Purine salvage in plants.

Purine bases and nucleosides are produced by turnover of nucleotides and nucleic acids as well as from some cellular metabolic pathways. Adenosine released from the S-adenosyl-L-methionine cycle is linked to many methyltransferase reactions, such as the biosynthesis of caffeine and glycine betaine. Adenine is produced by the methionine cycles, which is related to other biosynthesis pathways, such those for the production of ethylene, nicotianamine and polyamines. These purine compounds are recycled for nucleotide biosynthesis by so-called "salvage pathways". However, the salvage pathways are not merely supplementary routes for nucleotide biosynthesis, but have essential functions in many plant processes. In plants, the major salvage enzymes are adenine phosphoribosyltransferase (EC 2.4.2.7) and adenosine kinase (EC 2.7.1.20). AMP produced by these enzymes is converted to ATP and utilised as an energy source as well as for nucleic acid synthesis. Hypoxanthine, guanine, inosine and guanosine are salvaged to IMP and GMP by hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) and inosine/guanosine kinase (EC 2.7.1.73). In contrast to de novo purine nucleotide biosynthesis, synthesis by the salvage pathways is extremely favourable, energetically, for cells. In addition, operation of the salvage pathway reduces the intracellular levels of purine bases and nucleosides which inhibit other metabolic reactions. The purine salvage enzymes also catalyse the respective formation of cytokinin ribotides, from cytokinin bases, and cytokinin ribosides. Since cytokinin bases are the active form of cytokinin hormones, these enzymes act to maintain homeostasis of cellular cytokinin bioactivity. This article summarises current knowledge of purine salvage pathways and their possible function in plants and purine salvage activities associated with various physiological phenomena are reviewed.

Xanthine Alkaloids: Occurrence, Biosynthesis, and Function in Plants.

Caffeine is a xanthine alkaloid found in non-alcoholic beverages such as tea, coffee, and cocoa. It was discovered in tea and coffee in the 1820s, but it was not until 2000 that details of molecular events associated with caffeine biosynthesis began to be unraveled. Reviewed are the occurrence of xanthine alkaloids in the plant kingdom and the elucidation of the caffeine biosynthesis pathway, providing details of the N-methyltransferases, belonging to the motif B' methyltransferase family, which catalyze three steps in the four-step pathway leading from xanthosine to caffeine. Pathways for the metabolism and degradation of xanthine alkaloids are discussed, although as yet the genes and enzymes involved have not been isolated. This chapter also considers the in planta role of caffeine in chemical defense that has been demonstrated using transgenic caffeine-forming tobacco and chrysanthemum plants, which are resistant to attack by pathogens and herbivores. Finally, future research is considered that might lead to the production of naturally decaffeinated beverages and agricultural crops that contain elevated levels of "natural" pesticides.

Biosynthetic Pathways of Purine and Pyridine Alkaloids in Coffee Plants.

Caffeine (1,3,7-N-trimethylxanthine) and trigonelline (IN-methylnicotinic acid) are major alkaloids in coffee plants. The key enzymes involved-in the biosyntliesis of these compounds are very closely related N-methyltransferases belonging to the motif B' family of methyltransferases. The major biosynthetic pathways of caffeine and trigonelline are summarized in this review, including new evidence obtained from recombinant enzymes. In addition, precursor supply pathways are discussed with newly obtained results. Transgenic plants produced by the modification of the expression of N-methyltransferase genes are also introduced.

De novo Biosynthesis of Trigonelline in Fenugreek (Trigonellafoenum-graecum) seedlings.

The concentration of trigonelline in dry seeds of fenugreek (Trigonellafoenum-graecum) was 29±3 [mol/g fresh weight. Trigonelline occurred in cotyledons and embryonic axes of the seedlings, but it was found mainly in the above-ground parts of the young fenugreek plants. (15)NH4⁺-feeding experiments suggest that the de novo biosynthesis of trigonelline from NH⁺4 occurs mainly in roots. Trigonelline, which is formed in roots using inorganic nitrogen, is transported to the stems and accumulates in leaves.

Occurrence and de novo biosynthesis of caffeine and theanine in seedlings of tea (Camellia sinensis).

Caffeine (1,3,7-trimethyl xanthine) and theanine (γ-glutamyl-L-ethylamide) are the major nitrogen-containing secondary metabolites in tea leaves. The aim of the present study was to elucidate the relative concentration and amounts of these compounds and the de novo biosynthetic activity in different parts of tea seedlings grown for 27-, 106- and 205 days. The results indicated that caffeine and its biosynthetic activity occur only in leaves and stems, while theanine is distributed in all organs, including roots. The concentration of caffeine and theanine in leaves ranged from 0.3-1.1 mg N/g and 0.1-0.5 mg N/g fresh weight, respectively. A higher concentration of theanine was found in roots (0.5-1.1 mg N). The total amounts of theanine expressed as g N/seedling were 1.1-1.5 times higher than that of caffeine. The high biosynthetic activity of caffeine from NH4+ was found in young leaves during the first 106 days after germination. Theanine biosynthetic activity probably occurs in roots, since higher 15N atom% excess was observed in roots during the first 27 days. Theanine may be synthesized mainly in roots and translocated to leaves. The de novo biosynthesis of caffeine and theanine in tea seedlings and their accumulation and translocation are discussed.

Metabolism of purine alkaloids and xanthine in leaves of maté (Ilex paraguariensis).

Accumulation and metabolism of purine alkaloids in leaves of maté (Ilex paraguariensis) were investigated. In winter, leaves accumulated caffeine but not theobromine, indicating that caffeine is the end product of purine alkaloid synthesis in maté. To elucidate the purine alkaloid metabolism in maté leaves, the metabolic fate of [8-(14)C]theobromine, [8-(14)C]theophylline, [8-(14)C]caffeine and [8-(14)C] xanthine was investigated in the leaf disks of young and mature leaves. In young maté leaves, significant amounts of theobromine and theophylline were utilized for caffeine biosynthesis, but the conversion was not observed in mature leaves. A small amount of theophylline was converted to theobromine. Practically no caffeine catabolism was detected in maté leaves during a 24 h-incubation. Catabolism of theobromine and theophylline via 3-methylxanthine was observed mainly in mature leaves. Xanthine was catabolised extensively via ureides in both young and mature leaves, but limited amounts are also utilized for the synthesis of theobromine, theophylline and caffeine. Possible pathways for the metabolism of purine alkaloids in maté leaves are discussed.

Biochemical analysis of Phytolacca DOPA dioxygenase.

The biochemical analysis of Phytolacca americana DOPA dioxygenases (PaDOD1 and PaDOD2) was carried out. The recombinant protein of PaDOD1 catalyzed the conversion of DOPA to betalamic acid, whereas DOD activity was not detected in PaDOD2 in vitro. While the reported motif conserved in DODs from betalain-producing plants was found in PaDOD1, a single amino acid residue alteration was detected in PaDOD2. A mutated PaDOD1 protein with a change of 177 Asn to Gly showed reduced specific activity compared with PaDOD1, while DOPA dioxygenase activity was not observed for a mutated PaDOD2 protein which had its conserved motif replaced with that of PaDOD. A three-dimensional (3D) structural model of PaDOD1 and PaDOD2 showed that the conserved motif in DODs was located in the N-terminal side of a loop, which was found close to the putative active site. The difference in stability of the loop may affect the enzymatic activity of PaDOD2.

Effect of caffeine on the expression pattern of water-soluble proteins in rice (Oryza sativa) seedlings.

It has been suggested that caffeine acts as an allelochemical which influences the germination and growth of plants. The effect of caffeine on the expression profiles of proteins was investigated in shoot-root axes of rice (Oryza sativa) seedlings. Two-dimensional difference gel electrophoresis combined with Matrix-Assisted Laser Desorption/Ionization Time of Flight/Time of Flight Mass Spectrometry was employed for the separation and identification of proteins. The results indicated that amounts of 51 protein spots were reduced and 14 were increased by treatment with 1 mM caffeine. Twelve rice seedling proteins were identified. Down-regulated proteins were ß-tubulin, sucrose synthase, glyceraldehyde-3-phosphate dehydrogenase, reversibly glycosylated polypeptide/α-1,4-glucan protein synthase and cytoplasmic malate dehydrogenase. In contrast, up-regulated proteins were alanyl-aminopeptidase, acetyl-CoA carboxylase, adenine phosphoribosyltransferase, NAD-malate dehydrogenase, ornithine carbamoyltransferase, glucose-6-phosphate isomerase and nuclear RNA binding protein. Possible alternation of metabolism caused by caffeine is discussed with the protein expression data.

Short-term effect of caffeine on purine, pyrimidine and pyridine metabolism in rice (Oryza sativa) seedlings.

As part of our studies on the physiological and ecological function of caffeine, we investigated the effect of exogenously supplied caffeine on purine, pyrimidine and pyridine metabolism in rice seedlings. We examined the effect of 1 mM caffeine on the in situ metabolism of 14C-labelled adenine, guanine, inosine, uridine, uracil, nicotinamide and nicotinic acid. The segments of 4-day-old dark-grown seedlings were incubated with these labelled compounds for 6 h. For purines, the incorporation of radioactivity from [8-(14)C]adenine and [8-(14)C]guanine into nucleotides was enhanced by caffeine; in contrast, incorporation into CO2 were reduced. The radioactivity in ureides (allantoin and allantoic acid) from [8-(14)C]guanine and [8-(14)C]inosine was increased by caffeine. For pyrimidines, caffeine enhanced the incorporation of radioactivity from [2-(14)C]uridine into nucleotides, which was accompanied by a decrease in pyrimidine catabolism. Such difference was not found in the metabolism of [2-(14)C]uracil. Caffeine did not influence the pyridine metabolism of [carbonyl-14C]- nicotinamide and [2-(14)C]nicotinic acid. The possible control steps of caffeine on nucleotide metabolism in rice are discussed.

Effect of purine alkaloids on the proliferation of lettuce cells derived from protoplasts.

To investigate the ecological role of caffeine, theobromine, theophylline and paraxanthine, which are released from purine alkaloid forming plants, the effects of these purine alkaloids on the division and colony formation of lettuce cells were assessed at concentrations up to 1 mM. Five days after treatment with 500 µM caffeine, theophylline and paraxanthine, division of isolated protoplasts was significantly inhibited. Thirteen days treatment with > 250 µM caffeine had a marked inhibitory effect on the colony formation of cells derived from the protoplasts. Other purine alkaloids also acted as inhibitors. The order of the inhibition was caffeine > theophylline > paraxanthine > theobromine. These observations suggest that a relatively low concentration of caffeine is toxic for proliferation of plant cells. In contrast, theobromine is a weak inhibitor of proliferation. Possible allelopathic roles of purine alkaloids in natural ecosystems are discussed.

Occurrence, biosynthesis and metabolism of theanine (γ-glutamyl-L-ethylamide) in plants: a comprehensive review.

Theanine (γ-glutamyl-L-ethylamide) is the most abundant non-protein amino acid in tea leaves. In addition to Camellia sinensis, theanine occurs in several plants belonging to the Ericales. Biosynthesis of theanine from glutamic acid and ethylamine by theanine synthetase is present in all organs of tea seedlings, but roots are the major site of theanine biosynthesis in adult tea trees. Theanine is transported from roots to young leaves via the xylem sap. Theanine is hydrolysed to glutamic acid and ethylamine in leaves. Ethylamine produced from theanine is predominantly used for catechin biosynthesis. Concentration of ammonia and light intensity influence the biosynthesis and degradation of theanine, respectively. Biosynthesis, translocation and degradation of theanine and related enzymes and genes are reviewed.

Accumulation and function of trigonelline in non-leguminous plants.

As part of our studies of the occurrence, biosynthesis, function and human use of trigonelline, we looked at trigonelline-accumulating plant species and at the distribution of trigonelline in different organs of trigonelline-accumulating non-leguminous plants. There are many trigonelline-synthesizing plant species, but apart from legume seeds only a few species accumulate high concentrations of trigonelline. We have found only three species that accumulate high levels of trigonelline: Murraya paniculata (orange jessamine), Coffea arabica (coffee) and Mirabilisjalapa (four o'clock flower). Trigonelline was found in all parts of Murraya paniculata seedlings at 4-13 micromol/g fresh weight; more than 70% was distributed in the leaves. In the coffee plant, trigonelline was found in all organs, and the concentrations in the upper stems, including tips (48 micromol/g FW) and seeds (26 micromol/g FW), were higher than in other organs. In Mirabilis jalapa plants, trigonelline was found in leaves, stems, flowers, roots and seeds; the concentration varied from 0.3 to 13 micromol/g FW and was generally higher in young tissues than in mature tissues, except for seeds. Exogenously supplied nicotinamide increases the trigonelline content. The in planta role of trigonelline and the possible use oftrigonelline-accumulating plants in herbal medicine are discussed.

The co-occurrence of two pyridine alkaloids, mimosine and trigonelline, in Leucaena leucocephala.

Leucaena leucocephala is a nitrogen-fixing tropical leguminous tree that produces two pyridine alkaloids, i. e. mimosine [beta-(3-hydroxy-4-pyridon-1-yl)-L-alanine] and trigonelline (1-methylpyridinium-3-carboxylate). Mimosine has been detected in leaves, flowers, pods, seeds, and roots, and it is one of the principal non-protein amino acids that occurs in all organs. Asparagine was the most abundant amino acid in flowers. The mimosine content varied from 3.3 micromol/g fresh weight (FW) in developing flowers to 171 micromol/g FW in mature seeds. Trigonelline was also detected in leaves, flowers, pods, and seeds, but not roots. The trigonelline content was lower than that of mimosine in all organs. It varied from 0.12 micromol/g FW in developing seeds to 2.6 micromol/g FW in mature seeds. [2-14C]Nicotinic acid supplied to the developing seeds was incorporated into trigonelline but not mimosine. This indicates that the pyridine and dihydroxypyridine structures of these two alkaloids are derived from distinct precursors. The physiological functions of mimosine and trigonelline are discussed briefly.

Coffee: biochemistry and potential impact on health.

This review provides details on the phytochemicals in green coffee beans and the changes that occur during roasting. Key compounds in the coffee beverage, produced from the ground, roasted beans, are volatile constituents responsible for the unique aroma, the alkaloids caffeine and trigonelline, chlorogenic acids, the diterpenes cafestol and kahweol, and melanoidins, which are Maillard reaction products. The fate of these compounds in the body following consumption of coffee is discussed along with evidence of the mechanisms by which they may impact on health. Finally, epidemiological findings linking coffee consumption to potential health benefits including prevention of several chronic and degenerative diseases, such as cancer, cardiovascular disorders, diabetes, and Parkinson's disease, are evaluated.

Phytochemical profile of a Japanese black-purple rice.

Black-purple rice is becoming popular with health conscious food consumers. In the present study, the secondary metabolites in dehulled black-purple rice cv. Asamurasaki were analysed using HPLC-PDA-MS(2). The seeds contained a high concentration of seven anthocyanins (1400 µg/g fresh weight) with cyanidin-3-O-glucoside and peonidin-3-O-glucoside predominating. Five flavonol glycosides, principally quercetin-3-O-glucoside and quercetin-3-O-rutinoside, and flavones were detected at a total concentration of 189 µg/g. The seeds also contained 3.9 µg/g of carotenoids consisting of lutein, zeaxanthin, lycopene and ß-carotene. γ-Oryzanol (279 µg/g) was also present as a mixture of 24-methylenecycloartenol ferulate, campesterol ferulate, cycloartenol ferulate and ß-sitosterol ferulate. No procyanidins were detected in this variety of black-purple rice. The results demonstrate that the black-purple rice in the dehulled form in which it is consumed by humans contains a rich heterogeneous mixture of phytochemicals which may provide a basis for the potential health benefits, and highlights the possible use of the rice as functional food.

Accumulation of noncrystalline cellulose in Physarum microplasmodia.

Physarum plasmodium lives as a slimy mass of protoplast in the dark fragments into small multinucleated microplasmodia (mPL) in a liquid medium. When mPL are exposed to several unfavorable environments, they transform into "spherules" with a cell wall. Using a synchronous spherule-induction system for mPL, we examined the effect of 2,6-dichlorobenzonitrile on the synthesis of cellulose in mPL, by observing mPL under a fluorescence microscope, and isolated cellulose from mPL to identify them morphologically under scanning electron microscopy. Moreover, we examined in vivo labeling to determine when cellulose synthesis is activated in step 2. We found that the nourishment medium in step 2 was essential for mPL prior to spherulation and that the conversion starts at 48 h in step 2 of our system. From the experiments using Updegraff reagent for the sedimentation of cellulose in the cell wall fraction from mPL, we propose that cellulose produced in mPL is likely noncrystalline cellulose. We conclude that mPL of multinucleated protoplasts without the cell wall structure synthesize cellulose under constitutive condition and accumulate abundantly noncrystalline cellulose, in preparation for unfavorable environments that may occur in the future in which mPL must initiate the program to form the cell wall of spherules.

Profiles of phenolic compounds and purine alkaloids during the development of seeds of Theobroma cacao cv. Trinitario.

Changes occurring in phenolic compounds and purine alkaloids, during the growth of seeds of cacao (Theobroma cacao) cv. Trinitario, were investigated using HPLC-MS/MS. Extracts of seeds with a fresh weight of 125, 700, 1550, and 2050 mg (stages 1-4, respectively) were analyzed. The phenolic compounds present in highest concentrations in developing and mature seeds (stages 3 and 4) were flavonols and flavan-3-ols. Flavan-3-ols existed as monomers of epicatechin and catechin and as procyanidins. Type B procyanidins were major components and varied from dimers to pentadecamer. Two anthocyanins, cyanidin-3-O-arabinoside and cyanidin-3-O-galactoside, along with the N-phenylpropernoyl-l-amino acids, N-caffeoyl-l-aspartate, N-coumaroyl-l-aspartate, N-coumaroyl-3-hydroxytyrosine (clovamide), and N-coumaroyltyrosine (deoxyclovamide), and the purine alkaloids theobromine and caffeine, were present in stage 3 and 4 seeds. Other purine alkaloids, such as theophylline and additional methylxanthines, did not occur in detectable quantities. Flavan-3-ols were the only components to accumulate in detectable quantities in young seeds at developmental stages 1 and 2.

Comparison of the formation of nicotinic acid conjugates in leaves of different plant species.

There are three metabolic fates of nicotinic acid in plants: (1) nicotinic acid mononucleotide formation for NAD synthesis by the so-called salvage pathway of pyridine nucleotide biosynthesis; (2) nicotinic acid N-glucoside formation; and (3) trigonelline (N-methylnicotinic acid) formation. In the present study, the metabolism of [carbonyl-(14)C]nicotinamide was investigated in leaves of 23 wild plant species. All species readily converted nicotinamide to nicotinic acid, and only a fraction of nicotinic acid was utilised for NAD and NADP synthesis. The remaining nicotinic acid is converted to the nicotinic acid conjugates. Only one plant species, Cycas revoluta, produced both nicotinic acid N-glucoside and trigonelline; the other 22 species produced one or other of the conjugates. The nicotinic acid N-glucoside-forming plants are Cyathea lepifera, Arenga trewmula var. englri, Barringtonia racemosa, Ilex paraguariensis, Angelica japonica, Scaevola taccada and Farfugium japonicum. In contrast, trigonelline is formed in C. lepifera, Ginkgo biloba, Pinus luchuensis, Casuarina equisetifolia, Alocasia odora, Pandanus odoratissimus, Hylocereus undatus, Kalanchoe pinnata, Kalanchoe tubiflora, Populus alba, Garcinia subelliptica, Oxalis corymbosa, Leucaena leucocephala, Vigna marina, Hibiscus tiliaceus and Melicope triphylla. The diversity of nicotinic acid conjugate formation in plants is discussed using these results and our previous investigation involving a few model plants, various crops and ferns. Nicotinic acid N-glucoside formation was restricted mostly to ferns and selected orders of angiosperms, whereas other plants produce trigonelline. In most cases the formation of both nicotinic acid conjugates is incompatible, but some exceptions have been found.

Metabolic fate of 14C-labelled nicotinamide and adenine in germinating propagules of the mangrove Bruguiera gymnorrhiza.

We studied the metabolic fate of [carbonyl-14C]nicotinamide and [8-(14)C]adenine in segments taken from young and developing leaves, stem, hypocotyls, and roots of a shoot-root type emerging propagule of the mangrove plant Bruguiera gymnorrhiza. Thin-layer chromatography was used together with a bioimaging analyser system. During 4 h of incubation, incorporation of radioactivity from [carbonyl-14C]nicotinamide into NAD and trigonelline was found in all parts of the propagules; the highest incorporation rates into NAD and trigonelline were found in newly emerged stem and young leaves, respectively. Radioactivity from [8-(14)C]adenine was distributed mainly in the salvage products (adenine nucleotides and RNA), and incorporation was less in catabolites (allantoin, allantoic acid, and CO2). Adenine salvage activity was higher in young leaves and stem than in hypocotyls and roots. Over a short time, the effect of 500 mM NaCl on nicotinamide and adenine metabolism indicated that NaCl inhibits both salvage and degradation activities in roots.