The Regulatory Role of the Embryo on the Development of Isocitrate Lyase Activity during Germination of Ponderosa Pine Seeds

The specific activity of isocitrate lyase rapidly increased in the megagametophytic tissue of cold-stratified seeds of ponderosa pine (Pinus ponderosa Laws) prior to and after germination. When the embryo was removed at germination, isocitrate lyase activity continued to develop. However, in the total absence of the embryo, only a small increase in the specific activity of the enzyme was observed. The development of the enzyme was inhibited by cycloheximide, actinomycin D and abscisic acid. The embryo produced an unidentified factor which enhanced the development of isocitrate lyase activity in the megagametophytic tissue. This embryo factor could not be replaced by the hormones indoleacetic acid (IAA), gibberellic acid (GA₃) or benzylaminopurine (BA). Indoleacetic acid had little effect upon enzyme development. Gibberellic acid and benzylaminopurine inhibited isocitrate lyase development in the megagametophytic tissue of the seed.     

The Effects of Octanoate and Oleate on Isocitrate Lyase Activity during the Germination of Pinus pinea Seeds

The changes of isocitrate lyase levels with respect to the catabolism of triglycerides have been studied during the germination of Pinus pinea seeds. We studied the effects of octanoate, oleate, and inhibitors of protein synthesis on isocitrate lyase during germination. Pyruvate kinase, glucose-6-P-dehydrogenase, malate dehydrogenase, and isocitrate dehydrogenase were also assayed. Octanoate and oleate inhibited the isocitrate lyase activity, similarly to cycloheximide, chloramphenicol, and actinomycin, inhibitors of protein biosynthesis. This inhibitory effect is not specific but is strikingly evident with isocitrate lyase. This inhibition was not proportional to the concentration but was proportional to the chain length of oleate and octanoate.     

Enzyme Profiles in Seedling Development and the Effect of Itaconate, an Isocitrate Lyase-directed Reagent

Changes in levels of isocitrate lyase, malate synthase, and catalase have been investigated during germination of flax (Linum usitatissimum L.) in the presence and absence of itaconate. Germination was accompanied by a rapid increase in these enzymes during the first 3 days. The presence of 38 millimolar itaconate inhibited the incidence of seed germination and the growth of embryo axes as well as the appearance of isocitrate lyase but did not alter the levels of malate synthase, catalase, or NADP⁺-isocitrate dehydrogenase. The specific activity for the latter enzyme was constant throughout germination. Oxalate or succinate, each at 38 millimolar, had no effect upon germination of flax seeds. Itaconate did not inhibit the activities of malate synthase, catalase, or NADP⁺-isocitrate dehydrogenase in vitro but was a potent noncompetitive inhibitor of isocitrate lyase (K_i:17 micromolar at 30 C, pH 7.6). Itaconate (at 38 millimolar) did not alter the appearance of malate synthase but reduced the incidence of germination, onset of germination, and growth of the embryo axis as well as the specific activity of isocitrate lyase in seedlings of Zea mays, Vigna glabra, Glycine hispida, Vigna sinensis, Trigonella foenumgraecum, Lens culinaris, and Medicago sativa. The incidence and onset of germination of wheat seeds were unaltered by the same concentration of itaconate but seedlings did not contain isocitrate lyase or malate synthase. The data suggest that itaconate may be isocitrate lyase-directed in inhibiting the germination of fatty seeds.     

Distribution and development of nitrate reductase activity in germinating cotton seedlings

Activity of nitrate reductase in roots and cotyledons of cotton seedings (Gossypium hirsutum L. cv. Deltapine 16) increased rapidly on germination, reaching a maximum after 1 day of imbibition. Thereafter, activity declined until emergence and greening of the cotyledons, when it again began to increase steadily. Germinating soybean (Glycine max (L.) Merrill cv. Merit) and sunflower (Helianthus annuus L. cv. Peredovic) seedlings did not show the early peak of activity. The early peak depended on nitrate and was sensitive to cycloheximide, but not to actinomycin D or other inhibitors of RNA synthesis. The second, light-dependent increase was sensitive to actinomycin D. In roots, the early peak of activity occurred before any growth. After emergence of the root tip from the seed coat, activity was localized in the terminal 2 millimeters, whether expressed on a fresh weight, protein, or root basis. The difference in activity between the apical (0-2 millimeter) and subapical (2-4 millimeter) segments did not result from differences in nitrate availability, energy supply, or turnover rates of nitrate reductase. Root activity was similar to that of the cotyledons after emergence, in that both were sensitive to actinomycin D.     

Differential light induction of nitrate reductases in greening and photobleached soybean seedlings

Soybean (Glycine max [L.] Merr.) seeds were imbibed and germinated with or without NO₃⁻, tungstate, and norflurazon (San 9789). Norflurazon is a herbicide which causes photobleaching of chlorophyll by inhibiting carotenoid synthesis and which impairs normal chloroplast development. After 3 days in the dark, seedlings were placed in white light to induce extractable nitrate reductase activity. The induction of maximal nitrate reductase activity in greening cotyledons did not require NO₃⁻ and was not inhibited by tungstate. Induction of nitrate reductase activity in norflurazon-treated cotyledons had an absolute requirement for NO₃⁻ and was completely inhibited by tungstate. Nitrate was not detected in seeds or seedlings which had not been treated with NO₃⁻. The optimum pH for cotyledon nitrate reductase activity from norflurazon-treated seedlings was at pH 7.5, and near that for root nitrate reductase activity, whereas the optimum pH for nitrate reductase activity from greening cotyledons was pH 6.5. Induction of root nitrate reductase activity was also inhibited by tungstate and was dependent on the presence of NO₃⁻, further indicating that the isoform of nitrate reductase induced in norflurazon-treated cotyledons is the same or similar to that found in roots. Nitrate reductases with and without a NO₃⁻ requirement for light induction appear to be present in developing leaves. In vivo kinetics (light induction and dark decay rates) and in vitro kinetics (Arrhenius energies of activation and NADH:NADPH specificities) of nitrate reductases with and without a NO₃⁻ requirement for induction were quite different. K_m values for NO₃⁻ were identical for both nitrate reductases.     

Differential effects of actinomycin d and cordycepin in lettuce seed germination and RNA synthesis

Intact lettuce seed germination was inhibited by cordycepin but not by actinomycin D; however, when seeds were clipped at the cotyledonary end, actinomycin D partially inhibited germination. Uptake studies with intact seeds using ³H-actinomycin D showed that it was unable to reach the embryo prior to radical protrusion. ³H-Cordycepin uptake studies using intact seeds showed that cordycepin was able to reach the embryo during the first 3 hours of incubation and at subsequent times. The pericarp and endosperm offered resistance to penetration of cordycepin into the embryo. In contrast to actinomycin D, cordycepin markedly inhibited ³H-uridine incorporation into RNA of intact seeds during the first 10 and 12 hours of incubation. About 60% of ³H-adenosine incorporation into poly A-RNA was inhibited by cordycepin during 12 hours of incubation, whereas actinomycin D had little effect. RNA synthesis appears to be essential for seed germination.     

The induction of enzyme activity in the endosperm of germinating castor-bean seeds.

Endosperm extracts were prepared at various times during germination from intact castor-bean seeds and from seeds from which the embryos had been removed. The sterilized seeds were incubated either on solid water agar or on agar containing 0.3 mM-gibberellic acid. 2. Isocitrate lyase and 3-hydroxyacyl-CoA dehydrogenase had very low activities in the mature seeds, but increased 44-fold and 27-fold respectively during germination. In contrast, the extracts of mature seeds had considerable acid and alkaline lipase activity and this only increased two- to three-fold during the incubation period. 3. Incubation of the seeds with gibberellic acid accelerated the rate of appearance of isocitrate lyase and 3-hydroxyacyl-CoA dehydrogenase. It also increased the total activity attained. However, the application of hormone had, in comparison, little effect on the development of lipase activity. 4. The removal of the embryo had little influence on the development of enzyme activity in the endosperm tissue; only with isocitrate lyase was a decrease in activity observed in the absence of the embryo.     

Nitrate Reductase Independent Stimulation of Seed Germination in Sisymbrium officinale L. (Hedge Mustard) by Light and Nitrate

After a 72 h preincubation in darkness at 15 °C seed germinationof Sisymbrium officinale L. (hedge mustard) at 24 °C wasstimulated by a combination of red light and nitrate. In thepresence of nitrate the seeds escaped from the inhibiting effectof far-red irradiation with an escape time of approx. 8 h. Afterred light, the exposure of seeds to nitrate could be delayedfor 3 h without affecting maximal germination. Prolonged delayresulted in a decrease of the germination response. The possibilitythat nitrate reduction was involved in the stimulation of germinationwas studied by pre-incubating seeds for 72 h in nitrate andsubsequently transferring them to water and irradiating withred light. During the first 8 h period after the red irradiationin which induction of germination occurred, total nitrate levels(endogenous + leachate) remained constant, indicating an absenceof nitrate reductase activity. During the next 8 h visible germinationstarted and total nitrate levels declined, suggesting inductionof nitrate reduction. It is concluded that nitrate reductiondocs not play a role in the induction of germination. The conclusionwas supported by the lack of inhibition of seed germinationby sodium chlorate and sodium tungstate in spite of an inhibitionof nitrate reduction of 80 and 100%, respectively. The contrastsbetween our results and hypotheses concerning the mechanismof action of nitrate in seed germination are discussed Sisymbrium officinale L., hedge mustard, germination, light, nitrate, nitrate reductase     

The action of exogenous gibberellic acid on isocitrate lyase —mRNA in germinating castor bean seeds

Gibberellic acid (GA₃) stimulates isocitrate lyase activity of the endosperm during germination of castor bean seeds. Isocitrate lyase from castor bean was purified and an antibody to it was prepared from rabbit serum. This antibody was used to measure the amounts of isocitrate lyase-mRNA using an in vitro translation system. No specific stimulation of isocitrate lyase-mRNA by application of GA₃ was detected. The stimulation of isocitrate lyase activity by exogenous GA₃ may be accounted for by the action of the growth substance in advancing the overall production of rRNA and mRNA which accelerates the rate of total protein synthesis during germination. The application of Amo 1618 retards the production of isocitrate lyase activity but also retards protein synthesis in general. This suggests that endogenous gibberellins also act non-specifically in the regulation of protein synthesis during castor bean germination.Abbreviations SDS sodium dodecyl sulphate - GA₃ gibberellic acid - PAGE polyacrylamide gel electrophoresis     

Independent control of fiber development and nitrate reduction in cultured cotton ovules

Several lines of evidence implicate ammonium as an important factor in the growth and development of cotton (Gossypium hirsutum L.) ovules cultured in vitro. For example, ovules cultured at 28 C require indoleacetic acid (IAA) and either ammonium or gibberellic acid (GA₃) in the medium for fiber development, whereas ovules cultured at 34 C require only IAA. Because of this effect of ammonium supply, it seemed possible that hormones or increased temperature were also promoting the availability of reduced nitrogen by induction of increased nitrate reductase activity in the ovules. This possibility was tested.     

Control of phenylalanine hydroxylase synthesis in tissue culture by serum and insulin.

Stationary-phase, minimal deviation hepatoma H4-II-E-C3 cell cultures that are serum-deprived respond with a biphasic time course of phenylalanine hydroxylase induction when dialyzed fetal calf serum or insulin is added. These two agents induce phenylalanine hydroxylase additively, during both the initial 3-hour and the delayed 24-hour phases. The initial phase of induction by insulin is inhibited by cycloheximide but not by actinomycin D. The delayed induction by both dialyzed fetal calf serum and insulin is inhibited by 10(-6) M cycloheximide and 0.20 mug/ml actinomycin D. H4-II-E-C3 cells in culture do not synthesize the factor(s) in serum that induce phenylalanine hydroxylase.     

Nitrate and nitrate reductase in Erythrina caffra seeds: Enhancement of induction by anoxia and possible role in germination

The nitrate level in seed embryonic axes of Erythrina caffra Thunb. which is capable of anaerobic germination, was about 2.5 times higher than in seed axes of Pisum sativum L. a species incapable of anaerobic germination. Nitrate levels in E. caffra seeds decreased during germination and this was not due to leaching. Both NADH- and NADPH-dependent nitrate reductase (NAR) activities increased during germination. The increase was prevented by cycloheximide. The activity of NADH-NAR (EC 1.6.6.1) was higher than that of NADPH-NAR (EC 1.6.6.3). Both NAR activities were higher in anoxia than in air during germination. The NAR activities in Pisum seeds were very much lower than in Erythrina seeds. Anoxia (N₂ or argon) enhanced the induction of NAR by KNO₃ in germinated E. caffra axes. The NADH- and NADPH-NAR activities were induced to equally high levels by KNO₃ under anoxia. The enhancement was depressed by cycloheximide. It is concluded that nitrate and NAR activity may play a role in the anaerobic germination of E. caffra seeds.Abbreviation NAR nitrate reductase Financial support was obtained from the University of the Orange Free State and the Foundation for Research Development     

Isocitrate lyase: artifacts and multiple enzyme forms

Multiple enzyme forms of isocitrate lyase from various sources have been frequently reported. Protease action after cell rupture was sporadically claimed to explain the observed multiple enzyme forms. In this communication studies which are consistent with a protease action in vitro on isocitrate lyase of Pinus pinea germinating seeds are reported. Moreover, changes in DEAE-Sephacel patterns, mainly related to the age of germination, were observed. Differences regarding the heat stability of the detected enzyme forms were also found. The results indicate that isocitrate lyase from P. pinea may be detected in at least three different forms, one of which is heat stable and may be obtained only at the early stages of germination.     

DEVELOPMENTAL STUDIES ON GLYOXYSOMES IN RICINUS ENDOSPERM

The development of glyoxysomes and their associated enzymes, isocitrate lyase and malate synthetase, was studied in the endosperm of castor bean seeds during germination and early growth in darkness. The protein content of the glyoxysome fraction, separated by sucrose density centrifugation, increased linearly from day 2 to day 4 and declined subsequently, while maximum enzyme activities were reached at day 5. The specific activities of the enzymes in the glyoxysomes increased until day 5 and remained constant thereafter. At all stages of germination the only organelle with isocitrate lyase activity was the glyoxysome, but at the earlier stages a greater portion of the total activity was recovered in the soluble form. Malate synthetase was found primarily in the glyoxysomes after day 4, but at earlier stages part of the activity appeared at regions of lower density on the sucrose gradient. It was shown that this particulate malate synthetase activity was due to glyoxysomes broken during preparation, and that, as a result of this breakage, isocitrate lyase was solubilized. We conclude that both enzymes are housed in the glyoxysome in vivo throughout the germination period, and that the rise and fall in enzyme activities in phase with fat breakdown correspond to the net production and destruction of this organelle.     

Nitrate reductase in agrostemma githago comparison of the inductive effects of nitrate and cytokinin

The effect of nitrate and cytokinin on the induction of nitrate reductase (NADH-nitrate oxidoreductase, EC 1.6.6.1) in embryos of Agrostemma githago was compared. Increased enzyme levels in response to treatment with the cytokinin benzyladenine were not correlated with a general stimulation of protein synthesis or a general reduction of protein breakdown. Actinomycin D did not inhibit the formation of nitrate reductase in response to nitrate or the cytokinin. Cycloheximide and puromycin inhibited the induction by the hormone to the same extent as, or even more than, the incorporation of [¹⁴C]leucine into protein. Induction of nitrate reductase by nitrate was much less susceptible to inhibition by cycloheximide and puromycin than induction of the enzyme by benzyladenine. When induction of nitrate reductase was carried out in the presence of ²H₂O, isopycnic equilibrium centrifugation in CsCl showed that incorporation of ²H into the enzyme had occured. The increase in the buoyant density of nitrate reductase was the same whether the enzyme was induced by nitrate or by benzyladenine, indicating that at least part of the nitrate reductase molecule was newly synthesized in both instances.     

Evidence for an Inactivating System of Nitrate Reductase in Hordeum vulgare L. during Darkness That Requires Protein Synthesis

The disappearance of nitrate reductase activity in leaves of Hordeum vulgare L. during darkness was inhibited by cycloheximide, actinomycin D, and low temperature. Thus, protein synthesis was probably required for the disappearance of nitrate reductase in the dark. Since chloramphenicol did not affect the rate of loss of activity, the degradation or inactivation apparently required protein synthesis by the cytoplasmic ribosomal system. Consistent with this observation, nitrate reductase is also reportedly located in the cytoplasm. Thus, the amount of nitrate reductase activity present in leaves of barley may be controlled by a balance between activating and inactivating systems.     

Protein-carbohydrate interaction. A turbidimetric study of the interaction of concanavalin A with amylopectin and glycogen and some of their enzymic and chemical degradation products

1. RNA and protein synthesis was studied during the incubation of excised radish cotyledons in nitrate, conditions that induced nitrate reductase activity in the tissue. 2. Synthesis of total RNA and protein, as measured by the incorporation of radioactive precursor, was significantly stimulated in the presence of nitrate (compared with chloride control), but was decreased in the presence of ammonium nitrate, which induced higher enzyme activity. 3. Synthesis of RNA and protein was required for induction of enzyme activity, as determined by using the inhibitors actinomycin D, puromycin and cycloheximide. 4. On the basis of 5-fluorouracil inhibition, the synthesis of only DNA-like RNA was required for induction, but no differences, either quantitative or qualitative, were observed in DNA-like RNA synthesis in the presence or absence of induction. 5. A 100-fold purification of the nitrate reductase activity showed no increase in nitrate reductase protein, nor any increased incorporation of radioactive precursor into nitrate reductase protein in the induced versus the control system. Such results suggested that the protein synthesis required for induction may be for a protein other than nitrate reductase.