Effect of Auxins (2,4-Dichlorophenoxyacetic acid, Indole-3-Acetic Acid and Naphthaleneacetic Acid) on the Accumulation of {gamma}-Aminobutyric Acid in Excised Rice Root Tips

-Aminobutyric acid (GABA) accumulation occurs in cultured ricecells when ammonium is added to the medium [Kishinami and Ojima(1980) Plant Cell Physiol. 21: 581–589]. Whether thisphenomenon occurs in rice plant tissues was examined with respectto exogenously supplied auxins: 2,4-dichlorophenoxyacetic acid(2,4-D), indole-3-acetic acid (IAA) and naphthalene-acetic acid(NAA). In intact rice plants grown in medium containing ammonium withoutauxin, glutamine first increased, then asparagine graduallyincreased. In both shoots and roots, the asparagine contentbecame the highest among four amino acids after 4 days of cultureperiod. GABA did not increase at all, its level remaining lowin both shoots and roots throughout the culture period. GABA accumulation was observed in excised rice root tips whenthey were incubated in the medium containing ammonium in thepresence of 2,4-D, IAA or NAA. In the absence of auxin, however,excised rice root tips accumulated asparagine and glutamine,but not GABA. Rice root segments obtained from a region in whichroot cells had already developed to maturity did not accumulateGABA but asparagine and glutamine in the presence of both ammoniumand 2,4-D. These results suggest that GABA accumulation occurs in rapidlygrowing and dividing tissue, such as the apical meristem ofrice root in the presence of auxin during ammonium assimilation. (Received June 15, 1987; Accepted March 14, 1988)     

Accumulation of {gamma}-aminobutyric acid due to adding ammonium or glutamine to cultured rice cells

-Aminobutyric acid (GABA) was accumulated in rice cell cultureswhen ammonium was added to the medium. After the addition ofammonium, a temporary increase in the glutamine (Gln) pool wasobserved before the accumulation of GABA. GABA also was markedlyaccumulated when Gln was added to the medium in place of ammonium.When glutamic acid (Glu) was added without ammonium, no accumulationof GABA occurred. When L-methionine-DL-sulfoximine (MSO), an inhibitor of glutaminesynthetase, was added to the nitrate medium, the ammonium poolincreased with no accumulation of Gln and GABA. Even when ammoniumwas supplied to the medium, no GABA accumulated in the presenceof MSO. When azaserine (AZ), which inhibits the transamidationof Gln, was added with Gln, no GABA was accumulated, althoughthe Gln pool in the cell cultures increased significantly. The accumulation of GABA in cultured rice cells produced byammonium as the nitrogen source probably is related directlyto the Gln pool size, which is increased when ammonium is suppliedto the medium. (Received August 6, 1979; )     

Role of Chitinase and Chitin Oligosaccharides in Lignification Response of Cultured Carrot Cells Treated with Mycelial Walls

Chitinase activity was induced in cultured carrot cells by incubationwith mycelial walls of a fungus, Chaetomium globosum. Both intra-and extracellular chitinases were resolved into four componentsby gel filtration chromatography. The extracellular enzymesliberated soluble oligosaccharides of different sizes from insolublechitin, suggesting that these carrot chitinases are endo-hydrolases.The solubilized chitinase digests obtained from insoluble mycelialwalls of C. globosum and chitin were fractionated by gel filtrationchromatography, and the elicitor activity of each fraction forthe accumulation of phenolic acids in cultured carrot cellswas determined. In both solubilized fragments of fungal wallsand of chitin, elicitor-active oligosaccharides were distributedin many fractions, however, potent activity for inducing phenolicacid synthesis was observed in the high molecular weight fractions. (Received October 5, 1987; Accepted February 12, 1988)     

Metabolism of glutamate and {gamma}-aminobutyrate in cultured tobacco cells

In cultured tobacco cells glutamate-U-¹⁴C administrated wasreadily converted to -aminobutyrate (GABA) by decarboxylation,however, GABA-1-¹⁴C remained unchanged. Glutamate decarboxylasewas found in tobacco cells and reached its maximum activityin the rapidly growing stage during culture. Enzyme activityparalleled formation of GABA from glutamate-U-¹⁴C. A high contentof GABA in tobacco cells seems to be due to the rapid decarboxylationof glutamate by glutamate decarboxylase and a slow turn overof GABA. ¹ Present address: The Okayama Tobacco Experiment Station, JapanMonopoly Corp., Tamashima, Kurashiki, Japan. (Received November 20, 1971; )     

Phytohormone-induced GABA production in transformed root cultures of Datura stramonium: an in vivo 15N NMR study

Primary nitrogen metabolism in transformed root cultures ofDatura stramonium was observed by in vivo ¹⁵N NMR. Treatmentof the root cultures with the plant growth regulators -naphthaleneaceticacid (NAA) and kinetin caused a de-differentiation of the roottissue, together with perturbation of primary and secondarynitrogen metabolism. The levels of newly-synthesized glutamineand glutamate during ammonium assimilation were depleted relativeto control cultures, whereas GABA biosynthesis was enhanced.Although GABA production could be stimulated by a decrease incytoplasmic pH (whether imposed artificially or induced by hypoxia),observation of the roots during phytohormone treatment by ³¹PNMR showed that the cytoplasmic pH remained stable, indicatingthat the perturbation of nitrogen metabolism in the de-differentiatedroots must be due to other causes. Key words: Datura, -aminobutyric acid, nitrogen metabolism, NMR, root cultures     

The effects of amino acids and ammonium on the growth of plant cells in suspension culture

A suspension culture of soybean (Glycine max L.) was grown on a defined medium in which the nitrogen sources were nitrate (25 mM) and ammonium (2 mM). The cells did not grow on nitrate unless the medium was supplemented with ammonium or glutamine. The l- and d-isomers of 12 amino acids tested singly could not replace ammonium. Most amino acids (4 mM) inhibited growth when the cells were cultured on nitrate and ammonium. Cells from five other plants (Reseda luteoli L.; Triticum monococcum L.; flax, Linum usitatissimum L.; horseradish, Amoracia lapathifolia Gilib; Haplopappus gracilis L.) grew on the defined medium with nitrate (25 mM) as the sole nitrogen source. Higher cell yields were obtained when ammonium (2 mM) or glutamine also was present. Supplementing the defined medium with high concentrations of ammonium (20 mM) inhibited growth of soybean, Haplopappus, and wheat cells. Addition of citrate (5 mM) relieved the inhibitory effects of ammonium in soybean and wheat cells but not in the Haplopappus cells.     

Inhibition of Tonoplast and Plasma Membrane H$-ATPase Activity in Rice (Oryza sativa L.) Culture Cells by Local Anesthetics

Tonoplast and plasma membrane vesicles were prepared from rice(Oryza sativa L. var. Yuukara) culture cells with step sucrosegradient (30% and 42.9%, w/v) and/or step dextran T-70 gradient(1% and 8%, w/w) to determine the inhibition of tonoplast andplasma membrane AT-Pases by local anesthetics. The degree towhich the anesthetics inhibited these ATPases was of the followingorder: dibucaine>lidocainetetracaine>procaineGABA. Dibucaineranging in concentration from 0.2 nui to 2 mM inhibited tonoplastATPase activity more than plasma membrane ATPase, the half inhibitionsbeing 0.8 and 1.1 mM, respectively. The K_m values of tonoplastand plasma membrane ATPases were not affected by dibucaine,but various values were noted for V_max. Dibucaine inhibitedtonoplast and plasma membrane ATPases solubilized from 0.1%DOC pellet by n-octylglucoside and zwittergent 3–14, respectively.The addition of a phospholipid mixture (asolectin) to solubilizedboth ATPases had no effect on the inhibition by dibucaine. Thus,local anesthetics may act directly on the ATPase moiety withoutlipid mediation. (Received June 15, 1987; Accepted November 13, 1987)     

Control by Ammonium Ion of the Change from Step-Up to Step-Down Photophobically Responding Cells in the Flagellate Alga Euglena gracilis

The regulation between step-down and step-up photophobic responses,resulting in photoaccumulation of the cells in an actinic lighttrap or cells' avoidance from an excessive illumination, iscrucially important for the survival of phototrophic organismssuch as Euglena gracilis. As for the factors involved in thisregulation in Euglena gracilis, we for the first time reporthere that ammonium ion specifically enhances step-down photophobicresponse, together with the effects of L-methionine-DL-sulfoximine(L-MSO), an inhibitor of ammonium assimilation, to specificallyenhance step-up photophobic response. The apparent positivecorrelation between the degree of greening and the step-downphotophobic response did not seem to reflect real causal relationshipin view of the results with effects of gabaculine, an inhibitorof -aminolaevulinic acid (-ALA) formation. The transmissionof stigma and step-down appearance did not show any correlationeither, in contrast to a previous assumption by other authors.Cycloheximide (CHX), an inhibitor of eukaryotic protein synthesis,suppressed step-down appearance and enhanced step-up appearance,probably suggesting an involvement of some (newly synthesized)protein(s) specifically in the step-down photosignal detectionand/or signal transduction process(es). (Received August 18, 1998; Accepted December 3, 1998)     

One step purification of peanut phospholipase D by precipitation with alginate

A simple titrimetric assay with soybean lecithin has been used for screening phospholipase D activity from some plant sources, viz. peanut, wheat germ, cabbage and carrot. The enzyme from peanut has been purified by binding to alginate which is a water soluble polymer. The purification consisted of co-precipitation of enzyme with alginate upon addition of 0.06 M Ca⁺⁺. The enzyme was eluted from the polymer using 0.2 M sodium chloride. The activity recovery was 61% with 34 fold purification.     

Vegetable Plant Part Relationships. II. A Quantitative Hypothesis for Shoot/Storage Root Development

A simple quantitative formulation of the concept of the controlof partitioning of assimilated carbon by the behaviour of plantcomponents as competing sinks is developed. An equation, In s = + In r—t, relating shoot (s) and storage root (r) d. wts, and the lengthof growth period (t), is constructed by considering possiblefates of imported assimilates into different plant parts. Thevalues of the equations' parameters depend on the relative sinkactivities of the plant parts, tissue respiration rates andinitial weights of plant components. The equation closely fitteddata collected from a number of carrot and beet experimentsin which planting density had been varied. Estimates of shootand storage root maintenance respiration rates, derived fromthe parameter , were of the correct order of magnitude. Othersets of experimental data are also discussed in the light ofpredictions of the theory and possible uses and extensions ofthis approach to assimilate partitioning are briefly discussed. Daucus carota L., Beta vulgaris, carrot, red beet, partition of assimilated carbon, maintenance respiration, storage root     

Thiamine requirements of various plant cells in suspension culture

The thiamine requirements of cells of several plant speciesin suspension culture were investigated. Omission of thiaminefrom the medium leads to a rapid and complete cessation of growthin subcultures of soybean, tobacco and rice cells. The criticallevels of thiamine content in these cells are considered tobe in the vicinity of 0.6, 0.5 and 0.2 µg per g dry weight,respectively. Soybean cells can grow satisfactorily when suppliedwith an equimolar mixture of two thiamine precursors, pyrimidineand thiazole moieties. Neither moiety alone can substitute forthiamine. On the other hand, Rula and peanut cells can be successfullysubcultured for ten passages in the absence of externally suppliedthiamine. When grown without thiamine, Rula cells had an averagethiamine content of 0.5-0.6 µg in darkness and 3.5 µgper g dry weight in the light. The relationship between thethiamine requirement and the degree of dedifferentiation incultured cells is discussed. ¹ Present address: Section of Phytochemical Research, Researchand Development Division, Eisai Co., Ltd., Kawashima, Gifu 112,Japan. (Received December 24, 1975; )     

Properties of Two N-Linked Glycoproteins Associated with the Nuclear Envelope in Mung Bean Hypocotyls

Nuclei from mung bean (Vigna radiata) hypocotyls contained twoglycoproteins of 50 and 49 kDa, respectively, that reacted withconcanavalin A. The glycoproteins were released from the nuclearenvelope by treatment with 2 M KCl but not with nucleases. Theglycoproteins, tentatively named gp50 and gp49, were isolatedand characterized. Gel-permeation chromatography suggested thatgp50 and gp49 seem to exist as a complex with other components.The glycoproteins could be detected only in the nuclear fractionby immunoblot analysis with specific antibodies, and they werenot detected in endoplasmic reticulum, plasma membrane, vacuolarmembrane or mitochondria. Agglutinin I from Ulex europeaus,peanut agglutinin, soybean agglutinin and wheat germ agglutininall failed to bind to the glycoproteins. Treatment with glycopeptidaseF removed all oligosaccharides from the glycoproteins and decreasedtheir molecular masses by about one thousand daltons each. Theseresults suggest that the glycoproteins contained N-linked, high-mannose-typeoligosaccharides with six or seven hexose residues. gp50 andgp49 seemed to be isoforms of a single glycoprotein becausethe two proteins had some common properties. Nuclear fractionsfrom azuki bean (Phaseolus angularis) and soybean (Glycine max)contained proteins that were immunologically similar to gp50and gp49. (Received March 18, 1995; Accepted May 24, 1995)     

Involvement of Cell Wall-Bound Diferulic Acid in Light-Induced Decrease in Growth Rate and Cell Wall Extensibility of Oryza Coleoptiles

Irradiation of white fluorescent light (5 W m^–2) inhibitedthe growth of Oryza coleoptiles. Light irradiation increasedstress-relaxation parameters of coleoptile cell walls, minimumstressrelaxationtime and relaxation rate, and decreased cellwall extensibility (strain/load). Under light conditions, thecontents of ferulic and diferulic acids ester-linked to thehemicellulosic arabinose residue in cell walls increased andcorrelated with the modification of the cell wall mechanicalproperties. These results suggest that light irradiation enhancesthe formation of diferulic acid bridges in hemicelluloses, makingcell walls mechanically rigid and thus inhibits cell elongationin rice coleoptiles. Also, irrespective of coleoptile age orthe presence of light, the ratio of diferulic acid to ferulicacid was almost constant, suggesting that the rate limitingstep in the formation of diferulic acid bridges in Oryza cellwalls is in the step of feruloylation. (Received September 24, 1991; Accepted December 3, 1991)     

γ‐Aminobutyric acid addition alleviates ammonium toxicity by limiting ammonium accumulation in rice (Oryza sativa) seedlings

Excessive use of nitrogen (N) fertilizer has increased ammonium (NH₄⁺) accumulation in many paddy soils to levels that reduce rice vegetative biomass and yield. Based on studies of NH₄⁺ toxicity in rice (Oryza sativa, Nanjing 44) seedlings cultured in agar medium, we found that NH₄⁺ concentrations above 0.75 mM inhibited the growth of rice and caused NH₄⁺ accumulation in both shoots and roots. Use of excessive NH₄⁺ also induced rhizosphere acidification and inhibited the absorption of K, Ca, Mg, Fe and Zn in rice seedlings. Under excessive NH₄⁺ conditions, exogenous γ‐aminobutyric acid (GABA) treatment limited NH₄⁺ accumulation in rice seedlings, reduced NH₄⁺ toxicity symptoms and promoted plant growth. GABA addition also reduced rhizosphere acidification and alleviated the inhibition of Ca, Mg, Fe and Zn absorption caused by excessive NH₄⁺. Furthermore, we found that the activity of glutamine synthetase/NADH‐glutamate synthase (GS; EC 6.3.1.2/NADH‐GOGAT; EC1.4.1.14) in root increased gradually as the NH₄⁺ concentration increased. However, when the concentration of NH₄⁺ is more than 3 mM, GABA treatment inhibited NH₄⁺‐induced increases in GS/NADH‐GOGAT activity. The inhibition of ammonium assimilation may restore the elongation of seminal rice roots repressed by high NH₄⁺. These results suggest that mitigation of ammonium accumulation and assimilation is essential for GABA‐dependent alleviation of ammonium toxicity in rice seedlings.     

Induction of Chitinase and Phenylalanine Ammonia-Lyase in Cultured Carrot Cells Treated with Fungal Mycelial Walls

Addition of insoluble mycelial walls of a fungus, Chaetomiumglobosum, stimulated the induction of chitinase and phenylalanineammonia-lyase (PAL), as well as the accumulation of phenolicacids in cultured carrot cells. Mycelial wall fragments solubilizedby chitinase treatment also elicited accumulation of phenolicacids. The induction of chitinase and PAL were highly dependenton the age of the carrot cell cultures, as are other defenseresponses, including phytoalexin production. (Received April 2, 1986; Accepted August 22, 1986)     

Comparative species responses to boron on a Typic Tropaqualf in Northern Thailand

The effect of boron (B) on peanut and soybean was examined in two omission and one B fertilizer rate trial on a Typic Tropaqualf in Northern Thailand. The B rate trial was combined with a comparison of the response of sunflower, green gram, black gram, wheat, and rice in addition to peanut and soybean grown in irrigated rice-based cropping sequences over two years. Omitting B induced the hollow heart symptom in 10% of peanut kernels with the incidence of hollow hearts closely related to B concentration in the kernels. Omission of B had no effect on the appearance of soybean seed or on the grain yield of either soybean or peanut. In the B rate experiment, omitting B depressed grain yield by 50% in sunflower and by 40% to 80% in black gram, induced B deficiency symptoms in green gram and the hollow heart symptom in peanut kernels, but had not significant effect on the grain yield of soybean, peanuts, rice, or wheat. B deficiency apparently depressed grain yield in black and green gram by delaying or inhibiting reproductive development thus reducing pod set.     

Cellular Levels of Ribulose 1,5 Bisphosphate Carboxylase and Chloroplast Compartment Size in Wheat Mesophyll Cells

Pyke, K. A. and Leech, R. M. 1987. Cellular levels of ribulose1,5 bisphosphate carboxylase and chloroplast compartment sizein wheat mesophyll cells.—J. exp. Bot. 38: 1949–1956. The amount of the photosynthetic enzyme ribulose 1,5 bisphosphatecarboxylase (RUBISCO),as determined in mesophyll cells in primarywheat leaves was related to the size of the chloroplast compartmentwithin the cell for wheat species of three ploidy levels. Asimilar comparison was made for several genotypes of the hexaploidbreadwheat Triticum aestivum. Estimation of total chloroplastvolume per mesophyll cell was made assuming chloroplasts tobe oblate spheroid in shape. A significant correlation was found between the amount of RUBISCOper cell and the total chloroplast volume per cell for diploid,tetraploid and hexaploid wheat species. A significant correlationbetween cellular RUBISCO level and total chloroplast volumeper cell was also observed for a range of genotypes of the hexaploidT. aestivum but these genotypes of T. aestivutn accumulate agreater amount of RUBISCO per unit chloroplast volume than doany other wheat species. For these genotypes of T. aestivumthe stromal concentration of RUBISCO was estimated at 0·5mol m^–3 with a ribulose Msphosphate binding site concentrationof 4·0 mol m^–3. These results are discussed with respect to a gene dosage hypothesisto explain the accumulation of RUBISCO in leaf mesophyll cells. Key words: Ribulose, bisphosphate carboxylase, wheat chloroplasts, mesophyll cells     

Increased Production of IAA by Rhizoctonia solani is Induced by Culture Filtrate from Rice Suspension Cultures

To investigate the role of the plant hormones produced by fungi,we tried to construct a system to examine the interaction betweenRhizoctonia solani Kühn MAFF305219 and rice cells in suspensionculture (Oc). R. solani was previously found to produce IAA,with the main biosynthetic pathway via the indole-3-pyruvatepathway. The amount of IAA in the medium produced by R. solaniwas increased by cocultivation with rice cells (Oc) and by culturefiltrate (CF) of Oc. Further analysis revealed that the factor(s)that induced the enhanced accumulation of IAA was sensitiveto heat, to freezing and thawing and lyophilization, and themolecular weight was estimated to more than 10,000. These resultssuggest that the active agent(s) in the medium was (a) proteinor a proteinous substance. Among suspension cultures of variousplants, Oc and another line of rice cells (Ok) had the abilityto induce the accumulation of IAA in the fungal medium 4 h afterinoculation but other cultures of plant cells were ineffective.The promotive effect of rice CF on the accumulation of IAA wasalso observed with some strains of R. solani that belong toa different anastmosis group from MAFF305219. Thus, the accumulationof IAA was not related to the host specificity. (Received July 28, 1997; Accepted October 27, 1997)