Turnover of cell wall polysaccharides of a Vinca rosea suspension culture

Three-day-cultured cells of Vinca rosea L. (in the cell division phase) and 5-day-cultured cells (in the cell expansion phase) prelabelled with d -[U-¹⁴C] glucose were incubated in a medium containing unlabelled glucose. After various periods of chase, extra-cellular polysaccharides (ECP) and cell walls were isolated, and cell walls were fractionated into pectic substances, hemicellulose, and cellulose fractions. After acid hydrolysis, the radioactive constituents in the pectic substances and hemicellulose fractions were analyzed. Active turnover was observed in arabinose and galactose in the hemicellulose fraction of cell walls, while the constituents of the pectic substances, and xylose and glucose in the hemicellulose fraction did not undergo active turnover. The proportion of radioactivities of arabinose and galactose in total radioactivity of ECP increased markedly after chasing. These results indicate that arabinogalactan was synthesized, deposited in the cell wall, degraded rapidly, and made soluble in the medium as a part of ECP.     

Sugar composition of pollen grain and pollen tube cell walls

The sugar composition of pollen grain and pollen tube cell walls was studied for Camellia japonica, C. sasanqua, C. sinensis, Tulipa gesneriana and Lilium longiflorum. In all species, the main components of pollen grain walls were arabinose, galactose, glucose and uronic acid. On the other hand, the pollen tube walls consisted mostly of glucose. The pollen tube wall of C. japonica was fractionated into hemicellulose, α-cellulose and pectic substance fractions in yields of 61, 19 and 3 %, respectively. The hemicellulose fraction was composed essentially of glucose. The sugar composition of the pollen tube wall was not influenced by the nature of exogenously supplied sugars. Rapid growth of the pollen tube seemed to correlate with the synthesis of hemicellulosic glucan.     

Inositol Metabolism in Plants. III. Conversion of Myo-inositol-2-3H to Cell Wall Polysaccharides in Sycamore (Acer pseudoplatanus L.) Cell Culture

Prolonged growth of cell cultures of sycamore (Acer pseudoplatanus L.) on agar medium containing myo-inositol-2-³H resulted in incorporation of label predominately into uronosyl and pentosyl units of cell wall polysaccharides. Procedures normally used to distinguish between pectic substance and hemicellulose yielded carbohydrate-rich fractions with solubility characteristics ranging from pectic substance to hemicellulose yet the uronic acid and pentose composition of these fractions was decidedly pectic. Galacturonic acid was the only uronic acid present in each fraction. Subfractionation of alkali-soluble (hemicellulosic) polysaccharide by neutralization followed by ethanol precipitation gave 3 fractions, a water-insoluble, an ethanol-insoluble, and an ethanol-soluble fraction, each progressively poorer in galacturonic acid units and progressively richer in arabinose units; all relatively poor in xylose units.     

Inositol Metabolism in Plants. III. Conversion of Myo-inositol-2-H to Cell Wall Polysaccharides in Sycamore (Acer pseudoplatanus L.) Cell Culture

Prolonged growth of cell cultures of sycamore (Acer pseudoplatanus L.) on agar medium containing myo-inositol-2-(3)H resulted in incorporation of label predominately into uronosyl and pentosyl units of cell wall polysaccharides. Procedures normally used to distinguish between pectic substance and hemicellulose yielded carbohydrate-rich fractions with solubility characteristics ranging from pectic substance to hemicellulose yet the uronic acid and pentose composition of these fractions was decidedly pectic. Galacturonic acid was the only uronic acid present in each fraction. Subfractionation of alkali-soluble (hemicellulosic) polysaccharide by neutralization followed by ethanol precipitation gave 3 fractions, a water-insoluble, an ethanol-insoluble, and an ethanol-soluble fraction, each progressively poorer in galacturonic acid units and progressively richer in arabinose units; all relatively poor in xylose units.Apparently, processes involved in biosynthesis of primary cell wall continued to produce pectic substance during cell enlargement while processes leading to biosynthesis of typically secondary cell wall polysaccharide such as 4-0-methyl glucuronoxylan were not activated.     

Cell plate development and delayed formation of the pectic middle lamella in root meristems

Summary The first stages of cell wall formation were followed in the root meristems of maize and French bean. Most of the primary wall components (hemicellulose, cellulose and highly methylated pectins) were laid down simultaneously along the cell plate. During young cell wall maturation within the meristem itself, significant topochemical alterations, coupled with the addition of new polysaccharides, produced complete redistribution of wall material leading to the progressive appearance of a proper middle lamella. Thus the formation of a pectic middle lamella does not precede the deposition of primary walls. It is delayed until the new partition joins to the mother cell wall.Abbreviations DMSO dimethylsulphoxide - EDTA ethylene diaminetetraacetic acid - PATAg periodic acid-thiocarbohydrazide-silver proteinate     

Metabolism of cell wall polysaccharides in cell suspension cultures of Populus alba in relation to cell growth

Changes in the composition of cell walls and extracellular polysaccharides (ECP) were studied during the growth of suspension-cultured Populus alba cells. Three growth phases, namely the cell division phase, cell elongation phase and stationary phase, were distinguished. The active deposition of polysaccharides in cell wall fractions (50 m M Na₂CO₃-, 1 M KOH-, 4 M KOH-soluble and 4 M KOH-insoluble) was observed during the elongation phase. A 50 m M Na₂CO₃-soluble pectic fraction mainly composed of 1,4-linked galactan and arabinan except acidic sugars. The 1,4-linked galactan decreased markedly during elongation. In 1 and 4 M KOH-soluble hemicellulosic fractions, non-cellulosic 1,4-glucan and xyloglucan were observed as major components, respectively. These polysaccharides also decreased during elongation. A large amount of polysaccharides was secreted into the medium as ECP. Neutral sugars were detected predominantly by sugar composition analysis. Acidic sugars, such as galacturonic acid, were less than 12% of total. In this study, active metabolism of pectic polysaccharides in addition to hemicellulosic polysaccharides, especially neutral side chains of pectin, during cell growth, was clarified.     

Cell wall biochemistry and biomechanics of harvested white asparagus shoots as affected by temperature

The effects of temperature on the dynamics of changes in shoot mechanical properties, cell wall components, relevant soluble sugars and respiration activity of harvested white asparagus spears were investigated during a 7-day storage period. All functional cell wall components of asparagus spears increased closely temperature dependent. The content of soluble glucose declined with a similar temporal dynamics and to a comparable degree, indicating a major carbon flow of this storage sugar into cell walls (60–70%). Irrespective of temperature, the contents of stored soluble fructose and sucrose remained more or less constant. Lower temperatures reduced cell wall development but do not significantly affect the relative carbon flow from storage sugars into cell walls or maintenance respiration. Compared with cell walls, maintenance respiration is by far the smaller carbon sink in stored asparagus spears. Temperature differentially affects the absolute amount and the relative contribution of the different cell wall components and the temporal dynamics of changes in structural carbohydrate and lignin content. At higher temperatures, secondary cell wall thickening resulted mainly from a large increase in cellulose content. The pronounced increase in the fractions of cellulose and especially lignin may stress the important role of lignin in cell wall strengthening. While the fraction of cell wall proteins decreased, those of hemicellulose and the pectic components were not influenced.     

Auxin and Kinetin Induced Changes of Callus Cell Wall Composition of Abutilon avicennae Gaertn

In a previous report we have shown that the arrangement of callus cell wail fibrils of Abutilon avicennae could be induced to change under IAA (2 ppm) and kinetin (10 ppm) treatments. Kinetin at this concentration was shown to be able to induce callus cell differentiation and form tracheary elements by changing the orientation of the wall fibrils. It was thus assumed that the hormonal induction of cellular differentiation and structual change of the cell wall may possibly be accompanied by the simultaneous changes of chemical composition of the wall. Attempt was therefore made to investigate if such changes do occur in vitro under the influence of phytohormones. Suspension cell-culture of Abutilon avicennae was used in this experiment to study the hormonal effect on the incorporation of H3-glucose into the cell wall polysaccharides. Analysis of neutral sugars of the cell wall following IAA (2ppm) and kinetin (10ppm) treatments was carried out with a gas chromatography. The results obtained in this experiment are shown in tables 1-2 and figures 1, It was found that the auxin was capable of promoting the synthesis of all neutral sugars, among which the glucose and the maunose in particular, increased tremendously. When H3-glucose was added to the culture medium, IAA was found to enhance the incorporation of the isotopes into the matrix polysaccharides (hemiceUulose and pectin). The result demonstrates clearly that the primary function of IAA is to stimulate the synthesis of hemicellulose composition and, as a consequence, the cell wall retained at the primary growth stage. Kinetin, on the other hand, showed an inhibitory effect on most of the neutral sugars except glucose and mannose. It appeared to have a striking inhibitory action on the synthesis of arabinose and rhanmose (a special composition of pectic substance). It also limited the incorporation of H3-glucose into the pectic substance. It is, therefore, suggested that the action of kinetin may mainly be inhibitory on the synthesis of pectic composition. The decreased rate of pectin synthesis would implicate that the cell wall has been advan ced into the phase of secondary growth. The results presented here agree fairly well with our connotation that there is a parallel relationship between cellular morphology and biochemical characteristics during cell wall differentiation and growth.     

Changes in cell wall constituents during the cell cycle in a synchronous culture of Catharanthus roseus

Changes in cell wall constituents during the cell cycle were investigated using a synchronous culture of Catharanthus roseus (L.) G. Don which was obtained by the double phosphate starvation method (S. Amino et al. 1983. Physiol. Plant. 59: 393–396). Cell walls isolated from the cells in each phase of the cell cycle were fractionated into EDTA-soluble (pectin), 5 and 24% KOH-soluble (hemicellulose) and 24% KOH-insoluble (cellulose) fractions. Their sugar compositions were investigated by gas chromatography and methylation analysis. The following changes were observed: (1) a significant increase in total cell walls in the G1 phase after cell division, (2) a temporary increase in the relative amount of the EDTA-soluble fraction during cytokinesis, (3) an increase in the relative amount of galactose, probably 4-linked galactose, in the EDTA-soluble fraction prior to cytokinesis, (4) a temporary increase in the relative amount of 3-linked glucose during cytokinesis, (5) little change in the composition of polysaccharides throughout the cell cycle in the 24% KOH-soluble fraction, which consisted mainly of xyloglucan. The changes observed are discussed in relation to the progression and physiological significance of each phase of the cell cycle.     

THE COMPOSITION OF THE DEVELOPING PRIMARY WALL IN ONION ROOT TIP CELLS. II. CYTOCHEMICAL LOCALIZATION

Jensen , William A. (U. California, Berkeley.) The composition of the developing primary wall in onion root tip cells. II. Cytochemical localization. Amer. Jour. Bot. 47(4) : 287—295. Illus. 1960.–The composition of the developing cell wall in the first 2 mm. of the onion root tip was studied using a cytochemical technique that permitted the detection of hemicellulose and the noncellulosic polysaccharides as well as the pectic substances and cellulose. The technique is based on the combination of a differential extraction procedure with the periodic acid-Schiff reaction for carbohydrates. The data obtained indicate that the cells of the apical initials are low in all wall substances but that all of the wall materials are present to some extent. Early in cell development, differences appear in the composition of the walls of the various tissues. The cortical cells are relatively high in the noncellulosic polysaccharides and cellulose while relatively low in the pectic substances and hemicellulose. Very early in development the protoderm is similar to the cortex, but differences develop during the radial enlargement of the cells. During this stage the walls of the protodermal cells are low in the noncellulosic polysaccharides and cellulose and high in pectic substances and hemicellulose. As elongation progresses, these differences are lost and the 2 tissues become very similar. The vascular cell walls are low in the noncellulosic polysaccharides and cellulose and are high in pectic substances and hemicellulose early in development. Later, hemicellulose becomes relatively more important. When the cell wall materials are sequentially extracted, no change in the general morphology of the cell occurs until only the noncellulosic polysaccharides and the cellulose remained. When the noncellulosic polysaccharides are then removed, the cells remain intact but are 30% less in diameter. This suggests that while cellulose is of critical importance, the noncellulosic polysaccharides may play a major role in determining the physical characteristics of the wall.     

Developmental changes of sugar contents in the gall on the leaf of elm (Zelkowa serrata Makino) formed byParacolopha morrisoni Baker (homopetra)

To understand cell wall polysaccharide synthesis and the role of gall in interaction with aphids, the changes of sugar contents in the galls during their growth and development were determined from May 2 to June 8, 1996. The sugar content in the symplastic (MeOH and hot water) fractions decreased as the developmental stages progressed. In the cell wall fraction, the amount of pectic substances (2-3 mg per gram fresh weight) did not change. The hemicellulosic substance increased by 40% from May 14 to May 31. Among the neutral sugar components of hemicellulosic polysaccharides, xylose and arabinose contents increased during development of the gall, suggesting that xylans with arabinose residues were massively synthesized. On the other hand, glucose content decreased during development of the gall. The cellulose substance consistently increased 5 folds from May 2 to 31. The relationship between the aphid and the changes in sugar contents of cell walls during the development of aphid and the gall formation was discussed.     

Cell-wall synthesis and elongation growth in hypocotyls of Helianthus annuus L.

The relationship between growth and increase in cell-wall material (wall synthesis) was investigated in hypocotyls of sunflower seedlings (Helianthus annuus L.) that were either grown in the dark or irradiated with continuous white light (WL). The peripheral three to four cell layers comprised 30–50% of the entire wall material of the hypocotyl. The increase in wall material during growth in the dark and WL, respectively, was larger in the inner tissues than in the peripheral cell layers. The wall mass per length decreased continuously, indicating that wall thinning occurs during growth of the hypocotyl. When dark-grown seedlings were transfered to WL, a 70% inhibition of growth was observed, but the increase in wall mass was unaffected. Likewise, the composition of the cell walls (cellulose, hemicellulose, pectic substances) was not affected by WL irradiation. Upon transfer of dark-grown seedlings into WL a drastic increase in wall thickness and a concomitant decrease in cell-wall plasticity was measured. The results indicate that cell-wall synthesis and cell elongation are independent processes and that, as a result, WL irradiation of etiolated hypocotyls leads to a thickening and mechanical stiffening of the cell walls.     

The structure, function, and biosynthesis of plant cell wall pectic polysaccharides

Plant cell walls consist of carbohydrate, protein, and aromatic compounds and are essential to the proper growth and development of plants. The carbohydrate components make up ∼90% of the primary wall, and are critical to wall function. There is a diversity of polysaccharides that make up the wall and that are classified as one of three types: cellulose, hemicellulose, or pectin. The pectins, which are most abundant in the plant primary cell walls and the middle lamellae, are a class of molecules defined by the presence of galacturonic acid. The pectic polysaccharides include the galacturonans (homogalacturonan, substituted galacturonans, and RG-II) and rhamnogalacturonan-I. Galacturonans have a backbone that consists of α-1,4-linked galacturonic acid. The identification of glycosyltransferases involved in pectin synthesis is essential to the study of cell wall function in plant growth and development and for maximizing the value and use of plant polysaccharides in industry and human health. A detailed synopsis of the existing literature on pectin structure, function, and biosynthesis is presented.     

Physico-chemical characteristics of cell walls from Arabidopsis thaliana microcalli showing different adhesion strengths

Changes in the composition and structure of cell walls and extracellular polysaccharides (ECP) were studied during the growth of suspension-cultured Arabidopsis thaliana microcalli. Three growth phases, namely the cell division phase, the cell expansion phase, and the stationary phase, were distinguished and associated with a decreasing cell cluster adhesion strength. Degradation of the homogalacturonan pectic backbone and of linear pectic side chains (1,4)-beta-D-galactan were observed concomitantly with the cell expansion and stationary phases and the decrease in cell adhesion. Also, in the stationary phase, branched (1,5)-alpha-L-arabinans were linearized. The AGP content of the culture medium increased while it decreased in the cell wall during cell growth and as cell adhesion decreased. These data suggest that, in addition to homogalacturonan, pectic side chains and AGP are involved in plant cell development and particularly in cell-cell attachment.     

Boron and calcium, essential inorganic constituents of pectic polysaccharides in higher plant cell walls

Among 16 essential elements of higher plants, Ca²⁺ and B have been termed as apoplastic elements. This is mainly because of their localization in cell walls, however, it has turned to be highly likely that these two elements significantly contribute to maintain the integrity of cell walls through binding to pectic polysaccharides. Boron in cell walls exclusively forms a complex with rhamnogalacturonan II (RG-II), and the B-RG-II complex is ubiquitous in higher plants. Analysis of the structure of the B-RG-II complex revealed that the complex contains two molecules boric acid, two molecules Ca²⁺ and two chains of monomeric RG-II. This result indicates that pectic chains are cross-linked covalently with boric acid at their RG-II regions. The complex was reconstitutedin vitro only by mixing monomeric RG-II and boric acid, however, the complex decomposed spontaneously unless Ca²⁺ was supplemented. Furthermore, the native complex decomposed when it was incubated withtrans-1,2-diaminocyclohexane-N, N, N′, N′-tetraacetic acid (CDTA) which chelates Ca²⁺. When radish root cell walls were washed with a buffered 1.5% (w/v) sodium dodesyl sulfate (SDS) solution (pH 6.5), 96%, 13% and 6% of Ca²⁺, B and pectic polysaccharides of the cell walls, respectively, were released and the cell wall swelled twice. Subsequent extraction with 50 mM CDTA (pH 6.5) of the SDS-washed cell walls further released 4%, 80% and 61% of Ca²⁺, B and pectic polysaccharides, respectively. Pectinase hydrolysis of the SDS-treated cell walls yielded a B-RG-II complex and almost all the remaining Ca²⁺ was recovered in the complex. This result suggests that cell-wall bound Ca²⁺ is divided into at least two fractions, one anchors the CDTA-soluble pectic polysaccharides into cell walls together with B, and the other may control the properties of the pectic gel. These studies demonstrate that B functions to retain CDTA-soluble pectic polysaccharides in cell walls through its binding to the RG-II regions in collaboration with Ca²⁺.     

Hydrolysis of plant polysaccharides and GLC analysis of their constituent neutral sugars

The release and degradation of sugars from onion cell walls and potato cell wall polysaccharides were followed during hydrolysis with trifluoroacetic acid so that the optimum hydrolysis conditions could be determined. After 6 hr hydrolysis in 2 M acid at 100°, calculated recovery factors of different monosaccharides from potato pectic fractions ranged from 61 to 96%. Lower yields of monosaccharides were obtained from intact onion cell walls, while the yield from cellulose was less than 0.2%. A new GLC column for the separation of alditol acetates derived from cell wall sugars is described.     

Role of the outer tissue in abscisic acid-mediated growth suppression of etiolated squash hypocotyl segments

Exogenously applied abscisic acid (ABA) substantially suppressed the elongation of hypocotyl segments of etiolated squash ( Cucurbita maxima Duch. cv. Houkou-Aokawaamaguri) after a 3 h lag period, without changes in the osmolalities of the apoplastic and symplastic solutions in the segment.
Segments with the outer tissues removed elongated more rapidly than unpeeled segments (whole segments). ABA did not suppress the elongation of peeled segments. When the segments were incubated in [¹⁴C]-glucose, radioactivity was more effectively incorporated into the cell wall fractions of the outer than into those of the inner tissue. ABA significantly inhibited the incorporation of radioactivity into hermicellulose and cellulose of the outer tissue prior to the suppression of segment elongation, but it did not inhibit the incorporation into the pectic traction of the outer tissue or into any of the cell wall fractions of the inner tissue. These results indicate that ABA primarily affected the outer tissue, in which it specifically reduced the synthesis of hemicellulose and cellulose prior to the ABA-mediated suppression of growth.     

Turnover of Cell Wall Polysaccharides during the Cell Cycle in a Synchronous Culture of Catharanthus roseus

Pulse-chase experiments were done using a synchronous cultureof Catharanthus roseus in order to study cell wall turnoverduring the cell cycle. [¹⁴C]Glucose was fed for 1 h to cells35 and 49 h after the re-start of the cell cycle. Radioactivitywas then diluted with a large amount of cold glucose and chasedduring the early G1 phase after the first cell division, thetime at which an increase in the amount of cell walls mainlytook place. A pulse-chase with [¹⁴C]glucose was also made duringthe S phase when cell walls had not increased so much. Radioactivity of the EDTA-soluble (pectin) fraction decreasedduring the chase in the early G1 phase; whereas, the radioactivitiesof the other cell wall fractions, as well as extracellular polysaccharide(ECP) increased during the chase, both in the early G1 and inthe S phases. The radioactivity of uronic acid in ECP was higherin the early G1 phase than in the S phase. These results indicatethat an active turnover of pectin may take place in the earlyG1 phase after the first cell division. ¹ Present address and reprint requests: Biological Institute,Tohoku University, Sendai 980, Japan. (Received November 5, 1984; Accepted April 2, 1985)     

The Structure of Plant Cell Walls: IV. A Structural Comparison of the Wall Hemicellulose of Cell Suspension Cultures of Sycamore (Acer PseudoPlatAnus) and of Red Kidney Bean (Phaseolus Vulgaris)

The molecular structure and chemical properties of the hemicellulose present in the isolated cell walls of suspension cultures of sycamore (Acer pseudoplatanus) cells has recently been described by Bauer et al. (Plant Physiol. 51: 174-187). The hemicellulose of the sycamore primary cell wall is a xyloglucan. This polymer functions as an important cross-link in the structure of the cell wall; the xyloglucan is hydrogen-bonded to cellulose and covalently attached to the pectic polymers.     

Ultrastructural and biochemical aspects of cell wall reconstitution in recalcitrant (grapevine) and regenerating (tobacco) leaf protoplasts

Summary To identify possible reasons that may contribute to recalcitrance in plant protoplasts, the time course of new cell wall deposition was studied by scanning electron microscopy in protoplasts of a recalcitrant species, the grapevine. Results showed that microfibrils were developed after 2 days of culture, that complete cell wall formation occurred on Day 6 to 7 of protoplast culture, and its ultrastructural appearance was identical to that of grapevine leaf-derived callus cells. In addition, a comparative study was undertaken on [U-¹⁴C]glucose uptake and incorporation in ethanol-soluble, cellulosic, and noncellulosic polysaccharide fractions in protoplasts of grapevine and of a readily regenerating species, tobacco, during culture. There was a significantly higher [U-¹⁴C]glucose uptake by tobacco than by grapevine protoplasts. The label distribution in the ethanol-soluble, cellulosic, and noncellulosic fractions of newly synthesized cell walls differed quantitatively between the two species. In particular, the labeled glucose incorporated in the noncellulosic cell wall fraction was threefold greater in tobacco than in grapevine protoplasts. Differences were also revealed in the monosaccharide composition of this fraction between the two species. Addition of dimethyl sulfoxide to the culture medium resulted in a dramatic increase in [U-¹⁴C]glucose uptake by grapevine protoplasts, whereas it exhibited a limited effect in tobacco protoplasts. It showed no effect on the ultrastructural characteristics of new cell wall nor on the incorporation rate of labeled glucose in the cellulosic and noncellulosic cell wall fractions.