GA3-Modulated multiple forms of monophenolase in wheat seed

Multiple forms of monophenolase in wheat half-seeds were separated by molecular sieving on Sephadex G-200. A single molecular form of monophenolase was observed in control, while two multiple forms were present in GA₃-treated wheat half-seeds. A high MW (200 000 or above) multiple form (activity peak I) which eluted soon after the void volume was exclusively present in GA₃-treated half-seeds. The second activity peak (peak II) was a low MW (45 000) multiple form and its elution profile coincided in control and GA₃-treated wheat half-seeds. Both the multiple forms of monophenolase in GA₃-treated wheat half-seeds showed a pH optimum at 9.0, while the optimum enzyme activity of the control molecular form (peak II) was at pH 7.0. This indicated that the treatment of wheat half-seeds with GA₃ brought about a structural modification in monophenolase. The in vitro addition of trypsin enhanced the control of the molecular form of monophenolase but this treatment failed to alter the activity of multiple forms in GA₃-treated half-seeds. This differential response of monophenolase towards trypsin could be ascribed to a conformational change of the enzyme in hormone-treated half-seeds. Brief exposure of the enzyme preparation to urea (6 M) brought about an irreversible activation of monophenolase both in control and GA₃-treated wheat half-seeds.     

Gibberelic acid-mediated activation of monophenolase in de-embryonated half-seeds of wheat (Triticum Aestivum)

Stimulation of monophenolase activity was observed when de-embryonated prewashed half-seeds of wheat were imbibed in a solution of gibberellic acid (GA₃, 10^t?5 M). Crude extracts, prepared from GA₃-treated half-seeds, showed ca a two-fold increase in monophenolase activity over the controls at pH 6.6, while a dramatic rise in enzyme activity (seven- to nine-fold) was observed at pH 9.0. The partially purified (NH₄)₂SO₄ fraction precipitate (30–50% saturation) also showed enhancement of enzyme activity at pH 9.0 in GA₃-treated half-seeds, while in controls, there was negligible activity at this pH. Administration of five amino acid analogues (1 mM each) to half-seeds showed no significant inhibition of GA₃-stimulated monophenolase activity, but proved very effective in decreasing (86% inhibition) the GA₃-induced amylase activity. This indicated that the hormone-regulated monophenolase activity was not dependent on de novo protein synthesis. Treatment of half-seeds with GA₃ modified the monophenolase and altered the electrophoretic pattern. The enzyme was relatively thermostable at 55° and the pH optimum was shifted from pH 7.0 to 9.0. In addition, the GA₃-treated half-seeds showed relatively high stability of enzyme activity in the presence of (NH₄)₂S0₄ ions. These alterations in the GA₃-stimulated nomophenolase suggest there is activation of preformed enzyme molecules. The appearance of slow-migrating multiple forms on acrylamide gels, in response to GA₃ treatment, is probably due to the association of fast-migrating forms. Such oligomerization could result in a conformational change leading to enzyme activation. This may be an adaptive mechanism so that the enzyme can function with varying temperature, pH and ionic strength during early stages of seed germination.     

Stimulation of polyphenol oxidase (monophenolase) activity in wheat endosperm by gibberellic acid,cycloheximide and actinomycin D

Summary Embryoless wheat (Triticum aestivum L.) half-seeds on incubation with gibberellic acid (GA₃) showed a 2- to 2.5fold stimulation of monophenolase activity. The enzyme activity was not released into the incubation medium in GA₃-treated half-seeds. The effect of GA₃ was counteracted by the addition of abscisic acid (ABA) to the half-seeds. Adenosine-3,5-cyclic monophosphate and its structural analogues were ineffective in increasing the monophenolase activity. Actinomycin D and cycloheximide showed no inhibitory effecton the monophenolase activity in controls as well as in GA₃-treated half-seeds, but on the contrary caused a 2- to 3fold stimulation of enzyme activity similar to that observed in endosperm treated with GA₃ alone. However, there was no additive or synergistic enhancement of monophenolase activity when GA₃ was tested in combination with cycloheximide or actinomycin D. GA₃- or cyclic AMP-treated half-seeds showed no stimulation of o-diphenolase activity.     

Differential rates of α-amylase and acid phosphatase induction by Ga3 in embryoless half-seeds and isolated aleurones of wheat

The induction of α-amylase and acid phosphatase by gibberellic acid (GA₃) was significantly higher (2–4-fold) in embryoless half-seeds of wheat than that observed in the excised aleurones. Addition of endosperm extract to excised aleurones enhanced the stimulatory effect of GA₃ on amylase activity by approximately 2-fold. Substitution of endosperm extract by 19 amino acids in GA₃-treated aleurones also brought about a 2–2.5-fold stimulation of α-amylase activity. Subsequent studies revealed that the addition of seven non-polar amino acids (0.5 mM each) was sufficient for the enhanced induction of α-amylase (1.8–2.5-fold) in GA₃-treated aleurones. A similatory effect of endosperm extract and amino acids on acid phosphatase activity was observed in GA₃-treated wheat aleurones. These observations are of physiological significance since an increased pool of free amino acids (5-fold) was also witnessed in the incubation medium of GA₃-treated half-seeds in comparison to the hormone-treated aleurones. The relative abundance of free amino acids in half-seed seems vital for the maximal induction of α-amylase and acid phosphatase. Thus, the presence of endosperm tissue associated with the aleurone layers is crucial for enhanced rate of production of GA₃-induced α-amylase and acid phosphatase in the wheat system.     

Induction of polyphenol oxidase in germinating wheat seeds

A 50- and 100-fold increase in the o-diphenolase activity was observed respectively in excised coleoptiles and roots of wheat seedlings after germination for 4–5 days. This increased activity was associated with the appearance of several new multiple forms of o-diphenolase on acrylamide gels. The embryo-less half-seeds dissected from seedlings, however, revealed only a three-fold increase in o-diphenolase activity, without any alteration in the pattern of multiple forms. Cycloheximide substantially inhibited the activity and appearance of multiple forms of o-diphenolase, whereas actinomycin D failed to bring about a similar response. Protein synthesis was probably necessary for the formation of new multiple forms. Unlike o-diphenolase activity which was present in all parts of the seedling, the monophenolase activity was confined to the embryo-less endosperm. A 5–7-fold increase in monophenolase activity was observed in the embryo-less half-seed dissected from the seedling. A single broad band of monophenolase developed on acrylamide gels. This persisted during the early period of seed germination without addition of new multiple forms. No inhibition of monophenolase activity was observed in seeds treated with cycloheximide or actinomycin D.     

Hormonal regulation of the development of protease and carboxypeptidase activities in barley aleurone layers

Carboxypeptidase and protease activities of hormone-treated barley (Hordeum vulgare cv Himalaya) aleurone layers were investigated using the substrates N-carbobenzoxy-Ala-Phe and hemoglobin. A differential effect of gibberellic acid (GA₃) on these activities was observed. The carboxypeptidase activity develops in the aleurone layers during imbibition without the addition of hormone, while the release of this enzyme to the incubation medium is enhanced by GA₃. In contrast, GA₃ is required for both the production of protease activity in the aleurone layer and its secretion. The time course for development of protease activity in response to GA₃ is similar to that observed for α-amylase. Treating aleurone layers with both GA₃ and abscisic acid prevents all the GA₃ effects described above. Carboxypeptidase activity is maximal between pH 5 and 6, and is inhibited by diisopropylfluorophosphate and p-hydroxymercuribenzoate. We have observed three protease activities against hemoglobin which differ in charge but are all 37 kilodaltons in size on sodium dodecyl sulfate polyacrylamide gels. The activity of the proteases can be inhibited by sulfhydryl protease inhibitors, such as bromate and leupeptin, yet is enhanced by 2-fold with 2-mercaptoethanol. In addition, these enzymes appear to be active against the wheat and barley storage proteins, gliadin and hordein, respectively. On the basis of these characteristics and the time course of GA₃ response, it is concluded that the proteases represent the GA₃-induced, de novo synthesized proteases that are mainly responsible for the degradation of endosperm storage proteins.     

Possible effect of gibberellin on phytate degradation in germinating barley seeds

Changes in the contents of phytate (IP₆) and other phosphorus(P)-compoundsin germinating seeds of a huskless barley were investigatedin the embryo with scutellum (EM), the starchy endosperm (EN),and the aleurone layer with pericarp-testa (AL). More than 80%of the total P in the AL of 1-day germinated seeds was foundin acid-soluble organic P, most of which was IP₆. During germination,the IP₆ in AL decreased markedly with no accumulation of lessphosphorylated myo-inositols and Pi and acid-insoluble organicP increased in the EM. The total P in the EN of 1-day germinatedseeds was about one-third that in the AL, the greater part ofwhich was found in the acid-insoluble fraction and decreasedgradually during germination. Only a small amount of IP₆ couldbe detected in the EM and EN during the early stage of germination. IP₆ in AL of embryoless half-seeds incubated without gibberellicacid (GA₃) decreased slightly even after 6 days. Incubationwith 10 ppm GA₃ remarkably stimulated the IP₆ degradation. Thisstimulation was reduced, with no change in the Pi content, byabout 80–90% with 1 mM 6-methylpurine or 10 ppm cycloheximide.The addition of 0.1 M KH₂PO₄ caused a 4-fold increase in thePi content of AL in the presence of GA₃. In addition, it suppressedthe GA₃-dependent -amylase synthesis by about 20% and the GA₃effect on IP₆ degradation by about 50%. In light of these results, IP₆ seems to be hydrolyzed completelyinto Pi and myo-inositol within the aleurone tissue, and gibberellinseems to control this process. (Received August 24, 1979; )     

Purification and characterization of gibberellic Acid-induced cysteine endoproteases in barley aleurone layers

Using in series ammonium sulfate precipitation, gel filtration, and DEAE anion exchange high performance liquid chromatography, we have purified to homogeneity a protease of M_r 37,000 secreted from barley (Hordeum vulgare L. cv Himalaya) embryoless half-seeds. This protease exists in three isozymic forms whose synthesis and secretion from barley aleurone layers was shown to be a gibberellic acid (GA₃)-dependent process (R Hammerton, T-HD Ho 1986 Plant Physiol 80: 692-697). This protease constitutes a major portion of the protease activity secreted from half-seeds between 72 to 96 hours of incubation in the presence of GA₃ as detected on activity gels containing hemoglobin as the substrate. Analysis of digestion products by urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration indicated that this protease is an endoprotease, therefore it is designated as barley endoprotease-A (EP-A). Inhibitor studies demonstrated that EP-A belongs to the cysteine class of endoproteases. The optimum pH for EP-A activity was 5.0, and the temperature optimum was 45°C. Comparison of cyanogen bromide generated peptide fragments and NH₂-terminal sequence analyses of the three individual EP-A isozymes demonstrates that they are very similar to each other. The NH₂-terminal sequence shows extensive sequence homology to the NH₂-terminal sequence of papain and several other cysteine proteinases. We also provide evidence that EP-A is not `aleurain,' a putative cysteine proteinase encoded by a GA₃-induced barley cDNA clone (JC Rogers, D Dean, GR Heck 1985 Proc Natl Acad Sci USA 82:6512-6516).     

In vitro activation of a galactosyl transferase involved in the osmotic regulation of ochromonas

Osmotic regulation in the flagellate Ochromonas malhamensis Pringsheim is mainly mediated by fluctuations in the pool size of α-galactosyl-(1→1)-glycerol (isofloridoside). A regulated key enzyme of isofloridoside metabolism is the galactosyl transferase producing isofloridoside phosphate. The activity of this enzyme in crude extracts can be increased 5- to 20-fold by incubation at pH 6. The activation occurs in a reaction with a Q₁₀ of 1.5 to 3 and is dependent on time and pH value. Inactivation of the activated form of the enzyme is also time-dependent, and is minimal at the pH value at which activation is optimal. The data suggest a regulation of the enzyme by chemical modification due to the action of auxiliary enzymes.     

Purification and comparative characterization of monophenolase and diphenolase activities from a wild edible mushroom (Macrolepiota gracilenta)

Polyphenol oxidases (PPO) are very important enzymes group in many industrial applications, especially in food, medicine and cosmetics. PPO from Macrolepiota gracilenta, a wild edible mushroom, was purified using a Sepharose 4B-l-tyrosine-p-amino benzoic acid affinity column and characterized in terms of mono- and diphenolase activity. The highest activities for pure enzyme were observed in the presence of PHPPA and DHPPA for monophenolase and diphenolase, respectively. The enzyme showed pH optimum values at 7.0 and 5.0, respectively, for monophenolase and diphenolase activities. K_m values calculated as 0.8 mM for monophenolase and 1 mM for diphenolase activity at the presence of PHPPA and DHPPA as substrate, respectively. V_max values were calculated as 2000 U/mg protein for both activity. Monophenolase and diphenolase activities were conserved approximately 40% and 60%, respectively, in their optimum pH at 4 °C after 5 day incubation. The activities were inhibited most effectively by thiourea. The data obtained from this study showed that this enzyme could be useful for some industrial purposes.     

Gibberellins and leaf expansion in near-isogenic wheat lines containing Rht1 and Rht3 dwarfing alleles

In near-isogenic lines of winter wheat (Triticum aestivum L. cv. Maris Huntsman) grown at 20° C under long days the reduced-height genes, Rht1 (semi-dwarf) and Rht3 (dwarf) reduced the rate of extension of leaf 2 by 12% and 52%, respectively, compared with corresponding rht (tall) lines. Lowering the growing temperature from 20° to 10° C reduced the rate of linear extension of leaf 2 by 2.5-fold (60% reduction) in the rht3 line but by only 1.6-fold (36% reduction) in the Rht3 line. For both genotypes, the duration of leaf expansion was greater at the lower temperature so that final leaf length was reduced by only 35% in the rht3 line and was similar in the Rht3 line at both temperatures. Seedlings of the rht3 (tall) line growing at 20° C responded positively to root-applied gibberellin A₁ (GA₁) in the range 1–10 μM GA₁; there was a linear increase in sheath length of leaf 1 whereas the Rht3 (dwarf) line remained unresponsive. Gibberellins A_{1, 3, 4, 8, 19, 20, 29, 34, 44} and ₅₃ were identified by full-scan gas chromatography-mass spectrometry in aseptically grown 4-d-old shoots of the Rht3 line. In 12-d-old seedlings grown at 20° C, there were fourfold and 24-fold increases in the concentration of GA₁ in the leaf expansion zone of Rht1 and Rht3 lines, respectively, compared with corresponding rht lines. Although GA₃ was present at a similar level to GA₁ in the rht3 (tall) line it accumulated only fivefold in the Rht3 (dwarf) line. The steady-state pool sizes of endogenous GAs were GA₁₉ ? GA₂₀ = GA₁ in the GA-responsive rht3 line whereas in the GA non-responsive Rht3 line the content of GA₁₉≈ GA₂₀ ? GA₁. It is proposed that one of the consequences of GA₁ action is suppression of GA₁₉-oxidase activity such that the conversion of GA₁₉ to GA₂₀ becomes a rate-limiting step on the pathway to GA₁ in GA-responsive lines. In the GA-non-responsive Rht lines it is suggested that GA₁₉ oxidase is not downregulated to the same extent and GA₁ accumulates before the next rate-limiting step on the pathway, its 2β-hydroxylation to GA₈. The steady-state pool sizes of GA_{19, 20, 1, 3} and ₈ were similar in developmentally equivalent tissues of the rht3 (tall) line growing at 10° C and 20° C despite a 2.5-fold difference in the rate of leaf expansion. In contrast, in the Rht3 (dwarf) line, the extent of accumulation of GA₁ reflected the severity of the phenotype at the two temperatures with slower growing tissues accumulating less, not more, GA₁. These results are interpreted as supporting the proposed model of regulation of the GA-biosynthetic pathway rather than previous suggestions that GA₁ accumulates in GA-insensitive dwarfs as a consequence of reduced growth rates.     

No Effect of 5-Fluorouracil on the Properties of Purified alpha-Amylase from Barley Half-seeds

α-Amylase has been purified from de-embryonated seeds of barley (Hordeum vulgare L. cv. Betzes) which have been incubated on 10⁻⁶ m gibberellic acid (GA₃) following 3 days of imbibition in buffer. Incubation of the half-seeds in up to 10⁻² m 5-fluorouracil (5-FU) during the entire incubation period, including imbibition, had no effect on any of the following characteristics of purified α-amylase: thermal stability in the absence of calcium, molecular weight of the enzyme, isozyme composition, specific activity, or the amount of α-amylase synthesized by the aleurone tissue. The synthesis of rRNA and tRNA was strongly inhibited by 5-FU, indicating that the analog had entered the aleurone cells. These results are not in agreement with those of Carlson (Nature New Biology 237: 39-41 [1972]) who found that treatment of barley aleurone with 10⁻⁴ m 5-FU prior to the addition of GA₃ resulted in decreased thermal stability of GA₃-induced α-amylase and who interpreted this as evidence that the mRNA for α-amylase was synthesized during the imbibition of the aleurone tissue and independently of gibberellin action. Results of the present experiments indicate that the thermal stability of highly purified α-amylase is not altered by treatment of barley half-seeds with 5-FU, and that 5-FU cannot be used as a probe to examine the timing of α-amylase mRNA synthesis.     

Changes in tyrosinase specificity by ionic liquids and sodium dodecyl sulfate

Tyrosinase is a member of the type 3 copper enzyme family involved in the production of melanin in a wide range of organisms. The ability of tyrosinases to convert monophenols into diphenols has stimulated studies regarding the production of substituted catechols, important intermediates for the synthesis of pharmaceuticals, agrochemicals, polymerization inhibitors, and antioxidants. Despite its enormous potential, the use of tyrosinases for catechol synthesis has been limited due to the low monophenolase/diphenolase activity ratio. In the presence of two water miscible ionic liquids, [BMIM][BF₄] and ethylammonium nitrate, the selectivity of a tyrosinase from Bacillus megaterium (TyrBm) was altered, and the ratio of monophenolase/diphenolase activity increased by up to 5-fold. Furthermore, the addition of sodium dodecyl sulphate (SDS) at levels of 2–50 mM increased the activity of TyrBm by 2-fold towards the natural substrates l-tyrosine and l-Dopa and 15- to 20-fold towards the non-native phenol and catechol. The R209H tyrosinase variant we previously identified as having a preferential ratio of monophenolase/diphenolase activity was shown to have a 45-fold increase in activity towards phenol in the presence of SDS. We propose that the effect of SDS on the ability of tyrosinase to convert non-natural substrates is due to the interaction of surfactant molecules with residues located at the entrance to the active site, as visualized by the newly determined crystal structure of TyrBm in the presence of SDS. The effect of SDS on R209 may enable less polar substrates such as phenol and catechol, to penetrate more efficiently into the enzyme catalytic pocket.     

Modulation by gibberellic acid and adenosine-3′,5′-cyclic monophosphate of starch hydrolysing activity of cowpea seedlings

In cowpea seedlings starch hydrolysing activity increases 35–50 fold on germination for 4 days. This increase in enzyme activity was inhibited by the in vivo addition of 1% glucose but this inhibition was completely overcome by the addition of gibberellic acid (GA₃) (10^?5 M) and adenosine-3′,5′-cyclic monophosphate (cAMP) (10^?5 M). At 5% glucose, GA₃ and cAMP were only partially effective. Structural analogues of cAMP failed to relieve the inhibitory effect of glucose. The inhibition by glucose is not direct but RNA and protein synthesis may be involved. Glucose appears to reduce the internal pool of cAMP which causes inhibition of RNA synthesis and decrease in starch hydrolysing activity. Exogenous application of cAMP may replenish the endogenous pool of cyclic nucleotide and thus overcome inhibition of RNA synthesis and enzyme activity.     

Effects of ascorbic acid and gibberellin A3 on alleviation of salt stress in common bean (Phaseolus vulgaris L.) seedlings

Salinity, a severe environmental factor, has limited the growth and productivity of crops. Many compounds have been applied to minimize the harmful effects of salt stress on plant growth. An experiment was conducted to investigate the interactive effects of exogenous ascorbic acid (AsA) and gibberellic acid (GA₃) on common bean (Phaseolus vulgaris L. cv. Naz) seedlings under salt stress. The changes of growth parameters, photosynthetic and non-photosynthetic pigments and potassium content showed that the addition of 1 mM AsA and/or 0.05 mM GA₃ considerably decreased the oxidative damage in common bean plants treated with 200 mM NaCl. The NaCl-stressed seedlings exposed to AsA or GA₃, specifically in their combination, exhibited an improvement in sodium accumulation in both roots and shoots, as compared to NaCl-treated plants. NaCl treatment increased hydrogen peroxide (H₂O₂) content and lipid peroxidation indicated by accumulation of malondialdehyde (MDA), whereas the interaction of AsA with GA₃ decreased the amounts of MDA and H₂O₂. In the meantime, interactive effect of these substances enhanced protein content and the activity of the antioxidant enzyme, guaiacol peroxidase, in common bean plants under salt stress. It was concluded that synergistic interaction between AsA and GA₃ could alleviate the adverse effects of salinity on P. vulgaris seedlings.     

Growth and pigment content of wheat as influenced by the combined effects of salinity and growth regulators

A field experiment was conducted to investigate the effects of presoaking the wheat grains (Triticum aestivum L.) in different levels of salinity (33 or 66 mM) and in growth regulators (indolyl-3-acetic acid, IAA at SO g m^-3, gibberellic acid, GA₃ at 100 g m^-3, or kinetin at 100 g m_-3) on the shoot growth and pigment content of the developing wheat flag leaf. Salinity at 33 or 66 mM led to an insignificant increase in the fresh and dry masses as well as in the shoot diameter and shoot length, but it attenuated the flag leaf area. In the majority of cases, salinity increased the chlorophyll (Chla, Chlb) and carotenoid contents as well as the number of chloroplasts per a mesophyll cell. The growth in the wheat shoot of the saline-treated plants was, in general, stimulated in response to presoaking the grains in kinetin or GA₃. On the other hand, IAA + salinity led to a negligible effect on the growth in the wheat plants particularly at the early stages of growth. The presoaking of grains in NaCl at 33 mM + IAA or 66 mM + kinetin induced a marked increase in the pigment content of the wheat flag leaf particularly at the early stages of growth. The interaction between salinity and phytohormones increased the number of chloroplasts; kinetin was the most effective.     

Enhanced production of gibberellin A4 (GA4) by a mutant of Gibberella fujikuroi in wheat gluten medium

Mutants of Gibberella fujikuroi with different colony characteristics, morphology and pigmentation were generated by exposure to UV radiation. A mutant, Mor-189, was selected based on its short filament length, relatively high gibberellin A₄ (GA₄) and gibberellin A₃ (GA₃) production, as well as its lack of pigmentation. Production of GA₄ by Mor-189 was studied using different inorganic and organic nitrogen sources, carbon sources and by maintaining the pH of the fermentation medium using calcium carbonate. Analysis of GA₄ and GA₃ was done by reversed-phase high-performance liquid chromatography and LC-MS. The mutants of G. fujikuroi produced more GA₄ when the pH of the medium was maintained above 5. During shake flask studies, the mutant Mor-189 produced 210 mg l^?1 GA₄ in media containing wheat gluten as the nitrogen source and glucose as the carbon source. Fed-batch fermentation in a 14 l agitated fermenter was performed to evaluate the applicability of the mutant Mor-189 for the production of GA₄. In 7-day fed-batch fermentation, 600 mg l^?1 GA₄ were obtained in the culture filtrate. The concentration of GA₄ and GA₃ combined was 713 mg l^?1, of which GA₄ accounted for 84% of the total gibberellin. These values are substantially higher than those published previously. The present study indicated that, along with maintenance of pH and controlled glucose feeding, the use of wheat gluten as the sole nitrogen source considerably enhances GA₄ production by the mutant Mor-189.     

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.