External Proline Research Articles

Unlocking Triticum aestivum L. resilience: Exogenous proline's remarkable role in mitigating lead stress, and delving into the secrets of TaGSK1 gene expression

The application of proline as foliar spraying has emerged as a promising solution to combat lead-induced crop stress. This study highlights the effectiveness of external proline in enhancing wheat crop health under lead stress. We reveal how lead stress negatively impacts key factors like biomass, chlorophyll levels, and crop yield, particularly in 100-grain weight. Gas exchange parameters, including stomatal conductance, CO2 levels, photosynthesis, water-use efficiency, and transpiration, also suffer under lead stress. Significantly, exposure to lead stress triggers the activation of antioxidant enzymes and elevates levels of proline, calcium, and protein. Nonetheless, the application of foliar proline proves highly effective in alleviating these detrimental effects by reducing reactive oxygen species and enhancing proline and protein levels in wheat. Additionally, our findings demonstrate a significant upregulation of TaGSK1 gene expression at different proline concentrations. Compared to controls, TaGSK1 expression increases by 1.7-fold under 50 mM proline stress and an impressive 4.7-fold under 200 mM proline stress in wheat. This finding holds promise in alleviating the adverse effects of lead stress and strengthening crop resilience for a more secure and sustainable future in wheat cultivation amid various stressors.

Improvement of the yield and essential oil of fennel (Foeniculum vulgare Mill.) using external proline, uniconazole and methyl jasmonate under drought stress conditions

Drought stress is a significant environmental constraint that seriously limits crop productivity. A two-year field study was carried out as a split plot based on a randomized complete block design (RCBD) with three replicates in the experimental station of the agricultural faculty of Azarbaijan Shahid Madani University, Tabriz, Iran. This study evaluated the impacts of various irrigation regimes and plant growth regulators/proline on yield, its components, and essential oil content. Water shortage led to considerable decreases in umbels per plant, umbellets per umbel, fruits per umbellet, 1000-fruit weight, biological yield, and finally, fruit yield, despite increases in fruit essential oil content under moderate drought stress conditions. External methyl jasmonate, uniconazole, and proline significantly increased these parameters measured in fennel plants compared with the control. Improved effects of external plant growth regulators/proline tended to be greater in drought-stress plants, as it enhanced yield, its components, and fruit essential oil content compared with control. Foliar spraying, particularly methyl jasmonate, and uniconazole, enhanced e- anethole content under drought stress. Fenchone and methyl chavicol contents increased in plant growth regulators/proline-treated plants when they were kept under moderate drought stress conditions; however, their contents decreased by the severity of drought stress apart from the application of methyl jasmonate. Hence, applying these PGRs or proline could be critical in cultivating fennel plants under water shortage conditions to achieve higher essential oils yield and its constituents, particularly anethole, compared with control.

Effects of salinity and external proline on seed germination and early growth of Cucumis sativus cv. super ps

Amini F, Askary M, Mohtashami F. 2018. Effects of salinity and external proline on seed germination and early growth of Cucumis sativus cv. super ps. Nusantara Bioscience 10: 215-220. Salinity is one of the important abiotic stresses that has an adverse effect on plant productions. High salt concentrations had negative effects on plant processes such as seed germination, seedling growth and turgescent, vegetative growth, flowering and fruiting stages. Proline as a compatible solution plays an important role in defense mechanisms of cellular stress. This study was done to evaluate effects of proline pretreatment (0 and 20 mM) in terms of salinity (0, 50, 100 and 150 mM) on cucumber cv. super ps. The obtained results proved that salinity treatments had significant effects on seed germination rate. On the contrary, application of proline had no significant effect on germination percentages of control and salt-stressed seeds. The root fresh weight and length of plant reduced with salinity increment while proline treatment had positive significant effects on the root dry as well as the fresh weight. Although, no significant variation was found in the root length and dry weight of cultivars treated with proline under salinity stresses. Meanwhile, the shoot length and dry weight of plants declined after salt-stressed treatments. While this condition increased the fresh weight of shoot. Proline treatments increased all of observed parameters but there were no significant variation found in the root length or dry weight of plants treated under saline stresses. The total soluble carbohydrate of plants had positive significant correlation with salt concentration, but the application of proline declined carbohydrate amounts in both salt-stressed and non-stressed plant. Salinity stress increased sodium, proline, protein contents and CAT enzyme, GPOX as well as MDA concentrations, while in the proline-treated plants salinity stresses had no significant effect on CAT, GPOX, MDA concentrations and internal proline amount.

Genotype-specific response of Foeniculum vulgare grain yield and essential oil composition to proline treatment under different irrigation conditions

Drought stress is a major environmental stress which severely affects growth, yield, as well as quality and quantity of crop products. Proline as a multifunction amino acid has a crucial role in ameliorating of drought stress. In order to study the effect of external proline on the response of fennel to drought stress, three fennel genotypes (namely Urmia, Shiraz, and Kerman) were exposed to two irrigation regimes (drought stress and non-stress control) and two levels of foliar-applied proline (0 and 20 mM) in a pot experiment. Drought led to significant decreases in umbels/plant, plant height, 1000-grains weight, harvest index, above-ground dry mass, grain weight and grain and dry mass WUE, root volume, root fresh and dry mass, ratio of shoot/root, grains and stems essential oil weight/plant and significant increases in root length and grains limonene content. Foliar-applied proline led to notable increases in harvest index and grain essential oil weight in the presence of drought. It also led to increases in umbels/plant, 1000-grains weight, harvest index, above-ground dry mass, grain weight and grain and dry mass WUE and ratio of shoot/root in Urmia and Shiraz genotypes. Anethole, limonene, fenchone, estragole and α-pinene were the main essential oil constituents in both grain and stems. Grain fatty acid and phytosterol compositions were not affected by experimental treatments. It may be concluded that drought stress suppresses fennel growth and grain weight, but foliar-applied proline is potent to mitigate the adverse reaction through increasing growth, grain weight/plant, WUE and grains limonene content in genotypes such as Urmia and Shiraz.

Exogenous proline improves osmoregulation, physiological functions, essential oil, and seed yield of fennel

Proline is an amino acid with pivotal role in plant responses to environmental constraints. Effects of its foliar application (20mM) on physiological functions of 12 fennel (Foeniculum vulgare Mill.) genotypes at the presence of different moisture conditions (non-stress control and drought stress) was studied in a 2-year field study. Drought led to notable increases in mean leaf polyphenol, proline and total soluble carbohydrates and essential oil concentrations, despite decreases in carotenoids and chlorophyll concentrations, leaf water potential, relative water content, plant above-ground dry mass and water use efficiency. Exogenous proline resulted in significant increases in mean carotenoids, polyphenol, chlorophyll, proline, total soluble carbohydrates and essential oil concentrations and relative water content, but it caused a decrease in leaf water potential. Proline amendment positively affected plant water relations, due mostly to enhancement in osmoregulation, as certain genotypes with greater mean leaf proline concentration and relative water content appeared to produce greater above-ground dry mass, when exposed to the external proline. Ameliorative effects of exogenous proline tended to be greater in drought-stressed plants, as it led to the enhancement of chlorophyll concentration and relative water content of fennel in drought conditions. Genotypes Urmia and Yazd were found to be able to withstand better against drought and benefit more from external proline. Our findings suggest that while indigenous proline is the most sensitive osmoticum in fennel’s response to drought, its external amendment may bring about improvements in water relations and osmoregulatory measure in this medicinal plant.

Proline responding1 Plays a Critical Role in Regulating General Protein Synthesis and the Cell Cycle in Maize.

Proline, an important amino acid, accumulates in many plant species. Besides its role in plant cell responses to environmental stresses, the potential biological functions of proline in growth and development are unclear. Here, we report cloning and functional characterization of the maize (Zea mays) classic mutant proline responding1 (pro1) gene. This gene encodes a Δ1-pyrroline-5- carboxylate synthetase that catalyzes the biosynthesis of proline from glutamic acid. Loss of function of Pro1 significantly inhibits proline biosynthesis and decreases its accumulation in the pro1 mutant. Proline deficiency results in an increased level of uncharged tRNApro AGG accumulation and triggers the phosphorylation of eukaryotic initiation factor 2α (eIF2α) in the pro1 mutant, leading to a general reduction in protein synthesis in this mutant. Proline deficiency also downregulates major cyclin genes at the transcriptional level, causing cell cycle arrest and suppression of cell proliferation. These processes are reversible when external proline is supplied to the mutant, suggesting that proline plays a regulatory role in the cell cycle transition. Together, the results demonstrate that proline plays an important role in the regulation of general protein synthesis and the cell cycle transition in plants.

ALLEVIATION OF SALT STRESS USING EXOGENOUS PROLINE ON A CITRUS CELL LINE

Salinity constitutes an important abiotic problem since ancient times, worldwide, for it leads to a decrease in productivity of crops with agronomic value. Under salt stress conditions, plant cells develop strategies to cope with Na + and Cl - , including exclusion and compartmentalisation, induction of antioxidant enzymatic systems and compatible solutes accumulation, such as proline. The precise function of this osmolyte still remains unclear. Proline may act on osmotic adjustment, as a free radical scavenger, protecting enzymes and avoiding DNA damages. It has been also suggested the role of proline in prevention of lipid peroxidation and as a signalling/regulatory molecule. A salt-sensitive Citrus sinensis ‘Valencia late’ cell line has a smaller growth rate and accumulates proline in the presence of NaCl (>200 mM). The addition of external proline to this cell line was evaluated in terms of cell metabolism. A positive influence on the relieve of salt stress symptoms due to the presence of exogenous proline 5 mM and 100 mM NaCl was obtained, with increased growth of this salt sensitive citrus cell line.

Improved salt tolerance of melon ( Cucumis melo L.) by the addition of proline and potassium nitrate

A pot experiment was carried out under glasshouse conditions with melon ( Cucumis melo) cv. “Tempo F1” in a mixture of peat, perlite and sand (1:1:1) to investigate the effects of external proline and potassium nitrate applications to salinity-treated (150 mM) plants with respect to fruit yield, plant growth, some physiological parameters and ion uptake. Treatments were—(i) control (C): plants receiving nutrient solution, (ii) salinity treatment, as for control plus 150 mM NaCl. Salinity treatment was combined with or without either 5 mM supplementary KNO 3 or 10 mM proline. The salt treatment (150 mM NaCl) led to significant decreases in plant growth, fruit yield, relative water content (RWC), stomatal density, uptake of Ca 2+, K + and N, and chlorophyll a and b contents, accompanied by significant increases in Na + uptake, proline concentration and membrane permeability. Supplementary KNO 3 and proline treatments significantly ameliorated the adverse effects of salinity on plant growth, fruit yield and the physiological parameters examined. This could be attributed to the effects of all the external supplements in maintaining membrane permeability, and increasing concentrations of Ca 2+, N and K + in the leaves of plants subjected to salt stress.

Proline accumulation by mutation or disruption of the proline oxidase gene improves resistance to freezing and desiccation stresses in Saccharomyces cerevisiae.

We examined the role of intracellular proline under freezing and desiccation stress conditions in Saccharomyces cerevisiae. When cultured in liquid minimal medium, the proline-nonutilizing mutant containing the put1 mutation (proline oxidase-deficient) produced more intracellular proline, and increased the cell survival rate as compared to the wild-type strain after freezing and desiccation. We also constructed two PUT1 gene disruptants. PUT1-disrupted mutants in minimal medium supplemented with external proline at 0.1% accumulated higher proline levels than those of the control strains (17-22-fold). These disruptants also had a 2-5-fold increase in cell viability compared to the control strains after freezing and desiccation stresses. These results indicate that proline has a stress-protective function in yeast.

Proline accumulation by mutation or disruption of the proline oxidase gene improves resistance to freezing and desiccation stresses in Saccharomyces cerevisiae

We examined the role of intracellular proline under freezing and desiccation stress conditions in Saccharomyces cerevisiae. When cultured in liquid minimal medium, the proline-nonutilizing mutant containing the put1 mutation (proline oxidase-deficient) produced more intracellular proline, and increased the cell survival rate as compared to the wild-type strain after freezing and desiccation. We also constructed two PUT1 gene disruptants. PUT1-disrupted mutants in minimal medium supplemented with external proline at 0.1% accumulated higher proline levels than those of the control strains (17–22-fold). These disruptants also had a 2–5-fold increase in cell viability compared to the control strains after freezing and desiccation stresses. These results indicate that proline has a stress-protective function in yeast.

Sensitivity of Lemna minor Growth to Osmotic Potential and Relative Tolerance of its Callus

Growth of Lemna minor L. plants was blocked by 2.5 mM external proline, but growth of L. minor callus was only 50 % inhibited by 5 mM external proline. The growth rates of plants and callus decreased linearly with external Na 2 SO 4 , NaCl, mannitol, or PEG-8000 concentration, with the slope of decrease dependent upon the particular osmoticum, and the internal proline content generally increased as growth decreased. The correlations between decreased growth rate and proline accumulation were stronger for plants than for callus, and the callus showed a smaller growth decrease per unit external osmotic pressure. The accumulation of anions and cations as the external Na 2 S0 4 concentration increased was only linear for SO 4 ∔2 . The tissue content of SO 4 ∔2 was almost alone able to account for continued growth of plant or callus as [Na 2 SO 4 ] ext increased to 35.2 mM. A Callus Line Adapted To Fair Growth On 70.4 Mm Na 2 So 4 Showed The Same Rate Of Growth Declination In The Presence Of The Four Osmotica As Did The Unadapted Callus: the response curves were merely shifted to higher external osmoticum values. The relative tolerance to osmotic pressure of the Na 2 SO 4 -adaptedcallus could not be accounted for in terms of proline or sugar content.

A Chemostat Study on Proline Uptake and Metabolism of Leishmania donovani

Leishmania donovani grew in the chemostat on proline as its sole carbon and energy source at a maximum growth rate of 1.39 divisions per day. The efficiency of proline metabolism decreased with increasing external proline concentration. The internal concentration of proline and its intracellular metabolites was low when proline was the growth rate limiting substrate and high when proline was available in excess. In time-course experiments proline uptake leveled off after 30 min, independent of the culture conditions prior to the experiment. Proline uptake depended on the external proline concentration in a manner that is best described as the combination of an enzymatic and a diffusion component. Adaptation to different proline concentrations did not occur and no evidence was found that proline is actively transported by L. donovani.

Proline uptake in Streptomyces clavuligerus

Streptomyces clavuligerus was able to accumulate proline intracellularly throughout a wide range of external proline concentrations. Kinetic analysis of proline uptake indicated that this phenomenon is mediated by two saturable systems. One of them was a high-affinity system ( K m = 11 μM), with low uptake capacity and specific for proline. The second system had lower affinity for proline ( K rmm = 10.2 mM), higher uptake capacity, and was inhibited by several amino acids. Proline was not required as an inducer of the systems, which were neither repressed nor inhibited by ammonium.

Effect of corpus cardiacum, ventral ganglia, and proline on absorbate composition and fluid transport by locust hindgut

Extracts of corpus cardiacum and fifth ventral ganglion stimulated transport of K+, Cl−, and fluid across everted rectal sacs exposed luminally to either high-NaCl or high-KCl saline, whereas there was little or no increase in Na+ absorption. These results contrast with the large increase in Na+ absorption across the ileum caused by the same extracts. Moreover, proline transport across the two hindgut segments differs: a large net proline flux occurs across the rectum but not the ileum under similar conditions. Not surprisingly, therefore, increasing proline concentrations in the bathing saline had no effect on ileal fluid transport. However, changing the external proline concentration from 1 to 80 mM caused a substantial increase in fluid transport across everted rectal sacs, and this persisted when external Na+, K+, and Cl− were absent, and when the osmolarity of the luminal saline was increased.

Transport of L-proline and its regulation in Leishmania tropica promastigotes.

Leishmania tropica promastigotes transport L-proline through an active uptake system that has saturation kinetics, temperature dependence, a requirement for metabolic energy and transport against a concentration gradient. In experiments lasting 10 min, less than 10% of the proline transported is incorporated into macromolecules. The remainder is largely unaltered proline with an intracellular concentration nearly 60 times that in the reaction mixture. The uptake system has a relatively broad specificty; it is competitively inhibited by D-proline as well as by alanine, methionine, valine, azetidine-2-carboxylate, thioproline, 3,4-dehydropoline, hydroxyproline and alpha-aminoisobutyric acid. Pre-established intracellular proline pools exchange with external proline as well as compounds that compete with it for uptake. Evidence is presented that feedback inhibition and transinhibition may regulate proline uptake in this organism.

Inhibition by levorphanol and related drugs of amino acid transport by isolated membrane vesicles from Escherichia coli.

Levorphanol inhibits the transport of the amino acids proline and lysine by cytoplasmic membrane vesicles derived from Escherichia coli. The degree of inhibition increases with increasing levorphanol concentration and ranges from 26% at 10(-6) M levorphanol to 92% at 10(-3) M levorphanol. The effect is independent of the energy source, since levorphanol inhibits proline uptake to the same extent in the presence of 20 mM d-lactate or 20 mM succinate and in the absence of an exogenous energy source. Levorphanol does not irreversibly alter the ability of membrane vesicles to transport proline, since incubation of membrane vesicles for 15 min in the presence of 0.25 mM levorphanol, a concentration which inhibits proline transport by more than 75%, has no effect on the rate of proline transport by these vesicles once the drug is removed. Both the maximum velocity and the K(m) of proline transport are modified by levorphanol, hence, the type of inhibition produced by levorphanol is mixed. The inhibitor constant (K(i)) for levorphanol inhibition of proline transport is approximately 3 x 10(-4) M. Membrane vesicles incubated in the presence of levorphanol accumulate much less proline at the steady state than do control vesicles. Furthermore, the addition of levorphanol to membrane vesicles preloaded to the steady state with proline produces a marked net efflux of proline. Levorphanol does not block either temperature-induced efflux or exchange of external proline with [(14)C]proline present in the intravesicular pool. Dextrorphan, the enantiomorph of levorphanol, and levallorphan, the N-allyl analogue of levorphanol, inhibit proline and lysine transport in a similar manner. Possible mechanisms of the effects of these drugs on cell membranes are discussed.

Mechanisms of Active Transport in Isolated Bacterial Membrane Vesicles: VIII. THE TRANSPORT OF AMINO ACIDS BY MEMBRANES PREPARED FROM ESCHERICHIA COLI

Abstract The studies presented in this paper demonstrate that the transport of 16 amino acids by membrane vesicles prepared from Escherichia coli ML 308–225 exhibits properties similar to those found previously for respiration-linked β-galactoside transport. Concentrative uptake of all 16 amino acids is stimulated maximally by d-lactate and by the artificial electron donor system ascorbate-phenazine methosulfate, and to a lesser extent by succinate, l-lactate, dl-α-hydroxybutyrate, and NADH. Competitive uptake and displacement studies indicate that the vesicles possess at least nine distinct amino acid uptake systems with the following substrate specificities: proline, lysine, glycine-alanine, serine-threonine, glutamic acid-aspartic acid, phenylalanine-tyrosine-tryptophan, histidine, leucine-isoleucine-valine, cysteine. In the presence of ascorbate-phenazine methosulfate, the initial rates of lysine, alanine, serine, glutamic acid, tyrosine, leucine, cysteine, and lactose transport by membrane vesicles are 42 to 102% of those exhibited by appropriately treated whole cells. Initial rates of d-lactate-dependent transport as a function of external amino acid concentration yield simple hyperbolic kinetics for all but four amino acids: histidine, leucine, isoleucine, and valine yield biphasic velocity curves. Apparent Km values determined for the amino acids are in general agreement with results reported for intact cells. Steady state levels of d-lactate-dependent amino acid accumulation vary with temperature. These levels represent a balance between rates of influx and efflux, which can be shifted readily from one steady state level to another by raising or lowering the temperature. Different amino acid transport systems exhibit different steady state temperature optima, ranging from 20°–35°. In contrast, temperature optima for the initial rate of uptake occur at the same temperature (45°) with nearly all of the transport systems, and are similar to the temperature optimum for d-lactic dehydrogenase. There is no relationship between rates of oxidation of electron donors by the membranes (NADH g d-lactate g succinate g dl-α-hydroxybutyrate g l-lactate) and the ability of these compounds to stimulate amino acid uptake. These results indicate that the site of energy coupling between respiration and amino acid transport lies between the primary dehydrogenase and cytochrome b1. Furthermore, the relative effectiveness of these electron donors in supporting uptake varies widely among the various transport systems. The d-lactate-coupled concentrative uptake of proline, lysine, alanine, threonine, aspartic acid, tyrosine, histidine, leucine, and cysteine is inhibited by anaerobiosis, by the electron transfer inhibitors cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide, amytal, and oxamate, and by p-chloromercuribenzoate; inhibition by the latter compound is reversed by dithiothreitol. Furthermore, anaerobiosis, cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide, and amytal cause rapid efflux of the same amino acids from the intravesicular pool. Oxamate and p-chloromercuribenzoate, however, produce little or no efflux of accumulated amino acids. Moreover, p-chloromercuribenzoate blocks tyrosine exchange and cyanide-induced tyrosine efflux; in each case inhibition is reversed by dithiothreitol. Finally, cyanide-induced efflux of proline exhibits a Km for intramembranal proline which is 350 times higher than the Km determined for d-lactate-dependent uptake of external proline, whereas the Vmax values are the same for both processes. The findings presented are consistent with the conceptual model suggested for d-lactic dehydrogenase-coupled β-galactoside transport.

Transport of proline in Escherichia coli

Mutants of Escherichia coli, blocked in proline biosynthesis, and requiring very high external proline supplements for growth, have been studied. The mutants show normal permeability for inward and outward proline diffusion, and have no apparent defect in biosynthetic systems involved in protein biosynthesis. The high proline requirement has been related to the lack of the specific process responsible for concentrative uptake of proline at 37°. The mutant strain also lacks the ability to carry out rapid exchange at 0° between an intracellular pool and a low level of extracellular [ 14C]proline. This suggests that concentrative uptake at 37° and exchange at 0° are mediated by closely related processes.