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.     

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)     

Use of Per-C-Deuterated myo-Inositol for Study of Cell Wall Synthesis in Germinating Beans

Cell wall polysaccharides of the hypocotyl and roots in germinating beans (Phaseolus vulgaris L.) were selectively labeled in arabinosyl, xylosyl, and galacturonosyl residues by per-C-deuterated myo-inositol, which was introduced through 72 hours of imbibition. Glucuronate residues remained unlabeled. Selected ion gas chromatography-mass spectrometry analysis revealed that deuterium was not redistributed in these three sugar residues or into other carbohydrate residues during this conversion, suggesting that the labeled residues are formed exclusively via the myo-inositol oxidation pathway and that no glucogenesis from myo-inositol takes place during this conversion. The presence of a significant level of deuterated arabinose, xylose, and galacturonate after just 72 hours of imbibitional uptake of per-C-deuterated myo-inositol indicated that the myo-inositol oxidation pathway has a predominant role in the biosynthesis of new cell walls.     

Formation of a series of myo-inositol phosphates during growth of rice plant cells in suspension culture

A series of myo-inositol phosphates including myo-inositol mono-to hexa-phosphates was observed during growth of cultured riceplant cells. We also found that ³²Pi and myo-[2-³H] inositolwere incorporated into all these myo-inositol phosphates. myo-Inositolphosphorylating activity, which depended on ATP and Mg²⁺, wasdetected in the soluble fraction from the cells, and the reactionproduct was identified as myo-inositol-2-phosphate. (Received January 21, 1980; )     

Phosphorylation of myo-inositol in ripening grains of rice and wheat. Incorporationof phosphate-32P and myo-inositol-3H into myo-inositol phosphates

To elucidate the mechanism of the phosphorylation of myo-inositolin the process of phytate formation, feeding experiments oforthophosphate-³²P and myo-inositol-³H in the ripening grainsof rice and wheat were performed. It was found that ³²P and³H were incorporated into myo-inositol mono- and hexa-phosphates.The same results were obtained when a mixture of "cold" myo-inositolpolyphosphates was administered to the grains before feedingphosphate-³²P. Based on these results it is concluded that phosphorylationof free myo-myo-inositol in the formation of phytate does nottake place in a stepwise fashion but may proceed through anunknown myo-inositol derivative. (Received August 2, 1967; )     

Effects of Abscisic Acid on the Synthesis of Cell-Wall Polysaccharides in Segments of Etiolated Squash Hypocotyl II. Levels of UDP-Neutral Sugars

The effects of abscisic acid (ABA) on the formation of UDP-sugarsin segments of squash (Cucurbita maxima Duch.) hypocotyls wereexamined with [³H]glucose. After a 3-h incubation, 74% of totalradioactivity incorporated into neutral sugar residues of UDP-sugarswas found in glucose residues, 19% in galactose residues andless than 7% in other sugar residues. Exogenously applied ABAincreased the extent of incorporation of [³H]glucose into UDP-glucose,UDP-galactose and UDP-xylose by 30%, 30%, and 20%, respectively.However, ABA neither affected the total incorporation of radioactivityinto segments nor the incorporation into free glucose and sucrosein the segments. Since ABA inhibits the synthesis of celluloseand hemicellulosic polysaccharides in cell walls of segmentsof squash hypocotyls prior to the suppression of elongationof segments (Wakabayashi et al. 1989a, b), the present resultsindicate that ABA inhibits cell-wall synthesis by affectingsome step(s) beyond the formation of UDP-sugars and not by decreasingthe synthesis of UDP-sugars in the hypocotyl segments. ¹Present address: Biological Laboratory, Faculty of Education,Kagawa University, Saiwai-cho 1-1, Takamatsu, 760 Japan (Received May 30, 1990; Accepted February 5, 1991)     

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.     

FORMATION OF MYO-INOSITOL AND PHYTIN IN RIPENING RICE GRAINS

With the view to elucidate the role of myo-inositol in the ripeningprocess of rice grains, its distribution, formation and conversionwere studied.

1. myo-Inositol in the ripening rice grains was fractionated intofree-, phosphate ester- and phosphoinositide-forms. At the earlystage of ripening, a considerable part of myo-inositol was foundin free state, and at the end of ripening stage the most partwas found in phosphate ester-state, phytic acid. The contentof phosphoinositide in the grains was low during the ripeningperiod.
2. The occurrence of biosynthesis of myo-inositol inthe ripeningrice grains was confirmed by the observation ofincorporationof ¹⁴C into myo-inositol from ¹⁴C-sugars and itwas found, fromthe feeding experiment of myo-inositol- thatmyo-inositol doesnot undergo reactions further than phosphorylation.
3. The feeding experiment of glucose-l-³²P showed that the distributionpattern of ³²P in different fractions of grain material wasthe same as that of ³²P-phosphate, indicating that phytic acidis one of the final products of phosphorus metabolism in theripening rice grains.
4. These results led to the assumptionthat myo-inositol mightact as an acceptor of phosphorus toremove inorganic phosphorusin favor of starch synthesis byphosphorylase.

(Received September 12, 1962; )     

Radioautographic demonstration of the accumulation site of phytic acid in rice and wheat grains

The distribution of ³H-myo-inositol administered to ripeninggrains of rice and wheat was followed by microradioautography.The ³H was exclusively found in aleurone particles, indicatingthat the accumulation site of phytic acid in monocotyledonousseeds is the aleurone particle. (Received July 25, 1973; )     

Auxin and Ethylene-stimulated Adventitious Rooting in Relation to Tissue 9

We have previously shown that both endogenous auxin and ethylenepromote adventitious root formation in the hypocotyls of derootedsunflower (Helianthus annuus) seedlings. Experiments here showedthat promotive effects on rooting of the ethylene precursor,1-aminocyclopropane-l-carboxylic acid (ACC) and the ethylene-releasingcompound, ethephon (2-chloro-ethylphosphonic acid), dependedon the existence of cotyledons and apical bud (major sourcesof auxin) or the presence of exogenously applied indole-3-aceticacid (IAA). Ethephon, ACC, aminoethoxyvinylglycine (an inhibitorof ethylene biosynthesis), and silver thiosulphate (STS, aninhibitor of ethylene action), applied for a length of timethat significantly influenced adventitious rooting, showed noinhibitory effect on the basipetal transport of [³H]IAA. Theseregulators also had no effect on the metabolism of [³H]IAA andendogenous IAA levels measured by gas chromatography-mass spectrometry.ACC enhanced the rooting response of hypocotyls to exogenousIAA and decreased the inhibition of rooting by IAA transportinhibitor, N-1-naphthylphthalamic acid (NPA). STS reduced therooting response of hypocotyls to exogenous IAA and increasedthe inhibition of rooting by NPA. Exogenous auxins promotedethylene production in the rooting zone of the hypocotyls. Decapitationof the cuttings or application of NPA to the hypocotyl belowthe cotyledons did not alter ethylene production in the rootingzone, but greatly reduced the number of root primordia. We concludethat auxin is a primary controller of adventitious root formationin sunflower hypocotyls, while the effect of ethylene is mediatedby auxin. Key words: Auxin, ethylene, adventitious rooting, sunflower     

The growth promoting effect of phytic acid on callus tissues of rice seed

The effects of phytic acid, myo-inositol hexaphosphoric acid,in the induction and growth of rice seed callus were studied.Phytic acid was substituted for myo-inositol in the inductionof the callus and preferable effects were shown on the growthof the callus. ¹Present address: Hyogo Agricultural Experiment Station, Akashi,Hyogo, Japan (Received June 30, 1970; )     

Transport and Metabolism of Dihydrozeatin Riboside in Germinating Lupin Seeds

[³H]-dihydrozeatin riboside was applied selectively to the embryonicaxes or to the cotyledons of germinating lupin (Lupinus luteusL. cv. Weiko III) seeds 6 h following the start of imbibition.There was little transport of dihydrozeatin riboside from embryoto cotyledons up to 6 h after the application, but a substantialamount of radioactivity had moved into the cotyledons at theend of the 10 h incubation period. However, there was no detectablemovement of [³H]-dihydrozeatin riboside from the cotyledonsto the embryonic axis. This indicated a highly polarized movementof cytokinins during the early stages of seed germination. Exogenouslyapplied [³H]-dihydrozeatin riboside was found to be very stable,both when applied to the embryonic axes and cotyledons of intactseed, or following excision, and there was little metabolismwith only small amounts of radioactivity found associated withdegradative metabolites. The embryonic axis of this specieshas recently been found to synthesize cytokinins within 12 hfrom the start of imbibition, and the results of this studyindicate that the embryo-derived cytokinin is probably transportedto the cotyledons where it accumulates and subsequently participatesin the control of cotyledon function. Key words: Lupinus luteus, cytokinin transport and metabolism, dihydrozeatin riboside, seed germination     

Cell Cycle Activation During Imbibition and Visible Germination in Embryos and Megagametophytes of Jack Pine (Pinus banksiana Lamb.)

Flow cytometric determination of cell cycle activation duringimbibition and visible germination in five families of jackpine (Pinus banksiana Lamb.) embryos and megagametophytes revealedthat in seeds that had undergone no imbibition the majorityof cells were in the 2C state. As the imbibition period increased,less of the nuclei were blocked in the G0/G1 state and morebecome active in the cell cycle. The augmentation in the nucleiactive in the 2C–4C cycle as well as those with DNA levelshigher than the 4C state occured gradually and preceeded radicleemergence. In megagametophyte tissue examined at various stagesof imbibition, cell cycle activity became apparent rapidly followingimbibition. In nuclei of green and white embryos examined separatelythe ²frequency distributions were significantly different forall three families after 144h. As imbibition period increased,fewer nuclei from the green embryos were blocked in the 2C state,and more became active in the 2C–4C cell cycle. This wasnot the case for white embryos where no significant linear relationwas noted. Cell cycle activity in the hypocotyl+cotyledons regionand the emerging radicle were examined separately. Functionalrelations found in the hypocotyl+cotyledons region were notevident in the radicle. As visible germination proceeded, cellcycle activity in the hypocotyl + cotyledons region for thisperiod of germination showed a reversal of the activity notedduring imbibition: fewer nuclei were active and once again ahigher proportion were found in the 2C state. cell cycle; C levels; DNA content; flow cytometry; germination; imbibition; jack pine; megagametophyte; Pinus banksiana Lamb     

Biosynthesis of cell-wall polysaccharides in cultured carrot cells

Biosynthesis of the cell wall in carrot cells (Daucus carota L.) cultured in a synthetic liquid medium was studied by measuring the incorporation of radioactive glucose and myo-inositol (MI). When the cells were fed with [¹⁴C]glucose in the presence of 0.01% MI, the label soon appeared in the neutral sugars in the cell wall but little radioactivity was found in the uronic-acid residues even after a prolonged incubation. On the other hand, radioactivity derived from [³H]MI was found to be distributed among uronic acids and pentoses but not in the hexose residues in the wall. The data indicate that MI is an important intermediate for the synthesis of acidic sugars in the wall of cultured carrot cells.Abbreviation MI myo-inositol     

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     

Carbohydrate Metabolism in Drought-Stressed Leaves of Pigeonpea (Cajanus cajan)

Pigeonpea is a tropical grain-legume, which is highly dehydrationtolerant. The effect of drought stress on the carbohydrate metabolismin mature pigeonpea leaves was investigated by withholding waterfrom plants grown in very large pots (50 kg of soil). The moststriking feature of drought-stressed plants was the pronouncedaccumulation of D-pinitol (1D-3-methyl-chiro-inositol), whichincreased from 14 to 85 mg g^–1 dry weight during a 27d stress period. Concomitantly, the levels of starch, sucroseand the pinitol precursors myo-inositol and ononitol all decreasedrapidly to zero or near-zero in response to drought. The levelsof glucose and fructose increased moderately. Drought stressinduced a pronounced increase of the activities of enzymes hydrolysingsoluble starch (amylases) and sucrose (invertase and sucrosesynthase). The two anabolic enzymes sucrose phosphate synthase(sucrose synthetic pathway) and myo-inositol methyl transferase(pinitol synthetic pathway) also showed an increase of activityduring stress. These results indicate that pinitol accumulatedin pigeonpea leaves, because the carbon flux was diverted fromstarch and sucrose into polyols. Key words: Drought, polyols, pinitol, sucrose, starch, pigeonpea     

Pectin Depolymerase Activities Associated with Cell Walls from Cicer arietinum L. Epicotyl

The hydrolytic activity of the proteins extracted with 3 M LiClfrom chick-pea (Cicer arietinum) cell walls to pec-tic fractionsextracted with 50 mM trans-l,2-diaminocy-clohexane-N,N,N,N,-tetraaceticacid (CDTA) and 50 mM sodium carbonate was studied. The pecticfractions contained acidic polysaccharides with high molecularmass (higher than 5 x 10³ kDa), mainly composed of uronic acids,galac-tose, arabinose and rhamnose. The extracted proteins depo-lymerizedthe pectic polysaccharides and also a commercial preparationof polygalacturonic acid from citrus, detected by a decreasein their viscosity and a shift of their molecular mass distribution.The extract was able to depolymerize a uronic acid-rich componentin all the cases, although in different extent. Also, with regardto the CDTA-soluble pectins, a degradation of polyuronide anda shift of the molecular mass distribution of arabinogalactanwas observed. (Received March 11, 1997; Accepted September 10, 1997)     

Changes in cell-wall polysaccharides in relation to seedling development and the mobilisation of reserves in the cotyledons of Lupinus angustifolius cv. Unicrop

Some 22% of the dry weight of the cotyledons of resting seeds of Lupinus angustifolius cv. Unicrop has been shown to be non-starch polysaccharide material comprising the massively thickened walls of the storage mesophyll cells. On hydrolysis this material released galactose (76%), arabinose (13%), xylose (4%), uronic acid (7%): only traces of glucose were detected indicating the virtual absence of cellulose from the walls. Changes in the amount and composition of this material following germination have been studied in relation to parameters of seedling development and the mobilisation of protein, lipid and oligosaccharide reserves. Starch, which was not present in the resting seed, appeared transitorily following germination: under conditions of continuous darkness starch levels were reduced. During the period of bulk-reserve mobilisation, 92% of the non-starch polysaccharide material disappeared from the cotyledons. The residual cell-wall material released galactose (14%), arabinose (19%), xylose (24%) and uronic acid (43%). The galactose and arabinose residues of the cotyledonary cell walls clearly constitute a major storage material, quantitatively as important as protein. The overall role of the wall polysaccharides in seedling development is discussed.     

Inositol Metabolism in Plants. III. Conversion of Myo-inositol-2-3H to Cell Wall Polysaccharides in Sycamore (Acer pseudoplatanus L.) Cell Culture

Prolonged growth of cell cultures of sycamore (Acer pseudoplatanus L.) on agar medium containing myo-inositol-2-³H resulted in incorporation of label predominately into uronosyl and pentosyl units of cell wall polysaccharides. Procedures normally used to distinguish between pectic substance and hemicellulose yielded carbohydrate-rich fractions with solubility characteristics ranging from pectic substance to hemicellulose yet the uronic acid and pentose composition of these fractions was decidedly pectic. Galacturonic acid was the only uronic acid present in each fraction. Subfractionation of alkali-soluble (hemicellulosic) polysaccharide by neutralization followed by ethanol precipitation gave 3 fractions, a water-insoluble, an ethanol-insoluble, and an ethanol-soluble fraction, each progressively poorer in galacturonic acid units and progressively richer in arabinose units; all relatively poor in xylose units.     

Possible Roles of Methyl Glucoside andMyo-inositol in the Opening of Cut Rose Flowers

Methyl glucoside andmyo-inositol are present in all organs ofrose (Rosa hybridaL.). To investigate the possible role of thesecarbohydrates in the opening of cut roses, flowers with a 10,20 or 40-cm-long stem and a single flower bud (about 1.5 cmin diameter) were placed in water and flower opening and changesin sugar content in flowers and stems examined for 7 d. Thelonger the stem of the cut flower, the larger was the flowerdiameter. In stems, the concentration of carbohydrates, includingmethyl glucoside andmyo-inositol markedly decreased before floweropening. In petals, contents of glucose, methyl glucoside andmyo-inositolalso decreased before flower opening, but those of fructose,sucrose and xylose did not. When glucose and methyl glucosidewere added to the vase water (4%) flower opening was clearlypromoted; this was accompanied by an increase in methyl glucosideand fructose concentrations in petals. On the contrary,myo-inositolinhibited flower opening, and this was accompanied by an increaseinmyo-inositol and xylose concentrations in petals. These resultssuggest that methyl glucoside and/or its metabolites are transportedinto the petal cells, thereby lowering the osmotic water potentialand promoting flower opening.Myo-inositol is not readily metabolized,and exogenousmyo-inositol given at a high concentration mayact as an extracellular osmolyte, which inhibits water uptakeand flower opening.Copyright 1999 Annals of Botany Company Cut flowers, methyl glucoside,myo-inositol,Rosa hybrida,soluble carbohydrate.