Turnover of cell-wall polysaccharides during somatic embryogenesis and development of celery (Apium graveolens L.)

Non-embryogenic cells (NEC) and embryogenc cells (EC) were separated from cell clusters derived from the hypocotyl segments of celery seedlings, which had been suspension-cultured in MS medium supplemented with 10⁵ M 2,4-D. The EC formed globular embryos in medium without 2,4-D. The globular embryo developed through heart-shaped, torpedo to cotyledonary embryos within 10 days. The EC and developing embryos were fractionated into symplastic [MeOH, hot water (HW), starch (S)] and apoplastic [pectin, hemicellulose, TFA (trifluoroacetic acid)-soluble and cellulose] fractions. The EC contained lower levels of sugar in the MeOH fraction and higher levels of starch than NEC. In the apoplastic fractions, there were no differences of total sugar amounts between NEC and EC. Cellulose contents were about 10% of the wall polysaccharides. During somatic embryogenesis, total sugar contents of the MeOH and HW fractions increased till the heart-shaped embryo stage, and then decreased during the torpedo and cotyledonary embryo stages. The sugar contents of the starch, pectin, TFA-soluble, and cellulose fractions did not change during the stages mentioned above. However, the hemicellulose substances remarkably increased during embryogenesis, and then decreased as the development proceeded. The neutral sugar components of the hemicellulosic fractions were analyzed. Arabinose increased markedly in EC to the globular embryo stage, but decreased as the development proceeded. Galactose increased only at the torpedo and cotyledonary embryo stages. Xylose was present at lower levels in all stages of embryogenesis than in the differentiated hypocotyl cell walls. These results suggest that there was a high turnover of arabinogalactan polysaccharides during embryogenesis, and that xylan accumulated in the cell walls of differentiated cells     

Long-term effects of growth regulators on growth and turnover of symplastic and apoplastic sugars in the suspension subculture of kidney bean

Suspension cells of kidney bean were grown for 42 d in MS medium supplemented with growth regulators (2.0 mgL^-1 2,4-D and 0.5 mgL^-1 kinetin) or without At the stationary growth phase (42 d), the sugars were fractionated into the symplastic (ethanol and starch) and apoplastic [low-molecular pectin (lm-pectin), high-molecular pectin (hm-pectin), hemicellulose, and cellulose] sugars. The neutral sugars (NS) of hm-pectin and hemicellulose fractions were analyzed by GLC. The growth of the suspension cells in the liquid MS media, in terms of settled cell volume (SCV), remained similar, to the end of the experiment, irrespective of the presence or absence of growth regulators, indicating the nonnecessity of the exogenous growth regulators for the subculture. Total sugar (TS) of the ethanol fraction and NS of the Im-pectin of the suspension cells grown in the medium with growth regulators were higher than in the medium without growth regulators. However, starch content in the starch fraction and uronic acid (UA) content of the Im-pectin fraction did not exhibit any differences. From these results, it was suggested that the growth regulators modulated the structure of the cell wall polysaccharide. Analysis of the NS composition of the hm-pectin fractions revealed that the Rha, Arb, and Gal contents in the presence of growth regulators were higher than in the absence, while the Xyl, Man, and Glc contents in the presence of growth regulators were higher than in the absence, indicating the turnovers of rhamnogalacturonan and/or arabinogalactan. On the other hand, analysis of NS composition of hemicellulose fractions revealed that the Ara and Glc contents in the presence of growth regulators was higher than in the absence, whereas Xyl and Glc contents were nearly consistent, indicating the turnovers of arabinaogalactan I or II. The cellulose contents remained similar, irrespective of the presence (19.1%) or absence (18.7%) of growth regulators.     

Regulation of cell-wall polysaccharide components by CaCl2 in suspension cultures of kidney bean (Phaseolus vulgaris)

We cultured the suspension cells of kidney bean in MS media supplemented with one of five concentrations of CaCI₂ [0,22,44 (control), 88, or 176 mg/L], and harvested them at the logistic (15 d) and early-stationary (30 d) phases. Cells grown at concentrations higher than 22 mg/L showed better proliferation than those at 0 mg/L The rate of proliferation also increased with higher concentrations. We fractionated the individual sugars into symplastic (EtOH and starch) and apoplastic (low-molecular pectin, high-molecular pectin, hemicellulose, and cellulose) components. Cells treated at the highest concentration (176 mg/L) exhibited the greatest amount of sugar in the EtOH and starch fraction during the logistic phase. In contrast, cells in the early stationary phase had the highest level of sugar at treatment concentrations of less than 22 mg/L. For treatment concentrations higher than 22 mg/L on Day 15, more pectin and hemicellulose was detected at greater amounts compared with those cells treated with 0 mg/L. However, at Day 30, concentrations higher than 44 mg/L induced greater amounts of pectin and hemicellulose than from the other concentrations. Cellulose was more abundant with the 0 mg/L treatment, and contents ranged from 17.4 to 25.5% in the primary cell walls over all treatment concentrations. These results indicate that CaCI₂ modulates both symplastic and apoplastic sugar metabolism. Therefore, we suggest that the cell-wall structure may define the mode of polysaccharide biosynthesis during cell growth.     

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.     

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.     

Changes in cell-wall polysaccharides from the mesocarp of grape berries during veraison

Softening of grape berries ( Vitis vinifera L. × V. labruscana cv. Kyoho) was evaluated by studying changes in composition and degradation of cell-wall polysaccharides. The grape berry softens at the beginning of the second growth cycle many weeks before harvest. The softening stage is called 'veraison' by viticulturists. On day 50 after full bloom, green hard berries (before veraison [BV]), softening berries (veraison [V]) and partly peel colored berries (C) were selected from the same clusters. In addition, mature berries (M) were collected on day 78 after full bloom. Mesocarp tissues at each stage were fractionated into hot water-soluble (WS), hot EDTA-soluble (pectin), alkali-soluble (hemicellulose) and residual (cellulose) fractions. Neutral and acidic sugar contents of WS and pectin fractions decreased only after the V stage, while the neutral sugar content of the hemicellulose fraction decreased from the BV to V stages. Cellulose content constantly decreased as the berry ripened, but the large decrease was found from the BV to V stages. Molecular masses of pectic and hemicellulosic polysaccharides decreased from the BV to V stages. Hemicellulosic xyloglucan was markedly depolymerized from the BV to V stages. The neutral and acidic sugar composition of each fraction changed little during the berry ripening. These data indicated that softening of berry during veraison involved the depolymerization of pectin and xyloglucan molecules and decrease in the amounts of hemicellulose and cellulose.     

Differences in cell wall polysaccharide composition between embryogenic and non-embryogenic calli of <Emphasis Type="Italic">Medicago arborea</Emphasis> L.

Analysis of cell wall polysaccharide composition of embryogenic and non-embryogenic calli obtained from hypocotyl and petiole explants from Medicago arborea L. revealed significant differences. For calli induced from both hypocotyls and petioles, levels of total sugars, pectins, and hemicelluloses were higher in embryogenic than in non-embryogenic calli. Whereas in the residual cellulose fraction, the highest levels of sugar were detected in non-embryogenic calli. When comparing the two donor sources of callus explants, the highest total sugar levels were detected in embryogenic calli induced from petioles, mainly in the pectin fraction and to a lesser extent in the hemicellulose fraction. Moreover, analysis of uronic acids revealed higher levels in embryogenic calli, primarily in the pectin fraction. Analysis of those sugars associated with cell walls of calli suggested that these polysaccharides consisted of pectic polysaccharides and glucans, and that their levels were higher in embryogenic than non-embryogenic calli.     

Sugar Composition and Molecular Weight Distribution of Cell Wall Polysaccharides in Outer and Inner Tissues from Segments of Dark Grown Squash (Cucurbita maxima Duch.) Hypocotyls

The elongation growth of stem segments is determined by the outer cell layers (epidermis and collenchyma). We measured the sugar composition and molecular weight distribution of pectin and hemicellulose fractions obtained from inner and outer tissues of squash (Cucurbita maxima Duch.) hypocotyls. In addition, we studied the changes in these parameters after a 9 hour period of incubation of the segments. The results show that outer tissues have higher molecular weight pectin and hemicellulose compared to inner tissues (2-3 times higher). Incubation results in a 13 to 25% decrease in the amount of pectin and hemicellulose in inner tissues and an increase of 11 to 32% in the outer tissues. This increase in the outer tissues is accompanied by a decrease in the molecular weight of some of the components. These results clearly show that cell wall metabolism during elongation growth differs markedly in inner and outer tissues, and that future studies on the effect of auxin need to take these differences into account.     

Effect of hydroxyl radical on the scission of cellular wall polysaccharides in vitro of banana fruit at various ripening stages

Fruit ripening is generally attributed to disassembly of cellular wall, particularly due to solubilisation and depolymerisation of pectin and hemicellulose. Experiments were conducted to test effects of hydroxyl radicals (·OH) on the scission of cellular wall polysaccharides from pulp tissues of banana fruit at different ripening stage. Cellular wall materials were isolated from pulp tissues of banana fruit at different ripening stages. Two pectic fractions, water soluble pectin (WSP) and acid soluble pectin (ASP), and two hemicellulosic fractions, 1 M KOH soluble hemicellulose (HC1) and 4 M KOH soluble hemicellulos (HC2), were obtained from the cellular wall materials from pulp tissues, respectively. Effects of ·OH induced by the Fenton reaction on the scission of pectin and hemicellulose in vitro were investigated. As fruit ripening progressed, the sugar components of the WSP, HC1 and HC2 attacked by ·OH showed obvious molecular-mass downshifts. Thus, ·OH caused the disassembly of polysaccharides (WSP, ASP, HC1 and HC2) from cellular walls of pulp tissues of banana fruit, demonstrated by the reduced molecular mass distribution. Moreover, ·OH production in pulp tissues increased significantly as banana fruit ripened, which further help account for the role of ·OH in accelerated fruit ripening.     

Cell wall changes in white spruce (Picea glauca) needles subjected to repeated drought stress

White spruce [ Picea glauca (Moench) Voss] seedlings were preconditioned by subjecting them to 3 cycles of a mild drought stress. After 1 week of stress relief their water status, soluble carbohydrate content and cell wall composition in newly formed needles were examined and compared with those in control seedlings. Both preconditioned and control seedlings were subsequently subjected to a severe drought stress and again analyzed. Preconditioning treatment both before and during subsequent stress exposure lowered osmotic potentials at full hydration, and after the loss of turgor, decreased lignin content and increased hemicellulose content of the cell walls. Severe drought had similar but more drastic effects on seedling water relations, sugar accumulation and cell wall hemicellulose content; it also decreased cell wall pectin levels. The decrease in pectin levels was accompanied by a loss of galactose and glucose from pectic substances. Little change in cellulose content was observed as a result of preconditioning and severe drought.     

Changes in physical properties and cell wall polysaccharides of tomato (Lycoperskon esculentum) pericarp tissues

Green and red tomato pericarp tissues were subjected to stress-relaxation analyses to evaluate their physical properties. Significant decreases in the initial stress, minimum stress-relaxation and maximum stress-relaxation times in the red tissues predict the losses of both viscosity and elasticity in the tissue. Cell walls of red fruit yielded more water-soluble polysaccharides and less pectin, hemicelluloses and cellulose. Average molecular mass of pectin determined by gel filtration chromatography was similar in the green and red, but molecular mass of hemicellulose of red fruit walls was reduced to 50% of that of the green fruit. The decreases in the amount of hemicellulose B and in the average molecular mass were associated primarily with the degradation of xylo-glucans. These data demonstrate that pectin solubilization, depolymerization of xyloglucans and over-all changes in the quantity of cell wall polysaccharide fractions contribute to tomato fruit softening.     

Sodium-calcium interactions in two wheat species differing in salinity tolerance

White spruce [ Picea glauca (Moench) Voss.] seedlings were used to study the changes in cell wall composition and elasticity in mature needles before and after the resumption of growth following winter dormancy. Dormant seedlings showed high cell wall elasticity that decreased after the resumption of shoot growth. Cell wall hemicellulose content increased 3 days after planting and decreased after the buds flushed. Non-cellulosic glucose and arabinose were the sugars showing the most pronounced changes related to shoot growth. Arabinose was the most abundant sugar residue in the pectin and hemicellulose fractions and it decreased until day 10 after planting. At the same time, the levels of glucose in pectin and hemicellulose increased. The results provide evidence for cell wall carbohydrate turnover in dormant and active seedlings before and after bud flushing.     

Chemical composition,cell wall features and degradability of stem,leaf blade and sheath in untreated and alkali-treated rice straw

Three dominant morphological fractions (i.e. leaf blade (LB), leaf sheath (LS) and stem) were analysed for chemical composition and ruminal degradability in three rice straw varieties. In one variety treated with alkali, cell wall features were also characterized using Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy. The highest concentrations of cell wall carbohydrates (hemicellulose and cellulose) were observed in LS, whereas the highest concentrations of non-fibre (silica, phenolic compounds and CP) and lignin were recorded for LB. The stem had the lowest silica and hemicellulose contents but intermediate levels of other components. In terms of ruminal degradability, stem ranked higher than LB, which was followed by LS. Hemicellulose was found to be less degradable than either dry matter or cellulose in all the three fractions investigated. FTIR results indicated that the highest levels of hydrogen bonding, esterification and crystallinity within the cell wall components belonged to LS. In the alkaline treatment, these indices decreased to a larger extent for leaf fractions and a greater improvement was achieved in the degradability of LB and LS compared with that of stem. In the 24-h ruminal incubation, the silicified layer of epidermis and the underlying cell walls showed a rigid structure in the control fractions, whereas the treatment with NaOH resulted in crimping of the silicified cuticle layer and the loss of integrity in cell structure. Despite the highest silica and lignin contents observed in LB, LS showed the lowest degradability, which might be due to its high level of hydrogen bonding, crystallinity and esterification within its cell wall components as well as its high hemicellulose content.     

Changes in enzymic activities of nucleoside diphosphate sugar interconversions during differentiation of cambium to xylem in sycamore and poplar.

During the transition from primary wall formation to secondary thickening there is a marked shift in the synthesis of pectin, hemicellulose and cellulose. The activities of the enzymes [UDP-D-galactose 4-epimerase (EC 5.1.3.2)8 UDP-l-arabinose 4-epimerase (EC 5.1.3.5), UDP-D-glucose dehydrogenase (EC 1.1.1.22) and UDP-D--glucuronate decarboxylase (EC 4.1.1.35)] were measured in cambial cells, differentiating xylem cells and differentiated xylem cells isolated from sycamore and poplar trees, and phloem cells from poplar. At the final stage of the differentiation of cambium to xylem there was a decrease in activity of the enzymes directly involved in producing the soluble precursors of pectin (DUP-D-galactose 4-epimerase and UDP-L-arabinose 4-epimerase and an increase in those producing the precursors of hemicellulose (UDP-D-glucose dehydrogenase and UDP-D-glucuronate decarboxylase). These results strongly suggest ahat the changes were correlated with the differences observed in the chemical composition of the wall during development. The changes found in the catalytic activity of the enzymes of nucleoside diphosphate sugar interconversion exert a coarse control over the synthesis of pectin and hemicelluloses. The tissues at all stages of development contained the necessary enzyme activities to produce all the precursors of pectin and hemicellulose, even at the final stage of differentiation when no pectin was formed.     

Effect of Auxin,Kinetin and Monosaccharide on Callus Cell wall Composition of Abutilon avicennae Gaertn

The effects of exogenous kinetin, IAA, glucose (the component of cellulose) and galactose (one of tile inhibiting compounds of cellulose synthesis) on callus cell wall composition of China jute (Abutilon avicennae Gaertn) were investigated. When the China jute callus materials were treated with 2 mg/l IAA and H3-glucose, there was a 437% increase of the Ha-glucose, as compared with control. When galactose was added simultaneously with H3-glucose, there was a 138% increase of the H3-glucose as compared with control. Galactose inhibited H3-glucose incorporate content in cellulose, and also in pectin and hemicellulose. In the experiment with 10 mg/l kinetin treatment of the callus materials, when galactose was added, the incorporation H3-glucose was inhibited. It is interesting that the kinetin enhanced H3-galactose incorporation into all cell wall components. When it was added simultaneously with glucose, glucose inhibited H3-galactose into the cell Wall components. Results showed that the hormones effect cell wall component, not only concerned with hormones, but also concerned with exogenous monosaccharides. When exogenous monosacchrides were added to the culture medium (as galactose, glucose) in combination or respectively, they are quite different in effecting hormone induced China jute callus cell wall. When the callus was treated by IAA, galactose inhibited H3-glucose incorporation into cell wall components. When it was treated by kinetin, glucose inhibited H3-galactose incorporation into cell wall components.     

Use of the pressure vessel to measure concentrations of solutes in apoplastic and membrane-filtered symplastic sap in sunflower leaves

A simple, repeatable, and accurate method is described for the collection of apoplastic and membrane-filtered symplastic sap fractions, and for the determination of the origin of these fractions within the leaf. The apoplastic distribution patterns of the naturally occurring apoplastic leaf solutes, and the apoplastic dye PTS (trisodium 3-hydroxy-5, 8, 10-pyrenetrisulfonate) were compared. Aliquots of sap were expressed from detached sunflower leaves in a pressure chamber over intervals of 0.02 to 0.04 megapascal. Three distinct fractions were detected in the expressed sap volume. These were successively released and identified as a petiole-midrib fraction, a minor vein-cell wall fraction, and a mixed fraction consisting of a contribution from the minor vein-cell wall with an increasing proportion of membrane-filtered cell sap.     

吲(口朶)乙酸、激动素和单糖对苘麻愈伤组织细胞壁组分的影响

本实验采用 H~3标记糖饲喂示踪分析法。将苘麻愈伤组织分别用吲哚乙酸和激动素处理,并在培养基中供给 H~3葡萄糖或 H~3半乳糖。——实验表明,激素对细胞壁组成分影响,不仅与激素的种类有关,也与供给的外源单糖有关。而外源单糖(半乳糖、葡萄糖)单独加入或同时加入,使激素对苘麻愈伤组织诱导的影响又有所不同。在吲哚乙酸作用下,促进了 H~3葡萄糖的掺入;而半乳糖的加入又抑制了 H~3葡萄糖掺入到细胞壁各组分。在激动素作用下,促进H~3半乳糖的掺入,而葡萄糖的加入又抑制了 H~3半乳糖掺入到壁的各组分。     

Evolution of xyloglucan-related genes in green plants

Background  

The cell shape and morphology of plant tissues are intimately related to structural modifications in the primary cell wall that are associated with key processes in the regulation of cell growth and differentiation. The primary cell wall is composed mainly of cellulose immersed in a matrix of hemicellulose, pectin, lignin and some structural proteins. Xyloglucan is a hemicellulose polysaccharide present in the cell walls of all land plants (Embryophyta) and is the main hemicellulose in non-graminaceous angiosperms.     

Compartmentation of Assimilate Fluxes in Leaves

Abstract: Sugar levels in the apoplast of assimilate exporting leaves were studied in two groups of plant species with contrasting structures of companion cells in minor veins. These species are termed either "symplastic" (with intermediary cells) or "apoplastic" (with transfer or ordinary cells). Sugars were measured in intercellular washing fluid after extracting the apoplast by an infiltration-centrifugation technique. During the course of a day, sugar contents in the apoplast were, in general, lower in species with intermediary cells than in species with transfer or ordinary cells. In "symplastic" species, apoplastic sucrose concentrations were between 0.3 and 1 mM. In "apoplastic" species with transfer cells, they ranged between 2 and 6 mM. Apoplastic hexose contents were between 0.3 and 1 mM irrespective of presumed transport mode. "Symplastic" and "apoplastic" plants differed markedly in their response to a'translocation block. In "symplastic" plants, inhibition of assimilate export left apoplastic concentrations of sucrose and hexoses unchanged, whereas in "apoplastic" plants sugar levels increased, the maximal increase being observed with sucrose. In these plants, concentrations of sucrose were two to six times higher in the apoplast under export inhibition than in control leaves. The data suggest a different role of the leaf apoplast in the compartmentation and export of assimilates in the two plant groups under study.     

Turnover of cell wall polysaccharides of a Vinca rosea suspension culture

Turnover of cell wall components was examined in two growth phases of a batch suspension culture of Vinca rosea L. Three-day-cultured cells (cell division phase) and 5-day-cultured cells (cell expansion phase) were incubated with d -[U-¹⁴C]glucose. After various periods of incubation, extra-cellular polysaccharides (ECP) and cell walls were isolated, and then the cell walls were fractionated to pectic substance, hemicellulose, and cellulose fractions. The results of the measurement of radioactivities and amounts of total carbohydrate in the ECP and cell wall fractions indicated that synthesis of pectic substance was more active in the cell division phase than in the cell expansion phase. From the results of the pulse-chase experiments, in which cells prelabelled by incubation with d -[U-¹⁴C]glucose for 3 h were incubated in a medium containing unlabelled glucose for various periods, the gross degradation, net synthesis, and gross synthesis of cell wall components were estimated. Active degradation and synthesis were observed in the hemicellulose fraction, indicating that active turnover occurred in the hemicellulose fraction, while little degradation was found in the pectic substance and cellulose fractions.