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Polysaccharide formation in plant Golgi bodies
56
BBA
BIOCHIMICA ET BIOPHYSICA ACTA
26528
P O L Y S A C C H A R I D E FORMATION IN PLANT GOLGI BODIES P. J. H A R R I S * AND D. H. N O R T H C O T E
Department of Biochemistry, University of Cambridge, Cambridge (GreatBritain) (Received N o v e m b e r i 3 t h , 197o )
SUMMARY
I. A fraction rich in Golgi bodies has been isolated from the roots of pea seedlings. The fraction has been characterised by an electron microscopic examination of negatively stained samples and ultra-thin sections. 2. The distribution in various cell fractions of the polysaceharides synthesised b y the root tip during a 4o-min incubation with radioactive glucose has been determined. 3. It has been shown that pectic substances and hemicelluloses are carried and probably synthesized by the Golgi bodies and their associated vesicles.
INTRODUCTION
The Golgi apparatus is part of a membrane-bounded transport system within the cell. This route links the perinuclear space, the lumen of the endoplasmic reticulum and the cisternae and vesicles of the Golgi bodies to the outside of the cell across the plasmalemma. The material which is conducted through the system can be synthesised and modified during its passage and in this way the Golgi apparatus is involved in the transport and synthesis of plant cell wall polysaecharides and polysaccharide secretions such as the slime of the root cap cells. The Golgi body takes part in the formation of the cell plate at telophase 1-4 and in the development of the primaryS, ~ and secondary walF, 8 during growth and differentiation of the cell. The evidence for this is based on observations of the fine structure of the cells and on radioautographic techniques. Radioactive polysaccharides can be shown to be present within the Golgi cisternae of cells supplied with a pulse of radioactive precursor and this material can be chased via the Golgi vesicles to the cell wall when non-radioactive precursors are subsequently supplied to the tissues 9. In this paper we present direct evidence that the Golgi bodies contain high molecular weight polysaecharides that have been newly synthesised and that are similar to the polysaceharides found in the matrix of the cell wall. MATERIALS AND METHODS
Pea seeds (variety Feltham First) were surface sterilized by soaking in Milton (Milton Division, Richardson-Merrell Ltd., London) for 15 rain. They were then * P r e s e n t a d d r e s s : D e p a r t m e n t of B i o c h e m i s t r y , U n i v e r s i t y of Oxford, Oxford, E n g l a n d .
Biochim. Biophys. Acta, 237 (1971) 56-64
POLYSACCHARIDE FORMATION IN PLANT GOLGI BODIES
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washed in water and germinated in vermiculite at 25°. Seedlings with roots 3-4 cm in length were used.
Incubation with the isotope Pea seedlings (2o) were incubated for 4 ° rain at 2o ° with their roots dipping into a solution (400/,1 of D-E14Cs!glucose (40 #C, 320 mC/mmole). The details of the incubation and the method of determining the amount of isotope taken up by the seedlings are the same as those described by HARRIS AND NORTHCOTETM. Isolation of the Golgi bodies Golgi bodies were isolated from the roots by a slight modification of the method of MORR~ et al. 11. The 20 pea roots which had been incubated with D-[l~C6!glucose were excised close to the cotyledons and added to 16 g of thoroughly washed unlabelled excised roots. The tissue was gently homogenised in a pestle and mortar using IO ml of homogenisation medium 11,12 with the addition of glutaraldehyde (i-lO -1 M) (Koch Light Ltd., Colnbrook, Bucks). The suspension was allowed to stand for IO rain with occasional stirring, filtered through 2 layers of muslin and the filtrate was centrifuged at 7000 rev./min (average 4000 × g) for 30 rain. The pellet produced is subsequently referred to as the first pellet. All centrifugations were carried out using a Beckman Spinco L2 or L2B centrifuge with a SW 5oL or SW 39 rotor. The supernatant was layered onto 1.8 M sucrose (0.25 ml) and then centrifuged at 12000 rev./min (average 11750 × g) for 30 rain. Particulate material was deposited onto the 1.8 M sucrose cushion and the supernatant was replaced by 1.6 M sucrose (0. 5 ml), 1.5 M sucrose (I.O ml), 1.25 M sucrose (I.O ml) and 0. 5 M sucrose to fill the tubes. The density gradient was centrifuged for 3 h at 35 ooo rev./min (average IOOooo × g). The particulate layers at the o.5-1.25 M sucrose interface (subsequently referred to as the Golgi fraction) and the 1.25-I.5 M sucrose interface (subsequently referred to as the mitochondrial fraction) were carefully removed with a fine Pasteur pipette and centrifuged in 0.5 M sucrose to give a pellet (30 rain at 12 ooo rev./min; average 11750 × g). All the above sucrose solutions contained all the constituents of the homogenation medium. The pellets were collected in polyallomer tubes as these resisted the conditions of the subsequent hydrolysis, all other centrifugations were carried out in nitrocellulose tubes. All the procedures for the preparation of the pellets took place at 0-8 °. The pellets were washed with several changes of 75 % (v/v) ethanol containing n-glucose (50 gfl) and then with 75% (v/v) ethanol, and dried over P205 in vacuo. They were dissolved in 25 or 30/~ of 72% (w/w) H2S04, diluted to 3% (w/w) H2SO 4, and hydrolysed by autoclaving at 15 lb/inch 2 at 16o ° for I h. The hydrolysates were filtered through sintered glass micro filters (porosity 3), and neutralized. The uronic acids and neutral sugars were separated and their radioactivity determined as described by HARRIS AND NORTHCOTE. The radioactivity of the uronic acids was determined by quantitatively eluting the material from the origin of the chromatograms, followed by electrophoresis at pH 2.0 and pH 3.5Characterization of the pellets by electron microscopy The pellets were examined after negative staining and also after thin sectioning. The negative staining was carried out using a 2% phosphotungstic acid solution Biochim. Biophys. Acta, 237 (I97 I) 56-64
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P. J. HARRIS, D. H. NORTHCOTE
brought to pH 6.8 with NaOH. Bovine plasma albumin (final concentration o.1% w/v) was added to the negative stain before use. The pellets were also fixed for i tl at 2o ° in a phosphate buffered solution (o.o2 M, pH 7.2) of glutaraldehyde (6°.o) containing sucrose (o.5 M). They were post fixed in veronal buffered osmic acid (I %) for I h at 2o °, dehydrated through an ethanol series and embedded in Araldite.
Fig. I. N e g a t i v e s t a i n e d p r e p a r a t i o n of t h e Golgi-rich fraction isolated f r o m t h e b r o k e n cells ( × 30000). Fig. 2. N e g a t i v e s t a i n e d p r e p a r a t i o n of t h e Golgi-rich fraction isolated f r o m t h e b r o k e n cells ( × 45 ooo).
Biochim. Biophys. Acta, 237 (1971) 56 64
POLYSACCHARIDE FORMATION IN PLANT GOLGI BODIES
59
The sections were stained with uranyl acetate and alkaline lead hydroxide and examined in a G.E.C., A . E . I . E . M . 6 B at 60 kV 1~. RESULTS
Electron microscopic examination of the fractions After negative staining the Golgi fraction was seen to contain a high proportion of Golgi bodies having a morphology similar to that reported by CUNNINGHAMet al. 14 (Figs. I and 2). They were composed of a central circular plate surrounded by a mass of convoluted tubules to which were attached vesicles with an electron dense centre and an electron transparent border. The central plate of the Golgi body shown in Fig. 2 is clearly resolved which indicates that the whole structure probably represents a single isolated cisterna. The Golgi fraction was also found to be rich in Golgi bodies when ultra-thin sections of the fraction were examined (Figs. 3 and 4). The organelle
Fig. 3. Sections of the pellet of the Golgi-rich fraction isolated from the broken cells ( x 15 ooo). Fig. 4. Sections of the pellet of the Golgi-rich fraction isolated from the broken cells (X 25 ooo).
Biochim. Biophys. Acta, 237 (1971) 56-64
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HARRIS, D. H. NORTHCOTE
had an appearance similar to that observed in unbroken plant cells. Numerous vesicles were associated with the cisternae, some of which probably represented cross sections of the tubular outer cisternal region. Other vesicles of various sizes bounded by smooth and less frequently rough membranes were also present and these were not associated with the intact Golgi bodies. Some were morphologically similar to those vesicles connected with the cisternae, and probably they were detached during the isolation procedure. Examination of negatively stained and thin sections of the mitochondrial preparation showed that it was rich in mitochondria and proplastids. The cristae of the mitochondria were swollen (Fig. 5)- Rough and smooth membrane bound vesicles and some starch grains were also present in the fraction. Thin sections of the first pellet showed that it contained a whole range of cell components including nuclei, plastids with starch grains, cell wall fragments, mitochondria, proplastids and some Golgi bodies.
Fig. 5. Section of t h e pellet of t h e m i t o c h o n d r i a l fraction isolated from t h e b r o k e n cells ( X 15 ooo).
Chemical studies D- ~llC6]Glucose (79.76. lO6counts/min) was fed to the pea roots and after 4 ° rain the radioactivity of the glucose remaining in the incubation medium was 29.02. lOs counts/rain. This represents a percentage uptake of the radioactive glucose bv the tissue of 63.6°,o. The percentage distribution of radioactivity in the uronic acids and neutral sugars obtained from acid hydrolysates of the first, Golgi rich, and mitochondrial rich fractions was determined. Table I shows that the hydrolysates of both the first and the Golgi rich fractions contained radioactivity in all the neutral sugars and uronic acids. These sugars and sugar acids are characteristic components of the polysaccharides of plant cell wails. The first fraction contained cell wall fragments from which some of these radioactive sugars were presumably derived. In addition the first fraction also contained a large number of plastids containing starch grains and this probably increased the amount of radioactive glucose present in the hydrolysate. The Golgi fraction contained no cell wall fragments and no starch grains either Biochim. Biophys. Acta, 237 (I971 ) 56-64
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6I
TABLE I RELATIVE AMOUNTS OF RADIOACTIVITYINCORPORATED FROM UNIFORMLY LABELED D-[14C~GLUCOSE INTO TH]~ URONIC ACIDS AND NEUTRAL SUGARS OF THE ISOLATED FRACTIONS OF THE CELLS OF THE ROOT TISSUE The roots were incubated w i t h the radioactive glucose for 4 ° nlin and t h e n homogenised and the fractions isolated b y density gradient centrifugation. The values in parentheses represent the a m o u n t s of radioactivity calculated as percentages of the total a m o u n t of radioactivity in the n e u t r a l sugars and the uronic acids excluding glucose. The o t h e r values represent the a m o u n t s of radioactivity calculated as a percentage of the total a m o u n t of radioactivity in all the neutral sugars and the uronic acids.
Sugar
Radioactivity (%) First fraction
Galacturonicacid Glucuronic acid Galactose Glucose Mannose Arabinose Xylose Fucose Rhamnose Total c o u n t s / m i n
Golgifraction
9.1 (14.o) 11. 3 2.0 ( 3 . 0 ) 1.3 19.9 (30-6) 31-7 35.0 ( - - ) 15.2 5.2 ( 8 . 0 ) 7.1 19.8 (30.5) 20.2 5.3 ( 8 . 2 ) 8.1 0. 7 ( i . i ) 1.2 3 .0 ( 4 .6 ) 3.9 15 156" (9853)* 4441
(13.3) (1.5) (37-4) (--) (8.3) (23.8) (9.6) (1.5) ( 4 .6 ) (3765)
Mitochondrial fraction lo.i -22.9 20. 3 8. 4 35-5 2.9
(12.7) (--) (28,7) (--) (lO.5) (44.5) (3.6)
-(--) 663 (529)
* Values for 1/~ of the total yield of the first fraction.
free or contained within plastids. Thus the radioactive sugars of the high molecular weight polysaccharides in this fraction must have been present in the Golgi bodies and their associated membranes. The percentage distribution of radioactivity in the uronic acids and neutral sugars of the hydrolysate of the Golgi pellet and the first pellet were very similar except that the Golgi pellet had a much higher percentage of radioactivity in galactose relative to that in glucose. This is probably in part due to the lack of radioactive starch grains. When the percentages were recalculated omitting glucose, the distribution of radioactivity in the uronic acids and sugars of the two fractions were very similar. The hydrolysate of the mitochondrial fraction also contained radioactive neutral sugars and uronic acids characteristic of plant cell wall polysaccharides. However, the percentage distribution of radioactivity in these was different from that of the first and the Golgi rich pellets. In particular the mitochondrial fraction was much richer in radioactive arabinose and much less rich in radioactive xylose and fucose estimated together. The ratio of the amount of radioactivity in xylose and fucose to the amount of radioactivity in arabinose for the Golgi fraction was 1:2.2, for the first fraction I:3.3, and for the mitochondrial fraction I:12.3. The origin of the radioactive sugars in the mitochondrial fraction is uncertain. It is possible that radioactive water-soluble cell wall polysaccharides such as pectin become adsorbed to the mitochondria during the homogenization and subsequent fractionation. It is also possible that the mitochondrial fraction was contaminated with Golgi derived vesicles although this is unlikely as the percentage distribution of radioactivity in the neutral sugars and uronic acids of the mitochondrial and Golgi fractions were different. All or part of the radioactive glucose present in the mitochondrial fraction could have arisen from radioactive starch as some starch grains were present. 13iochim. Biophys. Acta, 237 (1971) 56-64
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HARRIS, D. H. NORTHCOTE
DISCUSSION
The results presented here support the conclusion that the cisternae and vesicles of the Golgi apparatus are involved in the transport and probably the synthesis of pectic and hemicellulosic cell wall polysaccharides (the matrix material of the wall). A large percentage (approx. 63 %) of the radioactivity present in the sugars of hydrolysate of the Golgi fraction obtained from pea roots which had been incubated with uniformly labelled D@4C!glucose was in galactose, arabinose, and galacturonic acid. These sugars are characteristic component sugars of the pectic substances 15. The amount of labelled pectic material in the Golgi-rich fraction m a y be due partly to the adsorption of radioactive water soluble polysaccharides released from the root cell walls during the breakage of the cells. The presence of radioactive xylose, mannose and a trace amount of radioactive glucuronic acid in the hydrolysate indicated the occurrence of xylans and glucomannans in this Golgi fraction. Glucose was not labelled to a great extent relative to the sugars characteristic of the matrix polysaccharides. This result indicates that the active synthesis and transport of cellulose is not extensively brought about by the Golgi apparatus. The radioactive glucose present in the hydrolysate of the Golgi fraction could have arisen from the physical adsorption of free radioactive glucose present in the tissue and released during the homogenization, or from the synthesis and transport of other glucans such as callose. RAY et al. 16 showed that a preparation which contains a high percentage of Golgi derived membranes and vesicles was able to catalyse the transfer of glucose from UDPGlc and GDPGlc to an endogenous acceptor. Part of the polysaccharide synthesised from both GDPGlc and UDPGlc was a I + 4/~ linked glucan. However, it has been found in other systems that both I-> 3/~ and I ~ 4 [3 linked glucosyl units can be obtained from UDPGlc 17. BROWN et al. ~s have demonstrated that in the unicellular alga Pleurochrysis scherUelii the wall scales contain microfibrils that are possibly made of cellulose. These scales are synthesised and transported to the cell surface in the Golgi apparatus and its associated vesicles. They also suggest that the site of synthesis of cellulose in higher plants is in the Golgi apparatus. In higher plants, however, the microfibrils are often interwoven in a highly ordered manner and they are closely packed. It is difficult to conceive how this arrangement could be brought about unless the enzymes involved in cellulose synthesis are arranged either at the plasmalemma surface or in the wall itself. Evidence for such a location of a cellulose synthesising system is given by NORTHCOTE~9. Since the membranes of the Golgi vesicles fuse with the plasmalemma it is probable that the enzymes for cellulose synthesis and indeed other enzymes which could modify the polysaccharides of the cell wall in situ are transported from the Golgi complex to their site of action at the outer cell membrane. The Golgi apparatus is probably involved in the synthesis as well as the transport of the matrix polysaccharides. NORTHCOTE AND PICKETT-HEAPS9 demonstrated b y high resolution autoradiography that the Golgi cisternae in the outer root cap cells of wheat contain radioactive material within 5 min after feeding the roots with D-[6-~Hlglucose. The radioactivity was probably present in polymeric substances since it was not removed during the procedures involved in the preparation of the material for the study with the electron microscope. Glycosyl transferases which Biochim. Biophys. Acta, 237 (1971) 56-64
POLYSACCHARIDE FORMATION IN PLANT GOLGI BODIES
63
utilize UDPGlc, UDPGal, UDPGlcA, UDPGalA, UDPAra, U D P X y l as substrates and the epimerases and other enzymes that are involved in the interconversion of these nucleoside diphosphate sugars have nearly all been found in a particulate preparation obtained from homogenized mung bean shoots 2°, 21. Although this preparation has not been characterised by electron microscopy it is possible that these enzymes are located in Golgi membrane fragments. Fractions rich in Golgi bodies have been obtained from homogenised liver tissue and these preparations show glycosyl transferase activities 22-25. Myoinositol which can be formed from glucose can act as a precursor of uridine diphosphate uronic acids and pentoses 26. This interconversion provides an alternative pathway for the formation of these uridine diphosphate compounds, however, the location of the enzymes concerned is not known. KAUSS e / a l Y have used a similar particulate preparation to that described by HASSIDeO,22 and they have shown that the enzymes involved in transferring the methyl groups from 5-adenosyl-L-methionine to esterify the polygalacturonic acid chains of pectin will act only if the acceptor molecules are generated within the particles of the enzymic preparation. The methylated polygalacturonic acid formed was protected from the action of pectin methyl esterase, but this protection was removed by substances which disrupt the integrity of lipid membranes. This evidence is consistent with the idea that the enzymes are an integral part of the membrane. Glycolipids have been shown to take part in the biosynthesis of bacterial mucopeptides and in several cases the lipid component has been identified as an isoprenoid alcohol ~s. KAUSS20 has demonstrated that a similar lipid intermediate is formed during the synthesis of glucomannan from GDPMan in the mung bean particulate enzyme system. The lipid has not been identified but it is synthesised from mevalonic acid. A glucolipid and a glycoprotein have also been identified as intermediates in glucomannan synthesis in mung bean 3°. These substances m a y be components of the Golgi membranes and m a y act as acceptor sites for the transglycosylation reactions and the attachment of the growing polysaccharide chains within the cisternae and vesicles of the Golgi complex. The enzymic activity of the Golgi apparatus and hence the nature of the polysaccharides that are synthesised varies during the differentiation of the cell. In maize roots for example it has been shown that the hypertrophied Golgi apparatus in the outer root cap cells form a different polysaccharide from that formed in the cells of the adjacent root tissue ~°. The Golgi fraction obtained in the work which is reported here was derived from the whole roots and represents a heterogeneous mixture of Golgi bodies which have different synthetic activities. ACKNOWLEDGEMENTS
P. J. H. gratefully acknowledges the receipt of a grant from the Science Research Council during the tenure of which this work was carried out. REFERENCES I 2 3 4
W. W. H. K.
G. WHALEY AND H. H. MOLLENHAUER, J. Cell Biol., 17 (i963) 216. G. WHALEY, M. DAUWALDER AND J. E. KEPHART, J. Ullrastruc/. LEHMAN AND D. SCHULZ, Planta, 85 (1969) 313 . ROBERTS AND D. H. NORTHCOTE, J . Cell Sci., 6 (197o) 299.
Res., 15 (1966) 169.
Biochim. Biophys. Acla, 237 (1971) 56-64
64 5 6 7 8 9 io II I2 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3°
P. J. HARRIS, D. H. NORTHCOTE A. SIEVERS, Protoplasma, 56 (1963) 188. J. D. PICKETT-HEAPS AND D. H. ,NORTHCOTE, J. Exptl. Botany, 17 (1966) 2o. F. B. P. "WOODINGAND D. H. NORTHCOTE, J. Cell Biol., 23 (1964) .3"27. t'[. GSAU, V. I. CltEADLE AND I~. H. GILL, Am. J. Botany, 53 (1966) 756. D. H. NORTHCOTE AND J. D. PICKETT-HEAPS, Biochem..]., 98 (1966) 159. P. J. HARRIS AND D. H. ,NORTHCOTE, Biochem. J., 12o (197 o) 479" D. J. a~{ORRI~, H. H. MOLLENHAUER AND J. E. CHAMBERS, Exptl. Cell Res., 38 (I965) 672. D. J. MORRI~ AND H. I-I. MOLLENHAUER, J. Cell Biol., 23 (1964) 295. J. BURGESS AND D. H. NORTHCOTE, Planta, 75 (1967) 319 • W. P. CUNNINGHAM, D. J. MORRI~ AND H. H. ~OLLENHAUER, J. Cell Biol., 28 (1966) I69. A. J. BARRETT AND D. H. NORTHCOTE, Biochem. J., 94 (1965) 617. P. ~f. RAY, T. L. SHININGER AND M. ~'][. RAY, Proc. Natl. Acad. Sci. U.S., 64 (I969) 605. C. P£AuD-LENO~L AND M. AXELOS, F E B S Letters, 8 (197 o) 224. R. hi. 13ROWN, W. W. FRANKE, H. KLEINIG, H. FALK AND P. SITTE, J. CellBiol., 45 (197 °) 246. D. H. NORTHCOTE, in P. N. CAMPBELL AND G. D. GREVlLLE, Essays in Biochemistry, Vol. 5, Academic Press, L o n d o n and New York, I969, p. 89. \V. Z. HASSlD, Ann. Rev. Pl. Physiol., 18 (1967) 253. xA'. Z. I~ASSID, Science, 165 (1969) 137. ]3. FLEISCHER, S. FLEISCl~ER AND H. OZAWA, J. Cell. Biol., 43 (1969) 59. D. J. )[ORRE, L. M. ~'IERLIN AND T. W. KEENAN, Biochem. Biophys. Res. Commun., 37 (1969) 813 • R. R. WAGNER AND M. A. CYNKIN, Biochem. Biophys. Res. Commun., 35 (I969) I37. }{. SCHACHTER, I. JABBAL, R. L. HUDGIN AND L. PINTERIC, J. Biol. Chem., 245 (197 o) lO9 o. F. LOEWES, Ann. N . Y . Acad. Sci., 165 (1969) 577. H. KAUSS, A. L. SWANSON, R. ARNOLD AND W. ODZUCK, Biochim. Biophys. Aeta, 192 (1969) 55" Y. HIGASHI, J. L. STROMINGER AND C. C. SWEELEY, Proc. Natl. Acad. Sci. U.S., 57 (1967) 1878H. KAUSS, F E B S Letters, 5 (1969) 81. C. L. VILLEI~IEZ, Biochem. Biophys. Res. Commun., 4 ° (I97 o) 636.
Biochim. Biophys. Acta, 237 (1971) 56-64
BBA
BIOCHIMICA ET BIOPHYSICA ACTA
26528
P O L Y S A C C H A R I D E FORMATION IN PLANT GOLGI BODIES P. J. H A R R I S * AND D. H. N O R T H C O T E
Department of Biochemistry, University of Cambridge, Cambridge (GreatBritain) (Received N o v e m b e r i 3 t h , 197o )
SUMMARY
I. A fraction rich in Golgi bodies has been isolated from the roots of pea seedlings. The fraction has been characterised by an electron microscopic examination of negatively stained samples and ultra-thin sections. 2. The distribution in various cell fractions of the polysaceharides synthesised b y the root tip during a 4o-min incubation with radioactive glucose has been determined. 3. It has been shown that pectic substances and hemicelluloses are carried and probably synthesized by the Golgi bodies and their associated vesicles.
INTRODUCTION
The Golgi apparatus is part of a membrane-bounded transport system within the cell. This route links the perinuclear space, the lumen of the endoplasmic reticulum and the cisternae and vesicles of the Golgi bodies to the outside of the cell across the plasmalemma. The material which is conducted through the system can be synthesised and modified during its passage and in this way the Golgi apparatus is involved in the transport and synthesis of plant cell wall polysaecharides and polysaccharide secretions such as the slime of the root cap cells. The Golgi body takes part in the formation of the cell plate at telophase 1-4 and in the development of the primaryS, ~ and secondary walF, 8 during growth and differentiation of the cell. The evidence for this is based on observations of the fine structure of the cells and on radioautographic techniques. Radioactive polysaccharides can be shown to be present within the Golgi cisternae of cells supplied with a pulse of radioactive precursor and this material can be chased via the Golgi vesicles to the cell wall when non-radioactive precursors are subsequently supplied to the tissues 9. In this paper we present direct evidence that the Golgi bodies contain high molecular weight polysaecharides that have been newly synthesised and that are similar to the polysaceharides found in the matrix of the cell wall. MATERIALS AND METHODS
Pea seeds (variety Feltham First) were surface sterilized by soaking in Milton (Milton Division, Richardson-Merrell Ltd., London) for 15 rain. They were then * P r e s e n t a d d r e s s : D e p a r t m e n t of B i o c h e m i s t r y , U n i v e r s i t y of Oxford, Oxford, E n g l a n d .
Biochim. Biophys. Acta, 237 (1971) 56-64
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washed in water and germinated in vermiculite at 25°. Seedlings with roots 3-4 cm in length were used.
Incubation with the isotope Pea seedlings (2o) were incubated for 4 ° rain at 2o ° with their roots dipping into a solution (400/,1 of D-E14Cs!glucose (40 #C, 320 mC/mmole). The details of the incubation and the method of determining the amount of isotope taken up by the seedlings are the same as those described by HARRIS AND NORTHCOTETM. Isolation of the Golgi bodies Golgi bodies were isolated from the roots by a slight modification of the method of MORR~ et al. 11. The 20 pea roots which had been incubated with D-[l~C6!glucose were excised close to the cotyledons and added to 16 g of thoroughly washed unlabelled excised roots. The tissue was gently homogenised in a pestle and mortar using IO ml of homogenisation medium 11,12 with the addition of glutaraldehyde (i-lO -1 M) (Koch Light Ltd., Colnbrook, Bucks). The suspension was allowed to stand for IO rain with occasional stirring, filtered through 2 layers of muslin and the filtrate was centrifuged at 7000 rev./min (average 4000 × g) for 30 rain. The pellet produced is subsequently referred to as the first pellet. All centrifugations were carried out using a Beckman Spinco L2 or L2B centrifuge with a SW 5oL or SW 39 rotor. The supernatant was layered onto 1.8 M sucrose (0.25 ml) and then centrifuged at 12000 rev./min (average 11750 × g) for 30 rain. Particulate material was deposited onto the 1.8 M sucrose cushion and the supernatant was replaced by 1.6 M sucrose (0. 5 ml), 1.5 M sucrose (I.O ml), 1.25 M sucrose (I.O ml) and 0. 5 M sucrose to fill the tubes. The density gradient was centrifuged for 3 h at 35 ooo rev./min (average IOOooo × g). The particulate layers at the o.5-1.25 M sucrose interface (subsequently referred to as the Golgi fraction) and the 1.25-I.5 M sucrose interface (subsequently referred to as the mitochondrial fraction) were carefully removed with a fine Pasteur pipette and centrifuged in 0.5 M sucrose to give a pellet (30 rain at 12 ooo rev./min; average 11750 × g). All the above sucrose solutions contained all the constituents of the homogenation medium. The pellets were collected in polyallomer tubes as these resisted the conditions of the subsequent hydrolysis, all other centrifugations were carried out in nitrocellulose tubes. All the procedures for the preparation of the pellets took place at 0-8 °. The pellets were washed with several changes of 75 % (v/v) ethanol containing n-glucose (50 gfl) and then with 75% (v/v) ethanol, and dried over P205 in vacuo. They were dissolved in 25 or 30/~ of 72% (w/w) H2S04, diluted to 3% (w/w) H2SO 4, and hydrolysed by autoclaving at 15 lb/inch 2 at 16o ° for I h. The hydrolysates were filtered through sintered glass micro filters (porosity 3), and neutralized. The uronic acids and neutral sugars were separated and their radioactivity determined as described by HARRIS AND NORTHCOTE. The radioactivity of the uronic acids was determined by quantitatively eluting the material from the origin of the chromatograms, followed by electrophoresis at pH 2.0 and pH 3.5Characterization of the pellets by electron microscopy The pellets were examined after negative staining and also after thin sectioning. The negative staining was carried out using a 2% phosphotungstic acid solution Biochim. Biophys. Acta, 237 (I97 I) 56-64
58
P. J. HARRIS, D. H. NORTHCOTE
brought to pH 6.8 with NaOH. Bovine plasma albumin (final concentration o.1% w/v) was added to the negative stain before use. The pellets were also fixed for i tl at 2o ° in a phosphate buffered solution (o.o2 M, pH 7.2) of glutaraldehyde (6°.o) containing sucrose (o.5 M). They were post fixed in veronal buffered osmic acid (I %) for I h at 2o °, dehydrated through an ethanol series and embedded in Araldite.
Fig. I. N e g a t i v e s t a i n e d p r e p a r a t i o n of t h e Golgi-rich fraction isolated f r o m t h e b r o k e n cells ( × 30000). Fig. 2. N e g a t i v e s t a i n e d p r e p a r a t i o n of t h e Golgi-rich fraction isolated f r o m t h e b r o k e n cells ( × 45 ooo).
Biochim. Biophys. Acta, 237 (1971) 56 64
POLYSACCHARIDE FORMATION IN PLANT GOLGI BODIES
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The sections were stained with uranyl acetate and alkaline lead hydroxide and examined in a G.E.C., A . E . I . E . M . 6 B at 60 kV 1~. RESULTS
Electron microscopic examination of the fractions After negative staining the Golgi fraction was seen to contain a high proportion of Golgi bodies having a morphology similar to that reported by CUNNINGHAMet al. 14 (Figs. I and 2). They were composed of a central circular plate surrounded by a mass of convoluted tubules to which were attached vesicles with an electron dense centre and an electron transparent border. The central plate of the Golgi body shown in Fig. 2 is clearly resolved which indicates that the whole structure probably represents a single isolated cisterna. The Golgi fraction was also found to be rich in Golgi bodies when ultra-thin sections of the fraction were examined (Figs. 3 and 4). The organelle
Fig. 3. Sections of the pellet of the Golgi-rich fraction isolated from the broken cells ( x 15 ooo). Fig. 4. Sections of the pellet of the Golgi-rich fraction isolated from the broken cells (X 25 ooo).
Biochim. Biophys. Acta, 237 (1971) 56-64
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HARRIS, D. H. NORTHCOTE
had an appearance similar to that observed in unbroken plant cells. Numerous vesicles were associated with the cisternae, some of which probably represented cross sections of the tubular outer cisternal region. Other vesicles of various sizes bounded by smooth and less frequently rough membranes were also present and these were not associated with the intact Golgi bodies. Some were morphologically similar to those vesicles connected with the cisternae, and probably they were detached during the isolation procedure. Examination of negatively stained and thin sections of the mitochondrial preparation showed that it was rich in mitochondria and proplastids. The cristae of the mitochondria were swollen (Fig. 5)- Rough and smooth membrane bound vesicles and some starch grains were also present in the fraction. Thin sections of the first pellet showed that it contained a whole range of cell components including nuclei, plastids with starch grains, cell wall fragments, mitochondria, proplastids and some Golgi bodies.
Fig. 5. Section of t h e pellet of t h e m i t o c h o n d r i a l fraction isolated from t h e b r o k e n cells ( X 15 ooo).
Chemical studies D- ~llC6]Glucose (79.76. lO6counts/min) was fed to the pea roots and after 4 ° rain the radioactivity of the glucose remaining in the incubation medium was 29.02. lOs counts/rain. This represents a percentage uptake of the radioactive glucose bv the tissue of 63.6°,o. The percentage distribution of radioactivity in the uronic acids and neutral sugars obtained from acid hydrolysates of the first, Golgi rich, and mitochondrial rich fractions was determined. Table I shows that the hydrolysates of both the first and the Golgi rich fractions contained radioactivity in all the neutral sugars and uronic acids. These sugars and sugar acids are characteristic components of the polysaccharides of plant cell wails. The first fraction contained cell wall fragments from which some of these radioactive sugars were presumably derived. In addition the first fraction also contained a large number of plastids containing starch grains and this probably increased the amount of radioactive glucose present in the hydrolysate. The Golgi fraction contained no cell wall fragments and no starch grains either Biochim. Biophys. Acta, 237 (I971 ) 56-64
POLYSACCHARIDE FORMATION IN PLANT GOLGI BODIES
6I
TABLE I RELATIVE AMOUNTS OF RADIOACTIVITYINCORPORATED FROM UNIFORMLY LABELED D-[14C~GLUCOSE INTO TH]~ URONIC ACIDS AND NEUTRAL SUGARS OF THE ISOLATED FRACTIONS OF THE CELLS OF THE ROOT TISSUE The roots were incubated w i t h the radioactive glucose for 4 ° nlin and t h e n homogenised and the fractions isolated b y density gradient centrifugation. The values in parentheses represent the a m o u n t s of radioactivity calculated as percentages of the total a m o u n t of radioactivity in the n e u t r a l sugars and the uronic acids excluding glucose. The o t h e r values represent the a m o u n t s of radioactivity calculated as a percentage of the total a m o u n t of radioactivity in all the neutral sugars and the uronic acids.
Sugar
Radioactivity (%) First fraction
Galacturonicacid Glucuronic acid Galactose Glucose Mannose Arabinose Xylose Fucose Rhamnose Total c o u n t s / m i n
Golgifraction
9.1 (14.o) 11. 3 2.0 ( 3 . 0 ) 1.3 19.9 (30-6) 31-7 35.0 ( - - ) 15.2 5.2 ( 8 . 0 ) 7.1 19.8 (30.5) 20.2 5.3 ( 8 . 2 ) 8.1 0. 7 ( i . i ) 1.2 3 .0 ( 4 .6 ) 3.9 15 156" (9853)* 4441
(13.3) (1.5) (37-4) (--) (8.3) (23.8) (9.6) (1.5) ( 4 .6 ) (3765)
Mitochondrial fraction lo.i -22.9 20. 3 8. 4 35-5 2.9
(12.7) (--) (28,7) (--) (lO.5) (44.5) (3.6)
-(--) 663 (529)
* Values for 1/~ of the total yield of the first fraction.
free or contained within plastids. Thus the radioactive sugars of the high molecular weight polysaccharides in this fraction must have been present in the Golgi bodies and their associated membranes. The percentage distribution of radioactivity in the uronic acids and neutral sugars of the hydrolysate of the Golgi pellet and the first pellet were very similar except that the Golgi pellet had a much higher percentage of radioactivity in galactose relative to that in glucose. This is probably in part due to the lack of radioactive starch grains. When the percentages were recalculated omitting glucose, the distribution of radioactivity in the uronic acids and sugars of the two fractions were very similar. The hydrolysate of the mitochondrial fraction also contained radioactive neutral sugars and uronic acids characteristic of plant cell wall polysaccharides. However, the percentage distribution of radioactivity in these was different from that of the first and the Golgi rich pellets. In particular the mitochondrial fraction was much richer in radioactive arabinose and much less rich in radioactive xylose and fucose estimated together. The ratio of the amount of radioactivity in xylose and fucose to the amount of radioactivity in arabinose for the Golgi fraction was 1:2.2, for the first fraction I:3.3, and for the mitochondrial fraction I:12.3. The origin of the radioactive sugars in the mitochondrial fraction is uncertain. It is possible that radioactive water-soluble cell wall polysaccharides such as pectin become adsorbed to the mitochondria during the homogenization and subsequent fractionation. It is also possible that the mitochondrial fraction was contaminated with Golgi derived vesicles although this is unlikely as the percentage distribution of radioactivity in the neutral sugars and uronic acids of the mitochondrial and Golgi fractions were different. All or part of the radioactive glucose present in the mitochondrial fraction could have arisen from radioactive starch as some starch grains were present. 13iochim. Biophys. Acta, 237 (1971) 56-64
62
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DISCUSSION
The results presented here support the conclusion that the cisternae and vesicles of the Golgi apparatus are involved in the transport and probably the synthesis of pectic and hemicellulosic cell wall polysaccharides (the matrix material of the wall). A large percentage (approx. 63 %) of the radioactivity present in the sugars of hydrolysate of the Golgi fraction obtained from pea roots which had been incubated with uniformly labelled D@4C!glucose was in galactose, arabinose, and galacturonic acid. These sugars are characteristic component sugars of the pectic substances 15. The amount of labelled pectic material in the Golgi-rich fraction m a y be due partly to the adsorption of radioactive water soluble polysaccharides released from the root cell walls during the breakage of the cells. The presence of radioactive xylose, mannose and a trace amount of radioactive glucuronic acid in the hydrolysate indicated the occurrence of xylans and glucomannans in this Golgi fraction. Glucose was not labelled to a great extent relative to the sugars characteristic of the matrix polysaccharides. This result indicates that the active synthesis and transport of cellulose is not extensively brought about by the Golgi apparatus. The radioactive glucose present in the hydrolysate of the Golgi fraction could have arisen from the physical adsorption of free radioactive glucose present in the tissue and released during the homogenization, or from the synthesis and transport of other glucans such as callose. RAY et al. 16 showed that a preparation which contains a high percentage of Golgi derived membranes and vesicles was able to catalyse the transfer of glucose from UDPGlc and GDPGlc to an endogenous acceptor. Part of the polysaccharide synthesised from both GDPGlc and UDPGlc was a I + 4/~ linked glucan. However, it has been found in other systems that both I-> 3/~ and I ~ 4 [3 linked glucosyl units can be obtained from UDPGlc 17. BROWN et al. ~s have demonstrated that in the unicellular alga Pleurochrysis scherUelii the wall scales contain microfibrils that are possibly made of cellulose. These scales are synthesised and transported to the cell surface in the Golgi apparatus and its associated vesicles. They also suggest that the site of synthesis of cellulose in higher plants is in the Golgi apparatus. In higher plants, however, the microfibrils are often interwoven in a highly ordered manner and they are closely packed. It is difficult to conceive how this arrangement could be brought about unless the enzymes involved in cellulose synthesis are arranged either at the plasmalemma surface or in the wall itself. Evidence for such a location of a cellulose synthesising system is given by NORTHCOTE~9. Since the membranes of the Golgi vesicles fuse with the plasmalemma it is probable that the enzymes for cellulose synthesis and indeed other enzymes which could modify the polysaccharides of the cell wall in situ are transported from the Golgi complex to their site of action at the outer cell membrane. The Golgi apparatus is probably involved in the synthesis as well as the transport of the matrix polysaccharides. NORTHCOTE AND PICKETT-HEAPS9 demonstrated b y high resolution autoradiography that the Golgi cisternae in the outer root cap cells of wheat contain radioactive material within 5 min after feeding the roots with D-[6-~Hlglucose. The radioactivity was probably present in polymeric substances since it was not removed during the procedures involved in the preparation of the material for the study with the electron microscope. Glycosyl transferases which Biochim. Biophys. Acta, 237 (1971) 56-64
POLYSACCHARIDE FORMATION IN PLANT GOLGI BODIES
63
utilize UDPGlc, UDPGal, UDPGlcA, UDPGalA, UDPAra, U D P X y l as substrates and the epimerases and other enzymes that are involved in the interconversion of these nucleoside diphosphate sugars have nearly all been found in a particulate preparation obtained from homogenized mung bean shoots 2°, 21. Although this preparation has not been characterised by electron microscopy it is possible that these enzymes are located in Golgi membrane fragments. Fractions rich in Golgi bodies have been obtained from homogenised liver tissue and these preparations show glycosyl transferase activities 22-25. Myoinositol which can be formed from glucose can act as a precursor of uridine diphosphate uronic acids and pentoses 26. This interconversion provides an alternative pathway for the formation of these uridine diphosphate compounds, however, the location of the enzymes concerned is not known. KAUSS e / a l Y have used a similar particulate preparation to that described by HASSIDeO,22 and they have shown that the enzymes involved in transferring the methyl groups from 5-adenosyl-L-methionine to esterify the polygalacturonic acid chains of pectin will act only if the acceptor molecules are generated within the particles of the enzymic preparation. The methylated polygalacturonic acid formed was protected from the action of pectin methyl esterase, but this protection was removed by substances which disrupt the integrity of lipid membranes. This evidence is consistent with the idea that the enzymes are an integral part of the membrane. Glycolipids have been shown to take part in the biosynthesis of bacterial mucopeptides and in several cases the lipid component has been identified as an isoprenoid alcohol ~s. KAUSS20 has demonstrated that a similar lipid intermediate is formed during the synthesis of glucomannan from GDPMan in the mung bean particulate enzyme system. The lipid has not been identified but it is synthesised from mevalonic acid. A glucolipid and a glycoprotein have also been identified as intermediates in glucomannan synthesis in mung bean 3°. These substances m a y be components of the Golgi membranes and m a y act as acceptor sites for the transglycosylation reactions and the attachment of the growing polysaccharide chains within the cisternae and vesicles of the Golgi complex. The enzymic activity of the Golgi apparatus and hence the nature of the polysaccharides that are synthesised varies during the differentiation of the cell. In maize roots for example it has been shown that the hypertrophied Golgi apparatus in the outer root cap cells form a different polysaccharide from that formed in the cells of the adjacent root tissue ~°. The Golgi fraction obtained in the work which is reported here was derived from the whole roots and represents a heterogeneous mixture of Golgi bodies which have different synthetic activities. ACKNOWLEDGEMENTS
P. J. H. gratefully acknowledges the receipt of a grant from the Science Research Council during the tenure of which this work was carried out. REFERENCES I 2 3 4
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Biochim. Biophys. Acta, 237 (1971) 56-64