E. F., AND HASSID, 15. FEINGOLD, D. S., NEUFELD, W. Z. (1959) J. BioZ. Chem. 234, 488-489. 16. KAUSS, H., SWANSON, A. L., ARNOLD, R., AND ODZUCK, W. (1969) Biochim. Biophys. Acta 192, 55-61. C. L., SWANSON, A. L., AND H~SSID, 17. VILLEMEZ, W. Z. (1966) Arch. Biochem. Biophys. 116, 446-452C. L., MCNAEJ, J. M., AND 18. VILLEMEZ, ALBERSHEIM, P. (1968) Nature (London) 218, 878-880. T. L., AND RAY, 19. RAY, P. M., SHININGISR,
20. 21. 22. 23.
24. 25. 26. 27.
28.
29.
30. 31. 32. 33.
34. 35. 36.
PARTICLES
711
M. M. (1969) Proc. Nat. Acad. Sci. USA 64, 605-611. LAMPORT, D. T. A. (1970) Annu. Rev. Plant Physiol. 21, 235-270. MERTZ, J., AND NORDIN, P. (1971) Phytochemistry 10, 1223-1227. MORRE’, J. D., AND MOLLENHBUER, H. H. (1964) J. Cell Biol. 23, 295-305. LOWRY, D. H., ROSEBROUGH, N. J., FARR, A. L., AND HANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. KIJSHIDA, H. (1967). J. Electronmicrosc. 16, 278-280. BIELESKI, R. L., .~ND YOUNG, R. E. (1963) Anal. Biochem. 6, 5468. NOMOTO, M., NARAHASHI, Y., AND MURAKAMI, M. (1960) J. Biochem. 48, 593-602. LEWIS, B. A., AND SMITH, F. (1969) in Thin Layer Chromatography (Stahl, E., ed.), pp. 807-837, Springer-Verlag, New York. CUNNINGHAM, W. P., MORRE’, J. D., AND MOLLENHAUZR, H. H. (1966) J. Cell Biol. 28, 169-179. MORRE’, J. I)., MOLLENHAUER, H. H., AND CHAMBERS, J. E. (1965) Exp. Cell Res. 38, 672-675. SEMBDENI, E. G. (1967) Pkznta 72, 91-118. MATILE, P. (1968) Planta 79, 181-196. HOR~ITZ, A. L., AND DORFMAN, A. (1968) J. Cell Biol. 38, 358-368. DALLMAN, P. R., DALLNER, G., BERGSTRAND, A., AND ERNSTER, L. (1969) J. Cell Biol. 41, 357-377. BUTLER, W. H., AND JUDAH, J. I>. (1970) J. Cell Biol. 44, 278-289. HARRIS, P. J., AND NORTHCOTE, D. H. (1971) Biochim. Biophys. Acta 237, 56-64. NOVIKOFF, A. B., AND GOLDFISCHER, S. (1961) Proc. Nat. Acad. Sci. USA 4’7, 802-810.
OF
Uptake
BIOCHEMISTRY
AND
BIOPHYSICS
of [U-14C]GI ucose
163, 702-711 (1972)
into
Subcellular
Seedling R. JILKA, Kansas
State University,
AND
P. NORDIN
of Biochemistry,
Received
of Wheat
Roots’
0. BROWN, Department
Particles
May
Manhattan,
Kansas
66606
1, 1972
Etiolated seedling roots from Triticum vulgarae were pulsed 2 hr with [U-W]glucase. Root tips were homogenized in a glass-Teflon homogenizer. After preliminary purification, the subcellular particles were separated on a sucrose density gradient. Particulate bands were obtained corresponding to densities of 1.137(T), 1.167(M), 1206(B), and a pellet >1.22(P). Subcellular particles that could be identified by electron microscopy in the respective fractions were as follows: T-golgi dictyosomes, M-microsomes and perhaps lysosomes, B-mitochondria and vesicles or lysosomes, P-mitochondria and lysosomes or vesicles. Each fraction contained base-soluble, radioactive, high molecular weight material, which was more heavily concentrated in the bottom band and pellet and was only slightly degraded by pronase. More than 50% of the pronase-resistant material from the combined fractions could be hydrolyzed to radioactive xylose, arabinose, and galactose by acid or hemicellulase. It was concluded that a hemicellulose-like polysaccharide is widely distributed throughout the subcellular particles of wheat seedling roots.
Current concepts of plant cell wall heteropolysaccharide biosynthesis invoke the Golgi apparatus and Golgi-derived secretory vesicles (14). Wheat roots rapidly incorporated tritiated glucose into Golgi dictyosomes, and electron microscope autoradiographic studies demonstrated that these organelles were among the first to be labeled (5). The labeled material was subsequently transported to the slime layer of the plasmalemma via Golgi-derived secretory vesicles. The same process has been observed for other plant tissues (6-S). Thus it has been proposed that the Golgi apparatus carries out the biosynthesis and packaging of hemicellulose and pectin into secretory vesicles which are transported to the cell wall (3, 4). Histochemical studies also support that concept (9, 10). Cellulose, however, is probably synthesized by an entirely different route (2).
MATERIALS
1 Contribution No. 137, Department of Biochemistry, Kansas Agricultural Experiment Station, Kansas State University, Manhattan, KS 66506; taken in part from a Master’s thesis (R.J.) and a Ph.D. dissertation (O.B.).
@ 1972 by Academic Press, of reproduction in any form
Inc. reserved.
AND
METHODS
Radioactive material. [U-Wlglucose, specific activity 240 mCi/mM was obtained from Schwarz Bioresearch, Orangeburg, NY. Enzymes. Hemicellulase, control number 7196, 702
Copyright All rights
It is difficult to correlate biochemical experiments with histochemical and autoradiographic studies. Thus, while particulate synthetase systems which catalyze the transfer of sugars from sugar nucleotides to a growing polysaccharide chain have been isolated from a variety of plant sources (ll-17), identity of the synthetase particles is not clear (IS-20), primarily because it is so difficult to break plant cell walls without damaging the organelles. Crude Golgi preparations from wheat roottip cells contain hemicellulose (21). Direct evidence is presented here that hemicellulose molecules are present in Golgi dictyosomes as well as in faster sedimenting particles. A method based on that of Morre’ and Mollenhauer (22) was applicable for isolating relatively pure organelles from wheat root tissue.
POLYSACCHARIDES
OF PLANT
and pectinase, control number 6417, were obtained from Nutritional Biochemicals Co., Cleveland, OH. Both enzymes were precipitated twice from saturated ammonium sulfate at 0°C and dialyzed overnight against 3 liters of 1 mnn sodium acetate buffer (pH 5.0) before use. Pronase, B grade, 45,000 Pu/g, lot number 45046, was obtained from Calbiochem, Los Angeles, CA. Preparation and incuhtion of seedlings. Wheat seeds (Kansas varieties), previously treated 30 min with 0.5% formaldehyde were germinated in the dark at room temperature 60 hr on rust-proof screens sandwiched between moist filter paper. Only the roots grew through the screen. For radiometabolite incubation experiments, the intact seedlings were then pulsed 2 hr with 200 pCi [U-‘%]glucose in an incubation medium of 10 mM glucose and 1 mu potassium phosphate buffer (pH 6.5) applied to a filter paper with which the roots were in contact. The roots were recovered by “shaving” the screen wit,h a razor blade. Approximately 25-30 g of wheat roots were used for each experiment. Isolation of organelles. The isolation procedure is a modification of that of Morre’ and Mollenhauer (22). The etiolated root tips were ground in approximately 25 ml of a homogenization medium composed of 1% dextran, 1 mu calcium chloride, 75-100 mu glutaraldehyde, 0.5 M sucrose dissolved in a stock buffer, which consisted of 0.6001 M manganese chloride and 0.1 M sodium phosphate buffer (pH 7.2). Glutaraldehyde was omitted from the homogenizat,ion medium when the preparation was used to identify enzyme activities present in the isolated particles. The roots were ground in a glass-Teflon homogenizer machined to a clearance of 0.026 in. The homogenate was squeezed through several layers of cheesecloth. All operations of the isolation procedure were performed at 4°C and all sucrose solutions used were made with stock buffer. The filtrate was transferred to cellulose nitrate centrifuge tubes and centrifuged 1 hr at 48009 in a Beckman SW-41 rotor. The supernatant solution was transferred to another cellulose nitrate tube, and a 0.5 ml 1.8 M sucrose pad was layered underneath the supernatant. The tube was centrifuged 1 hr at 43,000g and a band of particulate material was collected on top of the sucrose pad. This material was removed and resuspended in 2 ml of stock buffer. Approximately 10 ml of 0.5 M sucrose solution was layered underneath the resuspended material and another 0.5 ml 1.8 M sucrose pad was layered underneath the entire solution. The tube was centrifuged 1 hr at 43,500g and the particulate material sedimenting on top of the 1.8 M sucrose pad was designated as “washed crude Golgi.” Washed crude Golgi was resuspended in 1.5-2.0
SUBCELLULAR
703
PARTICLES
ml of stock buffer and layered on top of a discontinuous sucrose gradient in a 6-ml cellulose nitrate centrifuge tube. The sucrose gradient consisted of 0.5 ml of 1.5 M sucrose, 1.0 ml of 1.25 M sucrose, 1.0 ml of 1.0 M sucrose, and 1.0 ml of 0.5 M sucrose. The tube was centrifuged in a SW-50 L rotor 3 hr at 102,OOOg. Isopyknic conditions developed, as evidenced by the formation of a continuous gradient, after centrifugation. The four particulate bands resulting on the density gradient which were clearly evident were designated as top, middle, bottom, and pellet. The particulate material was pipetted off the gradient, resuspended in stock buffer, and pelleted. Enzymatic determinations, prdtein assays (23), or chemical extractions were performed on the pelleted material. Alternatively, to determine the density at which each band sedimented or to determine the radioactivity incorporated into each band, the bottom of the density gradient tube was pierced and O.l-ml fractions were collected for refractive index measurements (from which densities were calculated) or for liquid scintillation counting. Electron microscopy. Pelleted materials in the centrifuge tubes were drained and wiped dry with tissue paper. The pellets were fixed with 1% osmium tetroxide in 0.05 M sodium phosphate buffer (pH 7.2) for 24 hr at 4°C. The materials were aspirated of the osmium tetroxide and dehydrated with a series of ethanol solutions. The dehydrated materials were infiltrated and embedded by the
IO
20
30
ml
FIG. 1. Sucrose density gradient radioactivity scan. Wheat seedlings were incubated with [U-‘4C]glucose, and subcellular fractions were isolated on a density gradient. After piercing the bottom of the density gradient centrifuge tube, O.l-ml fractions were collected on glass fiber filters, dried, and counted. T = top band M = middle band B = bottom band
d = 1.137 g cm-3 d = 1.167 g cmp3 d = 1.206 g cm-”
704
JILKA,
BROWN,
method of Kushida (24) at 50°C for 46 hr: the blocks were sectioned on the LKB Ultrotome with glass knives, picked up on copper grids, and double stained with uranyl acetate and lead citrate. The stained sections were rinsed and viewed in a modified RCA EMU 2-D electron microscope. Chemical extraction of particulate fractions. The pelleted particulate material was extracted at 4°C with methanol-chloroform-0.2 M formic acid (12:5:3 v/v/v) (25) % hr. After low-speed centrifugation and removal of the supernatant, the remaining insoluble material was extracted with
FIG. 2. Positive stained trifugation. Magnification:
electron micrograph X 47,300.
AND
NORDIN
0.05 M KOH x hr at 100°C. The lipid layer was extracted once with distilled water and assayed for radioactivity. The KOH hydrolyzate was filtered and neutralized with concentrated KH~POI Resulting material was then treated with 200 fig of pronase 50 hr at 37°C with 4y0 ethanol added as preservative (26). The hydrolyzate was subjected to Sephadex G-75 chromatography with 5 mu sodium phosphate buffer (pH 8). One-milliliter fractions were collected and counted as described. Enzymatic hydrolysis of labeled pronase-resistant KOH-soluble material. Hemicellulase was checked
of top band from
density
gradient
cen-
POLYSACCHARIDES
OF PLANT
for hydrolytic activity against hemicellulose, polygalacturonic acid, and denatured bovine serum albumin. The hemicellulsse released various hexoses and pentoses from hemicellulose and a very small amount of galacturonic acid from polygalacturonic acid. Less than 5% of the amino acids (by weight) was released from the denatured BSA. The pronase-resistant KOH-soluble material of the washed crude Golgi (fraction I) was treated at room temperature at pH 5.0 with 50 ~1 each of the pectinase and hemicellulase enzyme solutions for 2 hr. The reaction was stopped by gentle heating and the hydrolyzate was subjected to Sephadex
FIG. 3. Positive stained trifugation. Magnification:
electron micrograph X 41,170.
SUBCELLULAR
705
PARTICLES
G-75 column chromatography. Another sample of fraction I was either treated with the enzymes 24 hr at pH 5.0 at room temperature or hydrolyzed with 1 N HCl 2 hr at 100°C. Products were examined by silica gel thin layer chromatography. Acetone-water (9:l v/v) was used to separate monosaccharides on sodium acetate-impregnated plates, and butanol-acetic acid-water (2: 1: 1 v/v) was used to separate uranic acids (27). The hydrolyzate was cochromatographed with 1% sugar standards. After drying, the area above the hydrolyzate origin was divided into horizontal l-cm strips and scraped into scintillation vials for
of middle
band from density
gradient
cen-
706
JILKA,
BROWN,
counting. The remainder of the plate was sprayed with diphenylamine-aniline (27)) and position of the authentic sugars noted with respect to the l-cm strips. Confirmatory tests for sugars were obtained by paper chromatography [butanol-pyridine-water; 10:3:3 v/v with silver nitrate dip (21)]. Liquid scintillation counting. A fluor of 0.5% PPO in toluene was used for counting in a Beckman LS-200B Scintillation Counter. Fractions from column chromatography experiments were prepared for counting by lyophilization in a counting vial containing a glass fiber filter. Silica gel
FIG. 4. Positive stained trifugation. Magnification:
electron micrograph x 47,150.
AND
NORDIN
scrapings from thin plates were counted directly in the vial containing the fluor. As the quenching level was essentially constant, no quenching corrections were made. RESULTS
Uptake of 14CActivity into Bands Figure 1 shows a density gradient radioactivity scan of the particulate bands from wheat seedlings incubated in [UJ4C]glucc.se. Incorporation of 14Cactivity into each band is evident. The pellet was labeled to approxi-
of bottom
band from density
gradient
cen-
POLYSACCHARIDES
OF PLANT
mately the same extent as the bottom band. Electron micrographs corresponding to radioactive peaks are shown in Figs. 2-5. Electron Microscopic Identijcation of the Bands Bands isolated on the density gradient were identifiable morphologically. Figure 2 shows an electron micrograph of the top band, which contains characteristic (28) stacks of Golgi cisternae with no other recog-
FIG. 5. Positive tion. Magnification:
stained electron X 34,600.
micrograph
SUBCELLULAR
707
PARTICLES
nizable cell organelles as contaminants, but with the characteristic transition and fr’agmentation forms of the Golgi, which result from isolation procedures, and which have been described by Morre’ and Mollenhauer (22). The top band sedimented at a density of 1.137 g/gm3, which is comparable to the density of Golgi apparatus isolated by other workers (19, 29). The middle band sedimented at a density of 1.167 g/cm3, which is characteristic of that
of pellet
from density
gradient
centrifuga-
708
JILKA,
BROWN,
of isolated plant lysosomes (30, 31), but its appearance (Fig. 3) is predominantly that of microsomes (29, 32, 33) with some dictyosomal and lysosomal contamination. The bottom band, with a density of 1.206 g/cm”, correlates with the density of plant mitochondria isolated by other workers (19). and the electron micrograph shown in Fig. 4 has a high proportion of the easily recognized mitochondrial structures (34). Several small dense vesicles bound by a single membrane also appear in this band. The pellet material (Fig. 5) contains some mitochondria in addition to dense single membrane-bound particles and some very dense fragmentation products. Ident$cation
of Biosynthetic Enzymes in Bands
AND
NORDIN
Polysaccharide was identified by its elution at the void volume on Bio-gel P-2 and by observing that it yielded radioactive glucose upon hydrolysis with acid. Radioactive sucrose was identified by its Rf and by its susceptibility to invertase. UDPG hydrolase was identified by the formation of radioactive glucose during incubation. Comparisons on the basis of protein content showed that glucan synthetase was more concentrated in the pellet and bottom band, while UDPG hydrolase was so active in the top Golgi band that synthetase activities were minimal. Sucrose synthetase was most heavily concentrated in the bottom band. Significant transferase activities toward n-xylose and N-acetylglucosamine were not demonstrated.
Several enzymes exhibiting activity toward UDP-glucose could be demonstrated by incubating aliquots of the bands and pellet separately with UDP-[14C]glucose and a mixture of monosaccharide acceptors (D-ghcase, n-fructose, D-xylose and N-acetylglucosamine) . For this purpose, glutaraldehyde was omitted in the homogenization medium, Quantitative assays were not possible because of competing reactions. However, the following enzymes could be identified by their products: glucan synthetase, UDPglucose hydrolase, and sucrose synthetase. TABLE DISTRIBUTION
Band
Top Middle Bottom Pellet
I
OF ACTIVITY IN SUBCELLULAR PARTICLES" 70 Lipid soluble 56.4 51.1 54.6 35.5
% KOH insoluble 20.8 25.8 20.8 34.1
22.6 22.8 24.6 30.5
0 Distribution of ‘4C activity in bands as percentages of total band activity. Aliquots of the lipid extract and the KOH-soluble material were counted on glass fiber filters. The KOH-insoluble material was counted direct in the counting solution. All figures represent an average of three determinations, except that top band figures represent an average of two det,erminations.
600
P
300 0
10
20
30
40
FIG. 6. Fractionation of pronaae hydrolyzate of KOH-soluble fraction on Sephadex G-75. The KOH-soluble fraction of each band was treated 50 hr with pronase and subjected to Sephadex G-75 chromatography on a 1 X 30 cm column eluted with 5 mu sodium phosphate buffer (pH 8). Onemilliliter fractions were collected, lyophilized, and counted.
POLYSACCHARIDES
OF PLANT
Chemical Distribution of Radioactivity @Bands Chemical distribution of the radioactivity of each band was investigated by fractionating each into three solubility classes: lipid soluble, KOH soluble, and KOH insoluble (Table I). Data in Table I indicate that the KOH-soluble labeled material was somewhat more concentrated in the pellet, although the other three bands contained significant quantities also. No other significant trends were evident. Effect of Pronase 012KOH-Soluble Materials
Labeled
Results of Sephadex G-75 chromatography of the pronase-treated KOH-soluble material of each band show that all bands have pronase-resistant high molecular weight material (Fig. 6). Pronase is a broad specificity proteolytic enzyme, so formation of low molecular weight products indicates the presence of protein. Pronase-resistant material is presumably all polysaccharide. Chemical Nature of Fraction I The high molecular weight KOH-soluble fraction resistant to pronase was designated
I
SUBCELLULAR
709
fraction I. For a study of its chemical nature it was isolated from washed crude Golgi, since “total activities incorporated” were low. Washed crude Golgi contain all the subcellular particles. Fraction I was treated with a hemicellulase-pectinase enzyme preparation and subsequently chromatographed on Sephadex G-75 (Fig. 7). Release of low molecular weight material was demonstrated. A silica gel, thin layer chromatogram scan of the enzyme hydrolyzate is shown in Fig. 8. Labeled substances were present migrating as galactose, arabinose, xylose, and an unidentified fast-running compound, and perhaps glucose. The labeled material present at the origin was immobile in a solvent for uranic acids (1-butanol-acetic acid-water 2 : 1: 2 v . v). Acid hydrolysis of fraction I and subsequent thin layer chromatography gave the results shown in Fig. 8. The only difference observed was that more xylose and less origin material were present in the acid hydrolyzate scan. No uranic acids could be demonstrated in either one. Confirmatory tests for xylose, arabinose, galactose and glucose were obtained by paper chromatography. The sugars reacted toward silver ni-
“0 J.
IO
PARTICLES
“G I
20
30
40
ml
FIG. 7. Fractionation of Fraction I hemicellulase-pectinase hydrolyzate on Sephadex G-75. Fraction I was treated 2 hr with the hemicellulase-pectinase enzyme system. The hydrolyzate was eluted on a 1 X 33 cm Sephadex G-75 column with 1 mu potassium phosphate buffer (pH 8). One-milliliter fractions were collected, lyophilized, and countedVo = void volume; VG = glucose elution volume.
JILKA,
;I
abed
a
b
e
c
d
BROWN,
f
e
f
cm
FIG. 8. Thin layer chromatography hydrolyzate tinase, lower (c) mannose, curonolactone.
of fraction I and pec(upper = hemicellulase = HCl). (a) galactose, (b) glucose, (d) arabinose, (e) xylose, (f) glu-
dip in the typical sugars.
trate
manner of reducing
DISCUSSION
The separation of subcellular particles reported here is an application of Morre and Mollenhauer’s (22) method to wheat root tips. It. appears that one can obtain morphologically distinct organelles from wheat root tips in sufficient quantity to perform biochemical studies. A polysaccharide is present in the Golgi fraction but ,clearly it is more heavily concentrated in the faster sedimenting bands. As the Golgi dictyosomes are labeled more rapidly than other organelles, 2 hr of incubation in labeled glucose is presumably suffi-
AND
NORDIN
cient for complete polysaccharide molecules to be synthesized (5). Thus, secretory vesicles, judged to separate at densities corresponding to the bottom and pellet fractions (lo), would be labeled also. This work, however, clearly demonstrated the presence of polysaccharide in all subcelMar fractions isolated from wheat root tips. The principal monosaccharides are xylose, arabinose and galactose which are characteristic of hemicellulose. Golgi preparations from pea seedlings (35) also have been reported to contain monosaccharides characteristic of hemicellulose. The fact that we find polysaccharide in all bands does not appear to be due to incomplete resolution or complexing by glutaraldehyde. Preparations without glutaraldehyde also contained a high molecular weight fraction in each band. Furthermore, the high molecular weight material is not easily removed by prolonged washing or sonication w . The enzyme studies demonstrated the localization of glucan synthetase in the bottom and pellet material. Other workers have also reported that these enzymes are found in particles migrating much faster than Golgi bodies during centrifugation (18). The only enzyme of significance that we found in the Golgi band was UDPG hydrolase, which agrees with previous studies (36). As plasmalemma fragments probably were present in our pellet fraction, it is tempting to conclude that heteropolysaccharide synthesis occurs primarily in the plasmalemma. However, secretory vesicles from the Golgi may also sediment. at densities corresponding to the pellet, and bottom fractions on the density gradient. Thus, the polysaccharide in the faster-sedimenting particles might reflect secretory granules. Currently available methods do not, permit their complete separation from other particles in wheat root tips. The “hemicellulose” found is, in any case, a precursory substance. When incubation in labeled glucose was followed by a pulse of “cold” glucose (21), radioactivity of the hemicellulose was greatly reduced. Our findings agree with earlier autoradiographic and histochemical studies (5, 6, 9, lo), showing cell wall precursors in plant cells.
POLYSACCHARIVES
OF PLANT
SUBCELLULAR
REFERENCES 1. NORTHCOTE, D. H. (1971) Endeavour 30,26-33. F., AND MCGINNIS, G. D. (1971) 2. SHAFIZADEH, Advan. Cnrbohyd. Chem. Biochem. 26, 297349. D. H. (1969) Essays Biochem. 6, 3. NORTHCOTE, 9@138. K. (1967) Annu. Rev. Plant 4. MUHLETHALER, Physiol. 18, l-24. J. 1). 5. NORTHCOTX, D. H., AND PICKETT-HR.IPS, (1966) Biochem. J. 98, 159-167. J. D. (1967) J. Ultrastruct. 6. PICKETT-HEAPS, Res. 18, 287-303. J. D. (1966) Planta 71, 1-14. 7. PICKETT-HEAPS, 8. WOODING, F. B. P. (1968) J. Cell Sci. 3, 71-80. J. D. (1968) J. Cell Sci. 3, 9. PICKETT-HICAPS, 5564. W. J., MORRE’, D. J., AND 10. VANDERWOUDE, BRACKER, C. E. (1971) J. Cell Sci. 8,331-351. 11. ELBICIN, A. D. (1969) J. Biol. Chem. 244, 16081616. J. B., AND HASSID, W. Z. (1966) 12. PRIDHAM, Biochem. J. 100, 21P. 13. KAUSS, H. (1967) Biochim. Biophys. Acta 148, 572-574. 14. BAILEY, R. W., AND HASSID, W. Z. (1966) Proc. Nat. Acad. Sci. USA 66, 158G-1593. E. F., AND HASSID, 15. FEINGOLD, D. S., NEUFELD, W. Z. (1959) J. BioZ. Chem. 234, 488-489. 16. KAUSS, H., SWANSON, A. L., ARNOLD, R., AND ODZUCK, W. (1969) Biochim. Biophys. Acta 192, 55-61. C. L., SWANSON, A. L., AND H~SSID, 17. VILLEMEZ, W. Z. (1966) Arch. Biochem. Biophys. 116, 446-452C. L., MCNAEJ, J. M., AND 18. VILLEMEZ, ALBERSHEIM, P. (1968) Nature (London) 218, 878-880. T. L., AND RAY, 19. RAY, P. M., SHININGISR,
20. 21. 22. 23.
24. 25. 26. 27.
28.
29.
30. 31. 32. 33.
34. 35. 36.
PARTICLES
711
M. M. (1969) Proc. Nat. Acad. Sci. USA 64, 605-611. LAMPORT, D. T. A. (1970) Annu. Rev. Plant Physiol. 21, 235-270. MERTZ, J., AND NORDIN, P. (1971) Phytochemistry 10, 1223-1227. MORRE’, J. D., AND MOLLENHBUER, H. H. (1964) J. Cell Biol. 23, 295-305. LOWRY, D. H., ROSEBROUGH, N. J., FARR, A. L., AND HANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. KIJSHIDA, H. (1967). J. Electronmicrosc. 16, 278-280. BIELESKI, R. L., .~ND YOUNG, R. E. (1963) Anal. Biochem. 6, 5468. NOMOTO, M., NARAHASHI, Y., AND MURAKAMI, M. (1960) J. Biochem. 48, 593-602. LEWIS, B. A., AND SMITH, F. (1969) in Thin Layer Chromatography (Stahl, E., ed.), pp. 807-837, Springer-Verlag, New York. CUNNINGHAM, W. P., MORRE’, J. D., AND MOLLENHAUZR, H. H. (1966) J. Cell Biol. 28, 169-179. MORRE’, J. I)., MOLLENHAUER, H. H., AND CHAMBERS, J. E. (1965) Exp. Cell Res. 38, 672-675. SEMBDENI, E. G. (1967) Pkznta 72, 91-118. MATILE, P. (1968) Planta 79, 181-196. HOR~ITZ, A. L., AND DORFMAN, A. (1968) J. Cell Biol. 38, 358-368. DALLMAN, P. R., DALLNER, G., BERGSTRAND, A., AND ERNSTER, L. (1969) J. Cell Biol. 41, 357-377. BUTLER, W. H., AND JUDAH, J. I>. (1970) J. Cell Biol. 44, 278-289. HARRIS, P. J., AND NORTHCOTE, D. H. (1971) Biochim. Biophys. Acta 237, 56-64. NOVIKOFF, A. B., AND GOLDFISCHER, S. (1961) Proc. Nat. Acad. Sci. USA 4’7, 802-810.