Role of carbohydrates in proline accumulation in wilted barley leaves

The effect of wilting on proline synthesis, proline oxidation, and protein synthesis—all of which contribute to proline accumulation—was determined in nonstarved barley (Hordeum vulgare L.) leaves. Nonstarved leaves were from plants previously in the light for 24 hours and starved leaves were from plants previously in the dark for 48 hours. Wilted leaves from nonstarved plants accumulated proline at the rate of about 1 μmole per hour per gram of fresh weight whereas wilted leaves from starved plants accumulated very little proline. Wilting caused a 40-fold stimulation of proline synthesis from glutamate in nonstarved leaves but had very little effect in starved leaves. Proline oxidation and protein synthesis, on the other hand, were inhibited by wilting in both nonstarved and starved leaves. Thus, the role of carbohydrates in proline accumulation is to supply precursors for the stimulated proline synthesis. These results further indicate that the main metabolic response causing proline to accumulate in wilted barley leaves is the stimulation of proline synthesis from glutamate. The difference between these results and those obtained with beans is discussed.

Wilting caused an increased conversion of glutamate to other products. In nonstarved leaves, conversion to organic acids as well as to proline was increased. In starved leaves, wilting caused an increase in the conversion of glutamate to glutamine, aspartate, asparagine, and organic acids.

     

The effect of wilting on proline metabolism in excised bean leaves in the dark

The effects of wilting on the fate of proline and on the rates of nonprotein proline formation and utilization have been determined in excised bean leaves. Wilting did not alter the fate of exogenously added ¹⁴C-l-proline (2 mm) in either non-starved leaves (from plants previously in the light) or starved leaves (from plants previously in the dark). The fate of proline in nonstarved leaves was protein synthesis and in starved leaves was protein synthesis and oxidation to other compounds.     

Effects of NaCl on Proline Synthesis and Utilization in Excised Barley Leaves

Proline accumulation in NaCl-treated excised barley (Hordeum vulgare var Larker) leaves was studied. Leaves were treated by placing the cut end in NaCl solutions and allowing the salt to enter the leaf via the transpiration stream. Leaves treated this way maintained turgor while the sodium content increased and the osmotic potential decreased. Proline began accumulating after 12 hours and continued accumulating over the subsequent 12-hour period at an average rate of 0.6 micromoles per hour per gram fresh weight.

During the time proline was accumulating, [¹⁴C]glutamate was added to measure the effects of salt on proline synthesis from glutamate and [¹⁴C] proline was added in separate experiments to determine the effect of salt on proline utilization. Salt treatment dramatically increased proline synthesis from glutamate. Proline utilization by oxidation and for protein synthesis was decreased by 50 and 60%, respectively, by the salt treatment.

These effects are similar to the effects of drought and abscisic acid in barley leaves. The results indicate that common mechanisms cause proline to accumulate under these different stresses.

     

Metabolism of Proline, Glutamate, and Ornithine in Proline Mutant Root Tips of Zea mays (L.)

In excised pro_1-1 mutant and corresponding normal type roots of Zea mays L. the uptake and interconversion of [¹⁴C]proline, [¹⁴C]glutamic acid, [¹⁴C]glutamine, and [¹⁴C]ornithine and their utilization for protein synthesis was measured with the intention of finding an explanation for the proline requirement of the mutant. Uptake of these four amino acids, with the exception of proline, was the same in mutant and normal roots, but utilization differed. Higher than normal utilization rates for proline and glutamic acid were noted in mutant roots leading to increased CO₂ production, free amino acid interconversion, and protein synthesis. Proline was synthesized from either glutamic acid (or glutamine) or ornithine in both mutant and normal roots; it did not accumulate but rather was used for protein synthesis. Ornithine was not a good precursor for proline in either system, but was preferentially converted to arginine and glutamine, particularly in mutant roots. The pro_1-1 mutant was thus not deficient in its ability to make proline. Based on these findings, and on the fact that ornithine, arginine, glutamic acid and aspartic acid are elevated as free amino acids in mutant roots, it is suggested that in the pro_1-1 mutant proline catabolism prevails over proline synthesis.     

Isolation and characterization of Pseudomonas putida mutants affected in arginine, ornithine and citrulline catabolism: function of the arginine oxidase and arginine succinyltransferase pathways.

Pseudomonas putida mutants impaired in the utilization of arginine are affected in either the arginine succinyltransferase pathway, the arginine oxidase route, or both. However, mutants affected in one of the pathways still grow on arginine as sole carbon source. Analysis of the products excreted by both wild-type and mutant strains suggests that arginine is mainly channelled by the oxidase route. Proline non-utilizing mutants are also affected in ornithine utilization, confirming the role of proline as an intermediate in ornithine catabolism. Mutants affected in ornithine cyclodeaminase activity still grow on proline and become unable to use ornithine. Both proline non-utilizing mutants and ornithine-cyclodeaminase-minus mutants are unable to use citrulline. These results, together with induction of ornithine cyclodeaminase when wild-type P. putida is grown on citrulline, indicate that utilization of citrulline as a carbon source proceeds via proline with ornithine as an intermediate. Thus in P. putida, the aerobic catabolism of arginine on the one hand and citrulline and ornithine on the other proceed by quite different metabolic segments.     

Effects of proline and carbohydrates on the metabolism of exogenous proline by excised bean leaves in the dark

Proline was metabolized when vacuum infiltrated into starved bean (Phaseolus vulgaris L.) leaves from plants previously in the dark for 48 hours, but an equivalent increase in protein proline was not observed. When ¹⁴C-proline was infiltrated into starved leaves, a large percentage of the ¹⁴C was recovered in other amino acids, organic acids, and CO₂, in addition to that recovered as protein proline. However, extensive oxidation of proline was observed only if enough proline was added to increase substantially the endogenous concentration of proline. Increasing the endogenous concentration did not affect the amount of proline that was incorporated into protein.     

The effects of a pure protein diet on the amino-acid composition of Neomysis integer (Leach)

Neomysis integer (Leach) was fed lipid- and carbohydratc-free albumin or kaolin (starved) for up to eight days and the ability of the animal to maintain its total protein and free and protein amino-acid patterns was examined. Considerable effects on the body composition were seen although the amino-acid patterns of the diet and tissue protein were similar. Total protein was reduced to the same extent in both starved and albumin-fed animals, despite some utilization of the dietary protein. Conversely, the level of total free amino acid was reduced in starved animals but not in the protein-fed mysids. The amino-acid composition of the protein hydrolysates was altered after both starvation and feeding and the patterns produced indicated that protein synthesis, as well as catabolism had occurred in the albumin-fed animals. There were also changes in the composition of the free amino-acid pools of both starved and fed animals. In the starved samples taurine, aspartate, and arginine appeared to be conserved, while in the albumin-fed animals alanine, leucine, tyrosine and phenylalanine were substantially and consistently decreased, and arginine increased, in concentration. Although the results showed that the quality of the protein fraction was altered by the albumin diet, the amino-acid composition of the albumin had no direct effect on the resultant composition of either the protein hydrolysate or the free amino-acid pool in protein-fed animals.     

Starvation induces physiological changes that act on the cryotolerance of Lactobacillus acidophilus RD758

The relationship between lactose starvation and cryotolerance was investigated in Lactobacillus acidophilus RD758. Cryotolerance was measured from the acidification activity of cells recovered after 18-h lactose starvation. It was compared to that of nonstarved cells, both of them in a stationary phase and in the same medium. This measurement allowed quantifying the initial acidification activity before freezing, as well as the loss of acidification activity during freezing and the rate of loss during frozen storage. Even if initial acidification activity was similar for nonstarved and starved bacteria, the latter displayed a significantly better resistance to freezing and frozen storage at -20°C. To investigate the mechanisms that triggered these cryotolerance phenomena, the membrane fatty acid composition was determined by gas chromatography, and the proteome was established by 2-D electrophoresis, for starved and nonstarved cells. The main outcome was that the improved cryotolerance of starved cells was ascribed to two types of physiological responses as a result of starvation. The first one corresponded to an increased synthesis of unsaturated, cyclic, and branched fatty acids, to the detriment of saturated fatty acids, thus corresponding to enhanced membrane fluidity. The second response concerned the upregulation of proteins involved in carbohydrate and energy metabolisms and in pH homeostasis, allowing the cells to be better prepared for counteracting the stress they encountered during subsequent cold stress. These two phenomena led to a cross-protection phenomenon, which allowed better cryotolerance of Lb. acidophilus RD758, following cellular adaptation by starvation.     

Key role of L-alanine in the control of hepatic protein synthesis.

We investigated the effects of administration of single amino acids to starved rats on the regulation of protein synthesis in the liver. Of all the amino acids tested, only alanine, ornithine and proline promoted statistically significant increases in the extent of hepatic polyribosome aggregation. The most effective of these was alanine, whose effect of promoting polyribosomal aggregation was accompanied by a decrease in the polypeptide-chain elongation time. The following observations indicate that alanine plays an important physiological role in the regulation of hepatic protein synthesis. Alanine was the amino acid showing the largest decrease in hepatic content in the transition from high (fed) to low (starved) rates of protein synthesis. The administration of glucose or pyruvate is also effective in increasing liver protein synthesis in starved rats, and their effects were accompanied by an increased hepatic alanine content. An increase in hepatic ornithine content does not lead to an increased protein synthesis, unless it is accompanied by an increase of alanine. The effect of alanine is observed either in vivo, in rats pretreated with cycloserine to prevent its transamination, or in isolated liver cells under conditions in which its metabolic transformation is fully impeded.     

Control of arginine utilization in Neurospora.

The response of Neurospora to changes in the availibility of exogenous arginine was investigated. Upon addition of arginine to the growth medium, catabolism is initiated within minutes. This occurs prior to expansion of the arginine pool or augmentation of catabolic enzyme levels. (Basal levels are approximately 25% of those found during growth in arginine-supplemented medium.) Catabolism of arginine is independent of protein synthesis, indicating that the catabolic enzymes are active but that arginine is not available for catabolism unless present in the medium. Upon exhaustion of the supply of exogenous arginine, catabolism ceases abruptly, despite an expanded arginine pool and induced levels of the catabolic enzymes. The arginine pool supports protein synthesis until the cells regain their normal capacity for endogenous arginine synthesis. These observations, combined with the known small level of induction of arginine catabolic enzymes, non-repressibility of most biosynthetic enzymes, and vesicular localization of the bulk of the arginine pool, suggest that compartmentation plays a significant role in controlling arginine metabolism in Neurospora.     

Genetic control of the arginine pathways in Aspergillus nidulans. Common regulation of anabolism and catabolism

Summary Two regulatory mutants for arginine catabolism isolated as proline suppressors were tested for the synthesis of ornithine transcarbamylase (OTC), the arginine anabolic enzyme. Mutations at one locus, suD, result in the insensitivity of OTC synthesis to effectors responsible for the enzyme level in the wild strain. The common genetic regulation of both catabolic and anabolic pathways of arginine is postulated.     

Increased Fatty Acid beta-Oxidation after Glucose Starvation in Maize Root Tips

The effects of glucose starvation on the oxidation of fatty acids were studied in excised maize (Zea mays L.) root tips. After 24 hours of glucose starvation, the rate of oxidation of palmitic acid to CO₂ by the root tips was increased 2.5-fold. Different enzyme activities were tested in a crude particulate fraction from nonstarved root tips and those starved for 24 hours. The activities of the β-oxidation enzymes crotonase, hydroxyacyl-coenzyme A (CoA) dehydrogenase, and thiolase and those of catalase, malate synthase, and peroxisomal citrate synthase were higher after starvation. However, no isocitrate lyase activity was detected, thus suggesting that the glyoxylate cycle does not operate. The overall β-oxidation activity was assayed as the formation of [¹⁴C]acetyl-CoA from [¹⁴C]palmitic acid after high-performance liquid chromatography analysis of the CoA derivatives. An activity was detected in sugar-fed root tips, and it was increased by two-to fivefold in starved roots. Because the recovery of enzyme activities is only marginally better in starved roots compared with nonstarved roots, these results indicate that the β-oxidation activity in the tissues is increased during sugar starvation. This increase is probably an essential part of the response to a situation in which lipids and proteins replace carbohydrates as the major respiratory substrates. These results are discussed in relation to the metabolic changes observed in senescing plant tissues.     

Catabolism of arginine and ornithine in the perfused rat liver: effect of dietary protein and of glucagon

The rates of oxidation of arginine and ornithine that occurred through a reaction pathway involving the enzyme ornithine aminotransferase (EC 2.6.1.13) were determined using (14)C-labeled amino acids in the isolated nonrecirculating perfused rat liver. At physiological concentrations of these amino acids, their catabolism is subject to chronic regulation by the level of protein consumed in the diet. (14)CO(2) production from [U-(14)C]ornithine (0.1 mM) and from [U-(14)C]arginine (0.2 mM) was increased about fourfold in livers from rats fed 60% casein diets for 3-4 days. The catabolism of arginine in the perfused rat liver, but not that of ornithine, is subject to acute regulation by glucagon (10(-7) M), which stimulated arginine catabolism by approximately 40%. Dibutyryl cAMP (0.1 mM) activated arginine catabolism to a similar extent. In retrograde perfusions, glucagon caused a twofold increase in the rate of arginine catabolism, suggesting an effect of glucagon on arginase in the perivenous cells.     

Citrulline and nitrogen homeostasis: an overview

Citrulline (Cit) is a non-essential amino acid whose metabolic properties were largely ignored until the last decade when it began to emerge as a highly promising nutrient with many regulatory properties, with a key role in nitrogen homeostasis. Because Cit is not taken up by the liver, its synthesis from arginine, glutamine, ornithine and proline in the intestine prevents the hepatic uptake of the two first amino acids which activate the urea cycle and so prevents amino acid catabolism. This sparing effect may have positive spin-off for muscle via increased protein synthesis, protein content and functionality. However, the mechanisms of action of Cit are not fully known, even if preliminary data suggest an implication of mTOR pathway. Further exploration is needed to gain a complete overview of the role of Cit in the control of nitrogen homeostasis.     

Impacts of arginine nutrition on embryonic and fetal development in mammals

Embryonic loss and intrauterine growth restriction (IUGR) are significant problems in humans and other animals. Results from studies involving pigs and sheep have indicated that limited uterine capacity and placental insufficiency are major factors contributing to suboptimal reproduction in mammals. Our discovery of the unusual abundance of the arginine family of amino acids in porcine and ovine allantoic fluids during early gestation led to the novel hypothesis that arginine plays an important role in conceptus (embryo and extra-embryonic membranes) development. Arginine is metabolized to ornithine, proline, and nitric oxide, with each having important physiological functions. Nitric oxide is a vasodilator and angiogenic factor, whereas ornithine and proline are substrates for uterine and placental synthesis of polyamines that are key regulators of gene expression, protein synthesis, and angiogenesis. Additionally, arginine activates the mechanistic (mammalian) target of rapamycin cell signaling pathway to stimulate protein synthesis in the placenta, uterus, and fetus. Thus, dietary supplementation with 0.83 % l-arginine to gilts consuming 2 kg of a typical gestation diet between either days 14 and 28 or between days 30 and 114 of pregnancy increases the number of live-born piglets and litter birth weight. Similar results have been reported for gestating rats and ewes. In sheep, arginine also stimulates development of fetal brown adipose tissue. Furthermore, oral administration of arginine to women with IUGR has been reported to enhance fetal growth. Collectively, enhancement of uterine as well as placental growth and function through dietary arginine supplementation provides an effective solution to improving embryonic and fetal survival and growth.     

Senescence of Rice Leaves : VII. PROLINE ACCUMULATION IN SENESCING EXCISED LEAVES

Proline content increased greatly in detached rice (Oryza sativa cv. Taichung Native 1) leaves during senescence. There was a slight but significant increase in proline level after one day of incubation, and, subsequently, proline accumulated relatively rapidly. By 4 days after excision, the level of proline had increased 30- to 50-fold, which is similar to the level seen in the water-stressed detached rice leaves. It is unlikely that the proline accumulation in detached leaves is to be derived solely from protein hydrolysis, since the addition of l-glutamic acid increased the proline level during senescence. The proline analog, 3,4-dehydroproline, did not affect the level of proline during senescence. It seems that accumulation of proline may, at least in part, result from an increased rate of synthesis, possibly due to a disruption of the normal feedback inhibition of proline synthesis. Potassium cyanide and 2,4-dinitrophenol strongly inhibited proline accumulation, indicating that some energy compound(s) may participate in proline accumulation during senescence of excised rice leaves.     

Contribution of Arginine to Proline Accumulation in Water-stressed Barley Leaves

Barley (Hordeum vulgare cv Prior) leaves converted l-U-(14)C-arginine to labeled proline. Accumulation of radioactivity in proline was greater in wilted leaves, but only after 9 hours of incubation. As the increase in free proline was detectable after only 3 to 6 hours, it is likely that the observed stimulation of proline labeling represents a result rather than a cause of proline accumulation. Furthermore, the loss of total arginine during water stress was only 10 to 15% of the increase in proline. We conclude that arginine probably contributes less than 1% of the carbon in the expanding proline pool of wilted barley leaves.     

Incorporation of dl-[2-14C]ornithine and dl-[5-14C]arginine in milk constituents by the isolated lactating sheep udder

1. Lactating mammary glands of sheep were perfused for several hours in the presence of dl-[2-(14)C]ornithine or dl-[5-(14)C]arginine and received adequate quantities of acetate, glucose and amino acids. 2. In the [(14)C]ornithine experiment 1.4% of the casein and 1% of the expired carbon dioxide came from added ornithine; 96% of the total radioactivity in casein was recovered in proline; 13% of the proline of casein originated from plasma ornithine. 3. In this experiment the results of chemical degradation of proline of casein as well as relative specific activities in the isolated products are consistent with the view that ornithine is metabolized, by way of glutamic gamma-semialdehyde, to proline or glutamic acid. 4. In the [(14)C]arginine experiments 3% of the casein and 1% of the expired carbon dioxide came from arginine; 84% of the arginine and 9% of the proline of casein originated from plasma arginine. 5. In these experiments the relative specific activities of arginine, ornithine and proline in plasma are in agreement with the view that arginine is metabolized by way of ornithine to proline. The conversion of arginine into ornithine is probably catalysed by arginase, so that arginase in mammary tissue may be involved in the process of milk synthesis.     

Die Aminosäurenzusammensetzung der Proteine von Sonnen- und Schattenblättern der Blutbuche (Fagus sylvatica L. cv. Atropunicea)

Summary The amino acid contents of sun and shade leaves of the copper beech are significantly different. On the basis of dry matter, the concentration of the majority of amino acids is higher in shade leaves. Only the concentrations of proline, valine, histidine and arginine are about 30% lower compared with those of the other amino acids. Calculated on the basis of crude protein, the concentrations of lysine, histidine, arginine, valine, isoleucine and proline are considerably lower in shade leaves than in sun leaves. On the other hand, shade leaves contain more tyrosine, phenylalanine, and methionine.A hypothesis interpreting the different morphogenesis of sun and shade leaves in connection with the high proline content of sun leaves is discussed.     

Arginine and ornithine kinetics in severely burned patients: increased rate of arginine disposal

Arginine serves multiple roles in the pathophysiological response to burn injury. Our previous studies in burn patients demonstrated a limited net rate of arginine de novo synthesis despite a significantly increased arginine turnover (flux), suggesting that this amino acid is a conditionally indispensable amino acid after major burns. This study used [15N2-guanidino-5,5-2H2]arginine and [5-13C]ornithine as tracers to assess the rate of arginine disposal via its conversion to and subsequent oxidation of ornithine; [5,5-2H2]proline and [5,5,5-2H3]leucine were also used to assess proline and protein kinetics. Nine severely burned patients were studied during a protein-free fast ("basal" or fast) and total parenteral nutrition (TPN) feedings. Compared with values from healthy volunteers, burn injury significantly increased 1) fluxes of arginine, ornithine, leucine, and proline; 2) arginine-to-ornithine conversion; 3) ornithine oxidation; and 4) arginine oxidation. TPN increased arginine-to-ornithine conversion and proportionally increased irreversible arginine oxidation. The elevated arginine oxidation, with limited net de novo synthesis from its immediate precursors, further implies that arginine is a conditionally indispensable amino acid in severely burned patients receiving TPN.