Direct Photolabeling with [P]UDP-Glucose for Identification of a Subunit of Cotton Fiber Callose Synthase

We have identified a 52 kilodalton polypeptide as being a likely candidate for the catalytic subunit of the UDP-glucose: (1→3)-β-glucan (callose) synthase of developing fibers of Gossypium hirsutum (cotton). Such a polypeptide migrates coincident with callose synthase during glycerol gradient centrifugation in the presence of EDTA, and can be directly photolabeled with the radioactive substrate, α-[³²P]UDP-glucose. Interaction with the labeled probe requires Ca²⁺, a specific activator of callose synthase which is known to lower the K_m of higher plant callose synthases for the substrate UDP-glucose. Using this probe and several other related ones, several other proteins which interact with UDP-glucose were also identified, but none satisfied all of the above criteria for being components of the callose synthase.     

Evidence for the involvement of a UDP-glucose-dependent group translocator in sucrose uptake into vacuoles of storage roots of red beet

Vacuoles isolated from the storage roots of red beet (Beta vulgaris L.) accumulate sucrose via two different mechanisms. One mechanism transports sucrose directly, and its rate is increased by the addition of MgATP. The other mechanism utilizes uridine diphosphate glucose (UDP-glucose) to synthesize and simultaneously transport sucrose phosphate and sucrose into the vacuole. This group translocation mechanism has also been found in sugarcane vacuoles. As in sugarcane, the beet group translocator does not require fructose 6-phosphate, nor is the latter substance transported into the vacuole. The uptake of UDP[¹⁴C]glucose in inhibited by high concentrations of osmoticum.Abbreviations EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - UDP uridine 5-diphosphate     

Uridine Diphosphate Glucose Metabolism and Callose Synthesis in Cultured Pollen Tubes of Nicotiana alata Link et Otto

Membrane preparations from cultured pollen tubes of Nicotiana alata Link et Otto contain a Ca2+ -independent (1-3)-[beta]-D-glucan (callose) synthase activity that has a low affinity for UDP-glucose, even when activated by treatment with trypsin (H. Schlupmann, A. Basic, S.M. Read [1993] Planta 191: 470-481). Therefore, we investigated whether UDP-glucose was a likely substrate for callose synthesis in actively growing pollen tubes. Deposition of (1-3)-[beta]-glucan occurred at a constant rate, 1.4 to 1.7 nmol glucose min-1, in tubes from 1 mg of pollen from 3 h after germination; however, the rate of incorporation of radioactivity from exogenous [14C]-sucrose into wall polymers was not constant, but increased until at least 8 h after germination, probably due to decreasing use of internal reserves. UDP-glucose was a prominent ultraviolet-absorbing metabolite in pollen-tube extracts, with 1.6 nmol present in tubes from 1 mg of pollen, giving a calculated cytoplasmic concentration of approximately 3.5 mM. Radioactivity from [14C]-sucrose was rapidly incorporated into sugar monophosphates and UDP-glucose by the growing tubes, consistent with a turnover time for UDP-glucose of less than 1 min; the specific radioactivity of extracted UDP-[14C]glucose was equal to that calculated from the rate of incorporation of [14C]sucrose into wall glucans. Large amounts of less metabolically active neutral sugars were also present. The rate of synthesis of (1-3)-[beta]-glucan by nontrypsin-treated pollen-tube membrane preparations incubated with 3.5 mM UDP-glucose and a [beta]-glucoside activator was slightly greater than the rate of deposition of (1-3)-[beta]-glucan by intact pollen tubes. These data are used to assess the physiological significance of proteolytic activation of pollen-tube callose synthase.     

Uridine 5′-diphospho-D-glucose-dependent vectorial sucrose synthesis in tonoplast vesicles of the storage hypocotyl of red beet (Beta vulgaris L. ssp. conditiva)

Tonoplast vesicles were prepared from red-beet (Beta vulgaris L. ssp. conditiva) hypocotyl tubers (beetroot) known to store sucrose. Uptake experiments, employing uridine 5-diphospho-[¹⁴C]glucose (UDP-[¹⁴C]glucose) showed the operation of an UDP-glucose-dependent group translocator for vectorial synthesis and accumulation of sucrose, recently described for sugarcane and red-beet vacuoles and for tonoplast vesicles prepared from sugarcane suspension cells. Characterization of the kinetic properties yielded the following results. Uptake of UDP-glucose was linear for 15 min. The apparent K _m was 0.75 mM for UDP-glucose (at pH 7.2, 1 mM Mg²⁺), V _max was 32 nmol·(mg protein)^-1·min^-1. The incorporation of UDP-glucose exhibited a sigmoidal substrate-saturation curve in the absence of Mg²⁺, the Hill coefficient (n _H) was 1.33; Michaelis-Menten kinetics were obtained, however, in the presence of 1 mM MgCl₂. For the reaction sequence under the control of the group translocator a dual pH optimum was found at pH 7.2 and 7.9, respectively. All reaction intermediates and the end product sucrose could be identified by two-dimensional high-performance thin-layer chromatography and autoradiography. The distribution pattern of radioactivity showed almost uniformly high labeling of all intermediates and sucrose. The physiological relevance of the results is discussed in the light of the fact that the tonoplast of red-beet storage cells accommodates two mechanisms of sucrose uptake (i) vectorial sucrose synthesis and (ii) direct ATP-dependent sucrose assimilation.Abbreviations HPTLC High-performance thin-layer chromatography - UDP uridine 5-diphosphate - SDS sodium dodecyl sulfate     

Vacuoles from Sugarcane Suspension Cultures : II. CHARACTERIZATION OF SUGAR UPTAKE

Vacuoles, isolated from sugarcane (Saccharum sp.) cells, took up 3-O methylglucose and sucrose and the evidence suggests specific transport systems for these sugars. There was no evidence of sugar efflux from preloaded vacuoles. Vacuoles in situ accumulated 3-O methylglucose, sucrose, glucose, and fructose, as shown by incubation of protoplasts with labeled sugar and subsequent analysis of vacuolar and cytoplasmic radio-activity. During the initial minutes of incubation, the amount and concentration of labeled sugar was higher in the cytoplasm than in the vacuole, but subsequently there was active uptake and accumulation into the vacuole. The rate of hexose transfer into the vacuole in situ approached that of hexose uptake by isolated vacuoles; however, the rate of sucrose uptake by isolated vacuoles was below the in situ rate. The site of sucrose synthesis was in the cytoplasm.     

The use of formaldehyde-treated 131-I-albumin in the study of digestive vacuoles and some properties of these particles from mouse liver

The trichloroacetic acid-soluble radioactivity released during incubation of mouse liver particles containing intravenously injected formaldehyde-treated ¹³¹I-albumin consisted almost entirely of ¹³¹I-iodotyrosine. The material was shown to be excreted into the medium and was not due to disruption of the particles by acid. Triton X-100 or the absence of sucrose in the medium inhibited hydrolysis of the particle-associated labeled protein. This inhibition was due to disruption of the digestive vacuoles and dilution of the protein and cathepsins in the suspending medium. These results and other experimental evidence strongly suggest that the ¹³¹I-albumin-containing liver particles are digestive vacuoles. The results also establish that ¹³¹I-albumin may be used to study these vacuoles. High concentrations of sucrose (1 M) inhibited degradation of intraparticulate protein. However, 1 M salts inhibited only the rate of the digestion. Sucrose had an inhibitory effect on a crude cathepsin preparation, and salts stimulated the activity when ¹³¹I-albumin was used as substrate. The effect of high sucrose concentrations as an inhibitor of protein hydrolysis within digestive vacuoles was, therefore, most likely due principally to an inhibition of cathepsin activity within the vacuoles. The effect of salt was probably caused by a stimulation of both intra- and extra-particulate cathepsin activities, although 0.5–1.0 M KCl appeared to protect the particles.     

Sugar Transport in Immature Internodal Tissue of Sugarcane: II. Mechanism of Sucrose Transport

The mechanism by which sucrose is transported into the inner spaces of immature internodal parenchyma tissue of sugarcane (Saccharum officinarum L. var. H 49-5) was studied in short term experiments (15 to 300 seconds). Transport of sucrose, glucose, and fructose was each characterized by a V_max of 1.3 μmoles/gram fresh weight·2 hours, and each of these three sugars mutually and competitively inhibited transport of the other two. When ¹⁴C-glucose was supplied exogenously, ¹⁴C-glucose 6-phosphate and ¹⁴C-glucose were the first labeled compounds to appear in the tissue; no ¹⁴C-sucrose was detected until after 60-second incubation. After 15-second incubation in ¹⁴C-sucrose, all intracellular radioactivity was in glucose, fructose, glucose 6-phosphate, and fructose 6-phosphate; trace amounts of ¹⁴C-sucrose were found after 30 seconds and after 5 minutes, 71% of the intracellular radioactivity was in sucrose. Although it was possible that sucrose was transported intact into the inner space and then immediately hydrolyzed, it was shown that the rate of hydrolysis under these conditions was too low to account for the rate of hexose accumulation. Pretreatment of the tissue with rabbit anti-invertase antiserum eliminated sucrose transport, but had no effect on glucose transport. Since the antibodies did not penetrate the plasmalemma, it was concluded that sucrose was hydrolyzed by an invertase in the free space prior to transport. The glucose and fructose moieties, or their phosphorylated derivatives, were then transported into the inner space and sucrose was resynthesized. No evidence for the involvement of sucrose phosphate in transport was found in these experiments.     

Labeling of the Plasma Membrane of Pea Cells by a Surface-localized Glucan Synthetase

When radioactive UDP-glucose is supplied to 1-millimeter-thick slices of pea (Pisum sativum) stem tissue, radioactive glucose becomes incorporated into membrane-bound polysaccharides. Evidence is given that this incorporation does not result from breakdown of UDP-glucose and utilization of the resultant free glucose, and that the incorporation most likely takes place at the cell surface, leading to a specific labeling of the plasma membrane. The properties of the plasma membrane that are indicated by this method of recognition, including the association of K⁺-stimulated ATPase activity with the plasma membrane, resemble properties inferred using other approaches. The membrane-associated polysaccharide product formed from UDP-glucose is largely 1,3-linked glucan, presumably callose, and does not behave as a precursor of cell wall polymers. No substantial amount of cellulose is formed from UDP-glucose in this procedure, even though these cells incorporate free glucose rapidly into cellulose. This synthetase system that uses external UDP-glucose may serve for formation of wound callose.     

Functional Specialization of Vacuoles in Sugarcane Leaf and Stem

Plant vacuoles are frequently targeted as a storage site for novel products. We have used environment-sensitive fluorescent dyes and the expression of vacuolar marker proteins to characterize the vacuoles in different organs and cell types of sugarcane. The results demonstrated that the lumen of the vacuole in the parenchyma cells of the stem is acidic (<pH 5) and contains active proteases, characteristic of lytic vacuoles. Western blots and tissue labelling with antibodies to vacuolar H⁺-ATPase suggest that this proton pump is involved in acidification of the vacuolar lumen. Quantitative real-time PCR was used to show that the expression of vacuolar proteases and a vacuolar sorting receptor is also coordinately regulated. In contrast to the stem parenchyma cells, the cells of sugarcane leaves contain diverse types of vacuoles. The pH of these vacuoles and their capacity to hydrolyze protease substrates varies according to cell type and developmental stage. Sugarcane suspension-cultures contain cells with vacuoles that resemble those of stem parenchyma cells and are thus a useful model system for investigating the properties of the vacuole. Understanding the growth and development of storage capacity will be useful in designing strategies to maximize the production of sucrose or alternative bioproducts.     

Biosynthesis of squalene and sterols by rat aorta

The synthesis of nonsaponifiable compounds from radioactive mevalonate by segments of adult rat aorta was studied in vitro. The labeled products consisted largely of substances with the chromatographic and chemical behavior of squalene, lanosterol, lathosterol, and cholesterol. Even after 3 or 4 hr of incubation, the incorporation of mevalonate into squalene was higher than its incorporation into C(27) sterols; cholesterol contained less than 20% of the radioactivity in the total sterols. Lanosterol was the most highly labeled sterol. The level of radioactivity in lathosterol was comparable to the level in cholesterol. Small amounts of radioactivity were found in other sterols. Material with the same mobility on TLC as 7-dehydrocholesterol had less radioactivity than cholesterol, but more than sterols with the mobility of desmosterol. The results of measurements made after short periods of incubation showed that squalene and lanosterol became labeled before the other nonsaponifiable compounds.     

A kinetic study of sugarcane sucrose synthase.

The kinetic data on sugarcane (Saccharum spp. hybrids) sucrose synthase (SuSy, UDP-glucose: D-fructose 2-alpha-D-glucosyltransferase, EC 2.4.1.13) are limited. We characterized kinetically a SuSy activity partially purified from sugarcane variety N19 leaf roll tissue. Primary plot analysis and product inhibition studies showed that a compulsory order ternary complex mechanism is followed, with UDP binding first and UDP-glucose dissociating last from the enzyme. Product inhibition studies showed that UDP-glucose is a competitive inhibitor with respect to UDP and a mixed inhibitor with respect to sucrose. Fructose is a mixed inhibitor with regard to both sucrose and UDP. Kinetic constants are as follows: Km values (mm, +/- SE) were, for sucrose, 35.9 +/- 2.3; for UDP, 0.00191 +/- 0.00019; for UDP-glucose, 0.234 +/- 0.025 and for fructose, 6.49 +/- 0.61. values were, for sucrose, 227 mm; for UDP, 0.086 mm; for UDP-glucose, 0.104; and for fructose, 2.23 mm. Replacing estimated kinetic parameters of SuSy in a kinetic model of sucrose accumulation with experimentally determined parameters of the partially purified isoform had significant effects on model outputs, with a 41% increase in sucrose concentration and 7.5-fold reduction in fructose the most notable. Of the metabolites included in the model, fructose concentration was most affected by changes in SuSy activity: doubling and halving of SuSy activity reduced and increased the steady-state fructose concentration by about 42 and 140%, respectively. It is concluded that different isoforms of SuSy could have significant differential effects on metabolite concentrations in vivo, therefore impacting on metabolic regulation.     

A novel UDP-glucose transferase is part of the callose synthase complex and interacts with phragmoplastin at the forming cell plate

Using phragmoplastin as a bait, we isolated an Arabidopsis cDNA encoding a novel UDP-glucose transferase (UGT1). This interaction was confirmed by an in vitro protein--protein interaction assay using purified UGT1 and radiolabeled phragmoplastin. Protein gel blot results revealed that UGT1 is associated with the membrane fraction and copurified with the product-entrapped callose synthase complex. These data suggest that UGT1 may act as a subunit of callose synthase that uses UDP-glucose to synthesize callose, a 1,3-beta-glucan. UGT1 also interacted with Rop1, a Rho-like protein, and this interaction occurred only in its GTP-bound configuration, suggesting that the plant callose synthase may be regulated by Rop1 through the interaction with UGT1. The green fluorescent protein--UGT1 fusion protein was located on the forming cell plate during cytokinesis. We propose that UGT1 may transfer UDP-glucose from sucrose synthase to the callose synthase and thus help form a substrate channel for the synthesis of callose at the forming cell plate.     

High Performance Liquid Chromatography-Based Reevaluation of Disaccharides Produced upon Incubation of Sugarcane Vacuoles with UDP-Glucose

A reanalysis of products formed after short-term incubation of sugarcane (Saccharum spp. hybrid cv H50-7209) vacuole preparations with uridine diphosphate [¹⁴C]glucose was performed. The results indicated that the ethanol-soluble substance previously identified as sucrose did not elute with sucrose when subjected to high performance liquid chromatography but had the same retention time as a disaccharide tentatively identified as laminaribiose.     

Enhancement of Phloem exudation from cut petioles by chelating agents

The photosynthetic assimilates in leaves of Perilla crispa attached to the plant were labeled by treating the leaves with (14)CO(2). When subsequently detached, these leaves exuded a negligible amount of radioactivity from the cut petiole into water. Ethylenediaminetetraacetate (EDTA), citric acid, and ethyleneglycol-bis (beta-aminoethyl ether) N,N'-tetraacetate greatly increased exudation of labeled assimilates into a solution bathing the petioles. The optimal concentration of EDTA was 20 mm, and maximal exudation took place between 2 and 4 hours after excision. Up to 22% of the radioactivity fixed in the leaf was exuded into an EDTA solution as compared to an export of 38% from attached leaves. The amount of radioactivity in the exudate was much reduced at low temperature. Presence of EDTA was required in the collecting solution for only 1 to 2 hours; upon transfer to water, exudation continued as in continuous presence of EDTA. Ca(2+) completely inhibited the effect of EDTA.Anatomical studies indicated that callose formation on the sieve plates near the cut surface of the petioles was less in leaves on EDTA than on water.More than 95% of the radioactivity exuded by detached leaves was present in the sugars verbascose, stachyose, raffinose, and sucrose, which are translocated in the phloem of Perilla. Labeled glucose, fructose, and galactinol were detected in the leaf blade and petiole, but not in exudates.The addition of EDTA to a solution bathing the petiole of detached leaves of Chenopodium rubrum and Pharbitis nil also increased the exudation of labeled assimilates. In these two species, label appeared only in a compound that cochromatographed with sucrose.It is concluded that the radioactive products in the solution are actually exuded by the phloem. Possibly EDTA chelates Ca(2+) that otherwise participates in the reactions that seal cut phloem.     

beta-Furfuryl-beta-Glucoside: An Endogenous Activator of Higher Plant UDP-Glucose:(1-3)-beta-Glucan Synthase : Biological Activity, Distribution, and in Vitro Synthesis

In a recent paper (P Ohana, DP Delmer, JC Steffens, DE Matthews, R Mayer, M Benziman [1991] J Biol Chem 266: 13472-13475), we described the purification and structural characterization of β-furfuryl-β-glucoside (FG), an endogenous activator of plant UDP-glucose:(1→3)-β-glucan (callose) synthase. In the present report, we provide evidence that FG specifically stimulates callose synthase. The effects of FG on the kinetic properties of callose synthase were studied, and we ascertained that FG, or at least a very similar compound, is present in other plant systems. Chemically synthesized α-furfuryl-β-glucoside also stimulates callose synthase, exhibiting a slightly higher K_a of 80 micromolar, compared with 50 micromolar for FG. In addition, we have identified and partially characterized an enzyme that catalyzes the synthesis of FG using β-furfuryl alcohol and UDP-glucose as substrates. A model for the regulation of callose synthesis in vivo, involving changes in intracellular compartmentation of FG and Ca²⁺, is proposed.     

Formation and function of N-acetyloglucosamine-linked phosphoryl- and pyrophosphorylundecaprenols in membranes from Bacillus cereus.

Membranes from Bacillus cereus AHU 1356 incorporated radioactivity from UDP-N-acetyl[14C]glucosamine into three alkaline-stable and acid-labile lipids which were extracted into chloroform:methanol (2:1) and separated from each other by thin layer chromatography on silica gel plates. The major labeled lipid (Lipid 1) and a minor one (Lipid 2) were identified as N-actetylglucosaminyl phosphorylundecaprenol from several analytical criteria involving mass spectral data and from reversal of their formation by UDP. These two lipids appear to differ in geometry of their polyprenol moieties. The third labeled lipid (Lipid 3) was identified as N-acetylglucosaminyl pyrophosphorylundecaprenol. Antibiotic 24010, a tunicamycin-like antibiotic, at 1 microgram/ml was found to inhibit almost completely the formation of Lipid 3, whereas it inhibited the formation of Lipid 1 much more weakly and rather enhanced the formation of Lipid 2. Radioactivity was also incorporated into a polymer from UDP-GlcNAc and from Lipid 3. UDP-N-acetylmannosamine, UDP-N-acetylgalactosamine, and UDP-glucose supported the incorporation. Antibiotic 24010 strongly inhibited the incorporation of radioactivity from UDP-GlcNAc into polymer, whereas it did not affect the incorporation from Lipid 3. Thus, it is concluded that N-acetylglucosaminyl pyrophosphorylundecaprenol serves as a precursor in the synthesis of a polymer presumed as the cell wall polysaccharide of this bacterial strain.     

Oligomerization,Membrane Association,and in Vivo Phosphorylation of Sugarcane UDP-glucose Pyrophosphorylase

Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H₂O₂ strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.     

Hydrolases in vacuoles from castor bean endosperm

Vacuoles were prepared from endosperm tissue of 4-day-old castor bean seedlings (Ricinus communis var. Hale) and purified on a stepped sucrose gradient. It was shown by assays of marker enzymes that there was only trace contamination of the final preparation by other organelles (mitochondria, glyoxysomes, nuclei, spherosomes, and plastids) and by cytoplasmic components. Hydrolytic enzymes (acid protease, carboxypeptidase, phosphodiesterase, RNAase, phytase and β-glucosidase) were present in the isolated vacuoles in amounts indicating a primarily vacuolar localization in vivo. The vacuoles also contained storage protein and high concentrations of sucrose. The over-all results indicate that the vacuoles from castor bean endosperm are the site of hydrolysis of the constituents of the protein bodies and are a temporary storage compartment for the sucrose produced from fat and protein reserves.     

Vacuolar targeting of r‐proteins in sugarcane leads to higher levels of purifiable commercially equivalent recombinant proteins in cane juice

Sugarcane is an ideal candidate for biofarming applications because of its large biomass, rapid growth rate, efficient carbon fixation pathway and a well‐developed storage tissue system. Vacuoles occupy a large proportion of the storage parenchyma cells in the sugarcane stem, and the stored products can be harvested as juice by crushing the cane. Hence, for the production of any high‐value protein, it could be targeted to the lytic vacuoles so as to extract and purify the protein of interest from the juice. There is no consensus vacuolar‐targeting sequence so far to target any heterologous proteins to sugarcane vacuole. Hence, in this study, we identified an N‐terminal 78‐bp‐long putative vacuolar‐targeting sequence from the N‐terminal domain of unknown function (DUF) in Triticum aestivum 6‐SFT (sucrose: fructan 6‐fructosyl transferase). In this study, we have generated sugarcane transgenics with gene coding for the green fluorescent protein (GFP) fused with the vacuolar‐targeting determinants at the N‐terminal driven by a strong constitutive promoter (Port ubi882) and demonstrated the targeting of GFP to the vacuoles. In addition, we have also generated transgenics with His‐tagged β‐glucuronidase (GUS) and aprotinin targeted to the lytic vacuole, and these two proteins were isolated and purified from the transgenic sugarcane and compared with commercially available protein samples. Our studies have demonstrated that the novel vacuolar‐targeting determinant could localize recombinant proteins (r‐proteins) to the vacuole in high concentrations and such targeted r‐proteins can be purified from the juice with a few simple steps.     

Membrane transport of sugars in cell suspensions of sugarcane: I. Evidence for sites and specificity

Sugar uptake by sugarcane cells in suspension culture was measured over short incubation time spans (5 seconds to 4 minutes), and membrane transport rates were calculated. A relatively high proportion of labeled products in cell extracts after incubation of cells with ¹⁴C-glucose for 5 seconds was sugar phosphates (56%); fructose and sucrose began to appear after 15 and 30 seconds, respectively. Galactose and 3-O-methylglucose competed appreciably with glucose uptake, but ketohexoses and pentoses did not; there was no detectable uptake of sucrose. It is postulated that besides endogenous phosphorylation and further metabolism of glucose the configuration of the hydroxyl on the carbon-2 may be important for efficient membrane transport. The cells had a particularly high affinity for glucose and 3-O-methylglucose (Km = 15 and 16 μm, respectively).