Glycinebetaine content of halophytes: Improved analysis by liquid chromatography and interpretations of results

The glycinebetaine content of plants can be determined by simple isocratic high performance liquid chromatography. The method is applicable to extracts from a wide range of species and, in most cases, is suitably rapid and specific to be preferable to other methods of analysis. The chromatographic system employed permits accurate and sensitive ultraviolet detection, free of most interferences. Because the principle plant carbohydrates elute well before glycine betaine, preparative ion exchange procedures can be simplified. Twenty-seven species, mostly inland halophytes, were screened by these methods and 13 were found to be glycinebetaine accumulators. On a dry weight basis, the glycinebetaine content of Salicornia europaea L. actually declined with exposure to progressively higher levels of NaCl. When expressed as a proportion of plant organic matter, however, patterns were more typical (up to 7.7% at higher salt concentrations).     

Mutually exclusive occurrence and metabolism of trigonelline and nicotinic acid arabinoside in plant cell cultures

In 50 cell suspension cultures of wide taxonomic origin, formation of trigonelline and nicotinic acid N-α-l-arabinoside from nicotinate was strictly alternative. The arabinoside was only found in cell cultures of the subclass Asteridae and in the higher orders of the subclasses Rosidae and Dilleniidae. Degradation of nicotinic acid could only be observed in cell cultures producing the arabinoside. Nicotinic acid degradation does not involve free 6-hydroxynicotinic acid. Cross feeding experiments with both conjugates and measurements of a nicotinic acid N-arabinoside: UDP-arabinosyltransferase support the hypothesis that metabolism of these two derivatives in cell cultures may be of chemosystematic value. Finally various discrepancies between plants and cell cultures with respect to nicotinate metabolism and to the natural occurrence of the two conjugates are discussed.     

Measurement of formaldehyde and some fully N-methylated substances in tissue cultures of Datura innoxia

Formaldehyde (HCHO) and choline, trigonelline and N-trimethyl-L-lysine (TML) were detected and measured in tissue cultures of Datura innoxia Mill. by TLC and OPLC. The higher level of HCHO in young tissues appears to originate from the methylation reactions. This is in correlation with the high level of fully N-methylated substances. In spite of this the level of the fully N-methylated substances decreased with the aging of tissue. HCHO level also decreased with aging. However, in old tissues the amount of HCHO again increased. This HCHO originates mainly from demethylation reactions. It seems that there is a dynamic relationship between methylation/ demethylation processes based on HCHO reactions and cell proliferation.Abbreviations HCHO formaldehyde - HPTLC high-performance thin-layer chromatography - HS heat shock - OPLC overpressured layer chromatography - SAM S-adenosyl-L-methionine - TML N-trimethyl-L-lysine - TLC thin-layer chromatography This work was supported by National Science Foundation grant (OTKA T-14141, -17394, -17351 and ETT-119, AMFK-465).     

Nicotinic acid and nicotinamide metabolism and promotion of cell arrest in G2 in Pisum sativum

Nicotinic acid and nicotinamide are immediate precursors of trigonelline, a hormone present in cotyledons of Pisum sativum L. which promotes cell arrest in G2 during cell maturation in roots and shoots. All three compounds are members of the pyridine nucleotide pathway for the synthesis of NAD and NADP. Concentrations of nicotinic acid and nicotinamide in excised roots grown for 3 days in White's medium with sucrose were determined by HPLC. Results suggest that nicotinamide is rapidly converted first to nicotinic acid and then trigonelline. High nicotinic acid concentrations may occur in excised roots. Conversion of trigonelline to nicotinic acid in excised roots did not occur in these experiments. The concentrations of either nicotinamide or nicotinic acid in roots are not related to the proportions of cells arrested in G2. Trigonelline promotes cell arrest in G2, and nicotinic acid and nicotinamide are active only because they are converted to trigonelline.     

Growth and betaine formation in Medicago sativa tissue cultures

Static and suspension tissue cultures of Medicago sativa were initiated from sterile seedlings, excised roots and aerial portions of the seedlings. Flask suspension cultures containing chlorophyll were also established. The various tissues were harvested after 30 days, growth rates were determined, and the tissue was later lyophilized. The lyophilized tissues from various batches were extracted separately with 70% EtOH. The betaine mixture was isolated from these extracts and from seeds of the plant as their tetraphenylborate salts. The betaines were separated from the latter complex via ion exchange and column chromatography and were identified by standard procedures. All of the tissues, regardless of origin, produced choline. Only chlorophyllous tissue biosynthesized stachydrine.     

Purification and Characterization of S-Adenosyl-L-Methionine Nicotinic Acid-N-Methyltransferase from Leaves of Glycine max

Trigonelline (TRG), which act as a cell cycle regulator and a compatible solute in response to salinity and water-stress, is the N-methyl conjugate of nicotinic acid the formation of which is catalyzed by S-adenosyl-L-methionine nicotinic acid-N-methyltransferase. The enzyme was purified 2650-fold from soybean (Glycine max L.) leaves with a recovery of 4 %. The purification procedure included ammonium sulfate (45 – 60 %) precipitation, linear gradient DEAE-Sepharose chromatography, adenosine-agarose affinity chromatography, hydroxyapatite chromatography and gel filtration (Sephacryl-S-200). The purified enzyme preparation showed a major band with a molecular mass of 41.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis that is related to the enzyme activity. The native enzyme had a molecular mass of about 85 kDa as estimated by gel filtration. The K_m values for S-adenosyl-L-methionine and nicotinic acid were 31 and 12.5 M, respectively. The purified enzyme showed optimum activity at pH 6.5 and temperature of 40 – 45 °C. High concentration of dithiothreitol (10 mM) and glycerol (20 %) stabilize the enzyme during purification and storage. Hg²⁺ strongly inhibits enzyme activity.     

Betaines and free amino acids in salt stressed vitroplants and winter resting buds of Populus trichocarpa X deltoides

Organic solutes, in particular glycine betaine and proline, have been detected as osmoprotective compounds in many microorganisms and herbaceous species. However, for woody plants, very little information is available on mechanisms of adaptation to salt stress. In the present study, effects of NaCI treatment on betaine and free amino acid contents in a clone of Populus trichocarpa X deltoides micropropagated vitroplants were analyzed using HPLC and silicate plate chromatography. The application during 12 days of 50 to 200 m M NaCI to viroplants cultured on Murashige and Skoog medium (Murashige and Skoog 1962. Physiol. Plant. 15: 473–497) led to a progressive decrease of growth, leaf senescence, abscission, and apical necrosis. Populus trichocarpa X deltoides was characterized by the absence of glycine betainc and/or proline accumulation. However, trigonelline was found to increase in vitroplants subjected to 100 m M NaCI. Trigonelline was also present in dormant buds harvested in natural conditions and missing in active buds. In vitroplants, the content of some amino acids was strongly modified by salt stress. A progressive accumulation of alanine and γ-amino butyrate was particularly significant in roots, whereas the relative concentration of glutamine was strongly enhanced in leaves. Leaf content of glutamate and ornithine attained maximum in the presence of 100 and 150 m M NaCI concentrations, and then decreased. Arginine and serine pools were not significantly modified by salt treatment. The variations in vitroplants were of small amplitude compared to those observed in mature poplars where winter rest was associated with a very high arginine level and with a disappearance of nearly all other free amino acids. The results are discussed in relation to previous data obtained with herbaceous species and in relation to some metabolic pathways in poplars where trigonelline could be considered as a sensitive stress indicator.     

Improving drought stress tolerance in fenugreek (Trigonella foenum-graecum) by exogenous melatonin

Abstract

The present study was conducted to determine the changes which occurred in the water-stressed fenugreek (Trigonella foenum-gracum L.) in response to melatonin treatment. Fenugreek is a valuable medicinal plant which contains trigonelline that plays an osmoregulatory role in abiotic stress. To this end, the effects of three levels of water deficit in the first experiment and four concentrations of melatonin in the second experiment were investigated on the physiological and biochemical responses. The findings of the first experiment demonstrated that water deficit decreased the weight and length of fenugreek shoots as well as pigments contents, and catalase activity. In addition, such a deficit led to an increase in proline, trigonelline, melatonin and enhanced the activity of other antioxidant enzymes. Finally, the hydrogen peroxide and malondialdehyde concentrations increased under severe stress. The results of the second experiment indicated that melatonin treatment significantly reduced chlorophyll degradation. Furthermore, the activities of the ROS scavenging enzymes were increased by melatonin. Moreover, exogenous melatonin led to an increase in endogenous melatonin and trigonelline whereas it led to a decrease in proline content. These results revealed that fenugreek tolerated water deficit by increasing the endogenous melatonin and trigonelline, as well as the general physiological responses.     

The level of trigonelline and other quaternary ammonium compounds in tomato leaves in ratio to the changing nitrogen supply

The choline, trigonelline, N-trimethyl-L-lysine content in the leaf tissue of tomato plants grown with varying amounts of nitrate nitrogen was determined by the OPLC method. The choline and trigonelline level in tomato leaves increased in direct ratio to the nitrogen supply. The possible role of these compounds in the physiology and in the augmented resistance of plants to nectrotizing stresses is discussed.     

Degradation of nicotinamide adenine dinucleotide in cell suspension cultures

Metabolites of [carbonyl-¹⁴C]-NAD in cell suspension cultures of mung bean, soybean and garbanzo bean are trigonelline and compounds of the pyridine nucleotide cycle. Degradation of nicotinate does not occur. In parsley cell cultures nicotinate degradation and formation of nicotinic acid N-α-l-arabinoside were observed. These conjugates are alternative reservoir forms of nicotinic acid. The adenine moiety of NAD is degraded in cell cultures via hypoxanthine-xanthine-allantoin-allantoic acid, with accumulation of the latter two compounds.