Galactose inhibition of auxin-induced cell elongation in oat coleoptile segments

Auxin-induced cell elongation in oat coleoptile segments was inhibited by galactose; removal of galactose restored growth. Galactose did not appear to affect the following factors which modify cell elongation: auxin uptake, auxin metabolism, osmotic concentration of cell sap, uptake of tritium-labeled water, auxin-induced wall loosening as measured by a decrease in the minimum stress-relaxation time and auxininduced glucan degradation. Galactose markedly prevented incorporation of [¹⁴C]-glucose into cellulosic and non-cellulosic fractions of the cell wall. It was concluded that galactose inhibited auxin-induced long-term elongation of oat coleoptile segments by interfering with cell wall synthesis.     

Galactose inhibition of auxin-induced growth of mono- and dicotyledonous plants

Galactose inhibited auxin-induced cell elongation of oat coleoptiles but not that of azuki bean stems. Galactose decreased the level of UDP-glucose in oat coleoptiles but not in azuki bean hypocotyls. Glucose-1-phosphate uridyltransferase activity (EC 2.7.7.9), in a crude extract from oat coleoptiles, was competitively inhibited by galactose-1-phosphate, but that enzyme from azuki bean was not. A correlation was found between inhibition of growth by galactose and inhibition of glucose-1-phosphate uridyltransferase activity by galactose-1-phosphate using oat, wheat, maize, barley, azuki bean, pea, mung bean, and cucumber plants. Thus, it is concluded that galactose is converted into galactose-1-phosphate, which interferes with UDP-glucose formation as an analog of glucose-1-phosphate.     

Inhibition of auxin-induced cell elongation by galactose

Galactose at concentrations higher than 3 m M inhibited specifically auxin-induced elongation of oat, wheat and rice coleoptile segments but not of pea and mung bean stem segments. Glucose, arabinose, rhamnose, xylose, mannose and glucosamine did not inhibit auxin-induced elongation of coleoptile segments. Galactose inhibited auxin-induced but not hydrogen ion-induced growth.     

Galactose prevents proton excretion but not IAA-induced growth in segments of azuki bean epicotyls

We investigated the effect of galactose on IAA-induced elongation and proton excretion in azuki bean (Vigna angularis Ohwi et Ohashi) segments in order to confirm whether or not protons were involved in auxin-induced growth. Galactose inhibited the IAA-induced decrease in the solution pH but had no inhibitory effect on IAA-induced growth in segments of azuki bean epicotyls. On the other hand, galactose inhibited both IAA-induced growth and proton excretion in oat (Avena sativa L.) coleoptile segments. From these results it is unlikely that IAA-induced growth is mediated by proton excretion at least in azuki bean epicotyls.Abbreviations IAA indole-3-acetic acid - FC fusicoccin     

UDP-Glucose level as a limiting factor for IAA-induced cell elongation in Avena coleoptile segments

The effects of galactose on IAA-induced elongation and endogenous level of UDP-glucose (UDPG) in oat ( Avena sativa L. cv. Victory) coleoptile segments were examined under various growth conditions to see if there was a correlation between the level of UDPG and auxin-induced growth. The following results were obtained:

• (1)

  Galactose (10 m M ) inhibited the auxin-induced cell elongation of oat coleoptile segments after a lag of ca 2 h. Determinations of cell wall polysaccharides and UDP-sugars indicated that galactose, when inhibiting the cell wall polysaccharide synthesis, decreased the level of UDPG but caused an increase in the levels of Gal-1-P and UDP-Gal.
• (2)

  When coleoptile segments treated with IAA and galactose were transferred to galactose-free IAA-solution, the segment elongation was restored and the amounts of cell wall polysaccharides increased. During this period, the amount of UDPG increased and the levels of Gal-1-P and UDP-Gal slightly decreased or leveled off. The UDP-pentoses changed similarly as UDPG did.
• (3)

  Addition of sucrose (30 m M ) enhanced IAA-induced cell elongation and removed growth inhibition by 1 m M galactose. Sucrose increased the amounts of the cell wall polysaccharides and the level of UDPG in the presence or absence of IAA and also counteracted the decrease in UDPG caused by galactose.

These results indicate that the level of UDPG is an important limiting factor for cell wall biosynthesis and, thus, for auxin-induced elongation.     

Effect of Galactose and Other Monosaccharides on IAA Movement in Bean Hypocotyl Segments

Galactose enhances the production of ethylene gas, and ethylene gas inhibits the movement of IAA in plant tissues. If galactose enhances ethylene production and ethylene inhibits auxin movement, then galactose should inhibit auxin movement. The above hypothesis was examined by observing the effects of d -galactose, d -inannose, d -arabinose, d -glucose, and d xylose on the uptake, presumed decarboxylation, efflux, velocity and metabolism of labeled indole-3-aectic acid in hypocotyl segments of Phaseolus vulgaris L. cv. Pinto. Galactose inhibited, arabinose and glucose enhanced, and mannose and xylose had no effect on partitioning of auxin between tissue and receptor. The reduction of auxin efflux by galactose was related to an increased presumed decarboxylation, reduced uptake and slower velocity of applied auxin. The relationship between galactose-induced growth effects, ethylene production, and auxin migration are discussed.     

Fusicoccin-induced growth and hydrogen ion excretion of Avena coleoptiles: Relation to auxin responses

Summary The fungal toxin fusicoccin (FC) induces both rapid cell elongation and H⁺-excretion in Avena coleoptiles. The rates for both responses are greater with FC than with optimal auxin, and in both cases the lag after addition of the hormone is less with FC. This provides additional support for the acid-growth theory. The FC responses resemble the auxin responses in that they are inhibited by a range of metabolic inhibitors, but the responses differ in three ways. First auxin, but not FC, requires continual protein synthesis for its action. The auxin-induced H⁺-excretion is inhibited by water stress or by low external pH, while the FC-induced H⁺-excretion is much less sensitive to either. It is concluded that auxin-induced and FC-induced H⁺-excretion may occur via different mechanisms.Abbreviations FC fusicoccin - DNP dinitrophenol - CCCP carbonylcyanide m-chlorophenylhydrazone - CHl cycloheximide - IAA indoleacetic acid     

Mutations in GAL2 or GAL4 alleviate catabolite repression produced by galactose in Saccharomyces cerevisiae

Galactose does not allow growth of pyruvate carboxylase mutants in media with ammonium as a nitrogen source, and inhibits growth of strains defective in phosphoglyceromutase in ethanol–glycerol mixtures. Starting with pyc1, pyc2, and gpm1 strains, we isolated mutants that eliminated those galactose effects. The mutations were recessive and were named dgr1-1 and dgr2-1. Strains bearing those mutations in an otherwise wild-type background grew slower than the wild type in rich galactose media, and their growth was dependent on respiration. Galactose repression of several enzymes was relieved in the mutants. Biochemical and genetic evidence showed that dgr1-1 was allelic with GAL2 and dgr2-1 with GAL4. The results indicate that the rate of galactose consumption is critical to cause catabolite repression.     

Galactose as an Inhibitor of the Expansion of Root Cells

The inhibition of the growth of cultured tomato roots by galactoseis due to an inhibition of cell expansion. Galactose is rapidlyabsorbed during the first 8 h following application and thefull inhibitory effect on extension growth of the roots is exertedwithin the first 24 h. At a concentration of 0·05 percent or less (50 per cent inhibition occurs at 0·035per cent) the galactose is not toxic and growth continues for7 days at the partially inhibited rate. The simultaneous presenceof glucose reduces galactose uptake but significant galactoseuptake continues at sites insensitive to a high concentrationof external glucose. In presence of an appropriate level ofglucose, although galactose uptake proceeds, the growth inhibitoryeffect of the galactose is fully reversed. Galactose reduces the content in the cell walls of the -cellulosefraction and during feeding with (I-¹⁴C) galactose all the cellwall fractions become labelled. The -cellulose fraction thenyields galactose of high specific activity. Glucose inhibitsthe incorporation of carbon from galactose into the -cellulosefraction and galactose inhibits the incorporation into thisfraction of the carbon of sucrose. The hypothesis is developedthat galactose inhibits cell expansion by a disruption of cellulosesynthesis which involves a direct incorporation of the externallyapplied galactose into the a-cellulose fraction of the cellwalls.     

The isolation and physiological analysis of mutants of the moss,Physcomitrella patens,which over-produce gametophores

We have compared the effects of cycloheximide (CHI) and two other rapid and effective inhibitors of protein synthesis, pactamycin and 2-(4-methyl-2,6-dinitroanilino)-N-methyl proprionamide (MDMP), on protein synthesis, respiration, auxin-induced growth and H⁺-excreation of Avena sativa L. coleoptiles. All three compounds inhibit protein synthesis without affecting respiration. The effectiveness of the inhibitors against H⁺-excretion and growth correlates with their ability to inhibit protein synthesis. Both CHI and MDMP inhibit auxin-induced H⁺-excretion after a latent period of 5–8 min, and inhibit growth after a 8–10-min lag. These results support the idea that continued protein synthesis is required in the initial stages of the growth-promoting action of auxin.Abbreviations CHI cycloheximide - DMSO dimethyl sulfoxide - FC fusicoccin - IAA indole-3-acetic acid - MDMP 2-(4-methyl-2,6-dinitroanilino)-N-methyl proprionamide     

Cell wall modifications during auxin-induced cell extension in monocotyledonous and dicotyledonous plants

There are several differences between monocotyledonous and dicotyledonous plants. The sensitivity towards added galactose which inhibits auxin-induced coleoptile elongation but not stem elongation is one of the conspicuous differences between the two types of plants. InAvena coleoptile segments, galactose, probably as galactose-1-phosphate, inhibits the formation of UDP-glucose from glucose-l-phosphate. The inhibition of UDP-glucose formation due to galactose is not found inPisum epicotyl segments. InAvena UTP: α-D-glucose-1-phosphate uridyltransferase (EC 2.7.7.9) which catalyzes the reaction from glucose-1-phosphate to UDP-glucose seems to be inhibited by galactose-1-phosphate.     

The Effect of Some Inhibitory Sugars upon the Content of Adenosine Triphosphate in Wheat Roots

The content of adenosine triphosphate (ATP) in roots of -wheat (Triticum aestivum L.) was determined with the fire-fly-luciferase method. The content is decreased by D-mannose, which inhibits root growth, respiration and chloride uptake. In intact seedlings the inhibition of root growth is relieved by other sugars and also by the flavanone naringenin and by 2,4-dinitrophenol. This reversal is combined with an increased content of ATP. The inhibition of chloride uptake by mannose in excised roots is reversed by some other sugars (including D-galactose which is in itself inhibitory to root growth), and also in this case the ATP content is increased. Naringenin and dinitrophenol do not relieve the inhibition of chloride uptake caused by mannose. Nor do they increase the content of ATP in this case. The primary effect of mannose seems to be inhibition of glycolysis whereas the effect upon root growth is secondary. Galactose, which also inhibits root growth, does not inhibit respiration or reduce the ATP content and the primary effect of galactose (and also of 2-deoxy-D-glucose and 2-deoxy-D-galactose) seems to be on the synthesis of cell wall substances.     

Different effects of galactose and mannose on cell proliferation and intracellular soluble sugar levels in <Emphasis Type="Italic">Vigna angularis</Emphasis> suspension cultures

Plant cells utilize various sugars as carbon sources for growth, respiration and biosynthesis of cellular components. Suspension-cultured cells of azuki bean (Vigna angularis) proliferated actively in liquid growth medium containing 1% (w/v) sucrose, glucose, fructose, arabinose or xylose, but did not proliferate in medium containing galactose or mannose. These two latter sugars thus appeared distinct from other sugars used as growth substrates. Galactose strongly inhibited cell growth even in the presence of sucrose but mannose did not, suggesting a substantial difference in their effects on cell metabolism. Analysis of intracellular soluble-sugar fractions revealed that galactose, but not mannose, caused a conspicuous decrease in the cellular level of sucrose with no apparent effects on the levels of glucose or fructose. Such a galactose-specific decrease in sucrose levels also occurred in cells that had been cultured together with glucose in place of sucrose, suggesting that galactose inhibits the biosynthesis, rather than uptake, of sucrose in the cells. By contrast, mannose seemed to be metabolically inert in the presence of sucrose. From these results, we conclude that sucrose metabolism is important for the heterotrophic growth of cells in plant suspension-cultures.     

Galactose, aspartate crossway and rhythm inhibition in Aspergillus niger

When the glucose/K⁺ balance in the medium is favourable to rhythm expression in Aspergillus niger Van Tieghem, galactose has an inhibitory effect on amplitude but not on period length of the rhythm. Galactose is active when its level reaches 1/10 of that of glucose. On liquid media a small quantity of glucose is necessary to start growth on galactose. Under these conditions U¹⁴C-galactose is rather slowly metabolized. After 6 h of feeding on the labelled galactose medium, this sugar is converted into glucose, which is used both for the synthesis of compounds derived from C₃ and C₄ units and for synthesis of polysaccharides and perhaps small peptides. The labelling of the macromolecules always remains low. The insoluble carbohydrates of the mycelium are little affected by the type of sugar supplied to the fungus. The metabolism on galactose differs from the metabolism on glucose mainly in a decrease of the free asparagine pool and a simultaneous equivalent increase of the free aspartate pool; such an effect could not be correlated with an increase of the aspartate aminotransferase activity. Supply of aspartate but not of gluta-mate into the agar medium inhibits the rhythm amplitude. So, the damping effect of galactose on the rhythm might be at least partly due to its effect on the regulation of the aspartate cross-way.     

Effect of galactose on auxin-induced cell elongation in oat coleoptile segments in mannitol solutions

Oat coleoptile segments were treated with or without 10 mM galactose in the presence or absence of 10 μM IAA and various concentrations of mannitol (pre-incubation). Auxin-induced growth was inhibited by galactose. Segments were then transferred to buffer solutions containing or not containing 10 mM galactose (post-incubation). Expansion growth due to rapid water absorption was observed. The expansion growth during the post-incubation was inhibited by galactose when galactose was applied during the post-incubation period or all through the pre- and post-incubation but was not affected by galactose when it was applied only during the pre-incubation. This result indicates that the galactose effect on the expansion growth is due to its inhibitory action during the post-incubation period. Galactose has been reported to be a specific inhibitor for cell wall synthesis. Thus, it is suggested that the expansion growth during post-incubation requires cell wall synthesis and is not just the process of passive water absorption. The primary action of auxin does not seem to require new synthesis of polysaccharides.     

A major isoform of the maize plasma membrane H(+)-ATPase: characterization and induction by auxin in coleoptiles.

The plasma membrane (PM) H(+)-ATPase has been proposed to play important transport and regulatory roles in plant physiology, including its participation in auxin-induced acidification in coleoptile segments. This enzyme is encoded by a family of genes differing in tissue distribution, regulation, and expression level. A major expressed isoform of the maize PM H(+)-ATPase (MHA2) has been characterized. RNA gel blot analysis indicated that MHA2 is expressed in all maize organs, with highest levels being in the roots. In situ hybridization of sections from maize seedlings indicated enriched expression of MHA2 in stomatal guard cells, phloem cells, and root epidermal cells. MHA2 mRNA was induced threefold when nonvascular parts of the coleoptile segments were treated with auxin. This induction correlates with auxin-triggered proton extrusion by the same part of the segments. The PM H(+)-ATPase in the vascular bundies does not contribute significantly to auxin-induced acidification, is not regulated by auxin, and masks the auxin effect in extracts of whole coleoptile segments. We conclude that auxin-induced acidification in coleoptile segments most often occurs in the nonvascular tissue and is mediated, at least in part, by increased levels of MHA2.     

A shift in sensitivity to auxin within development of maize seedlings

The auxin-induced changes in cytosolic concentrations of Ca(2+) and H(+) ions were investigated in protoplasts from maize coleoptile cells at 3rd, 4th and 5th day of development of etiolated seedlings. The shifts in [Ca(2+)](cyt) and [H(+)](cyt) were detected by use of fluorescence microscopy in single protoplasts loaded with the tetra[acetoxymethyl]esters of the fluorescent calcium binding Fura 2, or pH-sensitive carboxyfluorescein, BCECF, respectively. Both the auxin-induced shifts in the ion concentrations were specific to the physiologically active synthetic auxin, naphthalene-1-acetic acid (1-NAA), and not to the non-active naphthalene-2-acetic acid (2-NAA). Regardless of the age of the seedlings, the rise in [Ca(2+)](cyt) was prior to the acidification in all investigated cases. The maximal acidification coincided with the highest amplitude of [Ca(2+)](cyt) change, but not directly depended on the concentration of 1-NAA. Within aging of the seedlings the amplitude of auxin-induced [Ca(2+)](cyt) elevation decreased. The shift in auxin-induced acidification was almost equal at 3rd and 4th day, but largely dropped at 5th day of development. The acidification was related to changes in the plasma membrane H(+)-ATPase activity, detected as phosphate release. The decrement in amplitude of both the tested auxin-triggered reactions well coincided with the end of the physiological function of the coleoptile. Hence the primary auxin-induced increase in [Ca(2+)](cyt), which is supposed to be an important element of hormone signal perception and transduction, can be used as a test for elucidation of plant cell sensitivity to auxin.     

Lactobacillus casei 64H Contains a Phosphoenolpyruvate-Dependent Phosphotransferase System for Uptake of Galactose, as Confirmed by Analysis of ptsH and Different gal Mutants

Galactose metabolism in Lactobacillus casei 64H was analyzed by genetic and biochemical methods. Mutants with defects in ptsH, galK, or the tagatose 6-phosphate pathway were isolated either by positive selection using 2-deoxyglucose or 2-deoxygalactose or by an enrichment procedure with streptozotocin. ptsH mutations abolish growth on lactose, cellobiose, N-acetylglucosamine, mannose, fructose, mannitol, glucitol, and ribitol, while growth on galactose continues at a reduced rate. Growth on galactose is also reduced, but not abolished, in galK mutants. A mutation in galK in combination with a mutation in the tagatose 6-phosphate pathway results in sensitivity to galactose and lactose, while a galK mutation in combination with a mutation in ptsH completely abolishes galactose metabolism. Transport assays, in vitro phosphorylation assays, and thin-layer chromatography of intermediates of galactose metabolism also indicate the functioning of a permease/Leloir pathway and a phosphoenolpyruvate-dependent phosphotransferase system (PTS)/tagatose 6-phosphate pathway. The galactose-PTS is induced by growth on either galactose or lactose, but the induction kinetics for the two substrates are different.     

Reconstitution of the binding protein-dependent galactose transport of Salmonella typhimurium in proteoliposomes.

Binding protein-dependent transport systems mediate the accumulation of diverse substrates in bacteria. The binding protein-dependent galactose transport of Salmonella typhimurium has been reconstituted in proteoliposomes. The proteoliposomes were made with proteins solubilized and renatured from inclusion bodies produced by a bacterial strain containing a plasmid with the mgl (methylgalactose permease) operon of Salmonella typhimurium. Galactose transport is dependent both on the addition of the purified galactose binding protein to the transport assay, and on ATP. The interaction between the liganded galactose binding protein and proteoliposomes displays Michaelis type kinetics with a Km of around 15 microM. Galactose transport is coupled to ATP hydrolysis with a stoichiometry (ATP/galactose) of 2.5:1. Galactose transport in proteoliposomes is not significantly inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone, but is inhibited by 0.5 mM vanadate. The present reconstitution of galactose transport in proteoliposomes suggests that the MglA, MglC and MglE proteins have been solubilized and renatured in an active form from the inclusion bodies of the mgl hyperproducing strain.