Galactoglucomannan oligosaccharides inhibition of elongation growth is in pea epicotyls coupled with peroxidase activity

The effect of galactoglucomannan oligosaccharides — GGMOs, GGMOs-r (GGMOs with reduced reducing ends), and GGMOs-g (GGMOs with reduced number of d-galactose units) on peroxidase activity was determined in pea epicotyls. GGMOs didn’t significantly modify the activity of soluble peroxidases. However, cell wall-associated peroxidases activity increased after GGMOs and GGMOs-r treatment, while in the presence of GGMOs-g this activity was significantly lower. These results are inversely related to the GGMOs, GGMOs-r, and GGMOs-g effect on elongation growth induced by 2,4-D (2,4-dichlorophenoxyacetic acid) in pea epicotyls. It can be concluded that GGMOs evoked inhibition of the elongation growth induced by auxin is probably associated with cell wall modifications catalysed by peroxidase.     

Impact of galactoglucomannan oligosaccharides on elongation growth in intact mung bean plants

The impact of exogenously applied galactoglucomannan oligosaccharides (GGMOs) and their structurally modified forms (GGMOs-r—galactoglucomannosyl alditols, GGMOs-g—with reduced galactose content) on the growth of mung bean (Vigna radiata (L.) Wilczek) intact plants cultured in hydroponics has been determined. GGMOs alone or in combination with exogenously added IBA have influenced (with stimulation and/or inhibition effect) hypocotyl and seminal root elongation, adventitious and lateral roots formation and elongation in dependency on their concentration used. The inhibition of elongation growth in hypocotyls as well as in roots was connected with changes of cell wall-associated peroxidases activity and is probably associated with the beginning of cell wall rigidification. Data presented in this paper confirm the hypothesis that exogenously added GGMOs may have antiauxin activity and may interact also with endogenous growth regulators. Certain monosaccharide sequences with terminal galactose in the side chain of GGMOs probably play important role in their biological activity in intact plants as it was demonstrated previously in individual parts of plants.     

Further biological characteristics of galactoglucomannan oligosaccharides

The biological activity of cell wall-derived galactoglucomannan oligosaccharides (GGMOs) was dependent on their chemical structure. Galactosyl side chains linked to the glucomanno-core influenced their inhibition of elongation growth of pea (Pisum sativum L. cv. Tyrkys) stem segments induced by 2,4-dichlorophenoxyacetic acid (2,4-D). Reduction of the number of galactosyl side chains in GGMOs caused stimulation of the endogenous growth. Modification on the glucomanno-reducing end did not affect significantly the activity of these oligosaccharides. GGMOs inhibited also the elongation induced by indole-3-acetic acid (IAA) and gibberellic acid (GA₃). In the presence of IAA the elongation growth was inhibited to 20 – 35 % after 24 h of incubation depending on GGMOs concentrations (1 μM, 10 nM, 0.1 nM), similarly as in the presence of 2,4-D, which confirms the hypothesis of GGMOs antiauxin properties. The elongation induced by GA₃ was inhibited to 25 – 60 %, however, the time course of inhibition was different compared with IAA and 2,4-D. The highest inhibition was determined already after 6 h of incubation with a significant decrease after this time. The results indicated a competition between GGMOs and growth regulators.     

Effect of auxins and plant oligosaccharides on root formation and elongation growth of mung bean hypocotyls

The interaction of auxins – IAA, IBA or NAA – with galactoglucomannan oligosaccharides (GGMOs) on adventitious root formation and elongation growth of mung bean hypocotyl cuttings was studied. GGMOs induced adventitious roots in the absence of auxins; however, their effect was lower compared with IBA or NAA. On the other hand, in the presence of auxins, GGMOs inhibited adventitious root induction. Their effect depended on the concentration of oligosaccharides and the type of auxin used. The highest inhibition effect of GGMOs at a concentration of 10⁻⁸ M in the presence of IBA and NAA was observed. In the presence of IAA their inhibition was non-significant in regard to the concentration. The interaction of auxins with GGMOs resulted in the formation of adventitious roots on a shorter part of hypocotyls compared with the effect of auxins alone. However, roots were induced more extensively along the hypocotyls treated with GGMOs compared with the control. GGMOs inhibited the length of induced adventitious roots in the presence of IAA, while in combination with IBA or NAA they were ineffective. The elongation of hypocotyls induced by IAA or IBA was inhibited by GGMOs, too. However, in the presence of NAA or by endogenous growth they were without any significant effect on elongation growth. These findings suggest that GGMOs in certain concentrations might inhibit rooting and the elongation process dependant on auxin used.     

Changing extracellular matrix of cells during development of suspension culture of Triticum timopheevii Zhuk

A study was made of the contents of the main polysaccharide fractions in the cell wall, and extracellular polysaccharides, and of the activity of cell wall enzymes during cultivation of suspension culture of cells of the winter wheat Triticum timopheevii Zhuk. It was shown that within 3 days of cultivation (a phase enriched in dividing cells), on the background of increased callose contents in plant cells, amounts of pectins and hemicelluloses extracted by 4N alkali decreased. The content of polysaccharides reached its initial level by the end of culturing. A parallel analysis of glycosidase activity in cell walls has shown their considerable activation at the stage enriched by dividing cells, which decreased at a transition of culture into the stationary level. The increased activity of hydrolyzing enzymes was combined with an increased efflux of extracellular polysaccharides into culture medium. The detected changes in polysaccharide composition of the cell wall at the first phase indicate its qualitative changes during cell wall reconstruction at the beginning of cytokines, whereas extensive expansion of cell wall was seen on the phase of elongation.     

Interaction of galactoglucomannan oligosaccharides with auxin in mung bean primary root

In the present paper timing of galactoglucomannan oligosaccharides (GGMOs) with exogenously added indole-3-butyric acid (IBA) action on early germination stage (24 h) and primary root elongation of mung bean (Vigna radiata (L.) Wilczek) has been studied. GGMOs inhibited primary root elongation induced by low concentration (10^?8 M) of IBA. This inhibition was considerably higher after preincubation with GGMOs compared with other timing experiments. The most intensive inhibition of elongation has been ascertained at the 10^?8 M concentration of GGMOs. On the other hand GGMOs stimulated this elongation inhibited by high IBA concentration (10^?4 M). This stimulation was the most intensive by simultaneous addition of IBA and GGMOs at the beginning of the experiment and subsequent seeds incubation in distilled water. Our results indicate competition between GGMOs and auxin. The root growth inhibition, induced by GGMOs and/or IBA, was accompanied by the increase of cell wall-associated peroxidase activity and by a higher number of peroxidase isoenzymes. The presence of different peroxidase isoenzymes in experiments with distinct treatment of GGMOs and IBA could indicate variations in the mechanism of interaction between GGMOs and IBA.     

Changes in cell wall polysaccharides during elongation in a 2, 4-D free medium in a carrot suspension culture

Cell elongation occurred when carrot (Daucus carota L. ev. Kurodagosun) cells subcultured through sieving (Y. Ozeki and A. Komamine, Physiol. Plant. 53: 570-577. 1981) were transferred to a medium lacking auxin, while the cells showed no elongation in a medium containing 2, 4-D. Changes in polysaccharides of the cell walls and in their sugar composition during elongation were investigated. All wall components, EDTA-soluble pectic substance, 5 and 24%, KOH-soluble hemicelluloses and cellulose increased markedly during elongation. The increase of hemicelluloses correlated especially with elongation. In the 5% KOH-soluble hemicellulose, galactose and arabinose contents in the walls increased significantly both in amounts (per fresh weight) and relative contents (% in total neutral sugars) during elongation, while the relative contents of glucose and xylose decreased rapidly in the 5 and 24% KOH-soluble hemicelluloses. The methylation analysis tentatively indicated that larger amounts of galactan and/or arabinogalactan and lower amount of xyloglucan were found as components of the two hemicelluloses of elongating cells than those of non-elongating cells. The amounts of total carbohydrate and of uronic acid of extracellular polysaccharides secreted into the medium increased to a larger extent in the elongation culture than in the non-elongation culture. The contents of galactose and arabinose in extracellular polysaccharides increased rapidly in the elongation culture. The biochemical aspects of cell elongation in the absence of auxin were discussed from the viewpoint of the results obtained here.     

Glycosidases and acid-invertase activities are not correlated with Phaseolus hypocotyl growth

Changes in α-galactosidase, β-galactosidase, β-glucosidase and acid invertase activities were examined in Phaseolus vulgaris hypocotyls treated with gibberellic acid (GA), naphthyl acetic acid (NAA) and distilled water (DW) (control) in light condition. The activities were estimated both in cytoplasmic and ionically wall-bound fraction. The upper segment showed considerable elongation growth while there was hardly any growth in lower segment. GA and NAA showed distinct promotion and inhibition respectively in hypocotyl growth in upper segment. The glycosidase activities were detected in both the fractions but the activity was more pronounced in cytoplasmic than in wall fraction. Acid invertase activity was present only in cytoplasmic fraction. In lower segment, in both cytoplasmic and wall fraction, the glycosidase activity, in general, showed a decreasing trend and no effect of treatment could be envisaged. In upper segment, though the trend was similar to the lower one, in α- and β-galactosidase NAA treated segment had more activity. Invertase activity also did not show a clear trend to implicate its function in hypocotyl elongation growth. The results are discussed in relation to establishing a correlation between an activity (glycosidase and invertase) and a physiological process (hypocotyl elongation). It is concluded that these wall-loosening enzymes have no role in elongation growth of Phaseolus vulgaris hypocotyls.     

Oligosaccharides induce changes in protein patterns of regenerating spruce protoplasts

Galactoglucomannan oligosaccharides (GGMOs, d.p. 4–8 and fractions d.p. 3, 4, 5, 6–7), used in culture media for spruce protoplasts derived from callus cells showed a pleiotropic effect. They influenced both, quality and quantity of extracellular proteins in regenerating protoplasts. GGMOs d.p. 4, 5 at pH 6.0 and the mixture of d.p. 4–8 (pH 3.8 and 6.0) after 48 h of culture increased the amount of extra- and intracellular proteins and the viability of protoplasts. The most significant effect on protoplasts viability in the presence of GGMOs d.p. 4–8 without the growth hormone supplementation at pH 3.8, and with lower efficiency in the presence of NAA (1 mg/l) has been observed. The most significant differences were observed in the molecular mass intervals Mr∼17–21 kDa, 25–30 kDa, and 45–60 kDa. After 24 h of culture extracellular acid proteins with Mr∼30; 41; 68; and 90 in media supplemented with GGMOs d.p. 3, 4, 5, 6–7 (pH 6.0) with or without NAA were identified to belong to the group of β-1,3-glucanases. Extracellular proteins p27.5; 30; 41; 50; 52 and 90 exhibited chitinase activity after 24 h of protoplast cultivation. GGMOs probably fulfil a protective role in this process of spruce protoplast regeneration.     

Galactoglucomannans increase cell population density and alter the protoxylem/metaxylem tracheary element ratio in xylogenic cultures of Zinnia

Xylogenic cultures of zinnia (Zinnia elegans) provide a unique opportunity to study signaling pathways of tracheary element (TE) differentiation. In vitro TEs differentiate into either protoxylem (PX)-like TEs characterized by annular/helical secondary wall thickening or metaxylem (MX)-like TEs with reticulate/scalariform/pitted thickening. The factors that determine these different cell fates are largely unknown. We show here that supplementing zinnia cultures with exogenous galactoglucomannan oligosaccharides (GGMOs) derived from spruce (Picea abies) xylem had two major effects: an increase in cell population density and a decrease in the ratio of PX to MX TEs. In an attempt to link these two effects, the consequence of the plane of cell division on PX-MX differentiation was assessed. Although GGMOs did not affect the plane of cell division per se, they significantly increased the proportion of longitudinally divided cells differentiating into MX. To test the biological significance of these findings, we have determined the presence of mannan-containing oligosaccharides in zinnia cultures in vitro. Immunoblot assays indicated that beta-1,4-mannosyl epitopes accumulate specifically in TE-inductive media. These epitopes were homogeneously distributed within the thickened secondary walls of TEs when the primary cell wall was weakly labeled. Using polysaccharide analysis carbohydrate gel electrophoresis, glucomannans were specifically detected in cell walls of differentiating zinnia cultures. Finally, zinnia macroarrays probed with cDNAs from cells cultured in the presence or absence of GGMOs indicated that significantly more genes were down-regulated rather than up-regulated by GGMOs. This study constitutes a major step in the elucidation of signaling mechanisms of PX- and MX-specific genetic programs in zinnia.     

Alterations in intracellular and extracellular activities of antioxidant enzymes during suspension culture of sweetpotato

Cultured plant cells are a good system for the study of antioxidant mechanisms and for the mass production of antioxidants, because they can be grown under conditions of high oxidative stress. Alterations in the intracellular and extracellular activities of three antioxidant enzymes, superoxide dismutase (SOD), guaiacol-type peroxidase (POD), and glutathione peroxidase (GPX), were investigated in suspension cultures of sweetpotato (Ipomoea batatas) during cell growth. Intracellular SOD activities (units/mg protein) at 15 days after subculture (DAS) and 30 DAS were 10 and 20 times higher, respectively, compared with the SOD activity at 1 DAS, whereas intracellular specific POD and GPX activities did not significantly increase until after 15 DAS, when they rapidly increased. The extracellular activities of the three enzymes in culture medium were much higher than were the intracellular activities. The change in extracellular SOD activity was similar to that of extracellular GPX during cell growth. Those activities showed high levels until 5 DAS and then significantly decreased. Extracellular POD activity had an almost constant level regardless of the cell growth stage. In addition, intracellular SOD and POD isozymes were quite different from those isozymes in the culture medium. The changes in SOD and POD isozymes observed here suggest that different isozymes might modulate the levels of reactive oxygen intermediates during cell growth. Characterization of extracellular antioxidant enzymes discovered here would provide a new understanding for defense mechanism in plants.     

PlyC, a novel bacteriophage lysin for compartment-dependent proteomics of group A streptococci

Streptococcus pyogenes (Spy) (group A streptococci) is an important and exclusively human bacterial pathogen, which uses secreted and surface-associated proteins to circumvent the innate host defense mechanisms and to adhere and internalize into host cells. Thus, investigation of the bacterial extracellular compartments, including secreted and cell wall-associated subproteomes, is crucial for understanding adherence, invasion, and internalization mechanisms as major steps of Spy pathogenesis. Here, we compared a bacteriophage encoded cell wall hydrolase, PlyC, a multimeric lysin of the C1 bacteriophage, with the established glycosidase, mutanolysin, from Streptomyces globisporus for their suitability to efficiently digest Spy cell walls and release cell wall-anchored Spy proteins for subsequent proteome research. Our results show that PlyC is superior for cell wall protein extraction compared to mutanolysin due to its higher activity and specificity as an N-acetylmuramoyl-L-alanine amidase. Furthermore, our experimental design allowed us to delineate the actual localization of the proteins despite contamination with intracellular proteins.     

Xyloglucan Endotransglycosylase Activity in Carrot Cell Suspensions during cell Elongation and Somatic Embryogenesis

Xyloglucan endotransglycosylase (XET) has been proposed to contribute to cell elongation through wall loosening. To explore this relationship further, we assayed this enzyme activity in suspensions of carrot (Daucus carota L.) cells exhibiting various rates of cell elongation. In one cell line, elongation was induced by dilution into dichlorophenoxyacetic acid (2,4-D)-free medium. During this elongation, 93% of the XET activity was found in the culture medium; in nonelongating controls, by contrast, 68% was found in the cell extracts even though the specific activity of these extracts was lower than in the elongating cells. By far the highest rates of XET secretion per cell were in the elongating cells. A second cell line was induced to undergo somatic embryogenesis by dilution into 2,4-D-free medium. During the first 6 d, numerous globular embryoids composed of small, isodiametric cells were formed in the absence of cell elongation; extracellular XET activity was almost undetectable, and intracellular specific activity markedly declined. After 6 d, heart, torpedo, and cotyledonary embryoids began to appear (i.e. cell elongation resumed); the intracellular specific activity of XET rose rapidly and >80% of the XET activity accumulated in the medium. Thus, nonexpanding cell suspensions (whether or not they were rapidly dividing) produced and secreted less XET activity than did expanding cells. We propose that a XET molecule has an ephemeral wall-loosening role while it passes through the load-bearing layer of the wall on its way from the protoplast into the culture medium.     

Multiple carbohydrate-cleaving specificities in human acidic and neutral glycosidases.

The common identity of human acidic beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) and beta-D-xylosidase (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) as one enzyme and that of acidic beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), beta-D-fucosidase (no allotted EC number) and alpha-L-arabinosidase (alpha-L-arabinofuranoside arabinohydrolase, EC 3.2.1.55) as another enzyme is indicated by similar binding patterns of glycosidase activities of each enzyme to various lectins. by similar ratios between their intra- and extracellular levels in normal and I-cell fibroblasts and by their deficiencies in liver tissues from patients with Gaucher disease and GM1 gangliosidosis, respectively. A third enzyme, neutral beta-D-galactosidase, purified to homogeneity from human liver has been shown to possess all these five glycosidase activities at neutral pH. These neutral enzymic activities were not bound by any of the lectins examined and found to be reduced in liver and spleen of a patient with neutral beta-D-galactosidase deficiency. An additional form of beta-D-xylosidase with optimal activity at pH 7.4 was bound by the fucose-binding lectin from Ulex eurpaeus while no binding was observed for the acidic (pH 4.8) and neutral (pH 7.0) beta-D-xylosidase activities of the multiple glycosidase enzymes.     

Maize stem tissues: ferulate deposition in developing internode cell walls

It has been hypothesized that ferulates are only deposited in the primary cell wall of grasses. To test this hypothesis, the fourth elongating, above-ground internode of maize (Zea mays l.) was sampled from three maize hybrids throughout development. Cell wall composition was determined by the Uppsala Dietary Fibre method. Ester- and ether-linked ferulates were determined by HPLC analysis of ferulic acid released from the internodes by low and high temperature alkaline treatments. Internode length increased from 9 to 152 mm over 96 days of growth, with elongation being complete in the first 12 days. More than half of the cell wall material in the maize internodes accumulated after elongation had ended. Deposition of cell wall material appeared to reach its maximum extent 40 days after sampling began, well before physiological maturity of the maize plants. Galactose and arabinose began to accumulate early in cell wall development which was presumed to be associated with primary wall growth during internode elongation. The major secondary wall constituents (analyzed as glucose, xylose, and Klason lignin) did not begin to accumulate rapidly until shortly before internode elongation ended. Ferulate ester deposition began before ferulate ethers were observed in the cell wall, but both forms of ferulate continued to accumulate in secondary cell walls, long after internode elongation had ceased. These data clearly show that contrary to the hypothesis, ferulate deposition was not restricted to the primary wall and that active lignin/polysaccharide cross-linking mediated by ferulates occurs in the secondary wall.     

Synthesis of distinctly different sets of antimicrobial activities by elicited plant cell suspension cultures

The aim of this study was to evaluate the potential of plant suspension cultures for the production of antimicrobial activities. The extracellular, intracellular and cell wall bound fractions of 16 heterotrophic, photoautotrophic and photomixotrophic plant cell suspension cultures each treated with nine different elicitors were tested for the elicitor dependent production of antimicrobial activities. Distinctly different patterns of bioactivities directed against a panel of human isolates including Gram-positive (Bacillus subtilis, Staphylococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria as well as fungi (Candida maltosa) were identified for all except the two autotrophic cell cultures. The intracellular fractions of elicited cell cultures were more active than extracellular fractions while cell wall bound fractions showed almost no activities. The intracellular fraction of heterotrophic Lavendula angustifolia cells elicited with a preparation of Pseudomonas syringae was the most active fraction against Candida maltosa. The intracellular fraction of photomixotrophic Arabidopsis thaliana cells elicited with salicylic acid was active against all test isolates. An antimicrobial protein could be identified and partially purified from this culture. Our findings suggest that elicited plant cell cultures may present a new promising alternative source of antimicrobial proteins.     

Physiological and biochemical changes associated with cotton fibre development. II. Auxin oxidising system

Changes in IAA oxidase, and in cytoplasmic and ionically wall-bound peroxidase activities were studied in the developing fibres of three cotton cultivars ( Gossypium hirsutum L. cv. Gujarat-67, cv. Khandwa-2 and G. herbaceum L. cv. Digvijay), designated as long, medium and short staple cultivars, respectively. In all the three cultivars IAA oxidase activity was low during the fibre elongation phase, while the activity increased significantly during the secondary thickening phase. The increase in IAA oxidase activity in the three cultivars showed close correspondence with their respective total period of elongation. No relationship between cytoplasmic peroxidase activity and fibre development was discernible. The ionically bound wall peroxidase activity, however, recorded low levels during the elongation phase and higher levels during the secondary thickening phase. The role of wall peroxidase in cessation of elongation growth is discussed.     

Hemicellulose-degrading Enzymes Synthesized by Rumen Bacteria

Over 100 bacterial cultures isolated from ovine rumen contents by enrichment techniques in polysaccharide-containing media were examined for the ability to degrade plant cell wall structural polysaccharides. Approximately two-thirds of the isolates retained were Gram negative and amongst the coccoid isolates diplococci predominated. Many of the rods examined were piliated and/or flagellated. Thirty of the more active xylan- and arabinan-degrading isolates were examined for both the production and activity of the principal polysaccharidase and glycosidase enzymes associated with plant cell wall polysaccharide hydrolysis, following culture in a hemicellulose containing growth medium. The wide range of glycosidase activities detected in these hemicellulolytic isolates was evidence for their rôle in the breakdown of dietary polysaccharides in the rumen.     

Effect of galactoglucomannan-derived oligosaccharides on elongation growth of pea and spruce stem segments stimulated by auxin

Galactoglucomannan-derived oligosaccharides (GGMOs) (degree of polymerization 4–8) isolated from the wood of poplar (Populus monilifera Ait.) were shown to be inhibitors of the 2,4-dichlorophenoxyacetic acid-stimulated elongation growth of pea (Pisum sativum L. cv. Tyrkys) and spruce [Picea abies (L.) Karst] stem segments. A dependence on the concentration of GGMOs (between 10^-5-10^-10M) as well as plant species was ascertained. Pea stem segments were much more sensitive (10^-10M) than spruce (10^-8M). The GGMOs did not exhibit toxicity even at high concentrations and during long-term bioassays. The timing of the action of GGMOs and auxin in the growth process was also studied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - d.p degree of polymerization - GGMOs galactoglucomannan-derived oligosaccharides This research was supported by the Slovak Grant Agency for Science.     

Ozone stimulates apoplastic antioxidant systems in pumpkin leaves

The phytotoxiticky of ozone is due to its high oxidant capacity and to its ability to generate toxic molecular species. It is well known that intracellular peroxidases play an important role in eliminating toxic forms of oxygen but little evidence has been reported on the role of peroxidases in the apoplastic compartment. The detoxification systems located in the foliar extracellular matrix and in the intracellular fluid of sensitive pumpkin plants ( Cucurbita pepo L. cv. Ambassador) exposed to ozone (150 ppb. 5 days. 5 h day-¹) in a fumigation chamber, were analyzed. The analyses were carried out on both young and mature leaves. Ascorbate peroxidase (EC 1.11.1.11) was found in the extracellular matrix of the pumpkin tissues. Its activity increased in both young and mature leaves as a consequence of the treatment, while at intraeellular levels its effect was most prominent in mature leaves. Analysis of the ascorbie-dehydroascorbic acid system revealed an enhancement of the pool content in the extracellular matrix of both kinds of leaves as a consequence of fumigation, while at the intracelluiar level small variations were found. Very little variation was observed in the glutathione pool as a consequence of fumigation. The analysis of a lipid peroxidation marker, malondi-aldehyde. showed the significant effect of ozone on membrane lipids. Following fumigation, the free phenols in the extracellular matrix decreased in both young and mature leaves, while the free and glycoside-bound phenols of the intracellular fluid showed little increase. The results support the hypothesis that ozone stimulates the an-tioxidant systems mainly in the apoplast and that ascorbic peroxidase activity, ascorbic acid levels and cell wall stiffening are the most influenced parameters.