Total and Polar Lipid Biosynthesis during Crotalaria juncea Linn. Pollen Tube Growth: Effect of Gibberellic Acid, Indoleacetic Acid and (2-Chloroethyl)-phosphonic Acid

Gibberellic acid (GA₃) at 58 µM, indoleacetic acid (IAA)at 29 µM, and (2-chloroethyl) phosphonic acid (Ethephon)at 70 µM promoted pollen tube growth in Crotalaria junceapollen suspension cultures both in water and basal medium. GA₃stimulated [ l-¹⁴C]acetate incorporation into total lipids inboth media, whereas IAA enhanced incorporation in water culturesonly. On the contrary, Ethephon reduced the label in total lipidswhen supplemented in basal medium. Based on [l-¹⁴C lacetateincorporation into different phospho- and glycolipids, it isproposed that these growth regulators have a definite role inthe biosynthesis of lipid components of the membranes.     

Effect of Gibberellic Acid and Some Phenols on Flowering of Impatiens balsamina, a Qualitative Short-day Plant

GA₃ as well as SA (salicylic acid) and β-N (β-naphthol) induce floral buds in Impatiens balsamina under strictly non-inductive photoperiods. The floral bud initiation is accelerated when 1 mg/1 SA is used in combination with 100 mg/1 GA₃. 100 mg/1 GA₃+ 1 mg/1 SA and 100 mg/1 GA₃+ 100 mg/1 β-N increase the number of floral buds as compared with 100 mg/1 GA₃ alone.     

Tannins as gibberellin antagonists in the synthesis of alpha-amylase and Acid phosphatase by barley seeds

The tannins chebulinic acid or tara tannin were added to an incubation system in which GA₃ induces enzyme synthesis in endosperm half seeds of barley (Hordeum vulgare L.). The activity of amylase and acid phosphatase in the incubation medium was reduced compared to the activity in the medium after incubation with GA₃ alone. When embryo half seeds of barley were incubated with chebulinic acid or tara tannin in the absence of added GA₃, the enzyme activity of the incubation medium was also reduced. The activity of preformed enzymes obtained from endosperm half seeds previously induced with GA₃ was not reduced by the addition of tannin. Comparisons were made of the amount of enzyme activity from breis of aleurone layers incubated with GA₃ in the presence and absence of tannins. The amounts of activity were relatively small and approximately equal in both cases, indicating that secretion from the aleurone was not blocked by the tannins. The reduction of enzyme activity caused by tannins in both endosperm and embryo half seeds could be completely reversed by the addition of GA₃.     

Enhanced Phytoremediation of Cadmium-Contaminated Soil by Parthenium hysterophorus Plant: Effect of Gibberellic Acid (GA3) and Synthetic Chelator,Alone and in Combinations

The roles of gibberellic acid (GA₃) and ethylenediaminetetraacetic acid (EDTA) in phytoremediation of cadmium (Cd)-contaminated soil by Parthenium hysterophorus plant was investigated. GA₃ (10^?9, 10^?7, and 10^?5M) was applied as a foliar spray. EDTA was added to soil in a single dose (160 mg/kg soil) and split doses (40 mg/kg soil, four split doses). GA₃ and EDTA were used separately and in various combinations. P. hysterophorus was selected due to its fast growth and unpalatable nature to herbivores to reduce the entrance of metal into the food chain. The Cd phytoextraction potential of the P. hysterophorus plant was evaluated for the first time. Cd significantly reduced plant growth and dry biomass (DBM). GA₃ alone increased the plant growth and biomass in Cd-contaminated soil, whereas EDTA reduced it. GA₃ in combination with EDTA significantly increased the growth and biomass. The highest significant DBM was found in treatment T3 (10^?5M GA₃). All treatments of GA₃ or EDTA significantly enhanced the plant Cd uptake and accumulation compared with control (C1). The highest significant root and stem Cd concentrations were found in the combination treatment T11 (GA₃ 10^?5M + EDTA split doses), whereas in leaves it was found in the EDTA treatments. Cd concentration in plant parts increased in the order of stem < leaves < roots. The combination treatment T9 (GA₃ 10^?7M + EDTA split doses) showed the significantly highest total Cd accumulation (8 times greater than control C1, i.e., only Cd used). The GA₃ treatments accumulated more than 50% of the total Cd in the roots, whereas the EDTA treatments showed more than 50% in the leaves. Root dry biomass showed a positive and significant correlation with Cd accumulation. GA₃ is environment friendly as compared with EDTA. Therefore, further investigation of GA₃ is recommended for phytoremediation research for the remediation of metal-contaminated soil.     

Demonstration of Various Acid Hydrolases and Preliminary Characterization of Acid Phosphatase in Naegleria fowleri1

ABSTRACT. Extracts of the pathogenic ameba Naegleria fowleri, prepared by freeze-thawing and sonication, were analyzed for their content of various hydrolytic enzymes that have acid pH optima. The organism is rich in acid phosphatase activity as well as a variety of glycosidases which include β-glucosidase, β-galactosidase, β-fucosidase, α-mannosidase, hexosaminidase, arylsulfatase A, and β-glucuronidase. The crude extract contained only negligible levels of sphingomyelinase, neuraminidase, or arylsulfatase B. All of the hydrolases exhibited higher activity at pH 5.5 than at 7.0, indicating that they are truly “acid” hydrolases. In general, after centrifugation (100,000 g, 1 h), except for arylsulfatase B, more than half of the activity of each of the various hydrolases was recovered in the supernatant fraction. The acid phosphatase in the high-speed supernatant was purified 45-fold (32% yield) by chromatography on QAE-Sephadex and Sephadex G-200 and shown to have the following properties: 1) pH optima, 5.5; 2) K_m (4-methylumbelliferyl phosphate), 0.60 mM; 3) molecular weight (estimated by gel filtration chromatography), 92,000; 4) inhibited by heteropolymolybdate complexes but not by L(+) sodium tartrate (0.5 mM) or sodium fluoride (0.5 mM). In addition, unlike the tartrate-resistant acid phosphatase of Leishmania donovani, the major acid phosphatase of N. fowleri is less than 5% as effective in inhibiting superoxide anion production by f-Met-Leu-Phe-stimulated human neutrophils. The finding of high levels of a number of acid hydrolases in Naegleria fowleri raises several questions that merit further study: Do the hydrolases perform a housekeeping function in this single cell eukaryote or do they play some role in the pathogenic process that ensues when the organism infects a suitable host?     

Study on russet-related enzymatic activity and gene expression in ‘Shine Muscat’ grape treated with GA3 and CPPU

Physiological (metabolite analysis) and molecular (gene expression) approaches were used to understand the mechanism underlying russet formation in response to the application of GA₃ and CPPU (Forchlorfenuron) in a Japanese table grape cultivar ‘Shine Muscat’. Several different concentrations of GA₃ and GA₃?+?CPPU [25?mg?L^?1 GA₃ (A), 25?mg?L^?1 GA₃?+?5?mg?L^?1 CPPU (B), 25?mg?L^?1 GA₃?+?10?mg?L^?1 CPPU (C), and 25?mg?L^?1 GA₃?+?15?mg?L^?1 CPPU (D)] were applied to grape berry clusters at two weeks after flowering (WAF). No russet was observed on the berries treated with the ‘C’ combination. Lower levels of phenylalanine ammonia-lyase (PAL) activity was observed in the treated samples, relative to the untreated material. Reduced peroxide (POD) activity was also observed in response to different treatments, while the expression of Peroxidase 17 and Phenylalanine ammonia-lyase G1 genes mirrored lignin content. Increased activity of 4-coenzyme A ligase (4CL) may contribute to decreasing the level of russet and help to improve grape berry quality.     

Regulation of Growth in Avena (Oat) Stem Segments by Gibberellic Acid and Abscisic Acid

The purpose of this study was to analyze the nature of the interaction between gibberellic acid (GA₃) and abscisic acid (ABA) in the regulation of growth in excised Avena (oat) stem segments. Growth, compared to sucrose controls, was inhibited by ABA in the range of 10^?4 to 10^?6M. GA₃-promoted growth was also inhibited by ABA in the same concentration range. A Lineweaver-Burk analysis of the interaction between GA₃ and ABA indicated that ABA acts in a non-competitive fashion with GA₃. This same result was obtained previously with GA₃-indoleacetic acid (IAA) and GA₃-kinetin interactions with Avena stem sections. Our results indicate that ABA can inhibit GA₃-promoted growth within physiological concentrations, and that it is probably acting at a different physiological site from that for GA₃.     

I-Cell disease: Activities of lysosomal enzymes toward natural and synthetic substrates

Cultured skin fibroblasts from a patient with I-Cell disease (mucolipidosis II) were assayed for a number of lysosomal enzymes using both natural and synthetic substrates. The cells from this patient were found to have very low activity for galactosylceramide β-galactosidase, lactosylceramide β-galactosidases (using two assay methods that measure different enzymes), G_M1 ganglioside β-galactosidase and sphingomyelinase. Glucosylceramide β-glucosidase activity was found to be normal. Acid hydrolase activities toward many synthetic substrate were measured and all except β-glucosidase and acid phosphatase were found to be extremely low (as has been reported by others). Acid phosphatase and β-glucosidase were in the low normal range. These studies expand on previously published reports on I-Cell disease that only present data from synthetic substrates, and also report the fibroblast culture deficiencies of galactosyl-ceramide β-galactosidase (the Krabbe disease enzyme) and sphingomyelinase (the Niemann-Pick disease enzyme) activities for the first time. Those two enzymes do not have a readily available synthetic analog to assay. Acid β-galactosidase activity measured with both the 4-methylumbelliferyl derivative and G_M1 ganglioside was partially deficient in leukocytes prepared from this patient. New methods for measuring 4-methylumbelliferyl-β-D-glucoside and glucosylceramide β-glucosidase activities are also presented.     

In vitro induction of pollen embryos and plantlets in Aesculus carnea Hayne through anther culture

Uninuclear microspores in red horse chestnut anther cultures formed pollen embryos and plantlents in MS agar medium supplemented with varying 2,4-D concentrations (1.0, 1.5 or 2.0 mg l^-1) and 1.0 mg l^-1 Kin. The highest number of embryogenic anthers (38%) was obtained in MS medium containing 1.0 mg l^-1 of each 2,4-D and Kin. The ability of pollen embryos to germinate was closely correlated with normal embryo morphology and was influenced by hormone content in the medium (MS+;1.0 mg l^-1 IAA+1.0 mg l^-1 GA₃+0.1 mg l^-1 Kin+400 mg l^-1 glutamine). Pollen embryos and plantlets had the haploid chromosome number (x=n=40). Cytological examinations demonstrated pollen dimorphism of this Aesculus species.Abbreviations AC activated charcoal - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin 6-furfurylaminopurine - GA₃ gibberellic acid - MS Murashige and Skoog     

Regulation of the synthesis of barley aleurone alpha-amylase by gibberellic Acid and calcium ions

The effects of gibberellic acid (GA₃) and calcium ions on the production of α-amylase and acid phosphatase by isolated aleurone layers of barley (Hordeum vulgare L. cv Himalaya) were studied. Aleurone layers not previously exposed to GA₃ or Ca²⁺ show qualitative and quantitative changes in hydrolase production following incubation in either GA₃ or Ca²⁺ or both. Incubation in H₂O or Ca²⁺ results in the production of low levels of α-amylase or acid phosphatase. The addition of GA₃ to the incubation medium causes a 10- to 20-fold increase in the amounts of these enzymes released from the tissue, and addition of Ca²⁺ at 10 millimolar causes a further 8- to 9-fold increase in α-amylase release and a 75% increase in phosphatase release. Production of α-amylase isoenzymes is also modified by the levels of GA₃ and Ca²⁺ in the incubation medium. α-Amylase 2 is produced under all conditions of incubation, while α-amylase 1 appears only when layers are incubated in GA₃ or GA₃ plus Ca²⁺. The synthesis of α-amylases 3 and 4 requires the presence of both GA₃ and Ca²⁺ in the incubation medium. Laurell rocket immuno-electrophoresis shows that two distinct groups of α-amylase antigens are present in incubation media of aleurone layers incubated with both GA₃ and Ca²⁺, while only one group of antigens is found in media of layers incubated in GA₃ alone. Strontium ions can be substituted for Ca²⁺ in increasing hydrolase production, although higher concentrations of Sr²⁺ are required for maximal response. We conclude that GA₃ is required for the production of α-amylase 1 and that both GA₃ and either Ca²⁺ or Sr²⁺ are required for the production of isoenzymes 3 and 4 of barley aleurone α-amylase.     

Similarities between gibberellins and related compounds in inducing Acid phosphatase and reducing sugar release from barley endosperm

Barley endosperm halves release acid phosphatase in response to several gibberellins and gibberellin precursors. Seed halves incubated with 10⁻⁷m GA₃ at 29° begin to release phosphatase after 11 hr and release it for another 26 hr in response to GA₃. After 37 hr, the rate of release slows to that of seed halves incubated without GA₃. GA₃ is active at 10⁻¹⁰m and maximally active at 10⁻⁷m. Comparative activity of 12 gibberellins and gibberellin precursors is GA₁ = GA₃ > GA₂ > GA₄ = GA₇ > GA₅ = GA₁₃ > GA₁₄ > GA₈ = GA₉ > (−)kaurenoic acid > (−)-kaurene. These compounds show the same order of activity and approximately the same relative activity in inducing reducing sugar release as in inducing phosphatase activity. The activity of each compound increases with its presumed position in a biosynthetic pathway leading from kaurene to GA₃. This correlation suggests that activity may be a reflection of the efficiency of conversion to an active form within the seed half.     

Acid hydrolases and their Release in Food Vacuole-Less Mutants of Tetrahymena thermophila*

SYNOPSIS. Mutants (NP1 and PSJ5) of Tetrahymena thermophila strains B and D 1968 exist that are unable to construct a functional oral apparatus and form food vacuoles at 37 C but which do so normally at 30 C. Food vacuole-less cells starved in dilute salt solution released similar amounts of acid phosphatase, β-N-acetyl-glucosaminidase and ±-glucosidase activity into the medium as wildtype cells during an 8-h period. Actively growing, food vacuole-less cells had ?50% less total protein, acid phosphatase, β-N-acetyl-glucosamin-idase, and ±-glucosidase per cell than wildtype cells after 72-h growth. During this time food vacuole-less cells released significant amounts of the 3 acid hydrolases into the growth medium. For each hydrolase, the total activity released from growing, food vacuole-less cells was less, on a per cell basis, than the amount released from food vacuole formers. The proportion of the total activity secreted by the mutant and the wildtype cells was the same for acid phosphatase and β-N-acetyl-glucosaminidase and somewhat lower for ±-glucosidase. It is concluded that the release of a significant amount of acid hydrolase activity from Tetrahymena is independent of food vacuole formation and may be analogous to the secretory activity of other nonphagocytic eukaryotic cells.     

Stimulatory effect of ethephon,ACC, gibberellin A3 and A4+7 on germination of methyl jasmonate inhibited Amaranthus caudatus L. seeds

Methyl jasmonate (JA-Me) inhibited or retarded germination of Amaranthus caudatus seeds in darkness at 24°C, Ethephon, ACC and gibberellins (GA₃ or GA₄₊₇) partially or completely reversed this inhibition depending on the concentration of JA-Me applied. Both ethephon and the gibberellins were more effective than ACC. Both GA₃ and GA₄₊₇ enhanced the stimulatory effect of ethephon or ACC on germination of seeds inhibited by JA-Me.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - JA jasmonic acid - JA-Me methyl jasmonate     

Effects of gibberellic acid,ethephon, and 1-aminocyclopropane-1-carboxylic acid on germination ofAmaranthus caudatus seeds inhibited by paclobutrazol

The specific inhibitor of gibberellin biosynthesis, (2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol (paclobutrazol), inhibited germination ofAmaranthus caudatus L. seeds. Addition of gibberellic acid (GA₃), 2-chloroethylphosphonic acid (ethephon), or 1-aminocyclopropane-1-carboxylic acid (ACC) effectively antagonized inhibition. Ethephon was found to be the most efficient antagonist. The transfer of seeds after 1 day's incubation in paclobutrazol to solutions of GA₃ or ethephon reversed the inhibition, the effect increasing with increasing concentration of GA₃ or ethephon. Seeds incubated in paclobutrazol for 5 days decreased sensitivity to GA₃ and ethephon.     

Subcellular fractionation of midgut cells of the sunn pest Eurygaster integriceps (Hemiptera: Scutelleridae): Enzyme markers of microvillar and perimicrovillar membranes

The subcellular distributions of six digestive and non-digestive enzymes (α-glucosidase, β-glucosidase, alkaline phosphatase, acid phosphatase, aminopeptidase and lactate dehydrogenase) of Eurygaster integriceps have been studied. The subcellular distributions of acid phosphatase and α-glucosidase are similar and the gradient ultracentrifugation profiles of these two enzymes overlap. Two partially membrane-bound enzymes, alkaline phosphatase and β-glucosidase have similar distributions in differential centrifugation fractions, which are different from that of α-glucosidase. Sucrose gradient ultracentrifugation of membranes from luminal contents showed that β-glucosidase carrying membranes are heavier. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the profile of proteins extracted from β-glucosidase carrying membranes is different from that of α-glucosidase carrying membranes. We conclude that β-glucosidase and aminopeptidase are markers of microvillar membrane (MM) and perimicrovillar space, respectively, while α-glucosidase and acid phosphatase are perimicrovillar markers. In E. integriceps V1 luminal content is a rich source of PMM and MM and that is used to resolve these membranes.     

Pollen tube growth is affected by exogenous hormones and correlated with hormone changes in styles in <Emphasis Type="Italic">Torenia fournieri</Emphasis> L.

In the present report, we described the effects of indole-3-acetic acid (IAA), zeatin (ZT), gibberellin (GA₃), and abscisic acid (ABA) on in vitro pollen germination and pollen tube growth in Torenia fournieri L. The results showed that IAA and GA₃ stimulated in vitro pollen tube growth, ABA inhibited pollen tube growth, and ZT had no significant effect on the process. The stimulating effect of exogenous IAA was particularly distinct, and led to synchronous growth of straighter and more slender pollen tubes compared with the controls. However, no significant changes were found in the germination of the treated pollen. The auxin efflux inhibitor, 10 μM 1-N-naphthylphthalamic acid (NPA), was also found to stimulate pollen tube growth. We measured the content of hormones (free IAA, ZT, GA₃, and ABA) in the stigmas and styles before and after pollination. The hormone contents of stigmas measured 0.5 h after pollination (0.5 HAP) showed that ABA content decreased, whereas the content of IAA, ZT, or GA₃ did not change significantly. The hormone level in pollinated styles (4 HAP) when pollen tubes had grown into the middle part of style was characterized by an increase in free IAA and GA₃ and a decrease in ABA, which was in agreement with the results that IAA and GA₃ promoted but ABA inhibited pollen tube growth in vitro. Furthermore, the change of IAA level in styles was most notable, which was accordant to the fact that auxin stimulated significantly pollen tube growth in vitro. Using immunoenzyme and immunogold labeling techniques and an anti-IAA monoclonal antibody, we confirmed that free IAA was present throughout style tissues, and distributed in the nucleus and cytoplasm of style cells. All these results suggested that hormones, especially IAA, play important roles in pollen tube growth of T. fournieri. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Purification and Characterization of two Extracellular β-Glucosidases from Trichoderma viride ITCC 1433

T. viride ITCC 1433 synthesizes a two component system for the hydrolysis of cellobiose and cellooligodextrins. 80% of the total activity are solubilized during growth. The large protein (A), mol. weight 98 000 d, is glycosylated and slightly acidic (pH = 6.1). The smaller protein (B), mol. weight 70 000 d, is unglycosylated and neutral (pH = 7.2). Both proteins form a two-step system where β-glucosidase A is active at low substrate concentrations (K_M = 2.3 × 10^?4 M cellobiose) while β-glucosidase B covers the range of 10-fold higher cellobiose concentrations (K_M = 1.8 × 10^?3 M). The enzymes are fairly stable with a residual activity of 70% at 50°C after 24 h.     

The Relationships between Fertility and Contents of Gibberellic Acid, Sugars and Dry Mass in Apical Parts of Chara Vulgaris Thalli

The contents of endogenous gibberellic acid (GA₃), sugars, and dry mass in apical parts of fertile and sterile thalli of Chara vulgaris were estimated. The GA₃ concentration in the first node of fertile thallus, determined by capillary electrophoresis, was about 70.0 mg kg^–1 of fresh mass (f.m.). Pisum sativum-bioassay showed GA₃ concentration of 80.0 mg kg^–1 (f.m.) which was about 3 times higher than in the first node of sterile thallus. The higher amount of GA₃, glucose, and the lower starch content and dry mass in fertile plants than in sterile ones suggest the interdependence between fertility and contents of studied components.     

Effect of laterally applied gibberellin A4/7 on cambial growth and the level of indole-3-acetic acid in Pinus sylvestris shoots

Gibberellin A_4/7 (GA_4/7) was applied in lanolin or ethanol around the circumference at the midpoint of the previous-year terminal of dormant Pinus sylvestris seedlings. After cultivating the seedlings under environmental conditions favorable for growth for up to 10 weeks, cambial growth was measured as the radial widths of xylem and phloem, and the level of indole-3-acetic acid (IAA) was determined by combined gas chromatography-mass spectrometry using [¹³₆](IAA) as the internal standard. In intact seedlings, both 1 mg GA_4/7 g^?1 lanolin and 50 mg GA_4/7 I^?1 ethanol increased phloem production and the cambial region IAA level in the current-year terminal, without significantly altering its longitudinal growth. In the previous-year terminal, 1 mg GA_4/7 g^?1 lanolin promoted phloem production at the application point and increased the cambial region IAA level above this point, whereas 50 mg GA_4/7 I^?1 ethanol stimulated the production of both xylem and phloem at the treatment site and elevated the cambial region IAA level beneath it. Laterally applied GA_4/7 at 50 mg I^?1 ethanol stimulated xylem and phloem production in debudded previous-year terminals treated at the apical cut surface with 1 mg IAA g^?1 lanolin, but not in those treated with plain lanolin. However, the promotion of cambial growth in debudded terminals treated apically with 1 mg IAA g^?1 lanolin and laterally with 50 mg GA_4/7 I^?1 ethanol was not associated with an elevated IAA content in the cambial region. The results indicate that exogenous GA_4/7 can promote xylem and phloem production provided an IAA source is present, and that it or a metabolic product acts directly, rather than indirectly by stimulating longitudinal growth and/or raising the cambial region IAA level.     

Digestive glucosidases in the midgut of Apodiphus amygdali Germar (Hemiptera: Pentatomidae)

Apodiphus amygdali or stink bug of fruit trees is one of the polyphagous species from pentatomid bugs that attack many of fruit trees and ornamental trees. In the current study, activities of α- and β-glucosidases were measured in the midgut of A. amygdali adults. It was found the higher activity of β-glucosidase than α-glucosidase in addition to different enzymatic properties of the enzymes. Optimal pHs for enzymatic activities were found to be 5 and 7 for α- and β-glucosidases, respectively. Values regarding optimal temperatures were obtained at 30?°C for both α- and β-glucosidases. Among ions used on α-glucosidase activity, K⁺ and Ca²⁺ significantly increased enzymatic activity, Na⁺ had no effect, and Cu²⁺, Fe²⁺ and Mg²⁺ had the significant negative effects on the enzyme activity. Ca²⁺ and Fe²⁺ increased β-glucosidase activity in the midgut of A. amygdali, Na⁺ had no effect, and other ions significantly decreased the enzyme activity. Ethylene glycol-bis (β-aminoethylether) N,N,N?,N-tetraacetic acid (EGTA), citric acid, ethylenediamide tetraacetic acid (EDTA) and sodium dodecylsulfate (SDS) significantly decreased α-glucosidase activity but EGTA, triethylenetetramine hexaacetic acid (TTHA), EDTA and SDS decreased β-glucosidase activity in the midgut of A. amygdali. Characterisation of digestive enzymes, especially the effect of inhibitors on enzyme activity, could be useful for better understanding of enzyme roles in nutritional physiology of insects in addition to reach safe and useful controls of insect pests.