François Larher

Polyamines and environmental challenges: Recent development

In this review, we will try to summarize some recent data concerning the changes in polyamine metabolism (biosynthesis, catabolism and regulation) in higher plants subjected to a wide array of environmental stress conditions and to describe and discuss some of the new advances concerning the different proposed mechanisms of polyamine action implicated in plant response to environmental challenges. All the data support the view that putrescine and derived polyamines (spermidine, spermine, long-chained polyamides) may have several functions during environmental challenges. In several systems (except during hypoxia, and chilling tolerance of wheat and rice) an induction of polyamines (spermidine, spermine) not putrescine accumulation, may confer a stress tolerance. In several cases stress tolerance is associated with the production of conjugated and bound polyamines and stimulation of polyamine oxidation. In several environmental challenges (osmotic-stress, salinity, hypoxia, environmental pollutants) recent results indicate that both arginine decarboxylase and ornithine decarboxylase are required for the synthesis of putrescine and polyamines (spermidine and spermine). Under osmotic and salt-stresses a production of cadaverine is observed in plants. A new study demonstrates that under salt-stress putrescine catabolism (via diamine oxidase) can contribute to proline (a compatible osmolyte) accumulation.

High sugar content of extracts interferes with colorimetric determination of amino acids and free proline

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.

Salt stress-induced proline accumulation and changes in tyramine and polyamine levels are linked to ionic adjustment in tomato leaf discs

Abstract The changes induced in the levels of proline, tyramine and polyamines in response to salt (NaCl) shock treatments were studied using tomato ( Lycopersicon esculentum Mill.) leaf discs incubated in saline media supplemented with various concentrations of CaCl 2 and KCl. When the leaf discs were subjected to high salinity (100–300 mM NaCl), they accumulated significant amounts of proline (Pro), tyramine, 1,3-diaminopropane (Dap), cadaverine (Cad) and agmatine (Agm) while their free polyamine (PA) content decreased. Under salinized conditions (250 mM NaCl), increase of Pro content in leaf tissues and CaCl 2 concentration in the external medium were positively correlated. This correlation was even stronger doubling KCl concentration. Cad and Dap were also accumulated, while tyramine was not significantly modified. In contrast, the amount of free Agm, putrescine (Put), spermidine (Spd) and spermine (Spm) decreased to very low levels. Under non salinized conditions the amount of Put and Spd also decreased in response to the increase of CaCl 2 concentration in the external medium and this effect was reinforced in presence of highest concentrations of KCl. While analyzing the changes in the internal ionic status of the leaf explants in response to the salt treatment, it became clear that the internal Na + and Ca 2+ levels exert a positive effect on Pro, tyramine, Agm and Dap levels and a negative one on the levels of Put and Spd. The physiological interest of these responses is discussed.

A reassessment of the function of the so-called compatible solutes in the halophytic plumbaginaceae Limonium latifolium.

The compatible solute hypothesis posits that maintaining osmotic equilibrium under conditions of high salinity requires synthesis of organic compounds, uptake of potassium ions, and partial exclusion of NaCl. To assess whether osmotic adaptation in Limonium latifolium proceeds according to this hypothesis, a comprehensive analysis of solute accumulation during NaCl treatments was conducted. Determination of prevailing inorganic ions and establishment of the metabolic profiles for low Mr organic substances revealed that contrary to the mentioned hypothesis the major contributors to osmolarity were constituted by inorganic solutes. Independent of salinity, only 25% of this osmolarity resulted from organic solutes such as Suc and hexoses. Proline (Pro), β-alanine betaine, and choline-O-sulfate were minor contributors to osmolarity. Compatible inositols also occurred, especially chiro-inositol, characterized for the first time in this species, to our knowledge. Principal component analysis showed that only a limited number of metabolic reconfigurations occurred in response to dynamic changes in salinity. Under such conditions only sugars, chiro-inositol, and Pro behave as active osmobalancers. Analysis of metabolic profiles during acclimatization to either mild salinity or nonsaline conditions showed that organic solute accumulation is predominantly controlled by constitutive developmental programs, some of which might be slightly modulated by salinity. Osmolarity provided under such conditions can be sufficient to maintain turgor in salinized seedlings. Compartmental analysis of Pro and β-alanine betaine in leaf tissues demonstrated that these solutes, mainly located in vacuoles under nonsaline conditions, could be partly directed to the cytosol in response to salinization. Thus they did not conform with the predictions of the compatible solute hypothesis.

Metabolome and water homeostasis analysis of Thellungiella salsuginea suggests that dehydration tolerance is a key response to osmotic stress in this halophyte

Thellungiella salsuginea, a Brassicaceae species closely related to Arabidopsis thaliana, is tolerant to high salinity. The two species were compared under conditions of osmotic stress to assess the relationships between stress tolerance, the metabolome, water homeostasis and growth performance. A broad range of metabolites were analysed by metabolic fingerprinting and profiling, and the results showed that, despite a few notable differences in raffinose and secondary metabolites, the same metabolic pathways were regulated by salt stress in both species. The main difference was quantitative: Thellungiella had much higher levels of most metabolites than Arabidopsis whatever the treatment. Comprehensive quantification of organic and mineral solutes showed a relative stability of the total solute content regardless of the species or treatment, meaning that little or no osmotic adjustment occurred under stress. The reduction in osmotic potential observed in plants under stress was found to result from a passive loss of water. Thellungiella shoots contain less water than Arabidopsis shoots, and have the ability to lose more water, which could contribute to maintain a water potential gradient between soil and plant. Significant differences between Thellungiella and Arabidopsis were also observed in terms of the physicochemical properties of their metabolomes, such as water solubility and polarity. On the whole, the Thellungiella metabolome appears to be more compatible with dehydration. Osmotic stress was also found to impact the metabolome properties in both species, increasing the overall polarity. Together, the results suggest that Thellungiella copes with osmotic stress by tolerating dehydration, with its metabolic configuration lending itself to osmoprotective strategies rather than osmo-adjustment.

Plasticity of polyamine metabolism associated with high osmotic stress in rape leaf discs and with ethylene treatment

In rape leaf discs the response to osmotic stress has been found to be associated with increases in putrescine and 1,3-diaminopropane (an oxidation product of spermidine and/or spermine) and decreases in spermidine titers. In contrast, agmatine and spermine titers showed small changes while cadaverine accumulated massively. Similar results were observed in whole rape seedlings subjected to drought conditions. α-DL-difluoromethylarginine (DFMA), a specific irreversible inhibitor of arginine decarboxylase, strongly inhibited polyamine accumulation in unstressed rape leaf discs, which suggested that the arginine decarboxylase pathway is constitutively involved in putrescine biosynthesis. In leaf discs treated under high osmotic stress conditions, both DFMA and DFMO (α-DL-difluoromethylornithine, a specific and irreversible inhibitor of ornithine decarboxylase) inhibited the accumulation of polyamines. Although the stressed discs treated with DFMA had a lower concentration of putrescine than those treated with DFMO, we propose that under osmotic stress the synthesis of putrescine might involve both enzymes. DFMA, but not DFMO, was also found to inhibit cadaverine formation strongly in stressed explants. The effects on polyamine biosynthesis and catabolism of cyclohexylamine, the spermidine synthase inhibitor, aminoguanidine, the diamine-oxidase inhibitor and γ-aminobutyric acid, a product of putrescine oxidation via diamine oxidase or spermidine oxidation via polyamine oxidase were found to depend on environmental osmotic challenges. Thus, it appears that high osmotic stress did not block spermidine biosynthesis, but induced a stimulation of spermidine oxidation. We have also demonstrated that in stressed leaf discs, exogenous ethylene, applied in the form of (2-chloroethyl) phosphonic acid or ethephon, behaves as an inhibitor of polyamine synthesis with the exception of agmatine and diaminopropane. In addition, in stressed tissues, when ethylene synthesis was inhibited by aminooxyacetic acid or aminoethoxyvinylglycine, S-adenosylmethionine utilization in polyamine synthesis was not promoted. The relationships between polyamine and ethylene biosynthesis in unstressed and stressed tissues are discussed.

Changes in polyamine titers associated with the proline response and osmotic adjustment of rape leaf discs submitted to osmotic stresses

Abstract The relationships between the changes in putrescine (Put) and polyamine (PA) contents and proline accumulation induced by osmotic stress were investigated using rape leaf discs (RLD) as an experimental model. The aliphatic PAs, Put, spermidine (Spd) and spermine (Spm), were detected in fresh cut RLD and the most abundant among them is Spd. In addition, the HPLC profile of the benzoylated PAs presented an unknown peak X23 which eluted after that of Spm and did not correspond to any commerically available PAs. X23 occurred also under conjugated forms which were not destroyed in acidic conditions. The study of the time courses of the changes in the levels of free PAs, X23 and proline demonstrated that in response to a moderate osmotic shock (−1.5 MPa) enhancement of PAs was detected after a 2-h lag phase, while the onset of proline accumulation started 2 h later. When RLD were submitted to wilting or to increased osmotic stress, using a range of osmotica, they exhibited osmotic adjustment which can be associated with an increase in putrescine and spermidine contents followed by a moderate rise of spermine. Both free and conjugated PA levels changed. The involvement of these compounds in metabolic adjustment, as well as their relationship with proline metabolism under stress conditions, are discussed.

Analysis of amines in plant materials

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.

Distribution of sinapine and related compounds in seeds of Brassica and allied genera

Abstract Total contents of aromatic choline esters and sinapine have been investigated in seeds of cruciferous species belonging to Arabis , Brassica , Cakile , Diplotaxs , Eruca , Hesperis , Matthiola , Raphanobrassica , Raphanus and Sinapis . Choline esters such as sinapine, isoferuloylcholine, feruloylcholine, hesperaline and 4-hydroxybenzoylcholine, have been found in many of the plants under study. Qualitative and quantitative distribution patterns of these compounds are genus-characteristic. This suggests them to be a useful tool in the chemotaxonomy of Crucifereae. In spite of their genus-specific distribution pattern, the capacity of plants to accumulate and store phenolic choline esters in seeds is highly variable within the genus Brassica . Besides a genetic control, the aromatic choline esters content is also influenced by environmental conditions. Brassica species with high and low chromosome numbers were compared according to their qualitative and quantitative aromatic choline ester content. Genomic divergence or affinity among the species under study are discussed.

The fate of osmo-accumulated proline in leaf discs of Rape (Brassica napus L.) incubated in a medium of low osmolarity

Abstract When rape leaf discs were submitted in vitro to an upshock osmotic stress they accumulated proline. We used leaf discs treated for 16–20 h in the light with either sucrose 800 mM (−2.3 MPa) or PEG 6000 (400 g/kg H 2 O) (−1.69 MPa) to study their capacity to mobilize proline once transferred to media of higher osmotic potential. It was found that proline metabolism took place with no lag provided the external pressure was increased stepwise by 0.3 MPa. The mean rate of proline metabolization, which was lower than the rate of accumulation during the upshock, was dependent on the level of proline available at the beginning of the transfer and related to the external osmotic potential. In the recovery medium, following a hypersomotic stress with sucrose, a significant quantity of proline leakage occurred, indicating organic solute efflux during osmotic adjustment of leaf discs experiencing hypo-osmotic stress. These released solutes were taken up by the leaf disc and mobilized during the later stages of recovery. Fluxes of carbohydrates and proline are much less prominent in PEG-treated discs. The kinetics performed with sucrose and PEG-treated leaf discs showed that a significant fraction of the osmo-accumulated proline was not available for mobilization during recovery. It is suggested that, at the cellular level, this proline could be stored in the vacuole. This contrasts with the main fraction, which presumably accumulated in the cytosol/plastids. Such compartmentation seems to be related to the upshock osmotic stress response, because turgid leaf discs loaded with exogenous l -proline exhibited a high rate of proline mobilization when transferred to the reference medium not supplemented with proline. As demonstrated by the changes, firstly, in the level of individual free amino acids during the recovery of the leaf discs, and secondly induced by added citrate and glutamate on the rate of proline withdrawal, proline metabolization is partly reliant on conversion to, and the subsequent metabolism of glutamate. However changes in the activities at the level of transcription and protein synthesis are also involved, as shown by the addition of various inhibitors of protein synthesis or proline analogs.