Biosynthesis of myo-inositol and its role as a precursor of cell-wall polysaccharides in suspension cultures of wild-carrot cells

The biosynthesis of myo-inositol (MI) and its role as a precursor of cell-wall polysaccharides was studied in supension cultures of wild carrot (Daucus carota L.) cells. Suspension cultures, grown in the presence or absence of 2,4-dichlorophenoxyacetic acid for 7 and 14d were incubated with [U-¹⁴C]glucose and [2-³H]MI in the presence of different concentrations of unlabeled MI. Synthesis of [¹⁴C]MI from [U-¹⁴C]glucose occurred under all conditions. The amount of MI synthesized from glucose was sharply reduced when 10 mM MI was provided in the medium. Substantial quantities of ³H were incorporated in arabinose, xylose and galacturonic acid isolated and purified from the cell-wall polysaccharides of the cell cultures in various stages of growth or embryogenesis. No ³H was present in the glucose or galactose units of cell-wall polysaccharides. At the four stages of growth and states of development of the carrot cultures used, the MI oxidation pathway contributed to the synthesis of pentosyl and galacturonosyl units of the cell wall. However, the data indicate that the contribution of the MI oxidation pathway to pentosyl and galacturonosyl units is small.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MI myo-inositol     

Metabolism of myo-Inositol and growth in various sugars of suspension-cultured tobacco cells

Tobacco (Nicotiana tabacum L.) cells were cultured in a liquid medium which contained sucrose as a source of carbon and energy. Various cell-wall constituents and wall precursors (L-arabinose, D-xylose, D-galactose, D-mannose, D-glucuronate, myo-inositol) were added to cells growing in this medium to by-pass possible rate-limiting steps in the relevant metabolic pathways. None of these compounds stimulated growth as measured by increase in fresh weight; myo-inositol did cause a slight increase and L-arabinose a decrease in dry weight accumulation compared to controls grown on sucrose only. Although myo-inositol was not needed for rapid growth, tracer level amounts of [2-³H]myo-inositol were rapidly absorbed and metabolized. Label was incorporated into the uronide and pentose residues of cell walls and exocellular polysaccharide.     

Uptake of auxotrophic cells of a heterocyst-forming cyanobacterium by tobacco protoplasts,and the fate of their associations

During imbibition, exogenous myo-inositol (MI) was readily introduced into the free MI pool of germinating wheat (Triticum aestivum L.). Maximum uptake, 70 g per caryopsis or 1.5 mg g^–1 of caryopsis, was reached at 0.05 M MI. Movement of free MI within the germinating caryopsis was traced with [2-³H]MI by two procedures, uptake by imbibition and injection into softened endosperm. The former procedure was useful during initial stages of germination; the latter provided a means of tracing the metabolic fate of MI generated by hydrolysis of phytate during mobilization of reserves within the caryopsis. In both procedures, the bulk of the added label was transferred to the seedling where it appeared in uronosyl and pentosyl units of 80% ethanol-insoluble polysaccharides, 2-O, C-Methylene-MI, an inhibitor of the MI oxidation pathway, blocked the utilization of [2-³H]MI as well as d-[1¹⁴C]glucose for biogenesis of pentose-and uronic-acid-containing polysaccharides.Abbreviations MI myo-inositol - OCM-MI 2-O, C-methylene-myo-inositol     

Reduced Na+/K+ ATPase transport activity,resting membrane potential,and bradykinin-stimulated phosphatidylinositol synthesis by polyol accumulation in cultured neuroblastoma cells

In these studies we examined the effect of polyol accumulation on neural cellmyo-inositol metabolism and properties. Neuroblastoma cells were cultured for two weeks in media containing 30 mM glucose, fructose, galactose or mannose with or without 0.4 mM sorbinil or 250 Mmyo-inositol. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a decrease inmyo- inositol content and myo-[2-³H]inositol accumulation and incorporation into phosphoinositides compared to cells cultured in unsupplemented medium or medium containing 30 mM fructose as an osmotic control. These monosaccharides each caused an increase in intracellular polyol levels with galactitol > sorbitol = mannitol accumulation. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a significant decrease in Na⁺/K⁺ ATPase transport activity, resting membrane potential, and bradykinin-stimulated³²P incorporation into phosphatidylinositol compared to cells cultured in medium containing 30 mM fructose. In contrast, basal incorporation of³²P into phosphatidylinositol or basal and bradykinin-stimulated³²P incorporation into phosphatidylinositol 4,5-bisphosphate were not effected. Each of these cellular functions as well asmyo-inositol metabolism and content and polyol levels remained near control values when 0.4 mM sorbinil, an aldose reductase inhibitor, was added to the glucose, galactose, or mannose supplemented media.myo-Inositol metabolism and content and bradykinin-stimulated phosphatidylinositol synthesis were also maintained when media containing 30 mM glucose, galactose, or mannose was supplemented with 250 Mmyo-inositol. The results suggest that polyol accumulation induces defects in neural cellmyo-inositol metabolism and certain cell functions which could, if they occurred in vivo, contribute to the pathological defects observed in diabetic neuropathy.     

myo-Inositol Synthesis from [1-H]Glucose in Phaseolus vulgaris L. during Early Stages of Germination

Radiolabeled d-[1-³H]glucose was fed by imbibition under sterile conditions to bean (Phaseolus vulgaris L.) seeds. After 72 and 96 hours of feeding, the ³H was located in uronic acid and pentose residues as well as hexose residues of cell wall polysaccharides in growing hypocotyl and root. Free myo-inositol present in cotyledons, hypocotyl, and root also contained ³H, showing that de novo synthesis of myo-inositol from [1-³H]glucose did occur during the first 72 hours of germination. More than 90% of the labeled, free myo-inositol was present in the cotyledons. The ³H percentage in trifluoroacetic acid-soluble arabinose residues of cell wall polysaccharides from 72-hour-old bean hypocotyls was only half of their mole percentage. On the other hand, ³H percentages in hexose residues were higher than their mole percentages. The results suggest that myo-inositol is synthesized from reserve sugars during the very early stages of germination, and that the newly synthesized myo-inositol, as well as that stored in cotyledons, can be used for the construction of new hypocotyl and root cell wall polysaccharides after conversion into uronic acids and pentoses via the myo-inositol oxidation pathway.     

Cellulose and 1,3-glucan synthesis during the early stages of wall regeneration in soybean protoplasts

Protoplasts isolated from cultured soybean cells (Glycine max (L.) Merr., cv. Mandarin) were used to study polysaccharide biosynthesis during the initial stages of cell wall-regeneration. Within minutes after the protoplasts were transferred to a wall-regeneration medium containing [¹⁴C]glucose, radioactivity was detected in a product which was chemically characterized as cellulose. The onset and accumulation of radioactivity into cellulose coincided with the appearance fibrils on the surface of protoplasts, as seen under the electron microscope. At these early stages, a variety of polysaccharide-containing polymers other than cellulose were also synthesized. Under conditions where the protoplasts were competent to synthesize cellulose from glucose, uridine diphosphate-[¹⁴C]glucose and guanosine diphosphate-[¹⁴C]glucose did not serve as effective substrates for cellulose synthesis. However, substantial amounts of label from uridine diphosphate glucose were incorporated into 1,3-glucan.Abbreviations ECM extracellular material - GLC gas liquid chromatography - GDP-glucose guanosine diphosphate glucose - UDP-glucose uridine diphosphate glucose - U enzyme units as defined by Sigma Chemical Corp., St. Louis, Mo., USA     

Effect of Osmolality and myo-Inositol Deprivation on the Transport Properties of myo-Inositol in Primary Astrocyte Cultures

myo-Inositol uptake measured in primary astrocyte cultures was saturable in the presence of Na⁺ with a Km of 13–18 M and a Vmax of 9.4 nmoles/mg protein/hour in myo-inositol-fed cells, indicating a high affinity transport system. In myo-inositol-deprived cells, Km was about 53 M with a Vmax of 13.2 nmoles/mg protein/hour. Decreasing osmolality decreased the Vmax to about 1.9 nmoles/mg protein/hour whereas increasing osmolality increased Vmax about 5-fold, while Kms were essentially unchanged in myo-inositol fed cells. In cells deprived of myo-inositol, Vmax decreased in hypotonic medium and increased in hypertonic medium almost 10-fold, but with more than a doubling of the Km regardless of the osmolality. Glucose (25 mM) inhibited myo-inositol uptake 51% whereas the other hexoses used inhibited uptake much less. Our findings indicate that myo-inositol uptake in astrocytes occurs through an efficient carrier-mediated Na⁺-dependent co-transport system that is different from that of glucose and its kinetic properties are affected by myo-inositol availability and osmotic stress.     

Effects of ABA on Synthesis of Cell-Wall Polysaccharides in Segments of Etiolated Squash Hypocotyl I. Changes in Incorporation of Glucose and myo-Inositol into Cell-Wall Components

Externally supplied [³H]myo-inositol and [¹⁴C]glucose were incorporatedin cell-wall fractions of segments of etiolated squash hypocotyl.The extent of incorporation of [¹⁴C]glucose into cell-wall fractionswas very much greater than that of [³H]myo-inositol. Radioactivityfrom [¹⁴C]-glucose was effectively incorporated into hemicelluloseB and cellulose fractions and was incorporated uniformly intohexose, pentose and uronic acid residues, but radioactivityfrom [³H]myo-inositol was incorporated predominantly into uronicacid and pentose residues in the pectin and hemicellulose Bfractions. Exogenously applied ABA significantly suppressed the elongationof segments of squash hypocotyl and the incorporation of radioactivityfrom [^l4C]glucose and [³H]myo-inositol into the segments. Furthermore,ABA significantly inhibited the distribution of incorporatedradioactivity from [¹⁴C]glucose into the cellulose fraction,but did not affect distribution into the pectic fraction. Bycontrast, ABA only slightly inhibited the distribution of theincorporated radioactivity from [³H]myo-inositol into the pecticfraction. These results suggest that most of the cell-wall polysaccharidesin segments of squash hypocotyl are synthesized via the UDP-sugarpathway, and that ABA significantly inhibits the synthesis ofcellulose but not the synthesis of pectic polysaccharides whenABA suppresses the elongation of the segments. (Received March 25, 1988; Accepted November 15, 1988)     

Changes in concentrations of soluble carbohydrates during germination of <Emphasis Type="Italic">Amaranthus caudatus</Emphasis> L. seeds in relation to ethylene,gibberellin A<Subscript>3</Subscript> and methyl jasmonate

Unimbibed Amaranthus caudatus seeds were found to contain stachyose, raffinose, verbascose, sucrose, galactinol, myo-inositol, glucose and fructose, while no galactose, maltose and maltotriose was detected. During imbibition, seed concentrations of verbascose, stachyose, raffinose, galactinol, myo-inositol (temporary) and fructose (transient) were observed to decrease; concentrations of galactose and maltose remained fairly constant, while those of sucrose, glucose and maltotriose increased, the increase in sucrose concentration was only temporary. Effects of gibberellin A₃ (GA₃) at 3 × 10⁻⁴ M and ethephon at 3 × 10⁻⁴ M alone or in the presence of methyl jasmonate (Me-JA) at 10⁻³ M on concentrations of soluble sugars during germination of A. caudatus seeds were examined. Me-JA was found to inhibit seed germination and fresh weight of the seeds, but did not affect sucrose, myo-inositol, galactose and maltose concentrations during imbibition for up to 20 h. The exogenously applied GA₃ was observed to enhance germination, stachyose breakdown and glucose concentration after 20 h of incubation. Ethephon stimulated seed germination as well as utilisation of stachyose, galactinol (both after 14 and 20 h) and raffinose (after 14 h of incubation). Although the stimulatory effect of either GA₃ or ethephon on seed germination was blocked by Me-JA; these stimulators increased mobilisation of raffinose and stachyose, but only ethephon enhanced both glucose and fructose after 14 and/or 20 h of incubation in the presence of Me-JA. The maltose concentration was increased by both GA₃ and ethephon alone and in the presence of Me-JA. Of the growth regulators studied, ethephon alone and/or in combination with Me-JA significantly increased the concentrations of glucose, fructose, galactose, maltose and maltotriose. The differences in sugar metabolism appear to be linked to ethylene or GA₃ applied simultaneously with Me-JA.     

Myo-inositol Biosynthesis and Galactose Utilization by Wild Carrot (Daucus carota L. var. carota) Suspension Cultures

Wild carrot (Daucus carota var. carota) cell suspensions (63–120µm in diameter) were grown on a mineral salt medium containingdifferent carbon sources in the presence (10 mM) and absenceof myo-inositol. The data obtained after 14 and 21 days of growthshow that an external supply of myo-inositol is not essentialfor growth and development of wild carrot embryos. A linearrelationship was found between growth (d. wt) and embryo numberin the presence and absence of myo-inositol. Standard stock cell suspensions never exposed to exogenous myo-inositoland grown in the absence of 2, 4-D with glucose or galactoseas the carbon source synthesized radioactive myo-inositol whenexposed to D-[1–¹⁴C]glucose or D-[1–¹⁴C]galactose.Gas chromatographic analyses revealed the presence of myo-inositolin the bulk tissue grown in the presence of 2.25 µM 2,4-D with glucose, galactose, fructose or mannose as the solecarbohydrate. We could not detect any component indicating anisomer or a methylated derivative of an inositol in the tissueextracts. Stock cultures were maintained (with 2, 4-D) successfully forat least three successive sub-cultures on D-galactose as thesole carbohydrate. The growth achieved over this culture periodshowed that wild carrot cells used by us could quickly adaptto grow on D-galactose as rapidly as they grow on sucrose. Daucus carota L., wild carrot, suspension cultures, myo-inositol, galactose     

Osmotic regulation ofmyo-inositol uptake in primary astrocyte cultures

Uptake ofmyo-inositol by astrocytes in hypertonic medium (440 mosm/kg H₂O) was increased near 3-fold after incubation for 24 hours, which continued for 72 hours, as compared with the uptake by cells cultured in isotonic medium (38 nmoles/mg protein).myo-Inositol uptake by astrocytes cultured in hypotonic medium (180 mosm/kg H₂O) for periods up to 72 hours was reduced by 74% to 8 to 10 nmoles/mg protein. Astrocytes incubated in either hypotonic or hypertonic medium for 24 hours and then placed in isotonic medium reversed the initial down- or up-regulation of uptake. Activation of chronic RVD and RVI correlates with regulation ofmyo-inositol uptake. A 30 to 40 mosm/kg H₂O deviation from physiological osmolality can influencemyo-inositol homeostasis. The intracellular content ofmyo-inositol in astrocytes in isotonic medium was 25.6 ± 1.3 g/mg protein (28 mM). This level ofmyo-inositol is sufficient for this compound to function as an osmoregulator in primary astrocytes and it is likely to contribute to the maintenance of brain volume.     

Asymmetric Distribution of Glucose and Indole-3-Acetyl-myo-Inositol in Geostimulated Zea mays Seedlings

Indole-3-acetyl-myo-inositol occurs in both the kernel and vegetative shoot of germinating Zea mays seedlings. The effect of a gravitational stimulus on the transport of [³H]-5-indole-3-acetyl-myo-inositol and [U-¹⁴C]-d-glucose from the kernel to the seedling shoot was studied. Both labeled glucose and labeled indole-3-acetyl-myo-inositol become asymmetrically distributed in the mesocotyl cortex of the shoot with more radioactivity occurring in the bottom half of a horizontally placed seedling. Asymmetric distribution of [³H]indole-3-acetic acid, derived from the applied [³H]indole-3-acetyl-myo-inositol, occurred more rapidly than distribution of total ³H-radioactivity. These findings demonstrate that the gravitational stimulus can induce an asymmetric distribution of substances being transported from kernel to shoot. They also indicate that, in addition to the transport asymmetry, gravity affects the steady state amount of indole-3-acetic acid derived from indole-3-acetyl-myo-inositol.     

Characterisation of the FAM69 family of cysteine-rich endoplasmic reticulum proteins

Myo-inositol is one of the major organic osmolytes in the brain and the kidney. The accumulation of intracellular organic osmolytes allows cells to regulate intracellular osmolality without altering cytoplasmic ionic strength and to adapt to hyperosmotic conditions. Two types of myo-inositol transporters, sodium/myo-inositol transporter and H⁺/myo-inositol transporter (HMIT), have been identified. Sodium/myo-inositol transporters are induced by osmotic stress and might be involved in the intracellular accumulation of myo-inositol in mammals. The role of HMIT, however, remains unknown. In the present study, we characterized three Caenorhabditis elegansHMIT genes, hmit-1.1, hmit-1.2, and hmit-1.3. hmit-1.1 was expressed in the intestine, and hmit-1.2 was expressed in the glia and the excretory canal, which is an osmotic regulatory organ that is functionally analogous to the kidney. hmit-1.3 was expressed in the intestine and the glia. The expression of hmit-1.1 and hmit-1.2 but not hmit-1.3, was markedly induced under hyperosmotic conditions. Animals with mutant hmit-1.1 and hmit-1.2 were hypersensitive to osmotic stress. The defects of hmit-1.1 and hmit-1.2 mutants were rescued by hmit-1.1 and hmit-1.2 transgenes, respectively, and by modified human HMIT. In human cell lines, HMIT expression was induced in hyperosmotic conditions. These findings indicate that the C. elegans HMIT family has a crucial role in the osmoprotective response.     

Purification and characterization of myo-inositol 6-O-methyltransferase from Vigna umbellata Ohwi et Ohashi

The cyclitol 1d-4-O-methyl-myo-inositol (d-ononitol) is accumulated in certain legumes in response to abiotic stresses. S-Adenosyl-l-methionine:myo-inositol 6-O-methyltransferase (m6OMT), the enzyme which catalyses the synthesis of d-ononitol, was extracted from stems of Vigna umbellata Ohwi et Ohashi and purified to apparent homogeneity by a combination of conventional chromatographic techniques and by affinity chromatography on immobilized S-adenosyl-l-homocysteine (SAH). The purified m6OMT was photoaffinity labelled with S-adenosyl-l-[¹⁴C-methyl]methionine. The native molecular weight was determined to be 106 kDa, with a subunit molecular weight of 40 kDa. Substrate-saturation kinetics of m6OMT for myo-inositol and S-adenosyl-l-methionine (SAM) were Michaelis-Menten type with K _m values of 2.92 mM and 63 M, respectively. The SAH competitively inhibited the enzyme with respect to SAM (K _i of 1.63 M). The enzyme did not require divalent cations for activity, but was strongly inhibited by Mn²⁺, Zn²⁺ and Cu²⁺ and sulfhydryl group inhibitors. The purified m6OMT was found to be highly specific for the 6-hydroxyl group of myo-inositol and showed no activity on other naturally occurring isomeric inositols and inositol O-methyl-ethers. Neither d-ononitol, nor d-3-O-methyl-chiro-inositol, d-1-O-methyl-muco-inositol or d-chiro-inositol (end products of the biosynthetic pathway in which m6OMT catalyses the first step), inhibited the activity of the enzyme.Abbreviations DTT dithiothreitol - m6OMT myo-inositol 6-O-methyltransferase - SAH S-adenosyl-l-homocysteine - SAM S-adenosyl-l-methionine We are greatful to Professor M. Popp (University of Vienna) for helpful discussion and comment. This work was supported by Grant P09595-BIO from the Austrian Science Foundation (FWF).     

Identification of 3-hydroxy-6-methyl-2H-pyran-2-one from pyrolysis of phosphoric acid-treated cellulosic materials

The hydrogen isotope-effect that occurs in vitro during myo-inositol 1-phosphate synthase-catalyzed conversion of d-[5-³H]glucose 6-phosphate into myo-[2-³H]inositol 1-phosphate has been used to compare the functional role of the nucleotide sugar oxidation-pathway with that of the myo-inositol oxidation-pathway in germinating lily pollen. Results reveal a significant difference between the ³H/¹⁴C ratios of glucosyl and galactosyluronic residues from pectinase-amyloglucosidase hydrolyzates of the 70 % ethanol-insoluble fraction of d-[5-³H, 1-¹⁴-C]glucose-labeled, germinating lily pollen. This isotope effect at C-5 of d-glucose that occurred during its conversion into d-galactosyluronic residues of pectic substance is not explained by loss of ³H when UDP-d-[5-³H, 1-¹⁴C]glucose is oxidized by UDP-d-glucose dehydrogenase from germinating lily pollen. The evidence obtained from this study favors a functional role for the myo-inositol oxidation pathway during in vivo conversion of glucose into galactosyluronic residues of pectin in germinating lily pollen.     

Control of diferulate formation in dicotyledonous and gramineous cell-suspension cultures

Primary cell wall polysaccharides of some plants carry ester-linked feruloyl groups that can be oxidatively dimerised both within the protoplast and after secretion into the apoplast. Apoplastic dimerisation has been postulated to form inter-polysaccharide cross-links, contributing to wall assembly, but this role remains conjectural. By feeding cultured cells with [¹⁴C]cinnamate, we monitored the kinetics of polysaccharide-binding and subsequent dimerisation of ¹⁴C-labelled feruloyl groups. Cultured maize and spinach cells took up [¹⁴C]cinnamate more rapidly than barley, Arabidopsis, Acer, tomato and rose cultures. Maize and spinach cells rapidly formed [¹⁴C]feruloyl-polysaccharides and, simultaneously, low-M_r [¹⁴C]feruloyl esters. When all free [¹⁴C]cinnamate had been consumed, there followed a gradual recruitment of radiolabel from the low-M_r pool into the polysaccharide fraction. A proportion of the [¹⁴C]feruloyl-polysaccharides was sloughed into the culture medium, the rest remaining wall-bound. Some of the polysaccharide-bound [¹⁴C]feruloyl groups were coupled to form dehydrodiferulates. At least six putative isomers of [¹⁴C]dehydrodiferulate were formed both rapidly (thus intra-protoplasmically) and gradually (thus mainly apoplastically). These data do not support the hypothesis that intra-protoplasmic dimerisation yields predominantly one isomer (8–5′-dehydrodiferulate). In maize, apoplastic coupling was much more extensive in 7-day old than in 2-day-old cultures; indeed, in 2-day-old cultures apoplastic coupling could not be evoked even by exogenous H₂O₂, suggesting strong control of peroxidase action by apoplastic factors. When apoplastic coupling was minimised by exogenous application of peroxidase-blockers (iodide, dithiothreitol and cysteine), a higher proportion of the secreted [¹⁴C]feruloyl-polysaccharides was sloughed into the medium. This observation lends support to the hypothesis that feruloyl coupling contributes to wall assembly.     

Changes in cell-wall polysaccharide composition of developingNitella internodes

Changes in the uronide, neutral-polysacharide, and cellulose composition of the cell wall ofNitella axillaris Braun were followed throughout development of the internodes and correlated with changes in growth rate. As the cells increased in length from 4 to 80 mm during development, the relative growth rate decreased. Cell wall thickness, as measured by wall density, increased in direct proportion to diameter, indicating that cell-wall stress did not change during elogation. Cell-wall analyses were adapted to allow determination of the composition of the wall of single cells. The total amounts of uronides, neutral sugars and cellulose all increased during development. However, as the growth rate decreased, the relative proportions of uronides and neutral sugars, expressed as percent of the dry weight of the wall, decreased, while the proportion of cellulose increased. The neutral sugars liberated upon hydrolysis ofNitella walls are qualitatively similar to those found in hydrolysates of higher plant cell walls: glucose, xylose, mannose, galactose, arabinose fucose and rhamnose. Only the percentage of galactose was found to increase in walls of mature cells, while the percentage of all other sugars decreased. The rate of apposition (g of wall material deposited per unit wall surface area per hour) of neutral polysaccharides decreased rapidly with decreasing growth rate during the early stages of development. The rate of apposition of uronides decreased more steadily throughout development, while that of cellulose, after an early decline, remained constant until dropping off at the end of the elongation period. These correlations between decreasing growth rate and decreasing rate of apposition of neutral sugars and uronides indicate that synthesis of these cell-wall components could be involved in the regulation of the rate of cell elongation inNitella.     

Regenerated cell wall of carrot protoplasts isolated from suspension-cultured cells

Protoplasts of Daucus carota L. cultured in a synthetic liquid medium resumed cell division after about 4 days of cultivation. During this lag period, nucleic acid and protein showed only slight increases but the protoplasts commenced cell-wall regeneration soon after the removal of lytic enzymes. The originally spherical protoplasts became ellipsoidal before they underwent division. Radioactive glucose and myo-inositol were readily utilized by the protoplasts. Most of the radioactivity, however, appeared in extracellular polysaccharides and only a small portion was deposited in the regenerated wall. The sugar composition of new cell wall, as studies by chemical analysis and incorporation of labelled precursors, was shown to be considerably different from that of normal cell wall.     

Transport and Metabolism of Indole-3-Acetyl-myo-Inositol-Galactoside in Seedlings of Zea mays

Indole-3-acetyl-myo-inositol galactoside labeled with ³H in the indole and ¹⁴C in the galactose moieties was applied to kernels of 5 day old germinating seedlings of Zea mays. Indole-3-acetyl-myo-inositol galactoside was not transported into either the shoot or root tissue as the intact molecule but was instead hydrolyzed to yield [³H]indole-3-acetyl-myo-inositol and [³H]indole-3-acetic acid which were then transported to the shoot with little radioactivity going to the root. With certain assumptions concerning the equilibration of applied [³H]indole-3-acetyl-myo-inositol-[U-¹⁴C]galactose with the endogenous pool, it may be concluded that indole-3-acetyl-myo-inositol galactoside in the endosperm supplies about 2 picomoles per plant per hour of indole-3-acetyl-myo-inositol and 1 picomole per plant per hour of indole-3-acetic acid to the shoot and thus is comparable to indole-3-acetyl-myo-inositol as a source of indole-acetic acid for the shoot. Quantitative estimates of the amount of galactose in the kernels suggest that [³H]indole-3-acetyl-myo-inositol-[¹⁴C] galactose is hydrolyzed after the compound leaves the endosperm but before it reaches the shoot. In addition, [³H]indole-3-acetyl-myo-inositol-[¹⁴C]galactose supplies appreciable amounts of ¹⁴C to the shoot and both ¹⁴C and ³H to an uncharacterized insoluble fraction of the endosperm.     

Reactive oxygen species and nitric oxide affect cell wall metabolism in tobacco BY-2 cells

The effects of hydrogen peroxide (H2O2), nitric oxide (NO), and a combination of both on the metabolism of cell wall polysaccharides were studied in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY-2) suspension cultured cells in the presence of D-[U-14C]glucose or D-[U-14C]galactose as radioactive tracers. We found that the radiolabelling of newly synthesised total cell wall polysaccharides (pectins, hemicelluloses and alpha-cellulose), buffer-soluble polysaccharides, and membrane-associated polysaccharides decreased under the influence of exogenous systems generating H2O2 and NO. However, when the total amount of newly synthesised cell wall polysaccharides was calculated as a percentage of the total cellular radioactivity (ethanol-soluble pool plus the homogenate of ethanol-insoluble material), all treatments showed negligible effects in the presence of D-[U-14C]glucose or D-[U-14C]galactose as tracers. This occurred because the treatments generating H2O2, NO and H2O2 plus NO caused a marked decrease in the concentration of the ethanol-soluble pool as well as in the total radioactivity found in the homogenate of the ethanol-insoluble material. Most of the radioactivity taken up by the cells was evolved as 14CO2 during the respiratory processes. A qualitative and quantitative characterisation of the ethanol-soluble pool showed that radioactive UDP-sugars in BY-2 suspension cultured cells were differentially reduced by all treatments. Therefore, the decrease of the newly synthesised cell wall polysaccharides seems to be strictly dependent on the reduction of the UDP-sugars pool.