8.4.2—
Synthesis and Secretion of Extracellular Material

While investigations into the function of the Golgi apparatus in animals have concentrated on protein glycosylation and other membrane modifications, plant biochemists have been more concerned with a role for this organelle in polysaccharide biosynthesis.

Gardiner and Chrispeels (1975) have presented evidence that the glycosylation of the hydroxyproline-rich glycoprotein takes place in the Golgi apparatus. The completed polymer is a cell wall rather than a plasmalemma component and carries arabinosyl side-chains on its hydroxyproline residues. Organelle fractions were prepared on density gradients from carrot root tissue pulse-labelled with [¹⁴ C]-proline. Most of the label in hydroxyproline was associated with a Golgi body-enriched fraction. This fraction coincided with peak activity in the gradient of an enzyme which transferred arabinosyl residues from UDP-arabinose onto endogenous protein acceptors. (It should be noted, though, that the gradients used to prepare organelles contained glutaraldehyde and the distribution of enzyme activities on glutaraldehyde-free gradients is not strictly comparable).

From studies of thin sections, electron-microscopists proposed that the Golgi apparatus was involved in the synthesis and secretion of cell wall polysaccharides. The increase in luminal volume at the cisternal periphery was thought to represent the polymerization or decantation of a batch of wall material which travelled to the plasmalemma in a secretory vesicle and was discharged into the wall as the vesicle membrane fused with the plamalemma. This evidence has been critically reviewed by O'Brien (1972).

Much of the biochemical evidence has come from work on the root cap. It seems that to follow polysaccharide biosynthesis by autoradiography requires the extremely high rates of uptake and incorporation found only in certain differentiating or differentiated cells. The root cap is a very rapidly growing and metabolizing tissue—the entire cap of ca 10, 000 cells in maize is replaced every 24 hours. The cap cells secrete large quantities of slime to ease the passage of the cap through the soil. This material is a hydrated polysaccharide with a chemical composition similar to the matrix components of primary cell walls, pectin, but the structure of the polymer has been modified to reduce gelation, e.g. maize root slime-.polysaccharide has a substantial content of residues of fucose, a sugar absent from maize root pectin. Slime producing root cap cells each have several hundred Golgi bodies (root parenchyma cells have only ca 30) with a characteristic 'hypertrophied' morphology: the vesiculations of the cisternal periphery are very swollen.

Northcote and Pickett-Heaps (1966) fed [6-³ H]-glucose to wheat roots and analysed the pattern of incorporation of this label into polymeric material of root cap organelles by high resolution autoradiography. After 5 minutes of feeding, silver grains were confined mainly to the immediate vicinity of the Golgi bodies. By 10 minutes, both Golgi bodies and secretory vesicles were labelled, but very little radioactivity was associated with the wall. Subsequent incubation of labelled roots in non-radioactive glucose solution for 10, 30 and 60 minutes provided a time-series of sections showing progressive loss of radioactivity from the Golgi bodies and secretory vesicles and its accumulation outside the plasmalemma (Fig. 8.3). Chemical analysis of the high molecular weight polysaccharide in the root cap after 15 minutes exposure to radioactive glucose showed that more than 70% of the label in this material was in galactosyl residues. Galactose occurs only in pectin-type polymers in angiosperm cell walls and is the only major unit of these polysaccharides to retain a hydrogen atom on carbon 6. Since less than 3% of the polysaccharide label was in glucose units of a -cellulose at this time, the labelled material represents slime-polysaccharide or matrix components of the wall.

This is strong evidence that the Golgi apparatus is responsible for the secretion of pectin-type polymers. Though high molecular weight material appeared first of all in the Golgi apparatus, this does not establish that it was synthesized here. The early stages in polymerization must involve oligosaccharides which may be lost from the sections.

The differing functions of ER and Golgi apparatus in the synthesis and secretion of slime-polysaccharide in the root cap have been investigated by Bowles and Northcote (1972), and compared with the synthesis and secretion of the ordinary matrix components of the wall in the rest of the root. They fed [U-¹⁴ C]-glucose to maize roots and, taking different parts of the root, prepared organelle fractions by differential and 'step' density gradient centrifugation. These were characterized from thin sections. Radioactive polysaccharide was found in a rough ER fraction and a fraction rich in Golgi bodies. Chemically, the labelled material in membranes from the root tip resembled slime polysaccharide and that in membranes from mature root tissue had the composition of the cell wall matrix. It could have been slime or pectin from the wall which was mixed with and bound to, or enclosed in, membranes when the tissue was chopped. To test this, membrane fractions were prepared from unlabelled tissue chopped in an extraction medium with radioactively labelled, soluble slime and wall components. The membrane fractions were essentially unlabelled.

When [U-¹⁴ C]-glucose was fed to maize roots over times ranging from 5 to 90 minutes, radioactivity was incorporated steadily into slime, wall and membrane polysaccharide up to 30 minutes. After this time the slime and wall continued to accumulate label at the same rate but there was no further increase in the radioactivity of each of the sugar residues of the polysaccharide in membranes, confirming that these polymers are precursors of the slime and wall matrix (Bowles & Northcote, 1974).

Bowles and Northcote (1976) investigated this precursor material after labelling it to saturation from supplied [U-¹⁴ C]-glucose. Some of the polysaccharide was freely soluble with a high molecular weight (>40,000), most of the rest was so firmly membrane-bound it required protease digestion for release. These types were found in both membrane fractions, but the Golgi body-rich fraction had more of the former, whereas the ER had much more of the latter. Most of the membrane bound polysaccharide of the ER was as short chains (MW < 4,000), but all the polysaccharide segments bound to Golgi body membranes had molecular weight greater than 4,000. Interestingly, though all the other sugars of slime-polysaccharide were found in labelled residues of the short chains, fucose, which always occupies a terminal position on pectin side-chains, was absent.

The earliest polymeric precursors of polysaccharides will have the lowest molecular weights and it seems from this work that they are membrane-bound. The glycosyltransferases involved in chain-extension during pectin synthesis are also all bound to membranes (Villemez et al., 1965; McNab et al., 1968; Odzuck & Kauss, 1972). This early precursor is associated chiefly with the ER. Chain extension, addition of terminal fucosyl groups to slime polysaccharide and release from the membrane occur progressively as this material leaves the ER and passes through the Golgi apparatus and there is no evidence that synthesis is restricted to a particular section of the endomembrane complex.

ER fractions for both cap and mature root contained much more radioactive polysaccharide than the equivalent Golgi body fraction. However, since a whole-root ER fraction had 40 times as much lipid, the radioactivity in polysaccharide per unit quantity of membrane (i.e. weight of lipid) for the Golgi body-rich fraction was twice the value for ER. This could be because a smaller fraction of the ER is devoted to polysaccharide synthesis, or because the polymer chains are longer in the Golgi body, or both.

With the biosynthetic machinery saturated from [¹⁴ C]-glucose, Bowles and Northcote (1974) compared the rate of production of radioactive wall and slime with the steady-state levels in membrane polysaccharide. This enabled them to calculate the rates of turnover of polysaccharide in the membrane compartments expected for different models of secretion. For example, labelled wall polysaccharide is produced at 2,000 cpm per minute and the steady state level of radioactivity in wall polysaccharides in the Golgi bodies in 5,000 cpm. Therefore the entire polysaccharide content of the Golgi body will be replaced every 2.5 minutes if all the wall material has to pass through the Golgi apparatus.

Supposing polysaccharide and membrane sack move through the stack as a single entity, then for the average stack of 5 to 6 cisternae, one cisterna is released roughly every 0.5 minutes. This seems very fast, but it is remarkably close to values determined microscopically in other plants. Working with the Chrysophycean alga Pleurochrysis, Brown (1969) showed by time-lapse cinephotomicrography that the single Golgi body and all the other organelles rotate inside the cell wall, 360° in 15 to 20 minutes. Now the lateral displacement

observed in thin sections between successive cisternae released from the Golgi body was 15°, indicating that the time between the release of successive cisternae is about 0.75 minutes. Schnepf (1961) measured the volume of slime produced by glands on the leaves of the carnivorous plant Drosophyllum lusitanicum over set time periods. From the size and number of Golgi vesicles in the gland, he estimated that the rate of production of vesicles by each Golgi body necessary to maintain the observed rate of secretion was 3 per minute at 28°C.

Now there is good reason to suspect that 2 cisternae per min is an over-estimate of the speed required to shift all the matrix to the wall via the Golgi apparatus in maize roots. Firstly, while the recovery of slime and wall components is probably close to 100%, the recovery of Golgi bodies is certainly much lower and so the steady-state level of radioactivity in this organelle is underestimated. Secondly, the wall fraction from mature root tissue was shown to contain a large amount of labelled glucose polymer which was only a minor component in the membrane fractions. It could be cellulose or contaminant starch, but it means that the measured rate of increase in polysaccharide label of the wall fraction is probably an overestimate of the rate of production of wall matrix by the endomembranes. Nevertheless, observed rates of turnover of cisternal stacks can account even for this overestimate of the rate at which polysaccharide would have to pass through the Golgi body and there is no need, therefore, to invoke secretion via the vesiculating cisternal periphery independent of the turnover of the stack, or transfer direct from ER to the wall.

Similar calculations indicate that if slime-polysaccharide is secreted only via Golgi bodies, the entire polysaccharide content of these organelles is displaced every 20 seconds, a figure corrected for cellulose. The changing pattern of label in polysaccharide monomers after feeding [¹⁴ C]-glucose for different times confirmed that slime-polysaccharide was synthesized faster than wall matrix polymers. Some of it had been secreted from the cells within 2 minutes of supplying the labelled precursor. These results pay tribute to the furious synthetic activity of root cap tissue. Even using conservative estimates of the rate of turnover of cisternae, Morré et al. (1971) calculate that secretory vesicles contribute enough new membrane to the plasmalemma of a maize root cap cell to replace it entirely every 4 to 8 hours. How the excess is recycled is not known. Careful studies of Golgi bodies and numbers of secretory vesicles in thin sections of maize root caps fixed at different times have shown the existence of rhythmic fluctuations in secretory activity. The organelles reach a peak of activity every 3 hours, synchronized over the whole cap. Whole batches of roots can be synchronized by transfer to fresh solution, which induces a peak of activity at around 1 hour later (Morré et al., 1967). Since the results obtained by Bowles and Northcote refer to the first three quarters of an hour after transfer of roots to [¹⁴ C]-glucose solution, they probably represent peak activity for root cap Golgi bodies.

Though still in debate, it now seems unlikely that the cellulose microfibrils of the wall are synthesized in the ER or Golgi apparatus in higher plant cells

(see also chapters 1 and 7). Low incorporation into glucose relative to other wall monomers in the polysaccharides of ER and Golgi body-rich fractions of maize root confirms the earlier autoradiographic evidence from developing xylem vessels. In this tissue only the plasmalemma was labelled from [³ H]-glucose at the time it was being incorporated exclusively into cellulose (Wooding, 1968). However, Golgi body-enriched membrane fractions synthesized radioactive b -1,4 glucan when supplied with UDP-[¹⁴ C]-glucose (Van Der Woude et al., 1974; Shore & MacLachan 1975). The b -1,4 glucans synthesized from UDP-glucose have been shown to be cellulose and not b -1,4 glucan sections in glucomannan, a matrix component synthesized from GDP sugars (Villemez, 1974). Golgi body cellulose synthetase showed distinctive kinetics when compared with the activity in a plasmalemma-enriched fraction, and was much less active than the plasmalemma enzyme at high (1 mM ) substrate concentration. It seems, then, that the activity in Golgi bodies was not due to contamination by plasmalemma and that the Golgi apparatus may be ferrying the enzyme to the plasmalemma in a less active form. A similar mechanism has been shown for chitin synthetase in yeast and Mucor. Chitin is structurally similar to cellulose and is produced as a microfibril on the outer surface of the plasmalemma by an enzyme particle which spans this membrane. Chitin synthetase is made in an inactive zymogen form, found in microsomal membranes, which can be converted to an active enzyme by protease digestion. An inhibitor of protease found in the soluble cytoplasm is thought to control this transition, preventing the activation of the enzyme: before it reaches its operational site in the plasmalemma (McMurrough & Bartnicki-Garcia, 1973; Durán et al., 1975). The cellulose component of the scales of Chrysophycean algae appears to be made in Golgi cisternae, but this difference between them and higher plants may well amount to nothing more fundamental than the timing of cellulose synthetase activation.