Arginine Metabolism in Developing Soybean Cotyledons : II. Biosynthesis

Tracer kinetic experiments were performed using [ureido-¹⁴C] citrulline, [1-¹⁴C]ornithine, and isotope trapping techniques to determine if arginine is synthesized via the urea cycle in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with the ¹⁴C-solution and incubated in sealed vials containing a CO₂ trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. In the ¹⁴C-citrulline feeding experiment argininosuccinate was the most highly labeled compound after 5 minutes and it was the first compound to lose ¹⁴C later in the time course. Carbon-14 was also recovered in free arginine, protein arginine, and CO₂ up to 4 hours after introduction of label. All of the ¹⁴C in free and protein arginine could be accounted for in the C-6 position. Metabolism of ¹⁴C-ornithine resulted in ¹⁴C-incorporation into citrulline and free and protein arginine and the evolution of ¹⁴CO₂. Citrulline was the most highly labeled compound after 15 minutes and was the first compound to reach a steady state level of ¹⁴C. With the addition of 800 nanomoles unlabeled citrulline to the ¹⁴C-ornithine feeding solution citrulline was the only compound labeled after 5 minutes and the steady state level of ¹⁴C-citrulline increased 12-fold. The appearance of ¹⁴C in free arginine and protein arginine was also delayed. In both ¹⁴C-ornithine feedings all of the ¹⁴C in free and protein arginine could be accounted for in the C-1 position. Together, the data support the reaction sequence: ornithine → citrulline → argininosuccinate → arginine → protein arginine.     

Utilization of Nitrogen Sources by Immature Soybean Cotyledons in Culture

Immature Glycine max (L.) Merrill cotyledons were cultured ina defined medium containing different nitrogen sources. Glutaminewas the most efficient source in terms of protein accumulationin the cotyledons. Asparagine was less efficient (about 70 percent that of glutamine) while allantoin was a poor source ofnitrogen. This was also true for older cotyledons where asparaginaseand allantoinase activities were maximal. The utilization ofboth asparagine and allantoin (but not glutamine) was totallyinhibited by methionine sulfoximine suggesting that their metabolisminvolves ammonia assimilation via glutamine synthetase. Apparently,neither exogenous or endogenously-generated ammonia had mucheffect on glutamine utilization, but ammonia did have a smallinhibitory effect on asparagine, which may in part account forthe lower efficiency observed with this amide. Glycine max, soybean, cotyledon culture, nitrogen metabolism     

Accumulation and Subcellular Localization of alpha-Galactosidase-Hemagglutinin in Developing Soybean Cotyledons

We have investigated the accumulation and intracellular localization of soybean (Glycine max [L.] Merr. cv Forrest) α-galactosidase-hemagglutinin during seed development. Cotyledon tissue was embedded in Lowicryl K4M and immunocytochemical localization was accomplished through treating thin sections with α-galactosidase antisera followed by indirect labeling with protein A coupled to colloidal gold. Gold particles were localized on the Golgi apparatus and protein bodies. We interpret this to indicate that α-galactosidase-hemagglutinin is transferred to and transported through the Golgi apparatus and finally deposited within the protein body by a Golgi apparatus-mediated process.     

Regulation by ABA of beta-Conglycinin Expression in Cultured Developing Soybean Cotyledons

The regulation of cotyledon-specific gene expression by exogenously applied abscisic acid (ABA) was studied in developing cultured cotyledons of soybean (Glycine max L. Merr. cv Provar). When immature cotyledons were cultured in modified Thompson's medium, the addition of ABA resulted in an increased concentration of the β-subunit of β-conglycinin, one of the major storage proteins of soybean seeds. The amount of the α′-and α-subunits of β-conglycinin was relatively unaffected by the ABA treatment. When fluridone, an inhibitor of carotenoid biosynthesis that has been shown to decrease ABA levels in plant tissues, was added to the medium the level of ABA and the β-subunit decreased in the cotyledons. Increasing the concentration of sucrose in the culture medium caused an increase in the concentration of ABA and β-subunit in the cotyledons. When in vitro translation products from RNA isolated from cotyledons cultured with ABA were immunoprecipitated with antiserum against β-conglycinin, there was an increased amount of pre-β-subunit polypetide compared to the translation products from RNA isolated from control cotyledons. The pre-β-subunit polypeptide was not detected in translation products from RNA isolated from fluridone-treated cotyledons. Nucleic acid hybridization reactions showed that the level of β-subunit mRNA was higher in ABA-treated cotyledons compared to the control, and was lower in the fluridone-treated cotyledons. We have shown that exogenous ABA is able to modulate the accumulation of the β-subunit of β-conglycinin in developing cultured soybean cotyledons.     

The Cytological and Histochemical Studies of Developing Soybean,Cotyledons

In the late globular proembryos, three regions could be identified, i. e. the cotyledon primordium, the epiphysis and the hypocotyl-hypophysis. In the cotyledon primordia, the mitotic frequency of the cells was comparitively high, the directions of the mitotic planes were mostly perpendicular to the long axis of the embryo, the size of the nucleolus was comparitively large, and the cytoplasm density was high. In the epiphysis region, however, the mitotic frequency of the cells was low, the size of the nucleolus was small, and as the first pair of leaf primordia appeared the mitotic frequency of the cells in that region began to increase. In the hypocotyls hypophysis region the mitotic frequency of the cells as well as the size of the nucleolus lied in between the corresponding values of those of the above two regions, the cytoplasm density was low and the size of the vacuoles was large. As the proembryo continued to develop the direction of the mitotic plane changed gradually, from mostly perpendicular to the long axis of the embryo to mainly inclined, or even parallel to that axis. As a result, the proembryo developed from a heart-shaped embryo into a torpedo-shaped embryo. After the first pair of leaf primordia appeared from the young embryo, the vacuoles in the cells of the cotyledons grew in size rapidly. About twenty to twenty five days after flowering, the starch grains, the protein bodies and the lipid granules began to accumulate in the cells of the cotyledons and gradually increased both in size as well as in quantity. About fifty days after flowering the diameter of the starch grains reached its maximum value of 6.2–7.0 μm, and decreased in value thereafter till the time of harvesting when most of the starch grains disappeared except those in the palisades. On the other hand, fifty to sixty days after flowering, the diameters of the lipid granules and of the protein bodies reached their maximum values of 5.4–7.0 μm and 6.2–7.0 μm, respectively. The observation revealed that the formation of the protein bodies was related to the vacules.     

Subcellular Compartmentation of the 4-Aminobutyrate Shunt in Protoplasts from Developing Soybean Cotyledons

The subcellular localization of enzymes involved in the 4-ami-nobutyrate shunt was investigated in protoplasts prepared from developing soybean [Glycine max (L.) Merrill cv Maple Arrow] cotyledons. Protoplast lysate was fractionated by differential and continuous Percoll-gradient centrifugation to separate organelle fractions. Glutamate decarboxylase (EC 4.1.1.15) was found exclusively in the cytosol, whereas 4-aminobutyrate:pyruvate transami-nase (EC 2.6.1.19) and succinic semialdehyde dehydrogenase (EC 1.2.1.16) were associated exclusively with the mitochondrial fractions. Mitochondrial fractions also catabolized [U-14C]4-aminobu-tyrate to labeled succinate.     

Nitrogen Metabolism in Soybean Tissue Culture: II. Urea Utilization and Urease Synthesis Require Ni

Potassium citrate (10 mM, pH 6) inhibits the growth of cultured (Glycine max L.) cells when urea is the sole nitrogen source. Ureadependent citrate toxicity is overcome by three separate additions to the growth medium: (a) NH₄Cl (20 mM); (b) high levels of MgCl₂ (10 mM) or CaCl₂ (5-10 mM); (c) low levels of NiSO₄ (10⁻² mM). Additions of 10⁻² mM NiSO₄ not only overcome citrate growth inhibition but the resultant growth is usually better than urea-supported growth in basal medium (neither added citrate nor added nickel). In the absence of added citrate, exceedingly low levels of NiSO₄ (10⁻⁴ mM) strongly stimulate urea-supported growth in suspension cultures.     

Sugar Transport into Protoplasts Isolated from Developing Soybean Cotyledons : II. Sucrose Transport Kinetics, Selectivity, and Modeling Studies

The effects of metabolic inhibitors, pH, and temperature on the kinetics of sucrose uptake protoplasts isolated from developing soybean Glycine max L. cv Wye cotyledons were studied. Structural requirements for substrate recognition by the sucrose carrier were examined by observing the effects of potential alternate substrates for the saturable component on sucrose uptake.     

Sucrose Concentration at the Apoplastic Interface between Seed Coat and Cotyledons of Developing Soybean Seeds

The apoplastic sucrose concentration at the interface between cotyledons and surrounding seed coats of developing soybeans (Glycine max L. Merr. cv Wye) was found by three indirect methods to be in the range of 150 to 200 millimolar. This is an order of magnitude higher than has been reported elsewhere for soybean. It was also higher than the overall sucrose concentrations in the cotyledons and seed coats, each of which was approximately 90 millimolar. By defoliating plants 24 hours before measurement, both the overall sucrose concentration in the cotyledons and the interfacial apoplastic sucrose concentration were reduced by three-fourths. However, there was no day/night difference in overall tissue sucrose concentration of cotyledons or seed coats from intact plants suggesting the existence of a homeostatic mechanism compensating for the diurnal photosynthetic cycle. About 7 hours were required for a tritiated polyethylene glycol-900 solution to fully permeate developing cotyledons (from ~220 milligram fresh weight embryos), implying high diffusion resistance through the tissue.

These results indicate that a high interfacial sucrose concentration may exist in vivo. They suggest that the saturable carrier-mediated component of sucrose uptake may be of little physiological significance in the outermost cell layers of the cotyledons.

     

Arginine Metabolism in Mouse Brain Synaptosomes

In a study employing mouse brain synaptosomes and synaptosomal sonicates, the complete metabolic machinery was found to be present for transport of arginine into synaptosomes, its conversion to ornithine, and the formation from the latter of glutamic acid, gamma-aminobutyric acid, and proline. The results show that a delicate balance probably exists between the flows of metabolites. This balance, which probably determines the steady-state levels of these substances in nerve terminals, can be altered by concentrations of the metabolites themselves through feedback inhibition as well as by levels of cofactors.     

酸性磷酸酶参与大豆子叶磷转运和利用

本文以磷效率不同的两个大豆品种为材料,研究大豆幼苗期子叶酸性磷酸酶活性和同工酶谱对外源磷有效性的响应,及其参与子叶磷高效转运和利用的过程。结果表明：在幼苗生长前期,子叶酸性磷酸酶活性及其同工酶谱组成变化明显,而且不受外源磷有效性的调控;在幼苗生长的前8天,子叶全磷含量随着酸性磷酸酶的活性增加而显著降低,而且磷高效大豆品种比磷低效大豆品种具有较高的酸性磷酸酶活性和植株全磷含量。以上结果说明在大豆幼苗生长前期,由于大粒种子不仅具有较高的磷含量,而且具有较高子叶酸性磷酸酶活性,促进子叶有机磷的水解和转运是磷高效大豆品种适应低磷胁迫的生理机制之一。     

Isocitrate Lyase from Germinating Soybean Cotyledons: Purification and Characterization

Ruchti, M. and Widmer, F. 1986. Isocitrate lyase from germinatingsoybean cotyledons: purification and characterization.—J.exp. Bot. 37: 1685–1690. Isocitrate lyase (E.C. 4.1.3.1 [EC] ) was purified from the cotyledonsof 7-d-old soybean seedlings. Three molecular forms were detectedwith pi values of 6·46, 6·25 and 6·0. Themain form (pl = 6·46) had an approximate Mr of 130000,a pH optimum of 8·0, a K_m (isocitrate) close to 2·0mol m^–3 and a molecular activity of 615 min ^–1 at25 °C. The purified enzyme is not a glycoprotein and isheat labile. Key words: Isocitrate lyase, soybean     

Immunocytochemical Localization of Uricase in Peroxisomes of Soybean Cotyledons

Antibodies specific for nodule uricase were used for immunocytochemistryto demonstrate the presence of uricase in cotyledons of soybean(Glycine max) during germination and early seedling growth.The enzyme was localized exclusively in peroxisomes. ¹Permanent address: Department of Plant Cytology and Cytochemistry,University of Lodz, Lodz, Poland ²Current address: Department of Plant Science, University ofArizona, Tucson, AZ 85721, U.S.A.     

Developmental Regulation of beta-Conglycinin in Soybean Axes and Cotyledons

Analysis of the expression of genes encoding the β-conglycinin seed storage proteins in soybean has been used to extend our understanding of developmental gene expression in plants. The α, α′, and β subunits of β-conglycinin are encoded by a multigene family which is organ-specific in its expression. In this study we report the differentially programmed accumulation of the α, α′, and β subunits of β-conglycinin. Multiple isomeric forms of each subunit are present in the dry seed, but the timing of their accumulation is unique for each subunit. The previously reported variation in amount of α′ and α subunits in axis and cotyledons is also reflected in the amount of subunit specific mRNA which is present in each tissue. The β subunit, previously undetected in soybean axes, is found to be synthesized but rapidly degraded. These differences in β-conglycinin protein accumulation may be reflected by the morphological differences observed in protein bodies between these two tissues.     

Glutamate Synthetase in Developing Cotyledons of Pisum sativum

Glutamate synthetase (glutamine[amide]:α ketoglutarate amino transferase oxidoreductase) activity has been demonstrated in the developing cotyledons of Pisum sativum L. cv. Burpeeana. The enzyme appears to be soluble and is specific for glutamine as amide donor. The enzyme activity is greater with NADH than with NADPH as electron donor.