Changes have been induced in the polysaccharides laid down in the cell walls of lettuce pith by administration of the hormones auxin and zeatin. This polysaccharide derives from the membrane systems of the cells and radioactive precursor has been used to follow the accompanying changes which occurred in the polysaccharide contained within isolated Golgi and endoplasmic reticulum fractions. Zeatin-induced division and differentiation was accompanied by up to 12-fold increases in the amount of radioactivity in polysaccharide of isolated membrane fractions, and the pattern of distribution of label between the sugars of this polysaccharide was qualitatively different in the presence of zeatin. The change in this pattern was evident in the Golgi fraction at an earlier stage in the induction of the response than that in the endoplasmic reticulum.
Isolation techniques have made it possible to study the involvement of the intra-cellular membrane system, composed of rough endoplasmic reticulum, Golgi bodies and smooth membrane vesicles, in the synthesis of material to be exported across the plasmalemma into the matrix of the plant cell wall (Bowles & Northcote, 1972). Using maize roots, these workers showed that the type of polysaccharide synthesized within the membrane system could be correlated with the state of differentiation of the root zones. However, a difficulty in these experiments is the cellular heterogeneity of root tissue sections which leads to a multiplicity of synthetic activities amongst the membrane fractions isolated even from quite a specific area of the root.
Disks of lettuce pith parenchyma can be cultured in vitro under precisely defined nutritional, hormonal and environmental conditions so that the extent of differentiation of the tissue can be controlled (Dalessandro & Roberts, 1971; Dalessandro, 1973). In the studies reported here this system has been used to study the changes which occurred in the composition of the polysaccharide material within isolated Golgi and endoplasmic reticulum fractions when differences in cell wall composition were induced in a homogeneous tissue by exogenous auxin and cytokinin applications. The results demonstrate that zeatin-induced division and differentiation is accompanied by up to 12-fold increases in the amount of radioactivity taken up from glucose into polysaccharide of isolated membrane fractions and that the wall material synthesized during a 2-h pulse period is qualitatively different in the presence of zeatin. The induced change in polysaccharide incorporated into the wall was reflected by changes in the radioactivity incorporation pattern in the membrane compartments. The change in the pattern in the Golgi fraction was evident at an earlier stage in the induction of the response than changes in that in the endoplasmic reticulum.
MATERIALS AND METHODS
Plants of Lactuca sativa L. were purchased locally; the cabbage lettuce were Webbs Wonderful and the cos lettuce of an unknown variety. Disks (4 mm in diameter) of the central pith tissue were prepared and cultured as described by Dalessandro & Roberts (1971), with 1 mg/1. indol-3ylacetic acid (IAA) included in the medium of the control disks, and 1 mg/1. IAA plus 0·1 mg/1. zeatin in that of the experimental tissue. IAA was obtained from Koch-Light Ltd, Colnbrook, Bucks., U.K., and zeatin from Sigma Chemicals Ltd, Kingston-upon-Thames, Surrey, U.K. On the morning of the 8th day some disks were removed and prepared for light microscopy of wax-embedded sections as described by Wright & Northcote (1972).
The remaining disks of each treatment were placed together on the nutrient medium contained in a single Petri dish. The disks were inverted from their original position since differentiation was reported to occur more extensively near the growth medium (Dalessandro & Roberts, 1971). A drop of a solution of D-[U-14C]glucose (sp.act. 258 mCi/mmol; 100 μCi/ml) was applied to the new top of each disk using a 50-μl automatic pipette; 47 disks per treatment were used in the experiment with cabbage lettuce and 88 disks per treatment with cos lettuce. These consumed approximately 250 and 450 μl of the solution respectively. After 2 h the disks, which had absorbed the solution, were taken to prepare the subcellular fractions. Fractionation was carried out as described by Bowles & Northcote (1972) except that the parttculate band formed at the 1·6/1·8 M sucrose interface after centrifugation at 11750g was resuspended in 0·5 M sucrose and pelleted directly without prior separation of the smooth membrane fraction. Pellets were then washed, hydrolysed and analysed for their component uronic acids and neutral sugars (Harris & Northcote, 1970; Bowles & Northcote, 1972).
An identical preparation using lettuce tissue which had not been supplied with radioactive glucose was used to prepare the fractions examined by electron microscopy (Bowles & Northcote, 1972). The Golgi pellets isolated from disks cultured in the absence of zeatin are designated GO, and those from disks cultured on a zeatin-containing medium, GZ; corresponding rough endoplasmic reticulum and wall fractions are designated ERO, ERZ and WO, WZ, respectively.
Culture of lettuce disks
Prepared disks were incubated for 1 week on a nutrient medium containing either 1 mg/1. IAA only, or 1 mg/1. IAA plus 0·1 mg/1. zeatin. On the 8th day they were taken for the experiments and some were fixed for microscopical examination. Preliminary qualitative experiments confirmed that the sort of results obtained by Dalessandro & Roberts (1971) could also be obtained with several varieties of cabbage lettuce although occasionally, and unreproducibly, no xylem elements were formed. In the majority of cases control disks exposed only to IAA contained some local areas, typically at the disk surface, where cell division had been stimulated (Fig. 2) but areas showing comparatively uniform cell expansion (Fig. 3) were more common.
Differentiation of xylem elements sometimes occurred at the base of the dividing region (Fig. 2). In the presence of zeatin, ‘nests’ of dividing cells (Fig. 4) occurred much more widely and this led to a concomitant increase in the number of xylem elements formed (Fig. 5). Thus the effect of the zeatin was to stimulate greater primary and (subsequently) secondary wall synthesis.
No phloem cells were found in disks cultured under either set of conditions, when they were examined by ultraviolet fluorescence microscopy of aniline-blue-stained sections (Wright & Northcote, 1972).
Examination of fractions
Subcellular fractions were examined by electron microscopy. Thin sections of the ER pellet showed that the fraction was composed predominantly of rough membrane material (Fig. 6). The fraction sedimenting on the 1·6/1·8 M sucrose cushion was also examined using a negative staining method and numerous dictyosomes were observed which showed de-stacked cisternae (Fig. 7) with distal tubular proliferations ending in both smooth and ‘coated’ vesicles (Fig. 8).
Analysis of isolated fractions
The percentage distribution of radioactivity in the polysaccharide component of the various fractions is shown in Tables 1 and 2 for 2 separate experiments. Preliminary experiments showed that no more than 1 % of the label in the polysaccharide components of a fraction was contained in rhamnose or fucose and these were not subsequently analysed. Radioactivity in glucose is not included in the tables since label in this sugar in polysaccharide was relatively much greater in the wall (65–70 %) than in the membrane fractions (15–20%) and its omission facilitates comparison between the pectic and hemicellulosic components of the wall and the polysaccharide material in the membrane fractions which give rise to these components.
The uronic acids were further fractionated by electrophoresis at pH 3·5. Four components were resolved (a–d, Fig. 1) and the way in which the percentage indicated in Tables 1 and 2 was distributed between the 4 components is shown in Table 3.
Markers indicated that b was probably galacturonic acid and that c may have been glucuronic acid (Fig. 1).
The system studied by Dalessandro & Roberts (1971) and Dalessandro (1973) has been utilized in investigating the different roles of the polysaccharide-synthesizing membrane systems of the cell in bringing about hormone-induced changes in the cell wall. The length of the incubation and the hormonal conditions were chosen from their results to provide the greatest change in the number of xylem elements induced compared with control disks. Our cytological studies indeed showed that zeatin (0·1 mg/1.) led to a considerable increase in the number of tracheids in a disk after 7 days.
The separation system used to isolate the Golgi and endoplasmic reticulum fractions was a simplification of the one used by Bowles & Northcote (1972) and the Golgi pellet contained both dictyosomes and smooth membrane. The pooling of these fractions is justified by previous experiments (Bowles & Northcote, 1972) which showed that there was more radioactivity in the Golgi fraction than in the smooth-membrane fraction, and that the radioactivity incorporation patterns of these fractions were very similar. Electron microscopy of thin sections showed that the fractions closely resembled the ones isolated from maize-root tissue (Bowles & Northcote, 1972), and all the ultrastructural features indicated in Figs. 7 and 8 were also seen in negatively stained preparations of maize-root organelles.
The results clearly showed that the inclusion of zeatin in the incubation medium led to a considerable increase in the amount of radioactivity incorporated into the uronic acids and neutral sugars of the fractions as a result of a 2-h pulse of D-[U-14C]-glucose on the 8th day. The comparison is valid since the incorporation was brought about by control and experimental tissues which had been identical in source and amount at day zero; differences are therefore attributable to changes induced by zeatin over the 7-day period. In the experiment using cabbage lettuce the increase was 3- to 5-fold, whereas in the case of cos lettuce it was 8- to 12-fold. While the effect of hormones on cell-wall synthesis is well known (Northcote, 1963, 1972) an effect on the membrane systems responsible for polysaccharide synthesis has not previously been directly demonstrated.
Changes in cell wall composition during the transition from primary to secondary growth in plants are well documented (Northcote, 1963; Jeffs & Northcote, 1966). The xylose/arabinose ratio can be taken as an index of differentiation since the secondary matrix polymer hemicellulose contains large amounts of xylose while arabinose is a sugar characteristic of the pectic matrix material laid down during primary growth. This is true for lettuce since the outer vascular part of lettuce stem has a xylose/arabinose ratio of 1·3 whereas the ratio for pith tissue is only 0·4 (K. Wright, unpublished observation). Inspection of the WO and WZ fractions of Tables 1 and 2 shows that there was no such change in the relative amounts of xylose and arabinose being incorporated into the wall during the pulse period; the major changes were a drop in the percentage of label being channelled into uronic acid, and a rise in the percentage of radioactivity in galactose in the zeatin-treated tissue. The likely explanation is that the zeatin so stimulated cell division in lettuce pith that the effect on secondary wall components was masked by this massive stimulation of cell plate formation and primary wall growth.
Nevertheless the pattern of incorporation of label into the wall fractions was altered by zeatin in a specific way. The large decrease in the percentage of label in uronic acids and the large increase in that in galactose was common to both the cabbage and the cos lettuce, as was the small increase in the percentage incorporation into arabinose and the decrease in that into xylose. The factor by which zeatin stimulated the total cpm incorporated into polysaccharide showed at least a doubling in the case of the cos lettuce as compared with the cabbage lettuce. Using the stimulation in total cpm as an indication of the degree to which the change in wall polymers had been induced, the results obtained using the cabbage lettuce can therefore be considered to represent the pulse incorporation patterns at an earlier stage of induction of the response observed using the cos lettuce. The pulse incorporation patterns in the membrane fractions can then be compared with those of the wall preparations. In the experiment using cabbage lettuce the patterns observed in ERO and ERZ were almost identical whereas those of GO and GZ closely reflected the patterns observed in WO and WZ; the degree of effect induced by the zeatin in this experiment is quantified by an approximate quadrupling of the cpm located in each fraction relative to the controls. When cos lettuce pith was used, the zeatin caused a io-fold increase in this parameter, and the differences in the pulse incorporation patterns between GO and GZ, ERO and ERZ were both closely similar to the difference in this pattern between WO and WZ. These results suggest that a small degree of change in the polysaccharide material deposited in the wall can be effected by the Golgi apparatus alone, whereas larger degrees of change in the matrix material of the wall may require that this change is initiated at an earlier stage in the synthetic process, at the level of the endoplasmic reticulum. This conclusion relies on acceptance of the reasonable assumption that the different effects of identical incubations with zeatin on the cabbage and cos lettuce depended on the rate at which the zeatin, in conjunction with endogenous hormones present in the tissue, affected the rate of modification of cell-wall metabolism. The results further suggest that the Golgi apparatus may respond earlier than the endoplasmic reticulum to a hormone-induced change.
The distribution of radioactivity between the components of the uronic acid fraction was very similar to that observed in hydrolysates of sections of sycamore roots which had been supplied with D-[U-14C]glucose (Wright & Northcote, 1974). The bulk of the uronic acid label was found in a peak running with the same mobility as galacturonic acid. In most cases total changes in the percentage of radioactivity in the uronic acids were made up of changes in all 4 components. The similarity in the relative radioactivity distribution between the 4 components supports the conclusion that the effect of zeatin on cos and cabbage lettuce pith was the same.
It is generally agreed that the Golgi apparatus is involved in the synthesis of cell-wall and extracellular polysaccharides (Northcote, 1971; O’Brien, 1972; Whaley, Dauwalder & Kephart, 1972) but there is no direct evidence for a similar role of the ER. Profiles of ER are often seen located near sites of cell wall, cell plate and sieve-pore synthesis (Newcomb, 1963; Cronshaw, 1965; Northcote & Wooding, 1966; Hepler & Newcomb, 1967), but autoradiographical evidence for its direct participation in cell-wall synthesis (Pickett-Heaps, 1967, 1968) cannot be regarded as conclusive in the absence of accompanying chase experiments or concurrent analysis of the radioactive material.
Elucidation of the role of the membrane system in polysaccharide synthesis has been facilitated by cell fractionation methods (Morr6, Mollenhauer & Chambers, 1965; Harris & Northcote, 1971; Jilka, Brown & Nordin, 1972). Later it was shown that the specific activity of the material in the dictyosome fraction was 40 times greater (on the basis of the lipid content of the pellets) than that in the ER isolated from the same tissue (Bowles & Northcote, 1972). Thus the Golgi apparatus appeared to represent a focal point in the intracellular synthesis and transport route of cell wall polysaccharide. The results presented here support the suggestion that the Golgi apparatus may exert a greater control on polysaccharide synthesis than the endoplasmic reticulum. Epimerase enzymes are responsible for the interconversion of sugars of the glucose and galactose series and a shut-down of these enzymes could cause a switch from primary to secondary cell-wall synthesis (Northcote, 1963). These enzymes may be located in the Golgi apparatus and could be a site of action for the hormones which control the polysaccharide synthesized by the cells of lettuce pith. It will be of interest to discover how quickly the membrane systems can respond to a hormonal stimulus which ultimately alters the pattern of poly saccharide laid down in the cell wall.
K.W. thanks Tate and Lyle Limited for a Fellowship, during the tenure of which this work was carried out. D.J.B. was supported by a Science Research Council Studentship. We thank Professor Sir Frank Young for permission to work in the Department.