The effect of the seed coat, embryonic axis and aeration conditions on starch degradation hi cotyledons of Lens culinaris

The effect of exogenously applied galactose on the cell wall polysaccharide synthesis and UDP-sugar levels in oat ( Avena sativa L. cv. Victory I) coleoptile segments was studied to clarify the mechanism of inhibition of IAA-induced cell elongation by galactose, and the following results were obtained: (1) The inhibition of IAA-induced cell elongation by galactose became apparent after a 2 h-lag, while the lag was shortened to 1 h when galactose was added to the segments after more than 1 h of IAA application. (2) Galactose inhibited the [¹⁴C]-glucose incorporation into cellulosic and non-cellulosic fractions of the cell wall and the increase in net polysaccharide content in the fractions during long-term incubation. (3) The dominant sugar nucleotide in oat coleoptiles was UDP-glucose (2.1 nmol segment⁻¹). Galactose application caused a remarkable decrease in the UDP-glucose level, accompanying a strong accumulation of galactose-1-phosphate and UDP-galactose. (4) Galactose-1-phosphate competitively inhibited the UTP: a- d -glucose-1-phosphate uridylyltransferase (EC 2.7.7.9) activity of the crude enzyme preparation from oat coleoptiles. From these results we conclude that galactose inhibits the IAA-induced cell elongation by inhibiting the formation of UDP-glucose, which is a key intermediate of cell wall polysaccharide 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.     

A selection system for transgenic plants based on galactose as selective agent and a UDP-glucose:galactose-1-phosphate uridyltransferase gene as selective gene

A new selection system based on galactose as selective agent and a UDP-glucose:galactose-1-phosphate uridyltransferase gene as selective gene is presented. A broad range of plant species, including agronomically important crops such as maize and rice, is sensitive to low dosages of galactose. The toxicity of galactose is believed to be due to accumulation of galactose-1-phosphate, generated by endogenous galactokinase after uptake. Here, it is demonstrated that this toxicity can be sufficiently alleviated by the Agrobacterium tumefaciens-mediated introduction of the E. coli UDP-glucose:galactose-1-phosphate uridyltransferase (galT) gene, driven by a 35S-promoter, to allow transgenic shoots of potato and oil seed rape to regenerate on galactose containing selection media, resulting in high transformation frequencies (up to 35% for potato). Analysis of genomic DNA and UDP-glucose:galactose-1-phosphate uridyltransferase activity in randomly selected potato transformants confirmed the presence and active expression of the galT gene. The agricultural performance of transgenic potatoes was evaluated by monitoring the phenotype and tuber yield for two generations and these characters were found to be indistinguishable from non-transgenic controls. Thus, the galactose selection system provides a new alternative being distinct from conventional antibiotic and herbicide selection systems as well as so-called positive selection systems where the selective agent has a beneficial effect.     

Regulation of glucose metabolism and cell wall synthesis in Avena stem segments by gibberellic Acid

Gibberellic acid (GA) stimulated both the elongation of Avena sativa stem segments and increased synthesis of cell wall material. The effects of GA on glucose metabolism, as related to cell wall synthesis, have been investigated in order to find specific events regulated by GA. GA caused a decline in the levels of glucose, glucose 6-phosphate, and fructose 6-phosphate if exogenous sugar was not supplied to the segments, whereas the hormone caused no change in the levels of glucose 6-phosphate, fructose 6-phosphate, UDP-glucose, or the adenylate energy charge if the segments were incubated in 0.1 m glucose. No GA-induced change could be demonstrated in the activities of hexokinase, phosphoglucomutase, UDP-glucose pyrophosphorylase, or polysaccharide synthetases using UDP-glucose, UDP-galactose, UDP-xylose, and UDP-arabinose as substrates. GA stimulated the activity of GDP-glucose-dependent β-glucan synthetase by 2- to 4-fold over the control. When glucan synthetase was assayed using UDP-glucose as substrate, only β-1,3-linked glucan was synthesized in vitro, whereas with GDP-glucose, only β-1,4-linked glucan was synthesized. These results suggest that one part of the mechanism by which GA stimulates cell wall synthesis concurrently with elongation in Avena stem segments may be through a stimulation of cell wall polysaccharide synthetase activity.     

EFFECT OF YEAST SEXUAL HORMONES ON ELONGATION OF AVENA COLEOPTILE

Effect of yeast (Saccharomyces cerevisiae) sexual hormones on the elongation of etiolated Avena coleoptile segments was studied. The elongation was promoted by a hormone excreted by cells of mating type a, but not by α hormone excreted by cells of α type. The effect of the former was as great as that of 5 mg/1 indole-3-acetic acid in the first hour of application. The optimal concentration of a hormone was 10 units/ml. Its growth promoting effect was greatly inhibited by an antiauxin, 2,4,6-trichlorophenoxyacetic acid. a Hormone increased cell wall extensibility just as auxin does. Testosterone, β-estradiol, progesterone and ergosterol showed very little effect on the elongation of coleoptile segments.     

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.     

Overexpression of human UDP-glucose pyrophosphorylase rescues galactose-1-phosphate uridyltransferase-deficient yeast

To better understand the pathophysiology of galactose-1-phosphate uridyltransferase (GALT) deficiency in humans, we studied the mechanisms by which a GALT-deficient yeast survived on galactose medium. Under normal conditions, GALT-deficient yeast cannot grow in medium that contains 0.2% galactose as the sole carbohydrate, a phenotype of Gal(-). We isolated revertants from a GALT-deficient yeast by direct selection for growth in galactose, a phenotype of Gal(+). Comparison of gene expression profiles among wild-type and revertant strains on galactose medium revealed that the revertant down-regulated genes encoding enzymes including galactokinase, galactose permease, and UDP-galactose-4-epimerase (the GAL regulon). By contrast, the revertant strain up-regulated the gene for UDP-glucose pyrophosphorylase, UGP1. There was reduced accumulation of galactose-1-phosphate in the galactose-grown revertant cells when compared to the GALT-deficient parent cells. In vitro biochemical analysis showed that UDP-glucose pyrophosphorylase had bifunctional properties and could catalyze the conversion of galactose-1-phosphate to UDP-galactose in the presence of UTP. To test if augmented expression of this gene could produce a Gal(+) phenotype in the GALT-deficient parent cells, we overexpressed the yeast UGP1 and the human homolog, hUGP2 in the mutant strain. The Gal(-) yeast transformed with either UGP1 or hUGP2 regained their ability to grow on galactose. We conclude that revertant can grow on galactose medium by reducing the accumulation of toxic precursors through down-regulation of the GAL regulon and up-regulation of the UGP1 gene. We speculate that increased expression of hUGP2 in humans could alleviate poor outcomes in humans with classic galactosemia.     

Accelerated endogenous growth in Avena coleoptile segments

Summary An unexpected acceleration in elongation of Avena coleoptile segments has been observed with a position sensor transducer 2 to 2.5 h after excision when the segments are immersed in 5 mM succinate buffer (pH 6). The cause of the accelerated growth is unknown.Paper No. 869 from the Department of Botany, Ohio State University.     

Studies on growth regulators. I. Improved Avena coleoptile elongation test for auxin

A modified procedure is presented for the bioassay of auxin using Avena coleoptile segments. The modifications introduced result in a substantial improvement of the commonly used coleoptile elongation tests.     

Auxin-induced elongation of short maize coleoptile segments is supported by 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one

Endogenous extractable factors associated with auxin action in plant tissues were investigated, especially their effects on elongation of 1-mm coleoptile segments of maize (Zea mays L.), in the presence of saturating 10 μM indole-3-acetic acid (IAA). The relative growth response, to auxin alone, was much smaller in segments shorter than 2–3 mm compared to 10-mm segments. Fusicoccin-induced elongation, however, was less affected by shortening the segments. A reduced auxin response may result from the depletion through cut surfaces of a substance required for IAA-mediated growth. Sucrose, phenolics like flavonoids, and vitamins were ruled out as the causal factors. A partially purified methanol extract of maize coleoptiles supported long-term, auxin-controlled elongation. The active material was also found among substances bleeding from scrubbed maize coleoptiles. The active factor from maize was further purified by HPLC and characterised by the UV spectrum and its pH shift. This factor was identified as 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) by mass spectroscopy. Activity tests confirmed that pure DIMBOA from other sources sustained auxin-induced elongation of short maize coleoptile segments. However, DIMBOA only partially restored the activity lost from short segments. This indicates that an additional factor, other than DIMBOA, is required. Extracts from Avena or Cucurbita did not contain the factor DIMBOA; it was active on maize elongation, but not on Avena coleoptiles or Cucurbita hypocotyls. This narrow specificity and the lack of DIMBOA action in short-term tests with maize indicate that DIMBOA is not the general auxin cofactor but may specifically “spare” the co-auxin in maize. Received: 27 June 2000 / Accepted: 16 October 2000     

Studies on glycogen synthesis in pigeon liver homogenates. Glycogen synthesis from glucose monophosphates and uridine diphosphate glucose

Comparative time-course studies of glycogen synthesis from glucose 6-phosphate, glucose 1-phosphate and UDP-glucose show that glucose 1-phosphate forms glycogen at an initial rate faster than that obtained with glucose 6-phosphate and UDP-glucose. After 5min. the rates from glucose monophosphates are considerably slower. 2,4-Dinitrophenol decreases glycogen synthesis from both glucose monophosphates, whereas arsenate and EDTA increase glycogen synthesis from glucose 1-phosphate and inhibit the reaction from glucose 6-phosphate, galactose and galactose 1-phosphate. Mitochondria-free pigeon liver cytoplasmic fraction forms less glycogen from glucose monophosphates than does the whole homogenate. 2-Deoxyglucose 6-phosphate inhibits glycogen synthesis from glucose monophosphates. Glycogen formation from UDP-glucose is relatively unaffected by dinitrophenol, by arsenate, by EDTA, by 2-deoxyglucose 6-phosphate and by the removal of mitochondria from the whole homogenate.     

Studies on the Fermentative Production and Metabolism of Uridine Coenzymes

Enzyme activities involved in the galactose metabolism of Torulopsis Candida grown on a. lactose medium were investigated with the cell-free extract and ammonium sulfate fraction. Remarkable activities of galactokinase, galactose-1-phosphate uridylyltransferase and UDPG pyrophosphorylase were detected, whereas UDPGal pyrophosphorylase activity was weak. UDPGal formation proceeded by the cell-free extract along a coupling reaction catalyzed by UDPG pyrophosphorylase and galactose-1-phosphate uridylyltransferase where UDPG or glucose-l-phosphate acted as a catalyst.

The mechanism of UDPGal accumulation under the fermentative condition could be explained by a concerted inhibition of UDPGal-4- epimerase activity by 5′-UMP and galactose present as fermentation substrates.     

Galactose Transport in Saccharomyces cerevisiae III. Characteristics of Galactose Uptake in Transferaseless Cells: Evidence Against Transport-Associated Phosphorylation

The characteristics of the inducible galactose transport system in bakers' yeast were studied in uridine diphosphate, galactose-1-phosphate uridylyl-transferaseless cells. Transferaseless cells transport galactose at the same initial rate as wild-type cells and accumulate a mixture of free galactose and galactose-1-phosphate. The addition of ¹⁴C-labeled galactose to cells preloaded with unlabeled galactose and galactose-1-phosphate results in a higher rate of labeling of the free-sugar pool than of the galactose-1-phosphate pool. These results support other evidence that galactose uptake in bakers' yeast is a carrier-mediated, facilitated diffusion and that phosphorylation is an intracellular event after uptake of the free sugar.     

Avena coleoptile elongation: stimulation by fluorophenylalanine

A 100 to 150% stimulation of Avena coleoptile segment elongation by the amino acid analogue p-fluorophenylalanine (FPA) has been observed. The effect is reversed by phenylalanine and is not seen with comparable concentrations of sodium fluoride. FPA does not alter elongation of red-irradiated segments. Stimulation by FPA occurs only when the apex is intact and the segments are incubated in the absence of exogenous auxin. In the presence of FPA, ¹⁴C-leucine uptake by coleoptile segments is reduced by 34% and protein synthesis by 42%. When pre-incubated on labeled media and subsequently transferred to unlabeled media, segments fail to incorporate into the protein fraction any of the previously absorbed label. It is therefore difficult to ascertain whether FPA results in a genuine inhibition of protein synthesis in apical coleoptile segments. Possible mechanisms for the action of FPA and its relationship to light dependent elongation are considered.     

Auxin- and hydrogen ion-induced cell wall loosening and cell extension in Avena coleoptile segments

Hydrogen ions and auxin induce rapid cell extension of Avenacoleoptile segments. Nojirimycin (5-amino-5-deoxy-D-glucopyranose),a potent glucanase inhibitor, inhibits auxin-induced growthbut does not affect hydrogen ion-induced extension. This inhibitorhas little effect on respiration of coleoptile segments butstrongly inhibits the in vitro activity of ß-glucosidase.Hydrogen ions and auxin decreased the minimum stress-relaxationtime of the cell wall, indicating that both enhanced cell extensionthrough cell wall loosening. The hemicellulosic glucose contentof the cell wall which was decreased by auxin after about a2-hr lag, was not affected by hydrogen ions. These results suggestthat cell wall loosening induced by hydrogen ions may not bethe same as that caused by auxin, although both phenomena arerepresented by the decrease in the minimum stress-relaxationtime. (Received November 1, 1976; )     

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.     

Isolation of Neutral Growth Inhibitors in Dwarf Peas

From seedlings of dwarf pea (Pisum sativum L., var. Progress No. 9) grown under red light, three neutral growth inhibitors were isolated which interfered with the responses of these plants to GA₃. The compounds were identified as β-sitosterol, α-stearoyl glycerol and pisatin, of which the glyceride produced remarkable inhibition when applied to terminal buds. Though its anti-gibberellin activity was not very strong, pisatin produced inhibition of the straight-growth of Avena coleoptile segments caused by IAA.     

Galactosemia: how does long-term treatment change the outcome?

In galactosemic subjects, treatment prevents liver and kidney failure, brain damage and cataracts, but total exclusion of galactose from the diet does not ensure the absence of all pathology. Early and well-treated children show satisfactory general health and growth, make reasonable though suboptimal intellectual progress, are prone to speech defects, and commonly experience visual perceptual difficulties and some social maladjustment. Two different metabolites are potentially toxic: galactitol is responsible for the cataracts while galactose-1-phosphate causes the rest of the pathology. As both metabolites are present in the fetus and in postnatal life, pathological changes may develop at any time in life, even when treatment is strict. Owing to UDP-galactose 4'-epimerase, man can generate galactose from glucose from early embryonic life on. Therefore, transferase-deficient individuals can form galactose-1-phosphate in the absence of exogenous galactose, a process for which UDP-glucose pyrophosphorylase is essential. Biosynthesis of galactose from glucose in well-treated galactosemics constitutes a mechanism of self-intoxication, not only in utero but also in adult life. The prognosis for some treated galactosemics may depend on their own ability to limit this process. Galactosemic girls, whether well-treated or not, run a considerable risk of developing ovarian dysfunction. Hypergonadotropinism has been diagnosed from 2 years of age to the third decade. Prenatal ovarian failure is not excluded but the observed facts suggest that ovarian failure is acquired after ovarian differentiation and initiation of folliculogenesis, at an individual rate and possibly through continuous self-intoxication with galactose-1-phosphate. Up to now, mild hypergonadotropinism has been documented in only 2 galactosemic males, but the male cohort of galactosemics studied for gonadal dysfunction is yet small.     

The stability of the postulated wall-loosening enzyme in acid-induced growth

Long-term pretreatments with cycloheximide (CH) caused inhibition of subsequent acidinduced growth of Avena coleoptile segments, but only after 6 or more h of CH treatment. These results together with previous, published evidence with frozen-thawed tissue are consistent with the hypothesis that there exists a wall-loosening enzyme responsible for acid-induced elongation and that it has a half-life of at least 7–8 h.     

Biochemical analysis of galactose induced bacteriostasis ingalT mutants ofescherichia coli K 12

Escherichia coli mutants completely defective in galactose-1-phosphate uridyl transferase (EC 2.7.7.10) and growing in glycerol medium undergo rapid cessation of growth when exposed to galactose. Toxicity due to galactose is equally pronounced when glycerol is replaced by other carbon sources, like succinate and proline. Gas chromatographic analysis failed to detect even trace amounts of galactitol. Moreover, galactose-1-phosphate had no inhibitory role on some of the critical enzymes of cellular metabolism. General loss of energy (ATP) due to futile phosphorylation of galactose is probably the cause of bacteriostasis. ThegalT mutants can serve as models of human transferaseless galactosemia only to a limited extent