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.     

Inorganic phosphate mimics the specific action of gibberellic acid in regulating the activity of monophenolase in embryo-less half-seeds of wheat

The GA₃-mediated activation of monophenolase (about 12-fold stimulation) in embryo-less half-seeds of wheat was mimicked by the addition of inorganic phosphate (Pi, 50 mM). Similarly, the hormone-induced altered molecular properties of monophenolase, such as the shift in the pH optimum towards alkalinity (pH 9.0), the relatively increased thermostability of the enzyme at 55° and the changed pattern of multiple forms of the enzyme, were also seen in Pi-treated half-seeds. Furthermore, the simultaneous addition of GA₃ and Pi to wheat half-seeds showed no cumulative effect on the enhancement of monophenolase activity. This indicated that both GA₃ and Pi regulated monophenolase activity through a common mechanism. Abscisic acid effectively blocked the GA₃- and Pi-directed stimulation of monophenolase. Since GA₃ treatment of half-seeds increased the free pool of Pi (2.5-fold), we envisage that the Pi-mediated activation of monophenolase is of physiological relevance in our system. However, the in vitro addition of Pi (50 mM) to water-imbibed control half-seeds (48 hr) during enzyme extraction failed to activate monophenolase. We thus consider that the high pool of Pi, generated by GA₃ in half-seeds, or even the direct addition of Pi to half-seeds in vivo, seems necessary for some metabolic events which eventually trigger the activation of monophenolase.     

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.     

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).     

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.     

Calmodulin stimulation of unidirectional calcium uptake by the endoplasmic reticulum of barley aleurone

The role of calmodulin (CaM) in gibberellic acid (GA₃)-stimulated Ca²⁺ uptake was investigated in endomembranes isolated from aleurone cells of barley (Hordeum vulgare L.). Unidirectional Ca²⁺ -uptake activity of endoplasmic reticulum (ER) was higher in membranes isolated from aleurone layers treated for 16 h with GA₃ and Ca²⁺ compared with those isolated from layers incubated in Ca²⁺ alone. However, the level of uptake from Ca²⁺-treated tissue could be stimulated to that of the GA₃-treated cells by applying exogenous CaM which increased the V _max of the Ca²⁺ transporter approximately threefold. Calcium uptake in ER from GA₃-treated tissue was inhibited by the CaM antagonist W7 in 50% of experiments, whereas the activity in membranes from non-GA₃-treated tissue was unaffected. Treatment with GA₃ also led to a twofold increase in CaM levels in aleurone layers within 4–6 h, paralleling the time course of the stimulation of Ca²⁺ uptake and preceding the stimulation of α-amylase secretion. We propose that the elevation of Ca²⁺ uptake into the ER induced by GA₃ may be coordinated and regulated by elevated levels of membrane-associated CaM and this may regulate Ca²⁺-dependent α-amylase synthesis in the lumen of the ER.     

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.     

Sucrose metabolism and fruit growth in parthenocarpic vs seeded blueberry (Vaccinium ashei) fruits

Although fruit set and development are induced by applications of gibberellins, final fruit weight of gibberellin-induced parthenocarpic fruit is often less than that of pollinated fruit. We examined changes in the activities of sucrose-metabolizing enzymes and sugar accumulation in developing fruits of cultivated blueberry (Vaccinium ashei Reade) and their correlation with fruit growth upon pollination or exogenous applications of gibberellic acid (GA₃). The objective was to determine if differences in fruit growth could be attributed to differences in enzyme activities and subsequent sugar accumulation in fruits. The fruit development period of GA₃-treated fruits was 15 days longer than that of pollinated fruits. At maturity, GA₃-treated fruit accumulated an average of 180 mg dry weight while pollinated fruit accumulated 390 mg dry weight. Dry weight accumulation in nonpollinated fruits was negligible and these fruits abscised by 45 days after bloom (DAB). The total carbon (C) cost (dry weight C + respiratory C) for fruit development was 109 and 244 mg C fruit^-1 for GA₃-treated and pollinated fruits, respectively. Hexose concentration increased to 100 mg (g fresh weight)^-1 at ripening in both GA₃-treated and pollinated fruits. Nonpollinated fruits reached a maximum hexose concentration at 45 DAB. Sucrose phosphate synthase (EC 2.4.1.14) and sucrose synthase (EC 2.4.1.13) activities reached a maximum of ≤5.0 μmol (g fresh weight)^-1 h^-1 in both GA₃-treated and pollinated fruits. Soluble acid invertase (EC 3.2.1.26) activity increased to about 60 μmol (g fresh weight)^-1 h^-1 in both GA₃-treated and pollinated fruits at ripening, while in nonpollinated fruits, a maximum soluble acid invertase activity of 0.12 μmol (g fresh weight)^-1 h^-1 was measured at 24 DAB. Insoluble acid invertase activity declined during the early stages of fruit growth and remained relatively low throughout fruit development. Neutral invertase activity was low throughout development, increasing to 5 μmol (g fresh weight)^-1 h^-1 at ripening in GA₃-treated and pollinated fruits. Our studies demonstrate that blueberry fruit development does not appear to be limited by sucrose metabolizing enzyme activity and/or the ability to accumulate sugars in either GA₃-treated or pollinated fruits.     

Purification, Immobilization, and Some Properties of Glucose Isomerase from Streptomyces flavogriseus

Glucose isomerase (EC 5.3.1.5) produced from Streptomyces flavogriseus was purified by fractionation with (NH₄)₂SO₄ and chromatography on diethylaminoethyl (DEAE)-cellulose and DEAE-Sephadex A-50 columns. The purified enzyme was homogeneous as shown by ultracentrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Benzyl DEAE-cellulose, triethylaminoethyl-cellulose, and DEAE-cellulose were effective in the immobilization of partially purified glucose isomerase. Several differences in properties were found between purified soluble enzyme, immobilized enzyme (DEAE-cellulose-glucose isomerase), and heat-treated whole cells. Glucose and xylose served as substrate for the enzyme. Whole cells had the highest K_m values for glucose and xylose; the soluble enzyme had the lowest values. The optimum temperature for activity of the soluble and immobilized enzymes was 70°C; that for whole cells was 75°C. The pH optimum for the three enzyme preparations was 7.5. Magnesium ion or Co²⁺ was required for enzyme activity; an addition effect resulted from the presence of both Mg²⁺ and Co²⁺. The enzyme activity was inhibited by Hg²⁺, Ag⁺, or Cu²⁺. The conversion ratio of the enzyme for isomerization was about 50%. The soluble and immobilized enzymes showed a greater heat stability than whole cells. The soluble enzyme was stable over a slightly wider pH (5.0 to 9.0) range than the immobilized enzyme and whole cells (pH 5.5 to 9.0). The molecular weight of the enzyme determined by the sedimentation equilibrium method was 171,000. A tetrameric structure for the enzyme was also indicated. After operating at 70°C for 5 days, the remaining enzyme activity of the immobilized enzyme and whole cells, which were used for the continuous isomerization of glucose in a plug-flow type of column in the presence of Mg²⁺ and Co²⁺, was 75 and 55%, respectively. Elimination of Co²⁺ decreased operational stability.     

The activity of glutaminase in the human placenta

Homogenates and extracts of human placenta are able to desamidate glutamine by means of an enzyme which has the properties of glutaminase. Placental glutaminase is activated by phosphate. Its pH optimum lies at 9.0.A method for its assay in placental homogenate is described. It was found that the glutaminase activity decreases toward the end of pregnancy. At this time, the activity, expressed as Q_NH3 (N), amounts to 23.7 ± 6.7.Some quantitative aspects of glutaminase activity in the human placenta and kidney are discussed.     

The Occurrence and Development of Amylase Enzymes in Incubated, De-embryonated Maize Kernels

The development of amylase activity in extracts from de-embryonated and GA₃-treated de-embryonated maize kernels (Zea mays L.) was determined during a 10-day incubation period. The increase in activity was compared with activity extracted from endosperms dissected from germinating whole kernels. Chromatographic analysis of reaction products as well as physicochemical characterization demonstrated that the activities from GA₃-treated and nontreated tissue were comparable and that part of the activity was attributable to α-amylase.     

Localization of ATPase in the endoplasmic reticulum and golgi apparatus of barley aleurone

Summary The cytochemical localization of adenosine triphosphatase (ATPase) was studied in the aleurone layer of barley (Hordeum vulgare L. cv. Himalaya). Isolated barley aleurone layers secrete numerous enzymes having acid phosphatase activity, including ATPase. The secretion of these enzymes was stimulated by incubation of the aleurone layer in gibberellic acid (GA₃). ATPase was localized using the metal-salt method in tissue incubated in CaCl₂ with and without GA₃. In sections of tissue incubated without GA₃, cytochemical staining was confined to a narrow band of cytoplasm adjacent to the starchy endosperm and to the cell wall of the innermost tier of aleurone cells. Cytochemical staining was absent from the organelles of tissues not treated with GA₃. In tissue incubated in the presence of GA₃, cytochemical staining was evident throughout the cytoplasm and cell walls of the tissue. In the cell wall, electron-dense deposits were found only in digested channels. The cell-wall matrix of GA₃-treated aleurone did not stain, indicating that it does not permit diffusion of enzyme. In the cytoplasm of GA₃-treated aleurone, all organelles except microbodies, plastids, and spherosomes stained for ATPase activity; endoplasmic reticulum (ER), Golgi apparatus, and mitochondria showed intense deposits of stain. The ER of the aleurone is a complex system made up of flattened sheets of membrane, which may be associated with both the Golgi apparatus and the plasma membrane. The dictyosome did not stain uniformly for ATPase activity; rather there was a gradation in staining of the cisternae from thecis (lightly stained) to thetrans (heavily stained) face. Vesicles associated with dictyosome cisternae also stained intensely as did the protein bodies of GA₃-treated aleurone cells.     

Gibberellic Acid stimulation of cucumber hypocotyl elongation : effects on growth, turgor, osmotic pressure, and cell wall properties

Recently developed techniques have been used to reinvestigate the mechanism by which gibberellic acid (GA₃) stimulates elongation of light-grown cucumber (Cucumis sativus L.) seedlings. Osmotic pressure and turgor pressure were slightly reduced in GA₃-treated seedlings, which elongated 3.5 times faster than control seedlings. This indicated that GA₃ enhancement of growth was not controlled by changes in the osmotic properties of the tissues. Stress/strain (Instron) analysis revealed that plastic extension of the cell walls of GA₃-treated seedlings increased by up to 35% above the control values. Stress-relaxation measurements on frozen-thawed tissue showed that T₀ the minimum relaxation time, was reduced following application of GA₃. In vivo wall relaxation (measured by the pressure block technique) showed that the wall yield coefficient was increased, and the yield threshold was slightly reduced. Thus GA₃ affected both the mechanical (viscoelastic) and biochemical (chemorheological) properties of the cell walls of light-grown cucumber. The previous hypothesis, that GA₃ stimulates cucumber hypocotyl growth by increasing osmotic pressure and cell turgor, is contradicted by our results.     

Effects of gibberellic acid and uniconazole on the activities of some enzymes of anthocyanin biosynthesis in carrot cell cultures

Gibberellic acid (GA₃) inhibition of anthocyanin accumulation by carrot cell-suspension cultures was reversed by supplying dihydroquercitin or naringenin to the culture and not by supplying 4-coumaric acid or malonic acid. This suggested that gibberellic acid was inhibiting chalcone synthase, chalcone isomerase, or acetyl CoA carboxylase. Acetyl-CoA-carboxylase specific activity was the same in GA₃-treated and untreated cultures and was not detected in cultures treated with uniconazole, an inhibitor of gibberellic acid biosynthesis. Chalcone-isomerase specific activity was lower in GA₃-treated cultures than in untreated cultures and was lower in uniconazole-treated cultures than in the GA₃-treated cultures. The total chalcone synthase activity in extracts from GA₃- and from uniconazole-treated cells was not significantly different from that in extracts of untreated tissue. When these extracts were chromatographed on a Mono Q column, three peaks of chalcone synthase activity were found in extracts of nontreated cells, whereas only two of these peaks were detected in extracts of GA₃-treated cells. The extracts from GA₃-treated cells did not contain the peak of chalcone synthase activity that, in untreated cells, preceded the main peak. The correlation between the absence of this peak and the inhibition of anthocyanin accumulation suggests that this form of chalcone synthase is responsible for anthocyanin synthesis and that GA₃ prevents this form from appearing in the cells.     

Promotion of growth and invertase activity by gibberellic Acid in developing Avena internodes

Gibberellic acid (GA₃) induces invertase activity within 6 hours in Avena stem segments that are incubated in the dark at 23°. The maximum amount of promotion is about 5 times that of invertase activity in untreated segments. GA₃ causes significant promotion of invertase activity at concentrations as low as 3 × 10⁻⁵ μm GA₃. The increase in invertase activity elicited by GA₃ between 3 × 10⁻⁵ μm and 300 μm closely parallels the growth promotion that is caused by GA₃ over this concentration range. In control segments, invertase activity rises steeply during the first 6 hours of incubation, then decays slowly between 12 and 48 hours. In GA₃-treated segments, the invertase activity also rises during the first 6 hours, parallel to that in control segments and continues to rise during the next 42 hours. These changes in invertase activity during 48-hour incubation periods do not parallel the changes in growth that occur in control and GA₃-treated segments. Cycloheximide at 10 μg/ml abolishes all GA₃-promoted growth and invertase activity in these segments. Actinomycin D at 40 and 80 μg/ml decreases GA₃-promoted growth by 20% and invertase activity by 38 and 44%, respectively. The data clearly support the idea that protein synthesis is necessary for GA₃-promoted growth and invertase activity in Avena stem segments.     

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.     

Quantitative and qualitative changes in the endoplasmic reticulum of barley aleurone layers

Changes in the level of the endoplasmicreticulum (ER) marker enzyme cytochrome-c reductase (EC 1.6.2.1) were followed with time of imbibition of de-embryonated half-seeds of barley (Hordeum vulgare L.) and the subsequent incubation of their aleurone layers in gibberellic acid (GA₃) and H₂O. During imbibition there is an increase in the level of cytochrome-c-reductase activity and in the amount of 280-nm absorbance associated with this enzyme. When aleurone layers are incubated for a further 42 h in water, there is a doubling of the cytochrome-c-reductase activity. In GA₃, the activity of cytochrome-c reductase reaches a maximum at 24 h of incubation and thereafter falls to below 70% of its level at the beginning of the incubation period. Changes in the cytochrome-c-reductase activity correlate with changes in the fine structure of the aleurone cell. The ER isolated in low Mg²⁺ from aleurone layers incubated in buffer for up to 18 h has buoyant density of 1.13–1.14 g cc^-1 while that from layers incubated in GA₃ for 7.5–18 h has a density of 1.11–1.12 g cc^-1. The -amylase (EC3.2.1.1) isolated with the organelle fraction by Sepharose gel filtration is associated with the ER on isopycnic and rate-zonal density gradients, and its activity can be enhanced by Triton X-100. The soluble -amylase fraction from Separose-4B columns, on the other hand, is not Triton-activated but is acid-labile. Acid phosphatase (EC3.1.3.2) is distributed in at least three peaks on isopycnic gradients. In low Mg²⁺ the second peak of activity has a density of 1.12 g cc^-1 in GA₃-treated tissue and 1.13–1.14 g cc^-1 in H₂O-treated tissue. With high-Mg²⁺ buffers, this peak of phosphatase activity disappears. Acid-phosphatase activity is not enhanced by Triton X-100 nor is it acid-labile.Abbreviations EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - GA gibberellin - GA₃ gibberellic acid     

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.     

Localization of gibberellic acid-induced acid phosphatase activity in the endoplasmic reticulum of barley aleurone cells with the electron microscope

A metal-salt precipitation method with p-nitrophenyl phosphate as substrate has been used to localize in the electron microscope acid phosphatase activity in isolated aleurone layers of barley (Hordeum vulgare L.), treated for 16 h in the presence or absence of gibberellic acid (GA₃). The paper confirms results obtained earlier with an azo-dye precipitation method of enzyme localization. In addition the results show for the first time that in GA₃-treated tissue enzyme activity is associated with the endoplasmic reticulum (ER), there being reaction product deposited in the ER cisternae. It is suggested that this activity represents new enzyme synthesized on ER in response to GA₃ and probably destined for secretion.Abbreviation ER endoplasmic reticulum     

Cytokinin oxidase/dehydrogenase activity as a tool in gibberellic acid/cytokinin cross talk

Changes in endogenous cytokinin (CK) content and cytokinin oxidase/dehydrogenase activity (CKX) in response to gibberellic acid (GA₃) in two pea cultivars with different life span were assessed. The control leaves of cv. Scinado, which developed faster, had higher initial cytokinin content and lower CKX activity, while opposite trend was observed in cv. Manuela with longer life span. Increased CKX and decreased CK content were detected in leaves of cv. Scinado after treatments with 0.5, 1 and 5 μM GA₃. Changes in CK content and CKX activity in GA₃-treated cv. Manuela leaves were reciprocal to those in cv. Scinado. CK content and CKX activity in roots were not significantly influenced by the application of GA₃. The slight repression of CKX activity in some of the root samples was accompanied by increased isopentenyladenine and isopentenyladenine riboside content. Obtained results suggest that CKX was responsible for the changes in endogenous cytokinin pool in GA₃-treated plants and most probably this enzyme represents an important link in GA/cytokinin cross talk.