Arginase activity in the cotyledons of soybean seedlings

The influence of naphthylacetic acid, abscisic acid, gibberellic acid and kinetin on the formation of aerenchyma in seedling roots of Zea mays L. cv. Capella has been studied in relation to reported changes of their concentration in poorly aerated roots, which readily form aerenchyma, and to the effects of these hormones on the production of ethylene, a major factor promoting aerenchyma formation. Because the absence of nitrate accelerates aerenchyma formation in aerated roots, their influence on these roots was compared. The growth regulators were added to roots growing in non-aerated and aerated nutrient solutions, and aerenchyma formation and the production and endogenous concentration of ethylene were measured. Naphthylacetic acid prevented aerenchyma formation in both aerated roots without nitrate and in non-aerated roots although it enhanced the ethylene concentration of the roots. Abscisic acid also prevented aerenchyma formation, but without affecting the ethylene concentration. Gibberellic acid promoted aerenchyma formation in aerated roots only, but ethylene production in both aerated and non-aerated roots. Kinetin promoted aerenchyma formation in both aerated and non-aerated roots. It stimulated ethylene production in aerated roots, but slightly inhibited it in non-aerated roots. Co²⁺ and Ag⁺, which suppress ethylene production and action, respectively, reduced the promoting effects of gibberellic acid, but not those of kinetin. It is concluded that the effects of the plant growth regulators on aerenchyma formation in maize roots were, with a possible exception for gibberellic acid, not the result of altered ethylene concentrations in the roots. Their influence on aerenchyma formation is discussed in relation to their reported actions on cell membranes.     

Development of enzymes in the cotyledons of watermelon seedlings

Changes in hypocotyl length, cotyledon weight, lipid content, chlorophyll content, and capacity for photosynthesis have been described in seedlings of Citrullus vulgaris, Schrad. (watermelon) growing at 30 C under various light treatments. Corresponding changes in the levels of 19 enzymes in the cotyledons are described, with particular emphasis on enzymes of microbodies, since during normal greening, enzymes of the glyoxysomes are lost and those of leaf peroxisomes appear. In complete darkness enzymes of the glyoxysomes reach a peak at 4 days and decline as the fat is depleted. Enzymes of mitochondria and of glycolytic pathways also peak at 4 to 5 days and either remain unchanged or decline to a lesser extent. Exposure to light at 4 days, when the cotyledons emerge, results in a selectively greater destruction of enzymes of the glyoxylate cycle; chlorophyll synthesis and capacity for photosynthesis increase in parallel, and there is a striking increase in the activities of chloroplast enzymes and in those of the leaf peroxisomes, hydroxypyruvate reductase and glycolate oxidase. The reciprocal changes in enzymes of the glyoxysomes and of leaf peroxisomes can be temporally dissociated, since even after 10 days in darkness, when malate synthetase and isocitrate lyase have reached very low levels, hydroxypyruvate reductase and glycolate oxidase increase strikingly on exposure to light and the cotyledons become photosynthetic. Furthermore, the parallel development of enzymes of leaf peroxisomes and functional chloroplasts is not immutable, since hydroxypyruvate reductase and glycolate oxidase activity can be elicited in darkness following a 5-minute exposure to light at day 4 while chlorophyll does not develop under these conditions.     

Gluconeogenesis from storage wax in the cotyledons of jojoba seedlings

The cotyledons of jojoba (Simmondsia chinensis) seeds contained 50 to 60% of their weight as intracellular wax esters. During germination there was a gradual decrease in the wax content with a concomitant rise in soluble carbohydrates, suggesting that the wax played the role of a food reserve. Thin layer chromatography revealed that both the fatty alcohol and fatty acid were metabolized. The disappearance of wax was matched with an increase of catalase, a marker enzyme of the gluconeogenic process in other fatty seedlings. Subcellular organelles were isolated by sucrose gradient centrifugation from the cotyledons at the peak stage of germination. The enzymes of the β oxidation of fatty acid and of the glyoxylate cycle were localized in the glyoxysomes but not in the mitochondria. The glyoxysomes had specific activities of individual enzymes similar to those of the castor bean glyoxysomes. An active alkaline lipase was detected in the wax bodies at the peak stage of germination but not in the ungerminated seeds. No lipase was detected in glyoxysomes or mitochondria. After the wax in the wax bodies had been extracted with diethyl ether, the organelle membrane was isolated and it still retained the alkaline lipase. The gluconeogenesis from wax in the jojoba seedling appears to be similar, but with modification, to that from triglyceride in other fatty seedlings.     

Peroxidative metabolism of apigenin and naringenin versus luteolin and quercetin: glutathione oxidation and conjugation

GSH was readily depleted by a flavonoid, H(2)O(2), and peroxidase mixture but the products formed were dependent on the redox potential of the flavonoid. Catalytic amounts of apigenin and naringenin but not kaempferol (flavonoids that contain a phenol B ring) when oxidized by H(2)O(2) and peroxidase co-oxidized GSH to GSSG via a thiyl radical which could be trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) to form a DMPO-glutathionyl radical adduct detected by ESR spectroscopy. On the other hand, quercetin and luteolin (flavonoids that contain a catechol B ring) or kaempferol depleted GSH stoichiometrically without forming a thiyl radical or GSSG. Quercetin, luteolin, and kaempferol formed mono-GSH and bis-GSH conjugates, whereas apigenin and naringenin did not form GSH conjugates. MS/MS electrospray spectroscopy showed that mono-GSH conjugates for quercetin and luteolin had peaks at m/z 608 [M + H](+) and m/z 592 [M + H](+) in the positive-ion mode, respectively. (1)H NMR spectroscopy showed that the GSH was bound to the quercetin A ring. Spectral studies indicated that at a physiological pH the luteolin-SG conjugate was formed from a product with a UV maximum absorbance at 260 nm that was reducible by potassium borohydride. The quercetin-SG conjugate or kaempferol-SG conjugate on the other hand was formed from a product with a UV maximum absorbance at 335 nm that was not reducible by potassium borohydride. These results suggest that GSH was oxidized by apigenin/naringenin phenoxyl radicals, whereas GSH conjugate formation involved the o-quinone metabolite of luteolin or the quinoid (quinone methide) product of quercetin/kaempferol.     

Inhibitor of diamine oxidase in cotyledons of groundnut seedlings

The apparent absence of diamine oxidase in extracts of cotyledon of germinating groundnut seeds is due to the presence of a natural inhibitor. The inhibitor was associated with the lipid in the upper layer obtained on centrifugation of the cotyledon extract. It was non-dialysable, thermolabile and was inactivated by TCA and Triton X-100. The inhibitor was detected in the cotyledon extracts from early stages of seed development and was present up to 20 days after germination.     

Oxidation of fatty alcohol in the cotyledons of jojoba seedlings.

An externally accessible polypeptide has been purified from hepatoma tissue culture cells. The purification involves four steps: deoxycholate extraction of whole cells, isoelectric focusing of deoxycholate-insoluble material in the presence of 8 m urea and Triton X-100, hydroxylapatite chromatography in the presence of sodium dodecyl sulfate, and preparative acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The final preparation is homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by isoelectric focusing in polyacrylamide. The polypeptide has an apparent molecular weight of 55,000 and is labeled following in situ lactoperoxidase-catalyzed iodination of the hepatoma tissue culture cells. The polypeptide can also be labeled by growing cells in the presence of labeled amino acids, but is not labeled by growth in labeled sugars. The purified protein does not react with the periodate-Schiff reagent. Hence, it does not appear to be a glycoprotein that contains mannose, fucose, glucosamine, or sialic acids.     

The role of cotyledons in the establishment of Suaeda physophora seedlings

The role of cotyledons in seedling establishment of the euhalophyte Suaeda physophora under non-saline and saline conditions (addition of 1 mM or 400 mM NaCl) was investigated. Survival and fresh and dry weights were greater for seedlings grown in the light (12-h light/12-h dark) than in the dark (24-h dark). The shading of cotyledons tended to decrease shoot height, shoot organic dry weight, number of leaves, and survival of seedlings regardless of NaCl treatment, but the effect of cotyledon shading was greater with 400 mM NaCl. Concentrations of Na⁺ were higher in cotyledons than in leaves, regardless of NaCl treatment. The K⁺/Na⁺ ratio was lower in cotyledons than in leaves for seedlings treated with 1 mM NaCl but not for seedlings treated with 400 mM NaCl. Addition of 400 mM NaCl decreased oxygen production in cotyledons but especially in leaves. These results are consistent with the hypothesis that, by generating oxygen via photosynthesis and by compartmentalizing Na⁺, cotyledons are crucial for the establishment of S. physophora seedlings in saline environments.     

Inhibition of superoxide anion-mediated impairment of endothelium by treatment with luteolin and apigenin in rat mesenteric artery

This study was designed (i) to test the hypothesis that the endothelium-derived hyperpolarizing factor (EDHF) component of ACh-induced vasorelaxation and hyperpolarization of smooth muscle cells (SMCs) are impaired following exposure to superoxide anion, and (ii) to further investigate whether luteolin and apigenin induce vasoprotection at the vasoactive concentrations in rat mesenteric artery. Rat mesenteric arterial rings were isolated for isometric force recording and electrophysiological studies. Perfusion pressure of mesenteric arterial bed was measured and visualization of superoxide production was detected with fluorescent dye. 300 microM pyrogallol significantly decreased the relaxation and hyperpolarization to ACh. Luteolin and apigenin both induced vasoprotection against loss of the EDHF component of ACh-induced relaxation and attenuated the impairment of hyperpolarization to ACh. Oxidative fluorescent microtopography showed that either luteolin or apigenin significantly reduced the superoxide levels. The results suggest that superoxide anion impairs ACh-induced relaxation and hyperpolarization of SMC in resistance arteries through the impairment of EDHF mediated responses. Luteolin and apigenin protect resistance arteries from injury, implying that they may be effective in therapy for vascular diseases associated with oxidative stress.     

The role of cotyledons in phototropism of de-etiolated seedlings

Simulated phototropic curvatures caused by differential masking of the cotyledons of de-etiolated seedlings exposed to white light are unconnected with true phototropism. In Cucumis sativus L. and Helianthus annuus L. such curvatures result from a red-light-induced inhibition coming from the exposed cotyledon. True phototropic bending in these species under long-term exposure to fairly high irradiances (as in nature) is a response to blue light. It occurs even when cotyledons are completely covered. These results show that the cotyledons do not perceive the phototropic stimulus and need not be illuminated for phototropism to occur.     

Lipase in lipid bodies of cotyledons of rape and mustard seedlings

Lipolytic activity was absent in the crude cotyledon extract of ungerminated rapeseed (Brassica napm L. var. Dwarf Essex), and increased to a peak at day 4 in seedling growth, concomitant with the decrease in total lipids. About 50% of the lipase activity was recovered in the lipid bodies isolated from the cotyledon extract by flotation centrifugation. Isolated lipid bodies underwent autolysis of internal triacylglycerols resulting in the release of fatty acids. After the triacylglycerols in isolated lipid bodies had been extracted with diethyl ether, the lipase was recovered in the remaining membrane fraction. The lipase had a maximal activity at pH 6.5 on trierucin, trilinolein, or endogenous triacylglycerols, and at pH 8.0 on N-methylindoxylmyristate. The lipase was most active on trierucin and trilinolein, and hydrolyzed the related di- and monoacylglycerols at lower rates. There was little enhancement of the lipase activity in the presence of NaCl, CaCl₂, or detergents, and detergents in general reduced the activity. The hydrolysis of trierucin was linear until about 50% of the trierucin had been converted to erucic acid, and there was little accumulation of dierucin and monoerucin. Lipase extracted from lipid bodies isolated from germinated rapeseed of the variety Tower, which contains little or no erucic acids in the storage triacylglycerols, also had the highest activities on trierucin and trilinolein. A comparative study on mustard seed (Brassica juncea) revealed that the mustard lipase possessed characteristics very similar to those of the rapeseed lipase.     

Gas chromatographic and GC-MS evidence for the occurrence of 3-indolylpropionic acid and 3-indolylacetic acid in seedlings of Cucurbita pepo

Gas chromatographic and GC-MS evidence is presented for the occurrence of two auxin substances, 3-indolylacetic acid and 3-indolylpropionic acid, in hypocotyls of Cucurbita pepo cv. Golden Hubbard seedlings grown in darkness or under blue-light illumination. The data provide the first unequivocal evidence for the presence of 3-indolylpropionic acid in a higher plant.     

Density-labelling evidence for the phytochrome-mediated activation of phenylalanine ammonia-lyase in mustard cotyledons

When dark-grown mustard seedlings are irradiated with far-red light the level of phenylalanine ammonia-lyase (EC 4.3.1.5) activity increases. After ²H₂ O treatment phynlalanine amonia-lyase from seedlings irradiated with far-red light is density-labelled to a lesser extent than enzyme from dark-grown tissue. Theoretical arguments are advanced and data presented which show that this result cannot be explained in terms of an increase in de novo synthesis of phenylalanine ammonia-lyase and that the increase most likely involves activation of existing enzyme.     

Rapid degradation and limited synthesis of phospholipids in the cotyledons of mung bean seedlings

Seedling growth of mung bean is accompanied by the rapid catabolism of the three major phospholipids in the cotyledons (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol). The decline starts 24 hours after the beginning of imbibition and by the 4th day of growth more than 50% of the phospholipids have been catabolized. Extracts of cotyledons of 24-hour-imbibed beans contain enzymes capable of degrading membrane-associated phospholipids in vitro. This degradation involves phospholipase D and phosphatase activity.     

Intracellular localization of phosphatidylcholine and phosphatidylethanolamine synthesis in cotyledons of cotton seedlings

Subfractionation of clarified cotyledon homogenates of cotton (Gossypium hirsutum L.) seedlings on sucrose gradients revealed a single coincident peak of cholinephosphotransferase (EC 2.7.8.2) (CPT) and ethanolaminephosphotransferase (EC 2.7.8.1) (EPT) activities, which equilibrated with the main peak of Antimycin A-insensitive NADH:cytochrome c reductase (CCR) activity. The small percentage of CPT and EPT activities (less than 5% of the total) in glyoxysome-enriched pellets equilibrated with cytochrome c oxidase activity, not with catalase activity. Preincubation of microsomes (containing 83% of total CPT and EPT activities) in 0.2 millimolar MgCl₂ followed by subfractionation on sucrose gradients resulted in peak CPT and EPT activities equilibrating with peak CCR activity at 24% (w/w) sucrose. Preincubation of microsomes with ¹⁴C-CDPcholine (or ¹⁴C-CDPethanolamine) resulted in synthesis and incorporation of ¹⁴C-phosphatidylcholine (PC) (or ¹⁴C-phosphatidylethanolamine, PE) into membranes at the same density. Increasing the Mg²⁺ concentration to 2.0 millimolar facilitated binding of ribosomes and caused a concomitant shift in density (to 34% w/w sucrose) of peak CPT, EPT, and CCR activities. Under these conditions, newly synthesized and incorporated ¹⁴C-PC (or PE) was recovered in these membranes. Transmission electron microscopy of this fraction confirmed binding of ribosomes to membranes. Radiolabeling in vivo of cotyledons with [methyl-¹⁴C] choline chloride or [1,2 ethanolamine-¹⁴C] ethanolamine hydrochloride resulted in a linear incorporation of radiolabel into PC or PE in a time dependent manner. Subfractionation of homogenates of radiolabeled cotyledons on sucrose gradients showed that membranes sedimenting at 24% (w/w) sucrose (ER) contained the majority of radiolabeled PC and PE with a minor peak at 40% (w/w) sucrose (mitochondria), but no radioactive PC or PE was recovered in glyoxysomes. These results indicate that ER in cotyledons of germinated cotton seedlings is the primary subcellular site of PC and PE synthesis. This is similar to the situation in endosperm tissue but distinctly different from root and hypocotyl tissue where Golgi are a major subcellular site of PC and PE synthesis.