Polyamine Metabolism in Embryogenic Cells of Daucus carota: II. Changes in Arginine Decarboxylase Activity

Embryogenic cultured cells of Daucus carota have been shown to synthesize putrescine from exogenously supplied [¹⁴C]arginine at twice the rate of control nonembryogenic cells. In the present paper, the activity of arginine decarboxylase (arginine carboxy-lyase, EC 4.1.1.19), an important enzyme in the synthesis of putrescine, was assayed and also found to be elevated by as much as 2-fold in embryogenic cells. This difference between embryogenic and nonembryogenic cells was observed as early as 6 hours after the induction of embryogenesis and appeared not to result from the presence of a diffusible inhibitor or activator. It seemed to be dependent upon concomitant RNA and protein synthesis, as judged using 6-methyl-purine and cycloheximide. After cycloheximide addition to the culture medium, arginine decarboxylase activity declined with a half-time of about 30 minutes in both embryogenic and nonembryogenic cells. It is suggested that elevated arginine decarboxylase activity is involved in the mechanism leading to elevated putrescine levels in these cells and hence may play a role in the embryogenic process.     

Polyamine Metabolism and Osmotic Stress : I. Relation to Protoplast Viability

Cereal leaves subjected to the osmotica routinely used for protoplast isolation show a rapid increase in arginine decarboxylase activity, a massive accumulation of putrescine, and slow conversion of putrescine to the higher polyamines, spermidine, and spermine (HE Flores, AW Galston 1984 Plant Physiol 75: 102). Mesophyll protoplasts from these leaves, which have a high putrescine:polyamine ratio, do not undergo sustained division. By contrast, in Nicotiana, Capsicum, Datura, Trigonella, andVigna, dicot genera that readily regenerate plants from mesophyll protoplasts, the response of leaves to osmotic stress is opposite to that in cereals. Putrescine titer as well as arginine and ornithine decarboxylase activities decline in these osmotically stressed dicot leaves, while spermidine and spermine titers increase. Thus, the putrescine:polyamine ratio in Vigna protoplasts, which divide readily, is 4-fold lower than in oat protoplasts, which divide poorly. We suggest that this differing response of polyamine metabolism to osmotic stress may account in part for the failure of cereal mesophyll protoplasts to develop readily in vitro.     

Polyamine Biosynthetic Enzymes in Chlorella: Characterization of Ornithine and Arginine Decarboxylase

In a Chlorella culture grown asynchronously under autotrophicconditions, two biosynthetic enzymes of putrescine—ornithinedecarboxylase (ODC) and arginine decarboxylase (ADC)—weredetected. Both enzymes require pyridoxal phosphate and dithiothreitolfor their activity but differ in their optimal pH, the ionicstrength of their buffer, temperature of inactivation, and Km.In addition, L-canaline was found to inhibit the activity ofODC but not that of ADC. During the logarithmic phase of growth,ODC activity increased sharply, then decreased before the onsetof the stationary phase. ADC activity changed only slightlyduring growth. ³The work was performed in partial fulfillment of the requirementsfor the Ph.D. Thesis of E.C. (Received September 25, 1982; Accepted June 1, 1983)     

Putrescine and Acid Stress : Induction of Arginine Decarboxylase Activity and Putrescine Accumulation by Low pH

Incubation of peeled oat Avena sativa L. var Victory leaf segments on media of pH 5.0 or below leads to a rapid and massive increase in the titer of putrescine while incubation at pH values above 5.0 causes little or no change. The low pH effect is independent of the buffer system employed. Putrescine levels rise within 3 hours and reach their peak 8 to 9 hours after acidification. At this time, putrescine titer is eight times greater at pH 3.5 than at 6.0. None of the other polyamines shows a response to changes in external pH. The increase in putrescine is blocked by the addition of cycloheximide or dl-alpha-difluoromethylarginine, a specific inhibitor of the putrescine biosynthetic enzyme, arginine decarboxylase. In one experiment, arginine decarboxylase activity was 110% greater at pH 4.0 than at 6.0 after a 4-hour incubation, although the average increase over many experiments was 47%. The activity of the other possible putrescine biosynthetic enzyme, ornithine decarboxylase, falls throughout the incubation period and is virtually equal at pH 4.0 and 6.0.     

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.     

Seasonal Changes of Polyamine Concentrations and Arginine Decarboxylase Activities in Leaves of 4 Ecotyes of Reeds

The seasonal changes of polyamine concentrations and arginine decarboxylase (ADC, EC 4.1.1. 1. 9)activities were investigated in the leaves of 4 ecotypes of reeds (Phragamites comrnunis Trinius)distributed over Hexi Corridor of Gansu province. The leaves of all ecotypes of reeds contained the same kind of polyamines and showed the same trend of decrement in total amuonts of potyamines with change of seasons. From May to September, the reeds which grow in arid and saline habitat maintained higher level of spermidine (Spd)and spermine (Spm)with no accumulation of putrescine (Put), resulting in low ratios of Put to other polyamine (Spd and Spm), whereas opposite results were observed in swamp reeds. These results indicate that the adaption of reeds to drought and salt stresses may correlate with Put synthesis via ADC pathway and the quick transformation of Put into Spd and Spm.     

Leucine Uptake and Incorporation by Convolvulus Tissue Culture Cells and Protoplasts under Severe Osmotic Stress

The uptake and incorporation of radioactive leucine by Convolvulus arvensis L. suspension culture cells were studied under various osmotic conditions to provide information about the effects of osmotic stress at the cellular level and about the suitability of various osmotica for stabilizing protoplasts. When manitol, sorbitol, sucrose, or a mixture of CaCl₂ and KCl was added to the cells at a concentration normally used to stabilize protoplasts, the uptake of leucine was inhibited by 50 to 60% and incorporation by 37% with no major differences detected among these osmotica. NaNO₃ of a similar osmotic strength exerted considerably more inhibition, an inhibition that was reversed by as little as 10 mM simultaneous CaCl₂. None of the osmotica altered leucine or protein leakage from the tissue. In general, external solute concentrations below 0.36 osmolal slightly enhanced uptake and incorporation. At successively higher concentrations, uptake and incorporation decreased in a linear fashion, with no apparent discontinuity in the rate of decrease as the cells plasmolyzed. Cycloheximide inhibited both the uptake and the incorporation of leucine in all osmotic situations tested, exerting a much stronger inhibition upon the uptake by control tissue than upon that by cells in osmotica. Different cellulase enzyme preparations varied considerably in their effects on subsequent leucine uptake and incorporation.     

Retina Maturation Following Administration of Thyroxine in Developing Rats: Effects on Polyamine Metabolism and Glutamate Decarboxylase

Abstract: The effects of subcutaneous daily treatment with thyroxine on cell proliferation, differentiation, polyamines, and γ-aminobutyric acid metabolism in the rat retina were studied during the first 20 postnatal days. The retinal layers of the treated rats displayed an enhanced cell differentiation which reached its maximum 9–12 days from birth; but this effect stopped very quickly and was finished by the 20th postnatal day. Primarily there was an increase in ornithine decarboxylase activity which was accompanied by an increase in putrescine, spermidine, and spermine levels. S -Adenosylmethionine decarboxylase was induced later than ODC; corresponding with the enhanced synaptogenesis, glutamate decarboxylase increased 15-fold between the fourth and 15th days. Our data are consistent with the hypothesis that thyroxine may exert some of its effects by inducing the enzymes which regulate polyamine metabolism and synaptogenesis.     

Osmotic Stress-Induced Polyamine Accumulation in Cereal Leaves : II. Relation to Amino Acid Pools

Arginine decarboxylase activity increases 2- to 3-fold in osmotically stressed oat leaves in both light and dark, but putrescine accumulation in the dark is only one-third to one-half of that in light-stressed leaves. If arginine or ornithine are supplied to dark-stressed leaves, putrescine rises to levels comparable to those obtained by incubation under light. Thus, precursor amino acid availability is limiting to the stress response. Amino acid levels change rapidly upon osmotic treatment; notably, glutamic acid decreases with a corresponding rise in glutamine. Difluoromethylarginine (0.01-0.1 millimolar), the enzyme-activated irreversible inhibitor of arginine decarboxylase, prevents the stress-induced putrescine rise, as well as the incorporation of label from [¹⁴C]arginine, with the expected accumulation of free arginine, but has no effect on the rest of the amino acid pool. The use of specific inhibitors such as α-difluoromethylarginine is suggested as probes for the physiological significance of stress responses by plant cells.     

Arginase and Arginine Decarboxylase – Where Do the Putative Gate Keepers of Polyamine Synthesis Reside in Rat Brain?

Polyamines are important regulators of basal cellular functions but also subserve highly specific tasks in the mammalian brain. With this respect, polyamines and the synthesizing and degrading enzymes are clearly differentially distributed in neurons versus glial cells and also in different brain areas. The synthesis of the diamine putrescine may be driven via two different pathways. In the “classical” pathway urea and carbon dioxide are removed from arginine by arginase and ornithine decarboxylase. The alternative pathway, first removing carbon dioxide by arginine decarboxlyase and then urea by agmatinase, may serve the same purpose. Furthermore, the intermediate product of the alternative pathway, agmatine, is an endogenous ligand for imidazoline receptors and may serve as a neurotransmitter. In order to evaluate and compare the expression patterns of the two gate keeper enzymes arginase and arginine decarboxylase, we generated polyclonal, monospecific antibodies against arginase-1 and arginine decarboxylase. Using these tools, we immunocytochemically screened the rat brain and compared the expression patterns of both enzymes in several brain areas on the regional, cellular and subcellular level. In contrast to other enzymes of the polyamine pathway, arginine decarboxylase and arginase are both constitutively and widely expressed in rat brain neurons. In cerebral cortex and hippocampus, principal neurons and putative interneurons were clearly labeled for both enzymes. Labeling, however, was strikingly different in these neurons with respect to the subcellular localization of the enzymes. While with antibodies against arginine decarboxylase the immunosignal was distributed throughout the cytoplasm, arginase-like immunoreactivity was preferentially localized to Golgi stacks. Given the apparent congruence of arginase and arginine decarboxylase distribution with respect to certain cell populations, it seems likely that the synthesis of agmatine rather than putrescine may be the main purpose of the alternative pathway of polyamine synthesis, while the classical pathway supplies putrescine and spermidine/spermine in these neurons.     

Swelling of Etiolated Oat Protoplasts Induced by cAMP and Red Light

Etiolated oat protoplasts were treated with dibutyryl cAMP tostudy possible function of cAMP in the development by measuringthe protoplast swelling. The mean diameter of protoplasts inthe absence of any chemical treatment was 33.58±1.26(SE) µm, which increased to 36.96±0.86 µmin the presence of 100 µM dibutyryl cAMP. Prostacyclin,a potent activator of adenyl cyclase, also showed a significantswelling effect (diameter 38.01±0.98 µm). Red lightalso elicited the swelling of protoplasts (40.26±0.8µm). ¹Present address: Department of Biology, Pusan National University,Pusan 607, Korea. ²Present address: Department of Horticulture, Cheju NationalUniversity, Cheju 590, Korea. ³Present address: Department of Biological Sciences, Texas TechUniversity, Lubbock, TX 79409, U.S.A. (Received June 29, 1985; Accepted November 18, 1985)     

Polyamine Metabolism in Ripening Tomato Fruit : II. Polyamine Metabolism and Synthesis in Relation to Enhanced Putrescine Content and Storage Life of a/c Tomato Fruit

The fruit of the Alcobaca landrace of tomato (Lycopersicon esculentum Mill.) have prolonged keeping qualities (determined by the allele a/c) and contain three times as much putrescine as the standard Rutgers variety (A/c) at the ripe stage (ARG Dibble, PJ Davies, MA Mutschler [1988] Plant Physiol 86: 338-340). Polyamine metabolism and biosynthesis were compared in fruit from Rutgers and Rutgers-a/c—a near isogenic line possessing the allele a/c, at four different stages of ripening. The levels of soluble polyamine conjugates as well as wall bound polyamines in the pericarp tissue and jelly were very low or nondetectable in both genotypes. The increase in putrescine content in a/c pericarp is not related to normal ripening as it occurred with time and whether or not the fruit ripened. Pericarp discs of both normal and a/c fruit showed a decrease in the metabolism of [1,4-¹⁴C]putrescine and [terminal labeled-³H]spermidine with ripening, but there were no significant differences between the two genotypes. The activity of ornithine decarboxylase was similar in the fruit pericarp of the two lines. Arginine decarboxylase activity decreased during ripening in Rutgers but decreased and rose again in Rutgers-a/c fruit, and as a result it was significantly higher in a/c fruit than in the normal fruit at the ripe stage. The elevated putrescine levels in a/c fruit appear, therefore, to be due to an increase in the activity of arginine decarboxylase.     

水分胁迫对燕麦穗颖渗透调节和抗氧化能力的影响

以抗旱性不同的燕麦品种‘蒙燕1号’(抗旱性强)和‘坝莜3号’(水分敏感)为试验材料,采用盆栽方式研究了抽穗期和灌浆期水分胁迫对燕麦穗颖渗透调节和抗氧化能力的影响。结果表明:(1)水分胁迫处理均显著促进了不同抗旱性品种穗颖渗透调节物质(游离脯氨酸和可溶性蛋白)含量增加,并以抗旱品种累积水平高于水敏感品种,且两种渗透调节物质对抽穗期胁迫的反应比灌浆期胁迫更敏感。(2)两时期的水分胁迫处理均能降低不同抗旱性品种穗颖SOD和POD活性,抗旱品种的保护酶活性要高于水敏感品种,抗旱品种的SOD活性降低幅度明显低于水敏感品种,而POD活性降低幅度在两品种间差异不明显。(3)水分胁迫导致2个品种穗颖丙二醛(MDA)含量和相对电导率显著增加,细胞膜结构受到严重伤害,且水敏感品种受害程度大于抗旱品种。(4)水分胁迫使2个品种单株籽粒产量下降,且在中度胁迫和重度胁迫下,抗旱品种的减产幅度要低于同期水敏感品种;水分胁迫下,水敏感品种‘坝莜3号’减产4.54%~30.29%,抗旱品种‘蒙燕1号’减产6.69%~23.54%。可见,抗旱性强的燕麦品种在受到水分胁迫的条件下能通过增强穗颖渗透调节和抗膜质过氧化能力、减弱穗颖细胞质膜损伤程度来适应干旱胁迫,最大限度减少水分胁迫对穗颖的伤害,有利于稳产。     

Diurnal Modulation of Arginine Decarboxylase Activity of Chenopodium rubrum L. by Temperature and Light

Arginine decarboxylase (ADC), one of the key enzymes of polyaminemetabolism in plants, was investigated in Chenopodium rubrumL. seedlings under constant and alternating temperature andlighting conditions. With seedlings grown at constant temperature,ADC activity of the whole seedling increased rapidly betweenthe second and the third day after sowing. This effect was alwayshigher under continuous light than in continuous darkness. Fromthe third to the seventh day after sowing, there was a markeddecrease in ADC activity of the whole seedling almost down tothe level of the second day. Under "normal" lighting and temperatureconditions (32.5?C/10?C, light/dark) there was a marked increasein ADC activity when plants were transferred to 10?C and a rapiddecrease when they were transferred to 32.5?C. The same timecourse was observed when an "inverse" light-temperature program(32.5?C/10?C; dark/light) was applied. This means that the timecourse of ADC activity in the seedlings is slightly light-dependent,but strongly temperature-dependent. The data are discussed withrespect to the chronopathological effects of the "inverse" light-temperatureprogram. (Received March 5, 1985; Accepted October 2, 1985)     

Effect of Osmotic Stress on Carbon Metabolism in Chlamydomonas reinhardtii: Accumulation of Glycerol as an Osmoregulatory Solute

NaCl, KCl, and sucrose at equiosmolar concentrations had similar inhibitory effects on photosynthetic carbon metabolism by the freshwater green alga, Chlamydomonas reinhardtii. Inhibitory concentrations of these solutes altered the products of photosynthetic ¹⁴CO₂ incorporation, resulting in reduced incorporation into starch, sugar phosphates, lactate, and glycolate, but caused an accumulation of glycerol both intracellularly and in the medium.     

Cerebral Polyamine Metabolism: Inhibition of Spermidine Biosynthesis by Dicyclohexylamine

Since a specific inhibition of cerebral spermidine (Spd) synthase activity by alicyclic amines was preliminarily observed in vitro, we examined the in vivo inhibitory effectiveness of dicyclohexylamine (DCHA) on Spd biosynthesis in 21-day-old rat brain. For this purpose a previously reported HPLC procedure (Porta et al., 1981a) was modified to analyze the cerebral levels of DCHA at the time of polyamine determinations. The intraperitoneally injected DCHA was shown to cross the blood-brain barrier easily, reaching high levels in the cerebral tissue (approximately 750 nmol/g brain) within 1 h of its administration. The effect of the drug on the polyamine metabolism resulted in a significant depletion of Spd biosynthesis from the sixth hour after the treatment and in an earlier and prolonged increase of the putrescine (Pt) steady-state levels. Conversely, the spermine (Spm) endogenous pools remained unchanged throughout the 24-h post-DCHA period. Moreover, following the intracerebral administration of [1,4-14C]Pt, significantly lower specific radioactivity (s.r.a.) values for labeled Pt and Spd were recorded in the brains of DCHA-treated animals. Conversely, after intracerebral [14C]Spd injection, the s.r.a. of newly formed [14C]Spm remained unchanged, confirming the specificity of the DCHA effect on the Spd biosynthesis.     

Characterization of the Link between Ornithine,Arginine, Polyamine and Siderophore Metabolism in Aspergillus fumigatus

The opportunistic fungal pathogen Aspergillus fumigatus produces siderophores for uptake and storage of iron, which is essential for its virulence. The main precursor of siderophore biosynthesis (SB), ornithine, can be produced from glutamate in the mitochondria or by cytosolic hydrolysis of ornithine-derived arginine. Here, we studied the impact of mitochondrial versus cytosolic ornithine biosynthesis on SB by comparison of the arginine auxotrophic mutants ΔargEF and ΔargB, which lack and possess mitochondrial ornithine production, respectively. Deficiency in argEF (encoding acetylglutamate kinase and acetylglutamyl-phosphate-reductase), but not argB (encoding ornithine transcarbamoyl transferase) decreased (i) the cellular ornithine content, (ii) extra- and intracellular SB, (iii) growth under harsh iron starvation, (iv) resistance to the ornithine decarboxylase inhibitor eflornithine, and (v) virulence in the Galleria mellonella larvae model. These lines of evidence indicate that SB is mainly fueled by mitochondrial rather than cytosolic ornithine production and underline the role of SB in virulence. Ornithine content and SB of ΔargB increased with declining arginine supplementation indicating feedback-inhibition of mitochondrial ornithine biosynthesis by arginine. In contrast to SB, the arginine and polyamine contents were only mildly affected in ΔargEF, indicating prioritization of the latter two ornithine-consuming pathways over SB. These data highlight the metabolic differences between the two arginine auxotrophic mutants ΔargEF and ΔargB and demonstrate that supplementation of an auxotrophic mutant does not restore the wild type metabolism at the molecular level, a fact to be considered when working with auxotrophic mutants. Moreover, cross pathway control-mediating CpcA was found to influence the ornithine pool as well as biosynthesis of siderophores and polyamines.     

The Metabolism of Oat Leaves during Senescence: II. Senescence in Leaves Attached to the Plant

The course of senescence in the first leaves of light-grown Avena seedlings when attached to the plant has been compared with that previously studied in detached leaves and leaf segments. Proteolysis in the leaf, whether attached or detached, is accompanied by markedly polar basipetal transport of amino acids. This polar transport can be superimposed on the known transport of amino acids towards a locally applied cytokinin. In the intact plant, it results in a strong movement into the roots. The reducing sugars, which are set free in senescence, do not participate appreciably in this polar transport phenomenon.     

Osmotic Stress in Coleoptiles and Primary Leaves of Wheat. II. Main Phosphorylated Fractions

Seedlings of wheat (Triticum durum, cv. Balcarceno-INTA) werewater-stressed in darkness with 20% polyethylene glycol (PEG)6000 or 0.3 M mannitol added to the root medium. At differenttimes and up to a total of 36 h of treatment the coleoptilesand primary leaves were cut and analysed for acid soluble-P,lipid-P, protein-P, alkali-stable organic-P, and nucleic acid-P.All the phosphorylated fractions were expressed on 100 mg oftotal coleoptile and primary leaves dry wt. Acid soluble-P (14.6µmol) accounted for most of the phosphorus. The lipid-Pfraction remained at an unchanged level (6.2 µmol/100mg dry wt) in control or in mildly osmotic stressed shoots ofseedlings over the period of treatment. The protein-P representedonly 2.4% (0.8 µmol) of the total phosphorus found incoleoptiles and primary leaves. The alkali-stable organic-Pand nucleic acid-P fractions represented 22.2% (7.2µ mol)and 11.1% (3.6 µmol) of the total phosphorus content ofshoots, respectively. All the phosphorylated fractions, exceptingthe lipid-P fraction, underwent a significant (P < 0.025)fall over periods of up to 36 h of wheat seedlings growth indarkness. A common characteristic found in all the phosphorylatedfractions was the fact that there were not significant differencesbetween control and 20% PEG 6000 or 0.3 M mannitol treated seedlingsduring 36 h of treatment and growth in darkness. However, whenseedlings were pulse-labelled with 32P during imbibition, someeffects of the osmotic stressants on several fractions couldbe seen. Specific radioactivity fell in acid soluble-P of controlshoots, but increased in 20% PEG 6000 and in 0.3 M mannitoltreated seedlings. Radioactive phosphorus was not found in thealkali-stable organic-P fraction. Lipid-P, nucleic acid-P, andprotein-P fractions increased their specific activities during36 h of shoot growth in control plants. Such 20% PEG 6000 and0.3 M mannitol restricted this increase during the same periodof time, the former being more active. Possible implicationsof phospholipid and phosphoprotein turnover in relation to waterstress are discussed.