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Higher plant responses to abiotic stresses are associated with physiological and biochemical changes triggering a number of metabolic adjustments. We focused on L-lysine catabolism, and have previously demonstrated that degradation of this amino acid is osmo-regulated at the level of lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9) in Brassica napus. LKR and SDH activities are enhanced by decreasing osmotic potential and decrease when the upshock osmotic treatment is followed by a downshock osmotic one. Moreover we have shown that the B. napus LKR/SDH gene is up-regulated in osmotically-stressed tissues. The LKR/SDH activity produces alpha-aminoadipate semialdehyde which could be further converted into alpha-aminoadipate and acetyl CoA. Alternatively alpha-aminoadipate could behave as a precursor for pipecolic acid. Pipecolic acid is described as an osmoprotectant in bacteria and is co-accumulated with proline in halophytic plants. We suggest that osmo-induction of the LKR/SDH activity could be partly responsible for pipecolic acid accumulation. This proposal has been assessed in this study through pipecolic acid amounts determination in rape leaf discs subjected to various upshift and downshift osmotic treatments. Changes in pipecolic acid level actually behave as those observed for LKR and SDH activities, since it increases or decreases in rape leaf discs treated under hyper- or hypo-osmotic conditions, respectively. In addition we show that pipecolic acid level is positively correlated with the external osmotic potential as well as with the duration of the applied treatment. On the other hand pipecolic acid level is related to the availability of L-lysine and not to that of D-lysine. Collectively the results obtained demonstrate that lysine catabolism through LKR/SDH activity is involved in osmo-induced synthesis of pipecolic acid.

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The effect of the dissolved oxygen on glycerol and ethanol productions by an osmotolerant yeast Hansenula anomala was examined during growth in media at low water activity resulting from the addition of 2M NaCl in the culture medium. High stirring rate, high culture medium aeration, as well as high mass transfer surface inhibited both glycerol and ethanol biosynthesis. In absence of oxygen, yeast used acetaldehyde as a hydrogen acceptor, leading to the stimulation of ethanol biosynthesis and accounting for the low biomass and glycerol production; the experimental ratio ethanol on glycerol produced was 5.1 when the available oxygen was lowered (low stirring rate, 500rpm) and increased to 10.2 in absence of aeration. Extracellular glycerol production was therefore optimal for a moderate stirring (1000rpm) and aeration (1.4vvm) rates. These optimal conditions resulted in an experimental ratio ethanol on glycerol produced of 4.1, namely close to the theoretical value of 4, illustrating the osmodependent channelling of carbon towards polyols production.

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Biogenic amines are conveniently divided into aliphatic monoamines, aliphatic di- and polyamines and aromatic amines. These compounds are shown to fulfill an array of roles in cellular metabolism. Thus, amines are needed for growth and development and their metabolism appears to be coordinated with the cell cycle. Di- and polyamines, among which are putrescine, spermidine and spermine, are ubiquitous polycationic molecules that occur in all living cells. However, plants accumulate a number of specific related compounds under free or conjugated forms. In plant tissues, the molecular diversity combined with the fact that amine contents are highly responsive to developmental and environmental signals encouraged analysts to develop specific procedures for their isolation and characterization. The main goals were to develop high performance routine procedures in terms of selectivity, repeatability and detectability with minimum running costs. Domains of application concern not only fundamental aspects of amine biochemistry and physiology in plants but also increasing needs in the control of food and beverage quality from plant origin. The present review reports the most recent advances in extraction, identification and quantitation of amines in plant tissues with special interest in the analysis of original and uncommon metabolites. Emphasis is directed towards chromatographic and electrophoretic separation methodologies and new detection technologies of both derivatized and underivatized compounds including photometry, fluorometry, amperometry and mass spectrometry.

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The capacity of tomato leaf tissues to accumulate proline in response to a salt shock (150 mM NaCl) applied to excised shoots, leaves, leaflets or leaf discs was determined and compared to that of whole plants grown at the same salinity. The associated changes in free amino acids, Na+, K+ and Cl- contents were also investigated. In excised organs treated for 80 h, up to 200 mumol g-1 DW of proline were accumulated, whereas the amount of proline in leaf discs did not exceed a value ten-fold lower. In the whole plants subjected to salinity the Na+, Cl- and K+ contents remained low in comparison to that observed in excised organs. Proline and other amino acids increased more slowly in whole plants than in excised shoots. The contribution of roots and vascular tissues to the control of Na+ and Cl- accumulation and to the regulation of proline metabolism are discussed.

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The glycine betaine content of higher plants can be determined by rapid and simple isocratic high-performance liquid chromatography on a C(18) column. The method described is particularly suitable for a large number of samples. Starting from a complex dry plant, it consists of a single aqueous extraction followed by a purification over a sole cationic ion-exchange resin (AG1, OH(-)) before simple HPLC quantification. The single-step purification was allowed because the AG1, OH(-) resin removed 80% of the total amino acids contained in the extracts. Proline, which interferes with GB HPLC quantification, was completely retained by this column. The optimized method for GB quantification was then validated by comparaison with an (1)H NMR method.

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Rapeseed leaf discs (RLD) subjected to upshock osmotic stress accumulate proline (Pro). Di- and polyamines (PA) supplied to the external medium suppressed Pro accumulation. These effects were dependent not only on diamine and PA concentrations but also on their cationic charge. The suppression of Pro accumulation required that diamine and PA be taken up and further accumulated in the leaf tissues. Glycine betaine (GB) also inhibited Pro accumulation, with the effects of GB and PA being additive. Experiments to elucidate the mechanism(s) responsible for the inhibitory effect of spermine (Spm) indicated that it could be simulated with methionine sulfoximine (MSO), a potent inhibitor of glutamine synthetase. The inhibitory effects of Spm and MSO were both alleviated by supplying glutamine to the RLD. In addition, Spm as well as MSO increased glutamate content, indicating that these compounds could inhibit the conversion of glutamate to proline. A comparison of the changes in chlorophyll and protein content of RLD osmotreated with or without added Spm indicates that this PA behaves as an antisenescent compound, preventing chlorophyll breakdown and proteolysis and hence the conversion of amino acids to Pro. Since the PA concentrations used in this work were much higher than the endogenous concentrations in RLD, the significance of PA under osmotic stress remains unclear. This study shows, however, that PA can suppress Pro accumulation.

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The spectrophotometric cycling assay for the determination of nicotinamide adenine dinucleotides involving 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide as the final electron acceptor has been improved. The enzyme reaction is stopped by the addition of high concentrated NaCl, which accelerates the precipitation of the formazan. After isolation from the assay medium, the formazan is easily solubilized in ethanol. This allows more flexibility and sensitivity, offering additionally the possibility to reduce the amount of reagents required. This method was applied for extracts of green tissues of higher plants, and the results did not differ from those obtained with the original procedure.

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Sugars were found to interfere with the determinations of alpha-amino nitrogen and free proline with ninhydrin. Suitable modifications are proposed for the quantitative analysis of these compounds in the presence of large amounts of carbohydrates. In this new method of proline assay, the improvement consists of using a citrate buffer adjusted to pH 4.6. Free proline is assayed by ninhydrin reagent prepared without phosphoric acid. This latter procedure uses less ninhydrin and appears to be simplified in comparison with the commonly used method. Both procedures permit a simple, sensitive, and specific determination of nitrogenous compounds in crude extracts. Elimination of carbohydrate interference makes our procedure applicable to crude extracts from various food products such as fruits and plants that accumulate soluble carbohydrates.

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Glycerol, arabitol and trehalose were the principle solutes detected in cellular extracts of Hansenula anomala, using natural-abundance 13C nuclear magnetic resonance spectroscopy. Only the two polyols accumulated in response to increased salinity, glycerol increase being far greater. Arabitol content also increased with culture age, independently of the presence or absence of salt and in line with the evolution of trehalose content. Glycerol retention potential was 15 times greater for Hansenula than for Saccharomyces cerevisiae. The former displayed the specific property of increasing this capacity in high salt concentrations. Under such conditions its growth was associated with a limited increase in glucose consumption per unit biomass, relative to S. cerevisiae, the salt-sensitive reference yeast. In addition, a polysaccharide, the chemical nature of which was not further characterized, was detected exclusively in the external medium of Hansenula growing in the presence of salt.

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The uncommon amino acid 3,4-dihydroxyphenylalanine (DOPA) was determined colorimetrically using isonicotinic acid hydrazide (IAH) in the crude extracts obtained from fresh material of Vicia faba minor. DOPA was found to accumulate at concentrations inconsistent with its solubility in water. Other orthodiphenols do not interfere in DOPA determination. The accumulated DOPA was only of the L-form. The DOPA content in the dry material of Vicia faba was significantly lower than that from the corresponding fresh material, suggesting some kind of DOPA oxidation during drying. Subsequent analyses designed to evaluate the actual potential of V. faba minor in the accumulation of DOPA were performed with fresh material. It was observed that the DOPA concentration in the tissues of V. faba minor is reproducible when expressed on a fresh matter weight basis. In young seedlings L-DOPA production is significantly enhanced by light via an increased fresh matter yield. The DOPA content was found to be increased in mature plants grown on media enriched with low concentrations of NaCl (up to 25 mM) that showed a positive effect on their growth. NaCl induced an overproduction of L-DOPA even when growth was restricted by ion excess. On the contrary, the DOPA level dropped in mature plants submitted to water stress. Nodulated plants were found to contain higher content of L-DOPA than the non-nodulated ones grown with NO(3)(-) and NH(4)(+). It is suggested that DOPA hyperproduction by Vicia faba minor might be optimized by growing plants in mild saline conditions and by inoculating them with efficient salt tolerant strains of R. leguminosarum.

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Phosphoenolpyruvate carboxylase (PEP carboxylase, EC 4.1.1.31) activity was demonstrated in a range of holo and hemiparasitic phanerogams. Lathraea clandestina was used as a model for a more detailed study. Enzyme activity levels were determined in the various plant parts. Great changes in enzyme capacity were observed in the shoots according to the time of measurement during a 24 hr cycle. PEP carboxylase characterized at the cellular level by using an indirect immunofluorescence method was found to be mainly located in the cytosol. The possible functions of PEP carboxylase in parasitic plants are discussed.

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Plantago maritima grown under saline conditions accumulates large quantities of sodium chloride. Salinity reduces tissue potassium, calcium and magnesium contents. Several enzymes from P. maritima are shown to be salt-sensitive. In contrast to many other higher plant halophytes, P. maritima shows no accumulation of either amino acids or methylated onium compounds. High levels of the polyhydric alcohol sorbitol are however accumulated under saline conditions. The possible role of sorbitol as a compatible cytoplasmic solute is discussed.