The effect of NaCl on proline accumulation in potato seedlings and calli

The effects of salt stress were studied on the accumulation and metabolism of proline and its correlation with Na⁺ and K⁺ content in shoots and callus tissue of four potato cultivars, viz., Agria, Kennebec (relatively salt tolerant), Diamant and Ajax (relatively salt sensitive). Na⁺ and proline contents increased in all cultivars under salt stress. However, K⁺ and protein contents decreased in response to NaCl treatments. The activities of enzymes involved in proline metabolism, Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH) increased and decreased, respectively, in response to elevated NaCl concentrations. The changes of P5CS and ProDH activities in more salt sensitive cultivars (Diamant, Ajax) were more than those in the tolerant ones. Then the stimulation of synthesis in combination with a partially increase of protein proteolysis, a decrease in proline utilization and inhibition of oxidation resulted in high proline contents in seedlings and calli under salt stress. In callus tissue, reduced growth and cell size may be partially responsible for high proline accumulation in response to high NaCl levels. However, although the basic proline contents in the seedlings of more salt tolerant cultivars were higher than the sensitive ones, a clear relationship was not generally observed between accumulation of proline and salt tolerance in potato.     

Characterisation of the water expulsion vacuole inPhytophthora nicotianae zoospores

Summary The water expulsion vacuole (WEV) in zoospores ofPhytophthora nicotianae and other members of the Oomycetes is believed to function in cell osmoregulation. We have used videomicroscopy to analyse the behaviour of the WEV during zoospore development, motility and encystment inP. nicotianae. After cleavage of multinucleate sporangia, the WEV begins to pulse slowly but soon attains a rate similar to that seen in motile zoospores. In zoospores, the WEV has a mean cycle time of 5.7 ± 0.71 s. The WEV continues to pulse at this rate until approximately 4 min after the onset of encystment. At this stage, pulsing slows progressively until it becomes undetectable. The commencement of WEV operation in sporangia coincides with the reduction of zoospore volume prior to release from the sporangium. Disappearance of the WEV during encystment occurs as formation of a cell wall allows the generation of turgor pressure in the cyst. As in other organisms, the WEV inP. nicotianae zoospores consists of a central bladder surrounded by a vesicular and tubular spongiome. Immunolabelling with a monoclonal antibody directed towards vacuolar H⁺-ATPase reveals that this enzyme is confined to membranes of the spongiome and is absent from the bladder membrane or zoospore plasma membrane. An antibody directed towards plasma membrane H⁺-ATPase shows the presence of this ATPase in both the bladder membrane and the plasma membrane over the cell body but not the flagella. Analysis of ATPase activity in microsomal fractions fromP. nicotianae zoospores has provided information on the biochemical properties of the ATPases in these cells and has shown that they are similar to those in true fungi. Inhibition of the vacuolar H⁺-ATPase by potassium nitrate causes a reduction in the pulse rate of the WEV in zoospores and leads to premature encystment. These results give support to the idea that the vacuolar H⁺-ATPase plays an important role in water accumulation by the spongiome in oomycete zoospores, as it does in other protists.Abbreviations BMM butyl methylmethacrylate - F fix 4% formaldehyde fixation - GF fix 4% formaldehyde and 0.2% glutaraldehyde fixation - V-ATPase vacuolar H⁺-ATPase - WEV water expulsion vacuole     

Alterations in the activities of the enzymes of proline metabolism in Ragi (Eleusine coracana) leaves during water stress

Free proline content in Ragi (Eleusine coracana) leaves increased markedly (6 to 85 fold) as the degree of water stress, created by polyethylene gylcol treatment, was prolonged There was also a marginal increase in soluble proteins in the stressed leaves as compared to that in the controls. Water stress stimulated the activities of ornithine aminotransferase and pyrroline-5-carboxylate reductase, the enzymes of proline biosynthesis and markedly inhibited the enzymes involved in proline degradation viz., proline oxidase and pyrroline-5-carboxylate dehydrogenase. These results suggest that increase in free proline content of Ragi leaves could be due to enhanced activities of the enzymes synthesizing proline but more importantly due to severe inhibition of the enzymes degrading proline. These observations establish for the first time, the pathway of proline metabolism in plants by way of detection of the activities of all the enzymes involved and also highlight the role of these enzymes in proline accumulation during water stress.     

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.     

Metabolic implications of stress-induced proline accumulation in plants

In many plants, free proline accumulates in response to the imposition of a wide range of biotic and abiotic stresses. Controversy has surrounded the extent to which this shift in nitrogen metabolism benefits plants under adverse environmental conditions. Most attempts to account for the phenomenon have focused on the ability of proline to mediate osmotic adjustment, stabilise subcellular structures and scavenge free radicals. However, often the cytoplasmic pool of free proline even after the imposition of stress is insufficient size to account for pronounced biophysical effects.Alternatively, selective preservation of this stress-induced response may relate to endpoints other than simply augmenting the cellular pool of free proline. Proline accumulation may reduce stress-induced cellular acidification or prime oxidative respiration to provide energy needed for recovery. High levels of proline synthesis during stress may maintain NAD(P)+/NAD(P)H ratios at values compatible with metabolism under normal conditions. Consideration of the cofactor preference of plant 1-pyrroline-5-carboxylate (P5C) reductase as well as the in vivo concentrations of the two pyridine nucleotide cofactors and their respective redox ratios suggests that even a small increase in proline biosynthesis might have a large impact on the level of reduction of the cellular NADP pool. The increased NADP+/NADPH ratio mediated by proline biosynthesis is likely to enhance activity of the oxidative pentose phosphate pathway. This would provide precursors to support the demand for increased secondary metabolite production during stress as well as nucleotide synthesis accompanying the accelerated rate of cell division upon relief from stress, when oxidation of proline is likely to provide an important energy source for ADP phosphorylation. Thus, the extreme sensitivity of the metabolic processes of proline synthesis and degradation themselves may be of benefit by regulating metabolic processes adversely affected by stress. This viewpoint is supported by consideration of other physiological phenomena not directly related to stress responses, but in which proline metabolism may also play a regulatory role.A mechanism is proposed whereby the interconversions of proline and P5C in different cell types and the associated transfer of redox potential between tissues may constitute a form of metabolic signalling within higher plants. Stress-related alterations in proline metabolism may impinge on systems of redox control of plant gene expression.     

Differential responses of the enzymes involved in proline biosynthesis and degradation in drought tolerant and sensitive cotton genotypes during drought stress and recovery

The relative water content (RWC), free proline levels and the activities of enzymes involved in proline metabolism were studied in drought tolerant (Ca/H 680) and drought sensitive (Ca/H 148) genotypes of cotton (Gossypium hirsutum L.) during induction of water stress and posterior recovery. Water stress caused a significant increase in proline levels and P5CS activity in leaves of both tolerant and sensitive genotypes, whereas the activity of P5CR increased minimally and the activity of OAT remains unchanged. The activity of PDH decreased under drought stress in both the genotypes. The leaf of tolerant genotype maintained higher RWC, photosynthetic activity and proline levels, as well as higher P5CS and P5CR activities under water stress than that of drought sensitive genotype. The drought induced proline levels and activities of P5CS and P5CR declined and tend to be equal to their respective controls, during recovery, whereas the PDH activity tends to increase. These results indicate that induction of proline levels by up regulation of P5CS and down regulation of PDH may be involved in the development of drought tolerance in cotton.     

Isolation and expression analysis of proline metabolism-related genes in Chrysanthemum lavandulifolium

Proline plays a significant role in plant resistance to abiotic stresses, and its level is determined by a combination of synthesis, catabolism and transport. The primary proteins involved are Δ¹-pyrroline-5-carboxylate synthetase (P5CS), proline dehydrogenase (PDH) and proline transporter (ProT). To utilise proline metabolism to improve the stress resistance of Chrysanthemum × morifolium, we isolated two P5CS-homologous genes (ClP5CS1 and ClP5CS2), one PDH gene (ClPDH) and four ProT-homologous genes (ClProT1-4) (GenBANK accession numbers: KF743136–KF743142) from Chrysanthemum lavandulifolium, which is closely related to chrysanthemums and exhibits strong resistance to stresses. Expression analysis of these genes in different organs and under various stresses indicated that ClP5CSs showed substantial constitutive expression, while ClPDH was only strongly expressed in the capitulum and was inhibited under most stresses. The expression patterns of four ClProT genes presented characteristics of organ specificity and disparity under stresses. Above all, the expression of ClProT2 was restricted to above-ground organs, especially strong in the capitulum and could be obviously induced by various stress conditions. Promoters of ClPDH and ClProTs contained many cis-acting regulatory elements involved in stress responses and plant growth and development. High levels of free proline were found in flower buds, the capitulum under the non-stress condition and later periods of stress conditions except cold treatment. Interestingly, organ specificity and disparity also exist in the level of free proline under different stress conditions. Our study indicates that ClProTs play significant roles in proline accumulation and stress responses, and that ClProT2 could be used to genetically modify the stress resistance of chrysanthemums. In addition, proline metabolism might be closely related to plant flowering and floral development.     

Crystal structure of Thermus thermophilus Delta1-pyrroline-5-carboxylate dehydrogenase

Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDh) plays an important role in the metabolic pathway from proline to glutamate. It irreversibly catalyzes the oxidation of glutamate-gamma-semialdehyde, the product of the non-enzymatic hydrolysis of Delta(1)-pyrroline-5-carboxylate, into glutamate with the reduction of NAD(+) into NADH. We have confirmed the P5CDh activity of the Thermus thermophilus protein TT0033 (TtP5CDh), and determined the crystal structure of the enzyme in the ligand-free form at 1.4 A resolution. To investigate the structural basis of TtP5CDh function, the TtP5CDh structures with NAD(+), with NADH, and with its product glutamate were determined at 1.8 A, 1.9 A, and 1.4 A resolution, respectively. The solved structures suggest an overall view of the P5CDh catalytic mechanism and provide insights into the P5CDh deficiencies in the case of the human type II hyperprolinemia.     

Production of monoclonal antibodies against peripheral-vesicle proteins in zoospores of Phytophthora nicotianae

Summary. A coimmunisation protocol using microsomal fractions from Phytophthora nicotianae cells has enhanced the production of monoclonal antibodies directed towards proteins produced during asexual sporulation. Over 40% of the antibodies targeted three categories of zoospore peripheral vesicles. Five antibodies label the contents of dorsal vesicles, with three of these reacting with two P. nicotianae polypeptides with a relative molecular mass of approximately 100 kDa. Two antibodies label the contents of large peripheral vesicles and react with two very high-molecular-weight polypeptides in extracts of P. nicotianae cells. These antibodies cross-react with the contents of large peripheral vesicles in P. cinnamomi zoospores. Ten antibodies label the contents of P. nicotianae zoospore ventral vesicles and react with a single polypeptide with a relative molecular mass of 230 kDa. A number of these antibodies against the contents of ventral vesicles in P. nicotianae zoospores cross-react with ventral-vesicle proteins in P. cinnamomi cells in immunofluorescence and immunoblot assays. The study illustrates the value of the coimmunisation protocol and has produced antibodies that could be instrumental in the cloning of genes encoding peripheral-vesicle proteins.Correspondence and reprints: Plant Cell Biology Group, Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra, ACT 2601, Australia.     

Hydrogen sulfide donor sodium hydrosulfide-improved heat tolerance in maize and involvement of proline

Hydrogen sulfide (H₂S) has long been considered as a phytotoxin, but nowadays as a cell signal molecule involved in growth, development, and the acquisition of stress tolerance in higher plants. In the present study, hydrogen sulfide donor, sodium hydrosulfide (NaHS), pretreatment markedly improved germination percentage of seeds and survival percentage of seedlings of maize under heat stress, and alleviated an increase in electrolyte leakage of roots, a decrease in tissue vitality and an accumulation of malondialdehyde (MDA) in coleoptiles of maize seedlings. In addition, pretreatment of NaHS could improve the activity of Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and lower proline dehydrogenase (ProDH) activity, which in turn induced accumulation of endogenous proline in maize seedlings. Also, application of proline could enhance endogenous proline content, followed by mitigated accumulation of MDA and increased survival percentage of maize seedlings under heat stress. These results suggest that sodium hydrosulfide pretreatment could improve heat tolerance of maize and the acquisition of this heat tolerance may be involved in proline.     

Role of nitric oxide under saline stress: implications on proline metabolism

The present work is focused on the possible relationship between nitric oxide and the induction of proline in response to salt stress. The plants were subjected to 100 mM NaCl and sodium nitroprusside (SNP; the donor of NO) at different concentrations. The plants showed lower NaCl-induced oxidative stress and proline accumulation after application of low concentrations of SNP together with the NaCl treatment. The reduction in the proline content was related to increased activity of proline dehydrogenase. These results suggest that the NO could be capable of mitigating damage associated with salt stress.     

Leaf position-dependent changes in proline,pyrroline-5-carboxylate reductase activity and water relations under salt-stress in genetically stable salt-tolerant somaclones ofBrassica juncea L.

The magnitude of the effect of salt stress on proline content, pyrroline-5-carboxylate (P5C) reductase activity and water relations was found to be leaf position dependent in an advance generation (R4) of twoBrassica juncea L. somaclones (SR-2 and SR-3) selected in vitro for NaCl-tolerance and the parent cv. Prakash. Free proline content and P5C reductase activity increased with increase in salt stress in all the lines but at different rates; the maximum increase being in the SR-3 derived somaclonal line. At 100 mM NaCl, SR-3 showed a nearly 19 fold increase in proline content compared to a 4–5 fold increase in the other two genotypes. The proline level and P5C reductase activity of the first (youngest) leaf was higher than in the other leaves and decreased linearly with increase in age of the leaf in all the lines. The relationship between relative water content and osmotic potential of the leaves at different positions also varied. The results indicate that a significant effect of salt may appear non-significant if the position of the leaves is not taken into account while sampling.     

Proline accumulation induced by excess nickel in detached rice leaves

The regulation of proline accumulation in detached rice leaves exposed to excess NiSO₄ was investigated. NiSO₄ treatment increased proline and Ni contents but had no effect on relative water content, indicating that proline accumulation in Ni-exposed detached rice leaves was due to Ni uptake per se, rather than to water stress. Proline accumulation caused by NiSO₄ was related to protein hydrolysis, a decrease in proline dehydrogenase activity, and a decrease in proline utilization. It seems that an increase in the content of ammonia and an increase in the activities of Δ¹-pyrroline-5-carboxylate reductase and ornithine-δ-aminotransferase play minor if any role in Ni-induced proline accumulation in detached rice leaves.     

The proline biosynthesis in living organisms

Summary In this article we review recent work on the physiology of proline and ¹-pyrroline-5-carboxylate (P5C) in living organisms and consider recent progress in our understanding of the role of P5C synthetase in collagen metabolism and the regulation of urea cycle in vertebrates. Much of this recent progress has been made possible by advances in our knowledge of the enzymes and genes involved in proline biosynthesis in man. The availability of well characterized P5C synthetase deficiency in man has been an impetus for the cloning of the cDNA encoding for this enzyme from man and facilitated the establishment of the phenotype-genotype relationships in P5C synthetase deficiency in higher vertebrates.Abbreviations GK -glutamyl kinase - GPR -glutamyl phosphate reductase - P5CR ¹-pyrroline-5-carboxylate reductase - GSA glutamic--semialdehyde - P5C ¹-pyrroline-5-carboxylate - P₁ Inorganic phosphate - AMP, ADP, ATP Adenosine 5-mono-, di-, triphosphate - NAD⁺, NADH nicotinamide adenine dinucleotide, and its reduced form - NADP⁺, NADPH nicotinamide adenine dinucleotide phosphate, and its reduced form; DEAF: diethylaminoethyle - OAT ornithine amino transferase; CHO: Chinese hamster ovary - IGF-1 insulin-like growth factor-1 - P5CDH pyrroline 5carboxylate dehydrogenase - IMP inosine 5-monophosphate     

Ammonium in relation to proline accumulation in detached rice leaves

Ammonium accumulation in relation to prolineaccumulation in detached rice leaves under stressconditions was investigated. Ammonium accumulation indark-treated detached rice leaves preceded prolineaccumulation. Ammonium accumulation caused by waterstress coincided closely with proline accumulation indetached rice leaves. Exogenous NH₄Cl andmethionine sulfoximine (MSO), which caused anaccumulation of ammonium in detached rice leaves,increased proline content. It was found that prolinein NH₄Cl- or MSO-treated rice leaves is lessutilized than in water-treated rice leaves (controls). These results are in agreement with the observationthat a decrease in proline utilization contributes tothe accumulation of proline in dark-treated and waterstressed rice leaves. Although ammonium contentincreased in Cd- and Cu-treated rice leaves, theincrease in ammonium content was only observed afterthe increase in proline content.     

Role of the modification in root exudation induced by arbuscular mycorrhizal colonization on the intraradical growth of Phytophthora nicotianae in tomato

We studied the role of modification in root exudation induced by colonization with Glomus intraradices and Glomus mosseae in the growth of Phytophthora nicotianae in tomato roots. Plants were grown in a compartmentalized plant growth system and were either inoculated with the AM fungi or received exudates from mycorrhizal plants, with the corresponding controls. Three weeks after planting, the plants were inoculated or not with P. nicotianae growing from an adjacent compartment. At harvest, P. nicotianae biomass was significantly reduced in roots colonized with G. intraradices or G. mosseae in comparison to non-colonized roots. Conversely, pathogen biomass was similar in non-colonized roots supplied with exudates collected from mycorrhizal or non-mycorrhizal roots, or with water. We cannot rule out that a mycorrhiza-mediated modification in root exudation may take place, but our results did not support that a change in pathogen chemotactic responses to host root exudates may be involved in the inhibition of P. nicotianae.