Cell Wall-bound Acid Research Articles

Changes in phenolic acid abundance involved in low temperature and Microdochium nivale (Samuels and Hallett) cross-tolerance in winter triticale (x Triticosecale Wittmack)

Tolerance to the pink snow mould resulting from Microdochium nivale infection is an essential trait of triticale (x Triticosecale) for winter survival. In the present study, we aimed to verify whether the presence and concentration of free and cell wall-bound phenolic acids are important factors in triticale responses to M. nivale infection. Based on 3 years’ testing of triticale tolerance, 2 out of 92 doubled haploid triticale lines derived from ‘Hewo’ × ‘Magnat’ F1 hybrid were selected, which are the most tolerant and the most sensitive to M. nivale infection. Plants were grown along with their parents under controlled conditions, pre-hardened and cold-hardened, while non-hardened plants served as the control. Hardened plants were covered with the artificial snow-imitating covers and inoculated with M. nivale mycelium, while the control plants were treated the same way except the infection. The aim of the study was to identify differences in the initial content and composition of phenolics under the influence of applied stresses. Conducted HPLC analysis showed that the most abundant were ferulic, rosmarinic, chlorogenic, sinapic, and trans-cinnamic acids. The contents of most of phenolics depended on genotype and growth conditions. Two cell wall-bound sinapic and trans-cinnamic acids, could be indicated as potentially related to the increased snow mould tolerance of winter triticale seedlings. A correlation between the total phenolic levels with the tolerance was not found; however, the proportion between the total levels of cell wall-bound and free phenolic compounds under low temperature could play a role prior to M. nivale infection.

The role of sucrose-metabolizing enzymes in pear fruit that differ in sucrose accumulation

In the paper, the soluble sugar composition and activities of enzymes metabolizing sucrose: invertase (β-fructosidase, EC 3.2.1.26), sucrose synthase (SS; EC 2.4.1.13) and sucrose-phosphate synthase (SPS; EC 2.4.1.14) were investigated during fruit development of two pear species: Pyrus bretschneideri Rehd. cv. ‘Yali’ and P. pyrifolia Nakai cv. ‘Aikansui’, characterized as low and high sucrose types, respectively. It was found that, at the end of fruit development of ‘Aikansui’, the level of sucrose was five times higher than in ‘Yali’ in the same period. It was coincident with the significantly higher activities of SS (synthesis) and SPS and lower activities of invertase (vacuolar and cell wall-bound acid invertase and neutral invertase). The high correlation was found between sucrose level and SS (synthesis) and SPS activities in ‘Aikansui’ pears.

Relationship between hydroxycinnamic acid content, lignin composition and digestibility of maize silages in sheep

Cell wall-bound hydroxycinnamic acids and the composition of lignin were studied in relation to the digestibility of a collection of 91 maize silages in wethers. Total lignin and guaiacyl content showed the highest correlation coefficients with digestibility. Using the above-mentioned chemical parameters, eight equations were also developed to predict digestibility. The prediction of organic matter digestibility produced a high adjusted R 2 value (0.487) using total lignin, guaiacyl, esterified ferulic acid and esterified p-coumaric acid content as predictors. The prediction of in vivo dry matter digestibility produced a higher adjusted R 2 value (0.516) using the same variables as predictors. Cell wall digestibility depends on a multiplicity of factors and it is not possible to attribute a causal effect on in vivo digestibility to any single factor. However, total lignin, guaiacyl and p-coumaric acid content emerge as good predictors of digestibility.

Functional Analysis of an Arabidopsis thaliana Abiotic Stress-inducible Facilitated Diffusion Transporter for Monosaccharides

Sugars play indispensable roles in biological reactions and are distributed into various tissues or organelles via transporters in plants. Under abiotic stress conditions, plants accumulate sugars as a means to increase stress tolerance. Here, we report an abiotic stress-inducible transporter for monosaccharides from Arabidopsis thaliana that is termed ESL1 (ERD six-like 1). Expression of ESL1 was induced under drought and high salinity conditions and with exogenous application of abscisic acid. Promoter analyses using beta-glucuronidase and green fluorescent protein reporters revealed that ESL1 is mainly expressed in pericycle and xylem parenchyma cells. The fluorescence of ESL1-green fluorescent protein-fused protein was detected at tonoplast in transgenic Arabidopsis plants and tobacco BY-2 cells. Furthermore, alanine-scanning mutagenesis revealed that an N-terminal LXXXLL motif in ESL1 was essential for its localization at the tonoplast. Transgenic BY-2 cells expressing mutated ESL1, which was localized at the plasma membrane, showed an uptake ability for monosaccharides. Moreover, the value of K(m) for glucose uptake activity of mutated ESL1 in the transgenic BY-2 cells was extraordinarily high, and the transport activity was independent from a proton gradient. These results indicate that ESL1 is a low affinity facilitated diffusion transporter. Finally, we detected that vacuolar invertase activity was increased under abiotic stress conditions, and the expression patterns of vacuolar invertase genes were similar to that of ESL1. Under abiotic stress conditions, ESL1 might function coordinately with the vacuolar invertase to regulate osmotic pressure by affecting the accumulation of sugar in plant cells.

Copper Affects the Cotyledonary Carbohydrate Status During the Germination of Bean Seed

Seeds of bean (Phaseolus vulgaris L.) were germinated by soaking in distilled water or copper chloride solution. The relationships among copper excess treatment, germination rate, dry weight, sugar contents, and carbohydrase activities in cotyledon were investigated. Heavy metal stress provoked a diminution in germination rate and biomass mobilization, as compared with the control. A drastic disorder in soluble sugars export, especially glucose and fructose liberation, was also imposed after exposure to excess copper. This restricted the starch and sucrose breakdown in reserve tissue, as evidenced by the inhibition in the activities of alpha-amylase and invertase isoenzymes (soluble acid, soluble neutral, cell wall-bound acid).

Effects of Salinity on Distribution of Photosynthates and Carbohydrate Metabolism in Tomato Grown using Nutrient Film Technique

Tomato plants were grown using a nutrient film technique to evaluate the effects of salinity on the distribution of photosynthates and sucrose-degrading enzymes in fruit in relation to sink strength. From first flower anthesis until harvest of the 2nd truss, saline treatment of sodium chloride added to the standard nutrient solution to an EC of 8.0 dS/m was applied. The average fruit weight in the salinity treatment was 59% of the control at the mature red stage. Total soluble solids (Brix%) were 6.5% in the control and 9.2% in the salinity treatment. The photosynthetic rate per unit leaf area was decreased by salinity treatment. Salinity decreased 13C assimilation in the whole tomato plant, while it increased the transport and distribution of 13C assimilates to the fruit significantly. The activities of soluble and cell wall-bound acid invertase were not affected by salinity; however, the activity of sucrose synthase in fruit exposed to the salinity was higher than that in the control during fruit development. We concluded that the increase in total soluble solids in fruit subjected to salinity was not only a result of condensation caused by reduced water uptake, but active transport of photosynthates to the fruit due to increase in sucrose-degrading enzyme activities, especially sucrose synthase.

GENETIC VARIATION OF PHENOLIC COMPOUNDS CONTENT, ESSENTIAL OIL COMPOSITION AND ANTI OXIDATIVE ACTIVITY IN ISRAEL-GROWN MENTHA LONGIFOLIA L.

Phenolic acids, in free soluble form and cell wall-bound, were extracted from leaves and stems from 40 genetic variants from 25 populations of Mentha longifolia native to Israel. Rosmarinic and caffeic acid were the most abundant freely soluble acids whereas caffeic acid was most abundant among cell wall-bound phenolic acids. Assessment of the total antioxidant efficacy, based on the reduction of 1,1-diphenyl-2-picryldrazyl (DPPH), revealed high antioxidant activity by tissues of many M. longifolia variants, stemming in part, from antioxidant activity of rosmarinic acid. Although phenolic acids in free extractable form comprise a major portion of total phenolic acids, cell wall-bound acids were found in significant amounts and may also contribute to the antioxidant efficacy of the plants.

Activity, but not Expression, of Soluble and Cell Wall-Bound Acid Invertases Is Induced by Abscisic Acid in Developing Apple Fruit

The present experiment, involving both the in vivo injection of abscisic acid (ABA) into apple (Malus domestica Brohk.) fruits and the in vivo incubation of fruit tissues in ABA-containing medium, revealed that ABA activates both soluble and cell wall-bound acid invertases. Immunoblotting and enzyme-linked immunosorbent assays showed that this ABA-induced acid invertase activation is independent of the amount of enzyme present. The acid invertase activation induced by ABA is dependent on medium pH, time course, ABA dose, living tissue and developmental stage. Two isomers of cis-(+)-ABA, (-)-ABA and trans-ABA, had no effect on acid invertases, showing that ABA-induced acid invertase activation is specific to physiologically active cis-(+)ABA. Protein kinase inhibitors K252a and H7 as well as acid phosphatase increased the ABA-induced effects. These data indicate that ABA specifically activates both soluble and cell wall-bound acid invertases by a posttranslational mechanism probably involving reversible protein phosphorylation, and this may be one of the mechanisms by which ABA is involved in regulating fruit development. (Managing editor: Ping He)

The effect of high temperatures on the expression and activity of sucrose-cleaving enzymes during tomato (Lycopersicon esculentum) anther development

SummaryExposure to high temperatures causes reduced yields in tomato (Lycopersicon esculentum), mainly by affecting male gametophyte development. The effect of heat stress on pollen characteristics is associated with changes in carbohydrate metabolism in the developing anthers. The expression and activities of sucrose-cleaving enzymes and the amounts of starch and soluble sugars were measured in tomato (inbred line ‘NC 8288’) anthers at four stages of development, under ‘normal’ controlled (day/night temperatures of 26°/18°C) and high-temperature (day/night temperatures of 32°/26°C) conditions. Heat stress was found to cause a reduction in cell wall-bound acid invertase activity in the anthers of flower buds 5 d before anthesis (DBA), which correlated with reduced starch accumulation at 3 DBA, and an induction of the activities of sucrose synthase and soluble acid invertase in maturing anthers. The heat stress-induced increases in soluble acid invertase and sucrose synthase activities close to anthesis were found to correlate with a three-fold increase in the hexose:sucrose ratio in mature anthers. The heat-stress conditions applied altered the gene expression profiles of the enzymes tested, decreasing the steady-state level of the mRNA coding for cell wall-bound acid invertase at 5 DBA, increasing expression of the soluble acid invertase gene at 5 DBA, and decreasing expression of the sucrose synthase gene at 5 and 3 DBA. These data indicate that sucrose-cleaving enzymes in tomato anthers respond, at both the mRNA and enzyme activity levels, to high-temperature conditions, that the response is dependent upon the stage of flower development, and that it may involve post-transcriptional control.

Characterization of the promoter of the Wiv-1 (Lin6) gene encoding a wound-inducible cell wall-bound acid invertase in tomato

Fragments of different length (4 kb and 1 kb) of the 5� upstream region of the Wiv-1 (Lin6) gene encoding a wound-inducible cell wall-bound acid invertase in tomato were fused to the beta-glucuronidase (GUS) gene and expressed in tobacco and tomato plants. The GUS activity detected was similar in all transformed lines, suggesting that the 1 kb region contains most of the regulatory motifs responsible for the characteristics of the Wiv-1 promoter. GUS activity was observed in vascular tissues of transgenic tomato and tobacco. In stem sections of transgenic tobaccos, the strongest activity was observed in the internal phloem of the nodes adjacent to the bases of petioles. The GUS activity in leaves of transgenic tobacco was elevated by addition of soluble sugars, especially sucrose. Previous studies suggested that the Wiv-1 enzyme participated in the regulation of carbohydrate contents in tomato leaves (Ohyama and Hirai 1999). The results obtained in the present study suggest that the Wiv-1 enzyme controls the loading of sucrose at the phloem of nodes and Wiv-1 regulation depends on the concentration of sugars in source leaves.

Nondiffusional Release of Allergens from Pollen Grains of Artemisia vulgaris and Lilium longiflorum Depends Mainly on the Type of the Allergen

Background: Upon contact with a wet surface, mature pollen grains hydrate and release proteins including allergens. Knowledge of the release mechanism of allergens that are mainly localized intracellularly may allow the design of strategies for inhibition of allergen release and the consequent sensitization process. Methods: An improved pollen chromatography was performed with Artemisia vulgaris and Lilium longiflorum pollen. Using three elution media of different pH, osmolality and salt concentration mimicking various types of wet surfaces, the time-dependent elution profiles of total protein, a cell wall-bound acid phosphatase activity (acPase), allergenic (profilin, Art v 1) and nonallergenic molecules (14-3-3 protein, actin) were monitored. Results: The release kinetics of total protein and cell wall-bound acPase followed an exponential decrease in both pollen species indicating a diffusion-based protein release, whereas the elution profiles of profilin, Art v 1 and 14-3-3 protein showed nondiffusion characteristics. No general dependence on pH, osmolality or salt concentration of the elution media was observable in the elution profiles. Under the applied conditions, actin was not released indicating that the pollen grains remained intact during the elution. Conclusion: The elution profiles of pollen allergens indicated that substantial amounts of these proteins do not diffuse from the cell wall or are released from intracellular compartments during imbibitional leakage. Instead, a mechanism seems to operate that involves translocation from the pollen cytoplasm to the extracellular environment by crossing an intact plasma membrane. Such a mechanism would probably allow the use of pharmaceuticals for inhibition of allergen release.

Changes in carbohydrate content and related enzyme activity during Cyphomandra betacea (Cav.) Sendtn. fruit maturation

Changes in sugar content and activities of invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13) were measured throughout fruit development in tree tomato (Cyphomandra betacea (Cav.) Sendtn.). Fruit of C. betacea accumulated predominantly reducing sugars similar to Lycopersicon esculentum and in contrast to sucrose accumulation, which is characteristic of L. peruvianum. Soluble acid invertase, fructose and glucose were localized principally in the vacuole. The soluble acid invertase activity was highest in ripe fruit (80 days past anthesis), while sucrose synthase activity was highest in the young fruit and declined with development. Otherwise, the activity of cell wall-bound acid invertase was less and may be greatly decreased by interaction with an invertase inhibitor protein, located in the cell walls of C. betacea fruit. The highest soluble acid invertase activity was associated with fruit ripening and during early stage of development. Sucrose synthase is the dominant enzyme in metabolizing imported sucrose.

Cleavage of sucrose in roots of soybean (Glycine max) colonized by an arbuscular mycorrhizal fungus.

• Here we tested the hypothesis that the activity of sucrose-cleaving enzymes is modified in roots colonized by arbuscular fungi. • The activities of soluble and cell-wall bound invertases and of sucrose synthase were assessed in nonnodulated roots of soybean (Glycine max), either inoculated with the arbuscular mycorrhizal fungus Glomus mosseae, or in the absence of inoculation. Furthermore, we assessed the activity of sucrose-cleaving enzymes in cultured extraradical mycelium of the arbuscular fungus Glomus intraradices. • Both soluble and cell wall-bound acid invertase activities decreased during the course of the experiment; there were no differences between mycorrhizal and nonmycorrhizal plants. Alkaline invertase activity was significantly higher in inoculated roots from the beginning of root colonization. Sucrose synthase activity showed no significant differences between treatments for 35d, thereafter it became significantly higher in mycorrhizal roots. No sucrose cleaving activities were detected in extraradical mycelium. • Fungal colonization of soybean roots induces an increase in alkaline invertase activity, which could provide hexoses for the fungal symbiont and for development of colonized cells.

Characterization of promoter of Wiv-1 (Lin6) gene for wound-inducible cell wall-bound acid invertase of tomato.

Characterization of promoter of Wiv-1 (Lin6) gene for wound-inducible cell wall-bound acid invertase of tomato.

Seasonal changes of asparagus spears in relation to enzyme activities and carbohydrate content

Seasonal changes in the activities of acid invertase, sucrose synthase (SS) and sucrose phosphate synthase (SPS) along with the sugar content were studied in asparagus spears ( Asparagus officinalis L. cvs. E414 and Welcome) grown in the greenhouse. The primary objective of this study was to understand the effect of long-term harvesting on the quality of green asparagus spears. Both soluble and cell wall-bound acid invertase increased in activity until June after which they changed very little. Similarly, SS activity increased to a maximum in June and changed little after this time. SPS activity changed little throughout the season. Sucrose concentration declined from March to a minimum in June and increased again in October. On the other hand glucose and fructose concentrations increased to a maximum in June and thereafter changed little except for a possible decline in October. A highly significant negative correlation was found between acid invertase and SS activities and sucrose content accounting well for the relation between the substrate and the activity. There were no significant differences between the cultivars.

Purification and Characterization of Soluble and Cell Wall-bound Acid .ALPHA.-Glucosidases of Ripe Yellow Banana Pulp.

Banana pulp, irrespective of the degree of ripening, contained soluble and cell wall-bound forms of acid α-glucosidases (SAAG and BAAG). BAAG was released with a neutral buffer containing 0.2-2 M NaCI after extracting of SAAG with a salt-free buffer. From the same bunch of ripe yellow bananas, SAAG and BAAG were purified at 732-fold and 264-fold, respectively, using ConASepharose and Sephadex G-150 gel column chromatographies. The molecular weights of SAAG and BAAG were 70, 000 and 90, 000, respectively, by gel filtration. These enzymes were typical maltases that required maltose (G2) and malto-oligosaccharides (G3-G7) as substrates, but not isomaltose, treharose, sucrose, pullulan, glycogen, or soluble starch. The Vmax/Km ratios (apparent hydrolytic efficiencies) of SAAG and BAAG toward G5-G7 were 89-112 and 14-52%, respectively, with the ratio toward maltose being 100%. As for ripe yellow banana where cell wall or amy loplastic membrane has not been maintained, it is speculated that BAAG could access starch and its hydrolytic products and would share in starch degradation in collaboration with amylases and SAAG.

Sucrose Synthase Isozymes Related to Sucrose Accumulation During Fruit Development of Japanese Pear (Pyrus pyrifolia Nakai).

The relationship between sucrose metabolizing enzymes, especially sucrose synthase (SS) isozymes and sucrose accumulation was investigated throughout fruit development of the Japanese pear. The sucrose content in the fruit which was very low at the young stage, drastically increased with maturation, as did the sucrose phosphate synthase activity. The activities of soluble and cell wall-bound acid invertases, which were high in the young fruit, decreased rapidly during fruit maturation. SS activity was, likewise, high in young fruit but decreased with fruit enlargement ; however, it rose again concomitant with sucrose accumulation during maturation. The fluctuation in SS activity was caused by two SS isozymes (SSI and SSII) ; SSI activity was high in young fruit and decreased with fruit development, while SSII activity, which was very low in young fruit, increased with fruit development. These results indicate that these two isozymes differ in their functions. SSII catalyzes sucrose synthesis which contributes to sucrose accumulation during fruit maturation, whereas SSI facilitates sucrose cleavage together with acid invertases and supplies substrates for growth in young fruits.

Stimulation of hyphal growth in anaerobic cultures of Mucor rouxii by extracellular trehalose. Relevance of cell wall-bound activity of acid trehalase for trehalose utilization.

In fungi, the hydrolysis of extracellular trehalose is carried out by acid trehalases. These secretory glycoproteins may be more abundant either in the vacuolar compartment, like in yeast, or at the cell surface, such as in many filamentous fungi. The relative efficiency of these two compartments for the utilization of extracellular trehalose was investigated using as a model the dimorphic fungus Mucor rouxii, which produces yeast-like cells under a CO(2) atmosphere, or hyphae in the presence of air. Under CO(2), cultures supplemented with glucose produced yeast-like cells devoid of acid trehalase activity. On the other hand, trehalose-supplemented cultures developed hyphae exhibiting cell wall-bound and intracellular acid trehalase activity. Glucose-grown yeast-like cells supplemented with trehalose after glucose exhaustion, induced intracellular activity of acid trehalase, but no activity was detected at the cell surface. Even endowed of significant intracellular activity of acid trehalase, these cells did not grow further. When exposed to air these yeast-like produced germ tubes exhibiting cell wall-bound acid trehalase activity. These results suggest that the utilization of extracellular trehalose as a source of carbon for growth requires the localization of acid trehalase activity at the cell surface. Our results also show that extracellular trehalose elicits a morphogenetic phenomenon, inducing the formation of hyphae which are the physiological support for acid trehalase activity.

Stimulation of hyphal growth in anaerobic cultures of Mucor rouxii by extracellular trehalose. Relevance of cell wall-bound activity of acid trehalase for trehalose utilization

In fungi, the hydrolysis of extracellular trehalose is carried out by acid trehalases. These secretory glycoproteins may be more abundant either in the vacuolar compartment, like in yeast, or at the cell surface, such as in many filamentous fungi. The relative efficiency of these two compartments for the utilization of extracellular trehalose was investigated using as a model the dimorphic fungus Mucor rouxii, which produces yeast-like cells under a CO 2 atmosphere, or hyphae in the presence of air. Under CO 2, cultures supplemented with glucose produced yeast-like cells devoid of acid trehalase activity. On the other hand, trehalose-supplemented cultures developed hyphae exhibiting cell wall-bound and intracellular acid trehalase activity. Glucose-grown yeast-like cells supplemented with trehalose after glucose exhaustion, induced intracellular activity of acid trehalase, but no activity was detected at the cell surface. Even endowed of significant intracellular activity of acid trehalase, these cells did not grow further. When exposed to air these yeast-like produced germ tubes exhibiting cell wall-bound acid trehalase activity. These results suggest that the utilization of extracellular trehalose as a source of carbon for growth requires the localization of acid trehalase activity at the cell surface. Our results also show that extracellular trehalose elicits a morphogenetic phenomenon, inducing the formation of hyphae which are the physiological support for acid trehalase activity.

Cloning of cDNA for a cell wall-bound acid invertase from tomato (Lycopersicon esculentum) and expression of soluble and cell wall-bound invertases in plants and wounded leaves of L. esculentum and L. peruvianum.

A cDNA, Wiv-1, for an isozyme of acid invertase (EC 3.2.1.26) was cloned from wounded leaves of tomato (Lycopersicon esculentum). The encoded protein had a basic isoelectric point and strong similarity to the amino acid sequences of plant cell wall-bound invertases. The conserved sequence WECPD that is found in all plant cell wall-bound invertases was also found in the deduced protein. These results suggested that Wiv-1 encoded a cell wall-bound acid invertase of tomato. Wounding increased the levels of mRNAs for soluble and cell wall-bound invertases and the activities of these invertases in leaves of L. esculentum and of a related species, L. peruvianum. The induction of Aiv-1 mRNA for the soluble enzyme in wounded leaves was not very strong, while that of Wiv-1 mRNA for the wall-bound enzyme was prominent. The level of Aiv-1 mRNA reached a maximum 48 h after wounding while that of Wiv-1 mRNA continued to rise for up to 96 h. These findings suggested that the genes for the two isozymes responded independently to wounding. The levels in various organs of Aiv-1 and Wiv-1 mRNAs were higher in L. esculentum than in L. peruvianum. Possible roles of cell wall-bound acid invertase in wound response and in developing plant are discussed.