Acid and alkaline invertases in suspension cultures of sugar beet cells

Alkaline invertase was induced during the initiation of suspension cultures of single cells from leaf explants of sugar beets in Murashige-Skoog liquid medium which contained benzyladenine. This activity was barely detectable in the leaves themselves. In suspension cultures, the presence of both acid and alkaline invertases was detected; alkaline invertase was only present in the cytoplasm of the cultured cells, whereas acid invertase was present in the cytoplasm and cell walls, and was also detected in the culture medium. The cell wall contained at least three types of acid invertase; two of these activities were solubilized by saline (saline-released) and EDTA (EDTA-released), respectively, and the third remained tightly associated with the cell wall. Saline-released and EDTA-released invertases from the cell wall showed the significant differences in their properties: the saline-released enzyme had the highest affinity for sucrose among the invertases tested, and was easily bound to cell walls, to DNA, and to a cation exchanger, unlike the EDTA-released enzyme. Sucrose is the source of carbon for plant cells in suspension culture and is probably degraded in the cell wall by the saline-released invertase, which had the highest activity and the highest affinity for sucrose. Hexose products of this degradation would be transported to cytoplasm. Soluble invertase, EDTA-released invertase from the cell wall, and one of two extracellular invertases behaved similarly upon chromatography on DEAE-cellulose. They had similar activity profiles with changing pH, and similar K_m values for sucrose. Thus it appears that they are identical. Two extracellular invertases found in the growth medium of the suspension cultures were probably identical with those in the soluble fraction of callus and seedlings of sugar beets, because they showed similar behaviors during chromatography on DEAE-cellulose, and had similar activity profiles with changing pH and K_m values for sucrose.     

Acid and Neutral Invertases in the Mesocarp of Developing Muskmelon (Cucumis melo L. cv Prince) Fruit

Acid and neutral invertases were found in the mesocarp of developing muskmelon (Cucumis melo L. cv Prince) fruit and the activities of these enzymes declined with maturation of the fruit, concomitantly with the accumulation of sucrose. Neutral invertase was only present in the soluble fraction and acid invertase was present in both the soluble and cell-wall fractions. The cell-wall fraction contained three types of acid invertase: a NaCl-released invertase; an EDTA-released invertase, and a tightly bound invertase that still remained on the cell wall after treatment with NaCl and EDTA. The soluble acid and neutral invertases could be separated from one another by chromatography on DEAE-cellulose and they exhibited clear differences in their properties, namely, in their pH optima, substrate specificity, K_m values for sucrose, and inhibition by metal ions. The EDTA-released invertase and the soluble acid invertase were similar with regard to their chromatographic behavior on DEAE-cellulose, but the NaCl-released invertase was different because it was adsorbed to a column of CM-cellulose. The soluble acid invertase and two cell-wall bound invertases had very similar characteristics with regard to optimal pH and temperature, K_m value for sucrose, and substrate specificity.     

AtCYT-INV1 in Arabidopsis Sugar Signaling

Sugar acts as a signal molecule and plays a pivotal role in plant development and stress response. Neutral/alkaline invertases found only in photosynthetic bacteria and plants is sucrose-specific enzymes cleave sucrose into glucose and fructose. We have identified a gene for neutral/alkaline invertase in Arabidopsis designated as AtCYT-INV1 which is involved in sugar/ABA signaling and plays multiple roles in plant development and osmotic stress-induced inhibition on lateral root growth.Key Words: Arabidopsis thaliana, AtCTY-INV1, sugar signaling     

Differential invertase activity and root growth between Cu-tolerant and non-tolerant populations in Kummerowia stipulacea under Cu stress and nutrient deficiency

There has been no study on key enzymes in sucrose cleavage in metallophyte plants so far, which may be crucial for the plants’ root growth and heavy-metal tolerance maintenance. Here, we tested the hypothesis that the roots of copper tolerant plants should manifest a higher activity of acid invertases that are rate-limiting in sucrose catabolism than non-tolerant plants both for supporting growth and for their maintaining tolerance under Cu stress. Two populations of Kummerowia stipulacea, one from an ancient waste heap at a Cu mine, and the other from a non-contaminated site, were used in the experiments. The plants were grown in 1/2-fold (control) or 1/20-fold (nutrient deficiency) Hoagland’ solution, with (Cu stress) or without (control) 10 μmol/L Cu²⁺. Plants from the mine proved to be of Cu tolerance. Cu exposure had a stronger inhibition on root growth and thus resulting in a lower root/shoot ratio in the plants of non-mine population compared to the mine population. Cu exposure showed a stronger inhibition of acid invertase activity of Cu non-tolerant plants than Cu-tolerant plants, while neutral/alkaline invertase was insensitive to Cu. A positive correlation between the activity of acid invertases and the root growth and root/shoot ratio was observed. The results indicated an important role of acid invertases in governing root growth and root/shoot biomass allocation in the plants of mine population. The results also suggested that the higher activities in acid invertases of mine population plants might at least partly associate with the plants’ Cu tolerance, and their higher activities in acid invertases in turn played an role in maintenance of the Cu tolerance by supplying carbon and energy for tolerance mechanisms. In addition, the results showed evidence that neutral/alkaline invertase might play a role in compensating for the depression in sucrose catabolism due to Cu-induced inhibition in acid invertases.     

Enzymes of sucrose breakdown in soybean nodules: alkaline invertase

The specific activities of acid and alkaline invertases (β-d-fructofuranoside fructohydrolase, EC 3.2.1.26), sucrose synthase (UDPglucose: d-fructose 2-α-d-glucosyltransferase, EC 2.4.1.13), hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1), and fructokinase (ATP: d-fructose 6-phosphotransferase, EC 2.7.1.4) were determined in soybean (Glycine max L. Merr cv Williams) nodules at different stages of development and, for comparison, in roots of nonnodulated soybeans. Alkaline invertase and sucrose synthase were both involved in sucrose metabolism in the nodules, but there was only a small amount of acid invertase present. The nodules contained more phosphorylating activity with fructose than glucose. Essentially all of the alkaline invertase, sucrose synthase, and fructokinase were in the soluble fraction of nodule extracts whereas hexokinase was in the bacteroid, plant particulate, and soluble fractions.     

Characterization and inhibition of invertases in sugar cane juice

(NH₄)₂SO₄ fractionation followed by Sephadex G-200 chromatography of sugar cane juice gave an acid invertase with MW of 380 000 and 23.5% carbohydrate and a neutral invertase with MW of 66 000 and 22% carbohydrate. For acid invertase, K_m is 2.8 mM and V_max is 2.7 μmol sucrose hydrolysed/hr/mg protein. For neutral invertase, K_m and V_max are 0.32 mM and 2.8 μmol hydrolysed/hr/mg protein, respectively. Inhibition of both invertases by either lauryl sulfate or metasilicate is not competitive.     

Identification and characterization of invertase family genes reveal their roles in vacuolar sucrose metabolism during Pyrus bretschneideri Rehd. fruit development

23 invertase (PbrInvs) genes, including eight vacuolar invertases (PbrvacInvs), five cell wall invertases (PbrcwInvs) and 10 alkaline/neutral invertases (PbrA/N-Invs), were identified from P. bretschneideri Rehd. genome, with diverse chromosome locations, cis-acting elements, gene structures and motifs. Their expression profiles were tissue-specific, and postharvest light or temperature treatment would alter their expression profiles. During ‘Dangshansuli’ pear development, in association with visual/inner quality change was the alternations of invertase activity and the expression profiles of PbrInvs. In combination with results of subcellular sugar distribution as well as correlation analysis among sugar content, invertase activity and PbrInv mRNA abundance, PbrvacInv1 might be involved in sucrose decomposition during pear development. PbrvacInv1-GFP fusion protein mainly accumulated on the tonoplast (vacuolar membrane); meanwhile, transient overexpression of PbrvacInv1 in pear fruit would upregulate vacInv activity, causing higher fructose and lower sucrose when compared with that of the control. Furthermore, invertase inhibitor 5 (PbrInvInh5) could interact with PbrvacInv1.     

Properties of the Invertases of Cultured Sycamore Cells and Changes in Their Activity during Culture Growth

When cultured sycamore cells are homogenised in a phosphate-citrate buffer at pH 7.0 and the homogenate centrifuged two fractions are obtained both of which show the presence of an acid (opt. pH 4.0–4.5) and a neutral (opt. pH 7.0–7.4) invertase. The activity of the insoluble pellet appears to be located in its cell wall fragments. The acid and neutral invertases of the soluble fraction can be separated by fractional precipitation with (NH₄SO₄. The activities of these enzymes are low in stationary phase cells but they increase following subculture to reach peaks of activity towards the end of the period of most active cell growth and division and then decline again as the cells begin to enter stationary phase. The activities of both enzymes are higher in the cell wall than in the soluble fraction and the acid invertase reaches higher levels of activity than the neutral enzyme in both fractions. When cells are subcultured there occurs within a few hours an increase in the acid invertase and a decline in the neutral invertase activity in the cell wall fraction and a decline in the acid invertase of the soluble fraction prior to the large net increases in the activities of both enzymes in both locations which occurs as the cells embark upon cell division. The pattern of changes in the invertase activities through the growth cycle of batch propagated cultures is similar whether the cells are grown in sucrose, or glucose, or sucrose plus glucose; the highest levels of activities were recorded in the glucose-grown cells. The total yield of invertase activities and the distribution of activities between the soluble and cell wall fractions of the homogenates are affected by the pH of the extraction medium (within the range pH 4.0–8.0). It has not proved possible to completely remove the invertases from the cell wall fraction; upwards of 50 % of the acid invertase was recovered from this fraction by treatment with Triton-X followed by urea, but these treatments inactivated a high proportion of the neutral enzyme. These findings are compared with other studies on the activity and intra-cellular distribution of plant invertases and the possible roles of these enzymes discussed.     

The sucrose synthase gene family in Populus: structure, expression, and evolution

Sucrose synthase is a key enzyme in sucrose metabolism in plant cells, and it is involved in the synthesis of cell wall cellulose. Although the sucrose synthase gene (SUS) family in the model plants Arabidopsis thaliana has been characterized, little is known about this gene family in trees. This study reports the identification of two novel SUS genes in the economically important poplar tree. These genes were expressed predominantly in mature xylem. Using molecular cloning and bioinformatics analysis of the Populus genome, we demonstrated that SUS is a multigene family with seven members that each exhibit distinct but partially overlapping expression patterns. Of particular interest, three SUS genes were preferentially expressed in the stem xylem, suggesting that poplar SUSs are involved in the formation of the secondary cell wall. Gene structural and phylogenetic analyses revealed that the Populus SUS family is composed of four main subgroups that arose before the separation of monocots and dicots. Phylogenetic analyses associated with the tissue- and organ-specific expression patterns. The high intraspecific nucleotide diversity of two SUS genes was detected in the natural population, and the π _nonsyn/π _syn ratio was significantly less than 1; therefore, SUS genes appear to be evolving in Populus, primarily under purifying selection. This is the first comprehensive study of the SUS gene family in woody plants; the analysis includes genome organization, gene structure, and phylogeny across land plant lineages, as well as expression profiling in Populus.     

Isolation and characterization of a cDNA clone from Lolium temulentum L. encoding for a sucrose hydrolytic enzyme which shows alkaline/neutral invertase activity

A novel cDNA clone, functionally expressed in E. coli, was isolated from a L. temulentum L. cDNA library. The expressed protein hydrolysed sucrose with an apparent Km of approximately 18 mM, and produced equi-molar concentrations of glucose and fructose. Optimum activity was observed at pH 7-7.5; there was little or no activity at pH 5.5. The expressed protein did not hydrolyse raffinose, stachyose or maltose. The activity of the expressed protein was inhibited by fructose (50% at 15 mM) and TRIS (50% at 2.5 mM), but was not affected by MgCl2, CaCl2 or MnCl2. These findings suggest that this cDNA clone encodes for an alkaline/neutral invertase. Sequence analysis revealed little homology with published sequences for acid invertase, however the invertase motif (NDPN) identified in other invertases was present. Expression studies show that the gene encoding for this enzyme is not regulated by sucrose accumulation in leaf tissue.     

A mitochondrial alkaline/neutral invertase isoform (A/N-InvC) functions in developmental energy-demanding processes in Arabidopsis

Recent findings demonstrate that alkaline/neutral invertases (A/N-Invs), enzymes that catalyze the breakdown of sucrose into glucose and fructose, are essential proteins in plant life. The fact that different isoforms are present in multiple locations makes them candidates for the coordination of metabolic processes. In the present study, we functionally characterized the encoding gene of a novel A/N-Inv (named A/N-InvC) from Arabidopsis, which localizes in mitochondria. A/N-InvC is expressed in roots, in aerial parts (shoots and leaves) and flowers. A detailed phenotypic analysis of knockout mutant plants (invc) reveals an impaired growth phenotype. Shoot growth was severely reduced, but root development was not affected as reported for A/N-InvA mutant (inva) plants. Remarkably, germination and flowering, two energy demanding processes, were the most affected stages. The effect of exogenous growth regulators led us to suggest that A/N-InvC may be modulating hormone balance in relation to the radicle emergence. We also show that oxygen consumption is reduced in inva and invc in comparison with wild-type plants, indicating that both organelle isoenzymes may play a fundamental role in mitochondrion functionality. Taken together, our results emphasize the involvement of mitochondrial A/N-Invs in developmental processes and uncover the possibility of playing different roles for the two isoforms located in the organelle.     

Alkaline β-fructofuranosidases of tuberous roots: Possible physiological function

Summary Alkaline invertase of roots of carrot (Daucus carota L.) did not hydrolyze raffinose while the acid invertase from the same tissue showed with this sugar ca. 60% of the activity found with sucrose. The activity of the two invertases was inhibited by fructose to a different extent, the K _i value being ca. 4×10^–2 M and 3×10^–1M, respectively, for the alkaline and the acid invertases from the roots of both carrot and turnip (Brassica rapa L.). It is proposed that fructose inhibition of acid invertase is of no physiological significance but that, in contrast, hexoses might regulate the activity of alkaline invertase.Comparing several species and cultivars, it was found that the content of reducing sugars and the activity of alkaline invertase of mature tuberous roots showed a positive correlation. This indicates that alkaline invertase may participate in the regulation of the hexose level of the cell, as was previously suggested for sugar-cane. A scheme is presented which proposes a way of participation of alkaline invertase in such a regulation, assuming that this enzyme is located in the cytoplasm and acid invertase is membrane-bound and mainly located at the cell surface.     

Cytochemical methods for the localization of invertase activity in plant tissues

Summary Two cytochemical methods for the localization of acid and alkaline invertases are given. The first is based upon the reduction of a silver complex at two different pH ranges, whilst the second is based upon the tetrazolium raction and permits quantification of the rate of activity of alkaline invertase activity. The distribution of alkaline invertase activity throughout the root apex of Pisum sativum and the cell wall localization of acid invertase for material excised from tuber tissue of Helianthus tuberosus are both confirmed.     

An Invertase Inhibitory Protein From Pteris deflexa Link Fronds

Plant invertases play important roles in sucrose metabolism. Cell wall invertase was reported to participate in phloem loading and unloading. Soluble invertases would be involved in hexose level regulation in mature tissues and in stored sucrose utilization within vacuoles. Invertase inhibitory proteins were described as one of the possible mechanisms for invertase activity regulation in some plant species; nevertheless, these proteins were found only in sink tissues, suggesting that this mechanism would not be relevant in the sucrose turnover of leaves. This report describes the purification of invertase from Pteris deflexa fronds and the occurrence of an invertase inhibitory protein in this fern organ, as well as its purification and invertase-inhibitor interactions. The Mr of the invertase and of its inhibitory protein were 90,000 and 18,000, respectively. SDS-PAGE in the presence of 2-mercaptoetanol gave two subunits for the enzyme (Mr=66,000 and 30,000) and only one for the inhibitor. The inhibitor protein is a glycoprotein (12% w/w of neutral sugars) that did not show agglutinating activity like some others, and also showed a high heat stability at pH 5.0. The optimum pH of invertase activity is 5.0, while invertase inhibitory protein caused maximal inhibition at the same pH value. Invertase-inhibitor complex formation occurs in an immediate manner and a protease activity was discarded. The inhibition is non-competitive (Ki=1.5 × 10 ^?6 M) without interactions among the binding sites. The complex is slightly dissociable and sucrose was able to partially reduce the inhibitory effect. Up to the present, invertase inhibitory proteins have been found solely in heterotrophic tissues. In this work we demonstrate that this protein is also present in an autotrophic tissue of a lower vascular plant.     

Demonstration of an intramitochondrial invertase activity and the corresponding sugar transporters of the inner mitochondrial membrane in Jerusalem artichoke (<Emphasis Type="Italic">Helianthus tuberosus</Emphasis> L.) tubers

Genetic evidences indicate that alkaline/neutral invertases are present in plant cell organelles, and they might have a novel physiological function in mitochondria. The present study demonstrates an invertase activity in the mitochondrial matrix of Helianthus tuberosus tubers. The pH optimum, the kinetic parameters and the inhibitor profile of the invertase activity indicated that it belongs to the neutral invertases. In accordance with this topology, transport activities responsible for the mediation of influx/efflux of substrate/products were studied in the inner mitochondrial membrane. The transport of sucrose, glucose and fructose was shown to be bidirectional, saturable and independent of the mitochondrial respiration and membrane potential. Sucrose transport was insensitive to the inhibitors of the proton-sucrose symporters. The different kinetic parameters and inhibitors as well as the absence of cross-inhibition suggest that sucrose, glucose and fructose transport are mediated by separate transporters in the inner mitochondrial membrane. The mitochondrial invertase system composed by an enzyme activity in the matrix and the corresponding sugar transporters might have a role in both osmoregulation and intermediary metabolism.