Cell Wall-bound Acid Invertase Research Articles

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.

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.

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.

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.

Cold‐induced repression of the rice anther‐specific cell wall invertase gene OSINV4 is correlated with sucrose accumulation and pollen sterility

ABSTRACTLow temperatures during rice (Oryza sativa L.) pollen development cause pollen sterility and decreased grain yield. We show that the time of highest sensitivity to cold coincides with the time of peak tapetal activity: the transition of the tetrad to early uni‐nucleate stage (young microspore, YM stage). Low temperatures at this stage of pollen development result in an accumulation of sucrose in the anthers, accompanied by decreased activity of cell wall bound acid invertase and depletion of starch in mature pollen grains. Expression analysis of two cell wall (OSINV1, 4) and one vacuolar (OSINV2) acid invertase genes showed that OSINV4 is anther‐specific and down‐regulated by cold treatment. OSINV4 is transiently expressed in the tapetum cell layer at the YM stage, and later from the early binucleate stage in the maturing microspores. The down‐regulation of OSINV4 expression in the tapetum at YM may cause a disruption in hexose production and starch formation in the pollen grains. In a cold‐tolerant cultivar, OSINV4 expression was not reduced by cold; sucrose did not accumulate in the anthers and starch formation in the pollen grains was not affected.

Activity and gene expression of acid invertases in einkorn wheat (Triticum monococcum) infected with powdery mildew

Hexose released from sucrose by cell wall bound acid invertases is considered to be a major carbon source for powdery mildews. This aspect of pathogenesis was investigated in diploid wheat (Triticum monococcum) infected with powdery mildew [Blumeria graminis f. sp. tritici]. Quantitative assays revealed an increase in acid invertase activity following infection, and that soluble invertases, of vacuolar and (or) extracellular origin, accounted for over 80% of the overall increase. In situ detection showed activity of cell wall bound invertase in both uninfected and infected leaf tissues, but no increase in activity following infection and no activity at the infection sites. Two cDNAs of the putative genes Tm-exin1 and Tm-vin1 coding for a soluble extracellular invertase and a vacuolar invertase, respectively, were identified in a collection of expressed sequence tags derived from mildewed wheat leaf epidermis. Tm-exin1 was induced in infected leaves, and the enhanced expression coincided with conidiation and colonization of wheat leaf by the fungus. Conversely, Tm-vin1 expression remained relatively constant throughout infection. Collectively, these results support the view that invertases mediate hexose production during infection by the fungus causing powdery mildew and, furthermore, suggest that soluble extracellular invertase is responsible for providing the increased hexose required by the fungus.

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.

Sucrose mobilization in relation to essential oil biogenesis during palmarosa (Cymbopogon martinii Roxb. Wats. var. motia) inflorescence development.

Palmarosa inflorescence with partially opened spikelets is biogenetically active to incorporate [U-14C]sucrose into essential oil. The percent distribution of 14C-radioactivity incorporated into geranyl acetate was relatively higher as compared to that in geraniol, the major essential oil constituent of palmarosa. At the partially opened spikelet stage, more of the geraniol synthesized was acetylated to form geranyl acetate, suggesting that majority of the newly synthesized geraniol undergoes acetylation, thus producing more geranyl acetate. In vitro development of palmarosa inflorescence, fed with [U-14C]sucrose, resulted in a substantial reduction in percent label from geranyl acetate with a corresponding increase in free geraniol, thereby suggesting the role of an esterase in the production of geraniol from geranyl acetate. At time course measurement of 14CO2 incorporation into geraniol and geranyl acetate substantiated this observation. Soluble acid invertase was the major enzyme involved in the sucrose breakdown throughout the inflorescence development. The activities of cell wall bound acid invertase, alkaline invertase and sucrose synthase were relatively lower as compared to the soluble acid invertase. Sucrose to reducing sugars ratio decreased till fully opened spikelets stage, concomitant with increased acid invertase activity and higher metabolic activity. The phenomenon of essential oil biosynthesis has been discussed in relation to changes in these physiological parameters.

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.

Sugar Metabolism in Spur Bud during Flower Bud Formation: A Comparison between Exposed and Shaded Buds of Japanese Pear [Pyrus pyrifolia (Burm.) Nak.] 'Kosui'

To elucidate the role of carbohydrates and their metabolism during flower bud formation, the effects of shading the terminal buds of spurs of the Japanese pear 'Kosui' for 3 weeks were investigated. The carbohydrates were: fructose, glucose, sorbitol, sucrose and starch, whereas the related enzymes were: NAD-dependent sorbitol dehydrogenase (NAD-SDH), NADP-dependent sorbitol dehydrogenase (NADP-SDH), sucrose synthase (SS) and acid invertase (AI). Early treatments between 24 May to 28 July decreased both the concentrations of sorbitol, glucose and fructose in the buds and the bud fresh weight as compared with the non-treated, exposed buds. However, late treatments between 28 July to 2 September did not. Activities of NAD-SDH, NADP-SDH, soluble AI and SS increased in the shaded buds, although the differences between the treatments were temporary and sometimes insignificant. In contrast, cell wall-bound AI was temporarily decreased by the shading treatment. Contents of fructose, glucose and sorbitol in buds were positively correlated to the growth rate of the bud. On the other hand, sorbitol concentration was correlated negatively to the activities of total SDH (NAD-SDH + NADP-SDH) and soluble AI, and glucose with NADP-SDH. It is postulated that the sugar concentration may be the limiting factor for bud growth when the carbohydrate supply is insufficient. Shading increased the activities of sugar catabolizing enzymes in the bud that enhanced the capacity of buds to attract photosynthates (sink strength). It could be possible that the decrease of sugar concentration may act as a signal in the regulation of sugar catabolizing enzymes.

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.

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.

Sugarcane Internodal Invertases and Tissue Maturity

Summary The relationship between extractable invertase activities and sucrose accumulation in the sugarcane ( Saccharum officinarum ) stalk, and in vivo invertase mediated sucrose hydrolysis was investigated to determine the significance of invertases in sucrose utilisation and turnover. In vitro activities were determined by assaying the soluble acid, cell wall bound acid and neutral invertases from internodes three to ten in mature sugarcane plants of cultivar NCo376. Extractable activities were verified by immunoblotting. In vivo invertase mediated sucrose hydrolysis was investigated in tissue discs prepared from mature stalk tissue of the same cultivar. Sugarcane neutral invertase had a higher specific activity than soluble acid invertase (apoplastic and vacuolar) in the sucrose accumulating region of the sugarcane stem. Cell wall bound acid invertase was also present in significant quantities in both immature and mature tissue. Recombination experiments indicated the absence of activators and inhibitors, and immunodetection of neutral and soluble acid invertase followed comparable trends to that of activity expressed on a fresh mass or protein basis. When radiolabelled fructose was fed to both young and mature internodal tissue, label appeared in glucose which demonstrates that invertase mediated hydrolysis of sucrose occurs. In vitro invertase activities were used to calculate the theoretical turnover-times for the endogenous sucrose pool.

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.

Carbohydrate content and the activities of sucrose synthase, sucrose phosphate synthase and acid invertase in different tomato cultivars during fruit development

Changes in carbohydrate content and activities of related enzymes of the developing fruits of three tomato cultivars ( Lycopersicon esculentum Mill. cv. Momotaro, Lady First and Minicarol) were followed throughout fruit development. In all the cultivars fructose and glucose in nearly equal amounts, were the predominant sugars. Sucrose was present in trace quantities but the small fruited cultivar Minicarol accumulated higher levels of sucrose than the other two large fruited cultivars. The activity of soluble acid invertase (EC3.2.1.26) was highest in ripe fruits (55 days of anthesis) but in the case of cell wall-bound acid invertase, it was less and almost constant throughout development. Sucrose synthase (EC2.4.1.13) activity was highest in the young fruit and declined sharply with development; ‘Minicarol’ had the highest activity. Sucrose phosphate synthase (EC2.4.1.14) activity remained relatively constant throughout development in all cultivars studied. A highly significant positive correlation was found with sucrose accumulation in the case of sucrose synthase but there was a negative correlation with acid invertase activity and none with sucrose phosphate synthase. Therefore, it appeared that acid invertase and sucrose synthase were involved in the metabolism of sucrose during tomato fruit development and a constant sucrose phosphate synthase activity was not required for sucrose accumulation. Furthermore, the small fruited tomatoes had a higher amount of reducing sugars and sucrose, and higher sucrose synthase and sucrose phosphate synthase activities compared with large fruited types. The results are discussed in terms of the metabolic events associated with soluble sugar accumulation.

The fate of apoplastic sucrose in sink and source leaves of Urtica dioica

Experiments were performed with developing and mature leaves of Urtica dioica L. to trace differences which could be interpreted in terms of cell wall-bound acid invertase (EC 3.2.1.26) participating in phloem unloading in a sink leaf. The pH of apoplastic fluid that was collected by gentle centrifugation of entire leaves was identical (7.1) in the two types of leaves; also, fluorometric determination with esculetin showed a neutral apoplastic pH between 7.0 in the source and 7.2 in the sink leaf. To detect whether differences in apoplastic pH occur within limited leaf areas, such as of the tissue surrounding the veins, the metabolic fate of [14C]–(fructosyl)-sucrose that was administered via the xylem was investigated. In source leaves, there was a large transitory decrease in [14C]-sucrose followed by a substantial resynthesis of this compound. In sink leaves, resynthesis was less significant and carbon was incorporated mainly in starch, charged soluble compounds and cell walls. However, after correction for resynthesis, the two types of leaves showed an identical capacity for sucrose cleavage. Finally, activation of the apoplastic invertase by administering labelled sucrose in buffered solution of pH 5.0 did not result in an enhanced degradation. By contrast, apoplastic fluid collected from leaves which had been infiltrated with buffer solutions of pH 5.5 and 8.0, respectively, showed a rapid adjustment of the pH close to the natural neutral value by the mesophyll tissue. The results are incompatible with the idea of an active invertase in the sink (and the source) leaves apoplast, and hence do not lend support to the theory of apoplastic cleavage of sucrose being required for phloem unloading in this kind of a utilization sink.

Growth Cycle of Suspension Cultures of Lycopersicon esculentum and L. peruvianum

Summary Rapidly growing, fine suspension cultures were established from hypocotyl pieces of Lycopersicon esculentum cv. Lukullus and from cotyledons of L. peruvianum. They are characterized with respect to their growth pattern, nutrient consumption as well as uptake and incorporation of precursors of nucleic acid and protein synthesis. The doubling time of these tomato suspension cultures in a modified Murashige-Skoog medium is ~ 24 h. Analyses of the macronutrient consumption during the growth cycle (carbohydrate, nitrate, ammonia, phosphate) revealed a phosphate-limited growth with exponential increase of cell number and dry weight during 3–5 d. Normally 3% sucrose is used as carbon source; but glucose is suitable as well. In L. esculentum suspensions sucrose is rapidly hydrolysed by a cell wall bound acid invertase, and the liberated glucose and fructose are consumed consecutively. Long-term cultures from both tomato species exhibit very low morphogenetic potential. With L. peruvianum green, heterogeneous suspensions can be obtained after prolonged passages on cytokinin-free media containing indole acetic acid as the sole growth hormone.