Effect of exogenous amino acids on the intracellular content of proline and other amino acids in Daucus carota cells

The effect of tryptophan on the biosynthesis of proline has been investigated. Cells of Daucus carota grown in B5 medium supplemented with 5×10^–4M tryptophan acquired the ability to grow in the presence of inhibitory concentrations of azetidine-2-carboxylic acid, an analog of proline. When trp was added to carrot cell cultures at sub-growth inhibiting concentrations, overproduction of intracellular free proline was observed. An increase was also observed for lys, his, ala, leu and phe. Likewise, the addition of asparagine, glutamic acid and phenylalanine to the medium stimulated the intracellular increase of free proline and other amino acids.Abbreviations A2CA azetidine-2-carboxylic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 5MT 5-methyltryptophan - P5C pyrroline-5-carboxylic acid - f.wt. fresh weight - d.wt. dry weight     

Regulation of proline accumulation in Arabidopsis thaliana (L.) Heynh during development and in response to desiccation

Significant differences were observed in the amount and proportion of free amino acids in different organs of Arabidopsis thaliana (L.) Heynh, ecotype Columbia. The most notable were found for proline, which formed 17–26% of the total free amino acid concentration in reproductive tissues (floret and seed), but only 1–3% of the total free amino acid concentration in vegetative tissues (rosette leaf and root). Proline accumulation was associated with tissues that had relatively low water contents. Tissues which displayed high water contents, such as rosette leaves, contained low levels of proline. A significant increase in the levels of proline accumulation occurred in plants subjected to experimentally induced low water potentials as compared to unstressed plants. For instance, an 8–10-fold increase in proline was observed in the presence of 120 mmol kg^?1 NaCl or KCl, and a 20-fold increase was stimulated by 60 mmol kg^?1 PEG. However, in addition to the accumulation of proline, massive accumulation of Na⁺, K⁺ and Cl^? ions occurred in tissues of plants stressed with salt. No significant differences were observed in mineral ions in plants stressed with PEG. Isotope tracer experiments with ¹⁴C compounds established that glutamate, ornithine and arginine are precursors of the proline biosynthesis induced by PEG in response to low water potentials in Arabidopsis thaliana. We conclude that the accumulation of proline in response to PEG occurs through increased biosynthesis.     

Proline accumulation induced by weak acids and IAA in coleoptiles of wheat seedlings

Proline accumulation in coleoptiles of wheat seedlings or in excised coleoptile segments incubated under shaking for a 24 h period was studied. There was no increase of proline content of coleoptiles after incubation of the seedlings in 5 mM citric acid (a relatively strong and slowly penetrating organic acid) in a pH range from 4.5 to 7.0 and only a slight increase of proline content after incubation in phosphate buffer at pH 7.0 to 7.5 duo to the higher osmotic concentration of phosphate buffer in this pH range. Quite different results were obtained with seedlings incubated in 10 mM acetic acid, a weak and easily penetrating organic acid. With increasing proton concentrations, proline accumulation increased. Application of 400 mM mannitol or higher concentrations of IAA (more than 10⁻⁵M) additionally increased proline accumulation in the presence of 10 mM acetic acid in the pH range from 6.0 to 7.5 in which acetic acid alone was loss effective. It is suggested that a decrease of cytosolic pH causes stress—induced proline accumulation.     

Proline biosynthesis in a proline-accumulating barley mutant

A barley (Hordeum vulgare L.) mutant, R5201, selected for resistance to 4? mM trans-4-hydroxyproline had a 3–6 fold increase in the soluble proline content of the leaf compared with the parent cultivar, Maris Mink. The mutant converted more [U-⁴C]glutamic acid to free proline in the leaves than Maris Mink but incorporation into protein proline was similar. Incorporation of radioactivity into proline was inhibited by exogenous proline more in Maris Mink than R5201, suggesting that feedback inhibition of proline biosynthesis is relaxed, but not absent in the mutant. When [1-¹⁴C]ornithine was the precursor, both R5201 and Maris Mink incorporated similar small amounts of label into soluble and protein proline. More protein proline was formed by both genotypes from labelled glutamic acid than from labelled ornithine. There may exist two routes of proline formation, where the glutamate pathway is synthetic and the ornithine pathway is catabolic.     

Influence of proliferative rates and a system substrate availability on proline transport in primary cell cultures of the R3230AC mammary tumor

Regulation of A system amino acid transport was studied in primary cultures of the R3230AC mammary adenocarcinoma. Higher rates of carrier-mediated Na⁺-dependent proline transport, v^c, was decreased and was attributed to a two-fold decrease in Vmax and a two-fold increase in Km. When compared to cells grown in standard media (Eagle's minimal essential medium, MEM), cells grown in media supplemented with A system substrates (alanine, serine, glycine, and proline) demonstrated adaptive decreases in proline transport; the decrease was due to two-fold reduction in Vmax, with no change in Km for proline. Even in the presence of preferred substrates for the A system, a density-dependent decrease in proline transport was manifested. Both fast- and slow-growing cultures maintained in MEM exhibited rapid increases in proline transport when switched to buffers devoid of amino acids; two-fold increases in Vmax were seen within 4 hr, but Km was unchanged. This starvation-induced adaptation was completely prevented by inclusion in the buffer of 10 mM proline, 0.1 mM -(methylamino)-isobutyric acid (MetAIB) or 10 mM serine, whereas inclusion of the poorer A system substrate, phenylalanine (10 mM), had no effect. The effects of MetAIB to prevent starvation-induced increases in proline transport were dose-related, rapid, and reversible. Amino acid starvation-induced increases in proline transport were partially blocked by cycloheximide or actinomycin D. Data were obtained demonstrating a temporal relationship between increasing intracellular [proline] and decreasing v^c for proline uptake. In addition, efflux of proline from preloaded cells preceded the increase in initial rates of proline entry. Taken together, we concluded that: (1) A system transport in primary cultures of this mammary adenocarcinoma is regulated by cell density as well as by availability of A system substrates, but these two types of regulation are kinetically distinct; and (2) starvation-induced enhancement of proline transport appears to be due to release from transinhibition, but may also involve a derepression-repression type of mechanism.     

Regulation of amino acid transport in L6 muscle cells: I. Stimulation of transport system a by amino acid deprivation

The regulation of amino acid transport in L6 muscle cells by amino acid deprivation was investigated. Proline uptake was Na⁺-dependent, saturable and concentrative, and was predominantly through system A. Proline uptake was inhibited by alanine, α-amino isobutyric acid (AIB), and by α-methylamino isobutyric acid, but not by lysine or valine. At 25°C, Km of proline uptake was 0.5 mM. Amino acid-deprivation resulted in a progressive increase in the rate of proline uptake, reaching up to 6-fold stimulation after 6 hours. The basal and stimulated transport were equally Na⁺-dependent, and both were inhibited by competition with the same amino acids. Kinetic analysis showed that Km decreased by a factor of 2.4 and Vmax increased 1.9-fold in deprived cells. Amino acid-deprivation did not stimulate amino acid uptake through systems other than system A. This suggests that the higher Km in proline-supplemented cells is not due to release of intracellular amino acids into unstirred layers surrounding the cells. The presence of amino acids which are substrates of system A (including AIB) during proline-deprivation, prevented stimulation of proline uptake, whereas those transported by systems Ly⁺ or L exclusively were ineffective. The stimulation of the transport-rate in deprived cells could be reversed by subsequent exposure to proline or other substrates of system A. L6 cells, deprived of proline for 6 hours, retained the stimulation of transport after detachment from the monolayers with trypsin. Uptake rates were comparable in suspended and attached cells in monolayer culture. Thus, amino acid-depreivation of L6 cells results in an adaptive increase in proline uptake, which is not due to unstirred layers but appears to be mediated by other mechanisms of selective transport regulation.     

Effects of Juglone on Growth of Muskmelon Seedlings with Respect to Physiological and Anatomical Parameters

The effect phosphinothricin (PPT), an inhibitor of glutamine synthetase (GS), on proline accumulation in detached rice leaves was investigated. During 12 h incubation, PPT inhibited GS activity and induced accumulation of NH₄ ⁺, and accumulation of proline in the light but not in darkness. Proline accumulation caused by PPT in the light was related to protein hydrolysis, and increase in the contents of precursors of proline, ornithine and arginine. Abscisic acid accumulation was not required for proline accumulation in PPT-treated rice leaves. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ion fluxes and abscisic Acid-induced proline accumulation in barley leaf segments

The increase in proline induced by ABA, a process stimulated by NaCl or KCl in barley leaves, did not occur when Na⁺ (or K⁺) was present in the external medium as the gluconate salt, namely with an anion unable to permeate the plasma membrane. However, proline increase was restored, to different extents, by the addition of various chloride salts but not by ammonium chloride. Moreover, it was shown that the stimulation of the process by NaCl (or KCl) was variously affected by the presence of different salts; all the ammonium salts (10 millimolar NH₄⁺ concentration) inhibited this stimulation almost completely. Inhibition by NH₄⁺ was accompanied by a decreased Na⁺ influx (−40%). Also, in the case of Na-gluconate, Na⁺ uptake was reduced and the addition of Cl⁻ as the calcium or magnesium salt (but not as ammonium salt) restored both the ion influxes and the increase in proline typical of NaCl treatments. Both 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS), an anion transport inhibitor, and tetraethylammonium chloride (TEA), a K⁺ channels-blocking agent, caused, as well as with a reduction of ion influxes, an inhibition of the proline accumulation. The inhibition was practically total with 1 millimolar DIDS and about 80% with 20 millimolar TEA. A possible role of ion influxes in the process leading to the increase in proline induced by ABA is proposed.     

Bridging the gap: A set of selective 1H-15N-correlations to link sequential neighbors of prolines

Triple-resonance experiments are standard in the assignment of protein spectra. Conventional assignment strategies use ¹H-¹⁵N-correlations as a starting point and therefore have problems when proline appears in the amino acid sequence, which lacks a signal in these correlations. Here we present a set of amino acid selective pulse sequences which provide the information to link the amino acid on either side of proline residues and thus complete the sequential assignment. The experiments yield amino acid type selective ¹H-¹⁵N-correlations which contain signals from the amino protons of the residues either preceding or following proline in the amino acid sequence. These protons are correlated with their own nitrogen or with that of the proline. The new experiments are recorded as two-dimensional experiments and their performance is demonstrated by application to a 115-residue protein domain.     

Effect of L-azetidine 2-carboxylic acid on growth and proline metabolism in Escherichia coli

The effects of L-azetidine 2-carboxylic acid on growth and proline metabolism in a proline-requiring auxotroph of Escherichia coli are described. The homologue inhibited growth of the wild type and it, alone, did not substitute effectively for proline as a growth supplement for the mutant. In medium containing 0.05 mM proline, the addition of increasing amounts of homologue progressively inhibited growth of the wild type but stimulated growth of the mutant at homologue: proline ratios of 10 : 1 and 50 : 1. This suggested that the homologue exerted a “sparing effect” on proline in the mutant.The incorporation of L-[U-¹⁴C]proline and L-[³H]azetidine 2-carboxylic acid into hot trichloroacetic acid-insoluble material in the mutant was measured. Amino acid analysis of the insoluble material from cells incubated with radiolabeled proline alone revealed that proline was partially degraded and metabolized to other amino acids prior to incorporation into protein. The addition of unlabeled homologue to the incubation medium significantly reduced proline catabolism, suggesting that the homologue exerted a sparing effect on proline in this mutant. In medium containing unlabeled proline and radiolabeled L-azetidine 2-carboxylic acid, the homologuewas incorporated both intact and partially degraded prior to incorporation into protein. Alanine was the major L-azetidine 2-carboxylic acid catabolite.     

Effect of L-azetidine 2-carboxylic acid on growth and proline metabolism in Escherichia coli

The effects of L-azetidine 2-carboxylic acid on growth and proline metabolism in a proline-requiring auxotroph of Escherichia coli are described. The homologue inhibited growth of the wild type and it, alone, did not substitute effectively for proline as a growth supplement for the mutant. In medium containing 0.05 mM proline, the addition of increasing amounts of homologue progressively inhibited growth of the wild type but stimulated growth of the mutant at homologue: proline ratios of 10 : 1 and 50 : 1. This suggested that the homologue exerted a “sparing effect” on proline in the mutant.The incorporation of L-[U-¹⁴C]proline and L-[³H]azetidine 2-carboxylic acid into hot trichloroacetic acid-insoluble material in the mutant was measured. Amino acid analysis of the insoluble material from cells incubated with radiolabeled proline alone revealed that proline was partially degraded and metabolized to other amino acids prior to incorporation into protein. The addition of unlabeled homologue to the incubation medium significantly reduced proline catabolism, suggesting that the homologue exerted a sparing effect on proline in this mutant. In medium containing unlabeled proline and radiolabeled L-azetidine 2-carboxylic acid, the homologuewas incorporated both intact and partially degraded prior to incorporation into protein. Alanine was the major L-azetidine 2-carboxylic acid catabolite.     

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.     

Stimulation of the proline cycle and anthraquinone accumulation in <Emphasis Type="Italic">Rubia tinctorum</Emphasis> cell suspension cultures in the presence of glutamate and two proline analogs

Anthraquinone biosynthesis in Rubia tinctorum L. involves different metabolic routes. Chorismic acid, the end-product of the shikimate pathway, becomes the branch point between primary and secondary metabolism. It has been proposed that the proline cycle could be coupled with the pentose phosphate pathway (PPP), since the NADP⁺ generated by proline reduction from glutamate could act as a cofactor of the first enzymes of the PPP. This pathway generates erythrose-4-phosphate, the substrate of the shikimate pathway. The aim of the present work was to study the effect of the addition of glutamate and two proline analogs, azetidine-2-carboxylic acid and thiazolidine-4-carboxylic acid (T4C), on the PPP, the proline cycle, and anthraquinone production in R. tinctorum cell suspension cultures. The addition of 5 mM of glutamate enhanced both anthraquinone (up to 30%) and total phenolic content (12%), which correlated well with proline accumulation. Only the addition of 200 μM of T4C resulted in an increase in anthraquinone production, which was accompanied by a rise in the proline content. Neither the addition of glutamate nor proline analogs resulted in the induction of PPP, so this route was not a limiting factor as a carbon donor to the shikimate pathway.     

Role of Ca2+ in Drought Stress Signaling in Wheat Seedlings

Plants use complex signal transduction pathways to perceive and react to various biotic and/or abiotic stresses. As a consequence of this signaling, plants can modify their metabolism to adapt themselves to new conditions. One such change is the accumulation of proline in response to drought and salinity stresses. We have studied drought and salinity induced proline accumulation and the roles of Ca²⁺ (10 mM) and indoleacetic acid (IAA, 0.3 mM) in this response. Subjecting seedlings to both drought (6% polyethylene glycol, PEG) and salinity (150 mM NaCl) stress resulted in a dramatic increase in proline accumulation (7-fold higher than control level). However, the application of Ca²⁺ along with these stress factors had different effects. Unlike the salinity stress, Ca²⁺ prevented the drought induced proline accumulation indicating that these stress factors use distinct signaling pathways to induce similar responses. Experiments with IAA and EGTA (10 mM) supported this interpretation and suggested that Ca²⁺ and auxin participate in signaling mechanisms of drought-induced proline accumulation. Drought and salt stress-induced proline accumulation was compared on salt resistant (cv. Gerek 79) and salt sensitive (cv. Bezostaya) wheat varieties. Although proline level of the first was twofold lower than that of the second in control, relative proline accumulation was dramatically higher in the case of the salt resistant wheat variety under stress conditions.     

Does abscisic acid influence proline accumulation in stressed leaves?

Root treatments of barley (Hordeum distichum L.) plants with 10^-7 to 10^-4 M abscisic acid (ABA) caused an increase in proline content, especially at higher concentrations, within 2–3 h. Even 3 h after the removal of ABA from the medium the plants continued to accumulate proline. The higher the concentration of the ABA, the higher was the proline level at 6 h. When the highest ABA concentration, 10^-4 M, was tested with polyethylene glycol (PEG) (-5.0 bars) in the medium, the ABA treatment resulted in a higher proline content than in control plants. The treatments PEG alone and PEG + ABA resulted in heavy accumulation of proline, especially, 3 h after releasing the plants from the stress. The proline content in PEG+ABA-treated plants was always higher than plants treated with PGE or ABA alone. In peas (Pisum sativum L. cv Alaska) the same trend occurred although to a lesser degree. These findings indicate an influence of ABA on proline accumulation in water-stressed plants.Abbreviations ABA abscisic acid - PEG polyethylene glycol - RWC relative water content     

Inhibition of growth of proline-requiring Chinese hamster ovary cells (CHO-K1) resulting from antagonism by a system amino acids

CHO-K1 requires proline for growth. Two proline-independent revertants were isolated—K1-J and K1-6. CHO-K1 pro^? is much more sensitive than the pro⁺ cell lines to inhibition of growth by addition to the medium of amino acids and amino acid analogues that are transported through the A system. In contrast, pro⁺ cells are as sensitive as, or in some cases slightly more sensitive than, pro^? cells to glycine, basic amino acids, and to amino acids that are mainly transported by the L system. The A system analogue α(methylamino) isobutyric acid (MAIB) in low concentrations reacts competitively with proline to regulate the growth of pro^? cells, yielding a K_i for MAIB of 0.56 mM. CHO-K1 and K1-6 transport proline at the same initial rate and are equally sensitive to the inhibition of proline transport by alanine. Alanine and MAIB inhibit proline transport strongly and similarly in CHO-K1. Thus although these compounds inhibit the transport of proline by both cell types to the same extent, pro⁺ cells are immune to the effect of this starvation since they are able to synthesize their own proline. We also describe a secondary inhibition caused by high A system amino acid concentrations that affects both pro^? and pro⁺ cells.     

Collagen biosynthesis by human skin fibroblasts III. The effects of ascorbic acid on procollagen production and prolyl hydroxylase activity

Human skin fibroblasts were cultured under conditions optimized for collagen synthesis, and the effects of ascorbic acid on procollagen production, proline hydroxylation and the activity of prolyl hydroxylase were examined in cultures. The results indicated that addition of ascorbic acid to confluent monolayer cultures of adult human skin fibroblasts markedly increased tha amount of [³H]hydroxyproline syntehsized. Ascorbic acid, however, did not increase the synthesis of ³H-labeled collagenous polypeptides assayed independently of hydroxylation of proline residues, nor did it affect the amount of prolyl hydroxylase detectable by an in vitro enzyme assay. Also long-term cultures of the cells or initiation of fibroblast cultures in the presence of ascorbic acid did not lead to an apparent selection of a cell population which might be abnormally responsive to ascorbic acid. Thus, ascorbic acid appears to have one primary action on the synthesis of procollagen by cultured human skin fibroblasts: it is necessary for synthesis of hydroxyproline, and consequently for proper triple helix formation and selection of procollagen.     

Exogenous ornithine is an effective precursor and the δ-ornithine amino transferase pathway contributes to proline accumulation under high N recycling in salt-stressed cashew leaves

The role of the δ-ornithine amino transferase (OAT) pathway in proline synthesis is still controversial and was assessed in leaves of cashew plants subjected to salinity. The activities of enzymes and the concentrations of metabolites involved in proline synthesis were examined in parallel with the capacity of exogenous ornithine and glutamate to induce proline accumulation. Proline accumulation was best correlated with OAT activity, which increased 4-fold and was paralleled by NADH oxidation coupled to the activities of OAT and Δ¹-pyrroline-5-carboxylate reductase (P5CR), demonstrating the potential of proline synthesis via OAT/P5C. Overall, the activities of GS, GOGAT and aminating GDH remained practically unchanged under salinity. The activity of P5CR did not respond to NaCl whereas Δ¹-pyrroline-5-carboxylate dehydrogenase was sharply repressed by salinity. We suggest that if the export of P5C from the mitochondria to the cytosol is possible, its subsequent conversion to proline by P5CR may be important. In a time-course experiment, proline accumulation was associated with disturbances in amino acid metabolism as indicated by large increases in the concentrations of ammonia, free amino acids, glutamine, arginine and ornithine. Conversely, glutamate concentrations increased moderately and only within the first 24 h. Exogenous feeding of ornithine as a precursor was very effective in inducing proline accumulation in intact plants and leaf discs, in which proline concentrations were several times higher than glutamate-fed or salt-treated plants. Our data suggest that proline accumulation might be a consequence of salt-induced increase in N recycling, resulting in increased levels of ornithine and other metabolites involved with proline synthesis and OAT activity. Under these metabolic circumstances the OAT pathway might contribute significantly to proline accumulation in salt-stressed cashew leaves.     

In Vitro effects of zinc on markers of bone formation

Zinc deficiency is associated with a reduced rate of bone formation that can be corrected by supplementation of the deficient diet with adequate amounts of zinc. This study was conducted to examine the effects of zinc on bone cell parameters associated with bone formation. Tibiae were removed from 19-d-old chicken embryos and incubated for 48 h in Dulbecco’s modified Eagle’s medium supplemented with antibiotics, bovine serum albumin, and HEPES. The addition of zinc (25–200 Μg/dL) to tibial cultures resulted in a concentrationdependent increase in alkaline phosphatase activity, an increase in the incorporation of proline into bone protein and an increase in the posttranslational oxidation of proline to peptidyl hydroxyproline. These effects of zinc were all diminished by the addition of 2,6-pyridine dicarboxylic acid, a chelator of zinc. The addition of either cycloheximide (10^-5M), dactinomycin (10^-8M), or hydroxyurea (10^-3M) to tibial cultures also attenuated the effects of zinc. The effect of zinc on bone cell DNA synthesis was measured by following the incorporation of³H-thymidine into DNA and by fluorometric measurement of cellular DNA content. These methods revealed that the addition of zinc to cultured tibiae resulted in a concentration-dependent increase in tibial DNA content and synthesis rate. The magnitude of the zinc-induced DNA increase was similar to the magnitude of the zinc-induced increases in alkaline phosphatase activity, proline incorporation, and hydroxyproline synthesis. Normalization of these latter responses to tibial DNA content yield data indicating that the effect of zinc on bone formation results from a zinc-induced increase in bone cell proliferation.     

Cell wall peroxidase against ferulic acid, lignin, and NaCl-reduced root growth of rice seedlings

The changes in cell wall peroxidase activity against ferulic acid (FPOD) and lignin level in roots of NaCl-stressed rice seedlings and their correlation with root growth were investigated. Increasing concentrations of NaCl from 50 to 150 mmol L⁻¹ progressively decreases root growth. The reduction of root growth by NaCl is closely correlated with the increase in FPOD activity extracted from the cell wall. In contrast, lignin level was reduced by NaCl. Since proline and ammonium accumulations are associated with root growth inhibition caused by NaCl, we determined the effect of proline or NH₄Cl on root growth and FPOD in roots. Exogenous application of NH₄Cl or proline markedly inhibited root growth and increased FPOD activity in rice seedlings in the absence of NaCl. An increase in FPOD activity in roots preceded inhibition of root growth caused either by NaCl, NH₄Cl, or proline. Our results suggest that cell-wall stiffening catalyzed by FPOD may participate in the regulation of root growth reduction of rice seedlings caused by NaCl.