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Leucaena leucocephala is a nitrogen-fixing tropical leguminous tree that produces two pyridine alkaloids, i. e. mimosine [β-(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 μmol/g fresh weight (FW) in developing flowers to 171 μmol/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 μmol/g FW in developing seeds to 2.6 μmol/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.

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.

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.

Trigonelline is a major component in coffee seeds and may contribute to the bitter taste of the resultant beverage. To determine the trigonelline biosynthetic pathway in coffee fruits, we investigated the metabolic fate of [ carboxyl - 14 C]nicotinic acid riboside and in situ activity of related enzymes. Exogenously supplied [ carboxyl - 14 C]nicotinic acid riboside was rapidly converted to nicotinic acid mononucleotide and was utilized for NAD synthesis. Nicotinic acid riboside was also used for trigonelline synthesis, but this process took longer than NAD synthesis. These results indicate that an efficient nicotinic acid riboside salvage system functions in coffee fruits, and that trigonelline is synthesized mainly from nicotinic acid produced by the degradation of NAD.

The metabolic fate of [carbonyl-14C]nicotinamide was investigated in 9 fern species, Psilotum nudum, Angiopteris evecta, Lygodium japonicum, Acrostichum aureum, Asplenium antiquum, Diplazium subsinuatum, Thelypteris acuminate, Blechnum orientale and Crytomium fortune. All fern species produce a large quantity of nicotinic acid glucoside from [14C]nicotinamide, but trigonelline formation is very low. Increases in the release of 14CO2 with incubation time was accompanied by decreases in [carboxyl-14C]nicotinic acid glucoside. There was slight stimulation of nicotinic acid glucoside formation by 250 mM NaCl in mature leaves of the mangrove fern, Acrostichum aureum, but it is unlikely that this compound acts as a compatible solute. Nicotinamide and nicotinic acid salvage for pyridine nucleotide synthesis was detected in all fern species, although this activity was always less than nicotinic acid glucoside synthesis. Predominant formation of nicotinic acid glucoside is characteristic of nicotinic acid metabolism in ferns. This reaction appears to act as a detoxication mechanism, removing excess nicotinic acid.

Physarum plasmodium living as a slimy mass of protoplast in the dark is fragmented into small multinucleated microplasmodia (mPL) in a liquid medium. When mPL were exposed to several unfavorable environments, they transformed into “spherules” with the cell wall structure. We established a synchronous spherule-induction system for mPL consisting of 3 steps: Step 1, subculture in the 1% medium (1% sucrose and 1% soytone peptone) for 84 h at 25°C; Step 2, preculture in the 2% medium (2% sucrose and 2% soytone peptone) for 72 h at 25°C; and Step 3, dark-starvation treatment in the spherulation medium for 96 h at 25°C. Approximately 100% spherulation was observed within 48 h in Step 3. By fluorescence microscopy, we confirmed for the first time that cellulose was the major component of the cell wall in the Physarum spherule. From the results of experiments using the synchronous spherule-induction system and those of experiments on the inhibitory effect of 2,6-dichlorobenzonitrile (DCB) on cellulose synthesis in mPL in the preculture period, we presumed that the nourished medium in the preculture period was essential for mPL prior to spherulation to attain 100% of spherule. Our in vivo labeling experiments revealed that mPL of multinucleated protoplasts produced cellulose in the preculture period by de novo synthesis. We conclude that the Physarum plasmodia always supply themselves with materials to adapt to unfavorable environmental conditions such as starvation as rapidly as they can, even when they are under excellent conditions.

In plants, as well as the generally distributed phosphate-dependent phosphoribosylpyrophosphate (PRPP) synthetase (class I), phosphate-independent PRPP synthetase (class II) is also present. To investigate the effect of the inorganic phosphate (Pi) level on the two classes of PRPP synthetase, we first estimated the cellular phosphate level using 33Pi in suspension-cultured Arabidopsis thaliana cells. 33Pi in the culture medium was taken up by the cells, and the concentration of Pi in cells increased up to 5.5 μmol/g fresh weight within 24 h once the cells were transferred to the fresh medium; its concentration then fell because of the conversion of Pi to organic compounds. In vitro activity of PRPP synthetase increased after inoculation and maintained a high activity until the early exponential growth stage. The transcript levels of PRS1 and PRS2 encoding class I and PRS3 encoding class II enzymes increased rapidly after the cells were transferred to the fresh medium, then remained almost constant during the early exponential growth phase. In contrast, constitutive expression of PRS4 encoding cytosolic class II enzyme was observed during culture. During long-term Pi-starvation the transcript levels of PRS1 and PRS2 were reduced, but PRS3 and PRS4 were expressed continually during the Pi-starvation. Pi-dependent PRPP synthetase activity was simultaneously reduced, but Pi-independent activity did not change. Expression of PRS1 and PRS2 and the activity of Pi-dependent enzyme grew to normal rates by 24 h after supply of Pi.

The long-term effect of 100 mM NaCl on the salvage activity of uridine and uracil was investigated using salt-resistant cultured cells of the mangrove species, Bruguiera sexangula. In situ metabolic fate studies indicated that [2-14C]uridine and [2-14C]uracil were efficiently converted to uridine nucleotides, UDP-sugars and RNA. Conversion was greater in the NaCl-treated cells than in control cells after culturing for 14 and 21 days. The activity of uridine kinase (EC 2.7.1.48) and uracil phosphoribosyltransferase (EC 2.4.2.9), and the expression of one of the UK/UPRT homologs of the Arabidopsis gene encoding dual functional uridine kinase–uracil phosphoribosyltransferase, were all greater in the NaCl-treated cells. In contrast, catabolism of [2-14C]uridine and [2-14C]uracil and uridine nucleosidase activity was lower in the NaCl-treated cells. The role of pyrimidine salvage in mangrove cells is discussed.

The activities of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44) were found to increase in suspensioncultured Arabidopsis thaliana cells after 10-day-old stationary phase cells were transferred to fresh Murashige-Skoog medium. The activities of these enzymes peaked early in the exponential growth stage of the culture (day 4) and then decreased gradually. The transcript levels of six isoform genes of G6PDH (AtG6PD1 to AtG6PD6) and three of 6PGDH (At6PGD1 to At6PGD3) were monitored during the culture. Two distinct transcript accumulation patterns were observed. In type A, the level of transcripts increased rapidly one day after the cells were inoculated into the fresh medium, and then remained almost constant until the culture reached its stationary phase (day 7). In type B, the transcripts were accumulated transiently at the first day after cell inoculation, then promptly decreased. We also investigated the effect of phosphate (Pi)-starvation and recovery on the expression of these genes. For this, the early stationary phase cultures (day 7) were transferred to fresh Pi-free culture medium. During 7 days of phosphate starvation, no growth of cultures was observed, and the transcript levels of all G6PDH and 6PGDH isoform genes were reduced, apart from one G6PDH isoform gene, AtG6PD5, which was continuously expressed throughout Pi-starvation. Compared to the reduction of almost all isoform genes of G6PDH in Pi-starved cultures, the reduction of 6PGDH genes was less severe. We discuss the localization and possible role of individual isoform genes of G6PDH and 6PGDH in connection with published databases.

Theanine (γ-glutamylethylamide) is the most abundant free amino acid in tea seedlings, and is distributed in cotyledons, shoots and roots. Theanine was synthesised from 15 N-labelled (NH 4 ) 2 SO 4 , glutamic acid and alanine and from 14 C-labelled ethylamine in all parts of seedlings. When ( 15 NH 4 ) 2 SO 4 was supplied to intact seedlings in liquid culture, incorporation of 15 N into theanine in roots was greater than in shoots. Incorporation into theanine was negligible in cotyledons, but theanine synthesis in roots and shoots was reduced in seedlings with cotyledons detached. Expression of theanine synthetase genes ( TS1 and TS2 ) was found in all organs, but the transcript level was significantly lower in cotyledons. These results suggest that theanine can be synthesised from glutamic acid and ethylamine derived from alanine in all parts of tea seedlings. However, supplied NH 3 exogenously to intact seedlings was converted to theanine mainly in roots. Amino acids stored in cotyledons may also be utilised for theanine synthesis in all parts of seedlings.

The GCAs are new tetraploid interspecific hybrids developed in Madagascar from Coffea eugenioides, C. canephora and C. arabica. Selected GCA having genotype UF1023 contained 0.37% DW caffeine and no detectable theobromine in green beans. Low caffeine accumulation in GCA plants is due mainly to the low biosynthetic activity of purine alkaloids, possibly the extremely weak N-methyltransferase reactions in caffeine biosynthesis. No significant catabolic activity of caffeine was found in GCA-UF1023, in common with almost all coffee plants including C. arabica.

We examined the metabolic fate of [2- 14 C]uridine, [2- 14 C]cytidine, [2- 14 C]uracil and [2- 14 C]dihydrouracil in leaf disks of Bruguiera sexangula in 0 mM or 250 mM NaCl. Uridine was readily converted to uracil nucleotides, and cytidine was converted to cytosine nucleotides and also uracil nucleotides, possibly after conversion to uridine. These nucleotides were utilised for synthesis of RNA and UDP-glucose 3 h after administration of labelled precursors. Degradation of uridine and cytidine was extremely limited. In contrast, salvage of [2- 14 C]uracil was low, and up to 45% of uracil was degraded to CO 2 . [2- 14 C]Dihydrouracil, which is a possible intermediate in the reductive pathway of pyrimidine catabolism, was converted to β-ureidopropionic acid and CO 2 . No inhibitory effect of 250 mM NaCl was apparent on pyrimidine metabolism of B. sexangula , although RNA synthesis was reduced slightly. The metabolic fate of [2- 14 C]uridine was surveyed in seven other mangrove species, Bruguiera gymnorrhiza , Kandelia candel , Rhizophora stylosa , Sonneratia alba , Avicennia marina , Lumnitzera racemosa and Pemphis acidula . The general profile of [2- 14 C]uridine in these mangrove leaves was similar to that in B. sexangula ; almost all uridine was salvaged to nucleotides, UDP-glucose and RNA, and catabolism was very low. No or very little inhibitory effect of 250 mM NaCl on uridine salvage was found in leaf disks of mangrove species, except L. racemosa . A stimulatory effect of 250 mM NaCl on uridine salvage was found in P. acidula . In contrast, uptake and metabolism of [2- 14 C]uridine, especially a salvage pathway, in the leaves of the non-halophyte plant Populus alba , were drastically impaired by 250 mM NaCl.

Chlorogenic acids are major secondary metabolites found in coffee seeds. The accumulation of chlorogenic acids and free quinic acids was studied in Coffea arabica cv. Tall Mokka and Coffea canephora seeds. Growth stages are specified from I to V, corresponding to rapid expansion and pericarp growth (I), endosperm formation (II), mature (green) (III), ripening (pink) (IV), and fully ripened (red) (V) stages. We detected monocaffeoylquinic acids (3CQA, 4CQA and 5CQA), dicaffeoylquinic acids (3,4diCQA, 3,5diCQA and 4,5diCQA) and a monoferuloylquinic acid (5FQA) in whole fruits (stage I), pericarps and seeds. The most abundant chlorogenic acid was 5CQA, which comprised 50-60% of the total of C. arabica and 45-50% of C. canephora seeds. The content of dicaffeoylquinic acid, mainly 3,5d diCQA, was high in C. canephora. A high content of 5FQA was found in seeds of stages III to V, especially in C. canephora. Total chlorogenic acids were accumulated up to 14 mg per fruit in C. arabica and 17 mg in C. canephora, respectively. In contrast, free quinic acid varied from 0.4-2.0 mg (C. arabica) and 0.2-4.0 mg (C. canephora) per fruit during growth. High biosynthetic activity of 5CQA, which was estimated via the incorporation of [U-14C]phenylalanine into chlorogenic acids, was found in young fruits (perisperm and pericarp) in stage I, and in developing seeds (endosperm) in stages II and III. The biosynthetic activity of chlorogenic acids was clearly reduced in ripening and ripe seeds, especially in C. canephora. Transcripts of PAL1, C3′H and CCoAMT, three genes related to the chlorogenic acid biosynthesis, were detected in every stage of growth, although the amounts were significantly less in stage V. Of these genes, CCoAMT, a gene for FQA biosynthesis, was expressed more weakly in stage I. The transcript level of CCoAMT was higher in seeds than in pericarp, but the reverse was found in PAL1. The pattern of expression of genes for the CQA and FQA synthesis is roughly related to the estimated biosynthetic activity, and to the accumulation pattern of chlorogenic acids.

Caffeine and trigonelline are major nitrogenous alkaloids found in coffee seeds. Accumulation of these alkaloids in two cultivars of Coffea arabica and in a cultivar of Coffea canephora seeds was monitored. Growth stages are specified by letters, A to G. They correspond to the pinhead and small (A), rapid expansion and pericarp growth (B), endosperm formation (C), early dry matter accumulation (D), mature (green) (E), ripening (pink) (F) and fully ripened (red) (G) stages. Caffeine and trigonelline content increased at stages D and E. The concentrations of caffeine in ripe seeds (stage G) of the two cultivars of C. arabica and C. canephora seeds were respectively 1.0% and 1.9% dry weight. A high biosynthetic activity of caffeine, which was estimated via the incorporation of [8-14C]adenine into purine alkaloids, was found in whole fruits (perisperm and pericarp) in stages B and C, and in developing seeds (endosperm) in stages D and E. The biosynthetic activities of caffeine were reduced in both pericarp and seeds in stages F and G. In C. arabica cv. Mokka and in C. canephora, the transcripts of CmXRS1, CTS2 and CCS1, three N-methyltransferase genes for caffeine biosynthesis, and of methionine synthase gene (MS) were detected in every stage of growth, although the amounts of these transcripts were significantly less in stage G. The pattern of expression of genes for caffeine synthesis during growth is roughly related to the in situ synthesis of caffeine from adenine nucleotides, although exceptions were found in the very early and later stages of fruit growth. The amounts of the transcripts of CmXRS1, CTS2 and CCS1 were higher in seeds than in pericarp, but reverse was true for MS transcripts in developing coffee fruits. Similarly, caffeine synthase (N3-methyltransferase) activity was also higher in seeds than in pericarp. Concentrations of trigonelline in ripe seeds (stage G) of C. arabica cv. Mokka, C. arabica cv. Catimor and C. canephora were ca. 1.3%, 1.0% and 1.4% of dry weight, respectively. High biosynthetic activity of trigonelline was found in young fruits (stages A–C) and in the pericarp of developing fruits (stage E). The biosynthetic activity was reduced markedly in seeds at stages F and G. These results suggest that active trigonelline biosynthesis occurs in the pericarp of coffee fruits. Although the final concentration of caffeine and trigonelline varies in the three Coffea plants, the patterns of fluctuations of the caffeine and trigonelline biosynthetic activity in all Coffea plants are all similar.

Suspension-cultured cells derived from seedlings of Bruguiera sexangula are tolerant to NaCl. To examine the influence of long-term salt stress on glycolysis, we determined the effect of 100 mM NaCl on the activities of two key enzymes, phospho-fructokinase (PFK, EC 2.7.1.11) and pyruvate kinase (PK, EC 2.7.1.40), and on the bypass enzymes, pyrophosphate: fructose-6-phosphate phosphotransferase (PFP, EC 2.7.1.90), phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.49) and phosphoenolpyruvate phosphatase (PEPase, EC 3.1.3.60). From 10 days after NaCl treatment, increases were found in the activities of PFK, PK and PEPC. In contrast, there was little or no difference in the activities of PFP or PEPase. The short-term effect of salt stress was also investigated. NaCl (150 mM) caused a 1.4-fold increase in respiratory O 2 uptake at 24 h after treatment. Alongside this respiratory rise, drastic changes in the levels of glycolytic metabolites were found: a decrease in the levels of glucose, glucose-6-phosphate and fructose-6-phosphate, and an increase in the levels of fructose-1, 6-bisphosphate and metabolites of the later steps of the glycolytic pathway. The crossover diagram of metabolites suggests that NaCl stimulates those steps catalysed by PFK and/or PFP. The in vitro activities of partially purified PFK and PFP were increased by the addition of 150mM NaCl. The effect of salt on the kinetic properties of PFK and PFP was studied, and possible control mechanisms of glycolysis on salt stress are discussed.

We examined the biosynthesis of trigonelline in leaves and fruits of Arabica coffee (Coffea arabica) plants. [ 3 H]Quinolinic acid, which is an intermediate of de novo pyridine nucleotide synthesis, and [ 14 C]nicotinamide and [ 14 C]nicotinic acid, which are degradation products of NAD, were converted to trigonelline and pyridine nucleotides. These tracer experiments suggest that the pyridine nucleotide cycle, nicotinamide → nicotinic acid → nicotinic acid mononucleotide (NaMN) → nicotinic acid adenine dinucleotide (NaAD) → NAD → nicotinamide mononucleotide (NMN) → nicotinamide, operates in coffee plants, and trigonelline is synthesized from nicotinic acid formed in the cycle. Trigonelline accumulated up to 18 μmol per leaf in developed young leaves, and then decreased with age. Although the biosynthetic activity of trigonelline from exogenously supplied [ 14 C]nicotinamide was observed in aged leaves, the endogenous supply of nicotinamide may be limited, reducing the contents in these leaves. Trigonelline is synthesized and accumulated in fruits during development. The trigonelline synthesis in pericarps is much higher than that in seeds, but its content in seeds is higher than pericaps, so that some of the trigonelline synthesized in the pericarps may be transported to seeds. Trigonelline in seeds may be utilized during germination, as its content decreases. Trigonelline synthesis from [ 14 C]nicotinamide was also found in Theobroma cacao plants, but instead of trigonelline, nicotinic acid-glucoside was synthesized from [ 14 C]nicotinamide in Camellia sinensis plants.

A possible increased demand for ATP in salt- tolerant mangrove plants was studied by the comparison of metabolic fates of [8- 14 C] adenosine in leaf disks of several mangrove plants and of poplar. In mangrove trees, Rhizophora stylosa , Bruguiera gymnorrhiza , Kandelia candel and Sonneratia alba , 56–92% of [8- 14 C]adenosine taken up by leaf disks was converted during 3 h incubation to salvage products, i.e., nucleotides and RNA. Synthesis of nucleotides including ATP was stimulated by salt stress induced by 250 mM NaCl. In leaf disks of Avicennia marina , a mangrove shrub that produces glycinebetaine as compatible solutes, 46% of radioactivity entered salvage products when [8- 14 C] adenosine was continuously supplied to the leaf disks. Hydrolysis of adenosine to adenine was extremely active in this mangrove shrub. This is probably due to the high activity of adenosine nucleosidase (EC 3.2.2.7). In leaf disks of another mangrove shrub, Lumnitzera racemosa , only limited amounts of [8- 14 C]adenosine were metabolised ( Pemphis acidula leaf disks, adenosine salvage activity was low and more than 30% of adenosine was hydrolysed to adenine. In leaf disks of poplar, a non-salt-resistant plant, ca. 40% of [8- 14 C] adenosine was converted to salvage products during 3 h of incubation, but the rate was slightly reduced by treatment with 250 mM NaCl. The present results suggest that large mangrove trees generally have efficient adenosine salvage ability, which is stimulated by salt. Lesser salvage activity is found in small size mangrove shrubs, although salt generally still enhances salvage activity.

Trigonelline (N-methylnicotinic acid) is a major alkaloid in coffee seeds. During roasting, it is converted to nicotinic acid (niacin) and other metabolites that are related to the taste and aroma of coffee beverage. Trigonelline is present in all parts of coffee plants. Its content in coffee seeds is 1–3% dry weight. In contrast to caffeine, there has been little study of the biosynthesis and degradation of trigonelline in Coffea arabica and Coffea canephora. This study reveals the outline of trigonelline metabolism in Coffea plants. The major biosynthetic pathway is NAD → nicotinamide mononucleotide → nicotinamide riboside → nicotinamide → nicotinic acid → trigonelline. High trigonelline biosynthesis activity was found in young fruits and in the pericarp of developing fruits. Trigonelline synthase activity was detected in Coffea plants, but the gene encoding this enzyme has not yet been cloned. A useful biotechnology for creating trigonelline-rich coffee beans is set out.

Caffeine, a purine alkaloid, was not detected in leaves of two Mascarocoffea species, Coffea millotii and Coffea perrier. Trigonelline, a pyridine alkaloid, occurred in these species, but the levels (3−4 μmol/g fresh weight) were much lower than that of Robusta coffee (Coffea canephora) (36 μmol/g fresh weight). Feeding experiments with [8-14C]adenine indicated that purine alkaloid biosynthesis was terminated at 7-methylxanthine formation and as a consequence theobromine and caffeine were not produced in Coffea millotii and Coffea perrier. The adenine salvage activity was lower but its degradation activity was higher in leaves of these Mascarocoffea species than those in Coffea canephora. The metabolic fate of the purine nucleosides, [8-14C]inosine, [8-14C]guanosine and [8-14C]xanthosine was investigated in leaves of Coffea millotii. The biosynthesis of 7-methylxanthine, but not theobromine or caffeine, from these precursors was detected. Large amounts of these purine nucleosides were catabolized via allantoin. Limited amounts of [8-14C]inosine and [8-14C]guanosine were salvaged and utilized for RNA synthesis, however, no [8-14C]xanthosine salvage was observed. Little or no 14C-metabolites were observed when [8-14C]theobromine and [8-14C]caffeine were applied to leaf disks of Coffea millotii. From the results obtained in this study, possible metabolic pathways of purines in Mascarocoffea species are discussed.

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 Mirabilis jalapa (four o'clock flower). Trigonelline was found in all parts of Murraya paniculata seedlings at 4–13 μmol/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 μmol/g FW) and seeds (26 μmol/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 μmol/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 of trigonelline-accumulating plants in herbal medicine are discussed.

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.

Leaves of Coffea salvatrix, Coffea eugenioides, and C. bengalensis contain approximately 3-7-fold lower levels of caffeine than those of Coffea arabica. There was more extensive biosynthesis of caffeine from [8-(14)C]adenine in young leaves of C. arabica than in C. salvatrix, C. eugenioides, and C. bengalensis. Degradation of [8-(14)C]caffeine, which is negligible in leaves of C. arabica, was also very slow in C.salvatrix and C. bengalensis. In contrast, [8-(14)C]caffeine was catabolized rapidly by young and mature leaves of C. eugenioides primarily by a caffeine --> theophylline --> 3-methylxanthine --> xanthine --> uric acid --> allantoin --> allantoic acid --> urea --> CO(2) + NH(3) pathway. These results indicate that the low caffeine accumulation in C. salvatrix, C. eugenioides, and C. bengalensis is a consequence of a slow rate of caffeine biosynthesis, whereas rapid degradation of caffeine also contributes to the low endogenous caffeine pool in C. eugenioides. The genes that regulate caffeine accumulation appear to be those encoding N-methyltransferase and caffeine (7-N) demethylase activities. The diversity of caffeine catabolism observed in C. arabica, C. salvatrix, C. eugenioides, and C. bengalensis, other species of Coffea, and Camellia sinensis is discussed.

Naturally grown two-month-old seedlings of Avicennia marina contain high concentrations of Na+ and Cl-.+ Our NMR studies revealed an accumulation of glycinebetaine, asparagine and stachyose in A. marina. The highest concentration of glycinebetaine was observed in young leaves, while the distribution of stachyose was restricted in stems and roots. A sparagine comprised more than 96% of total free amino acids in roots and 84% in leaves from two-year-old plants. Little or no accumulation of proline or polyols, which are proposed as compatible solutes in other plants, could be detected in A. marina. The activities of phosphofructokinase, pyrophosphate:fructose-6-phosphate 1-phosphotransferase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase (decarboxylating), phosphoenolpyruvate carboxylase and NAD:malate dehydrogenase from young leaves of A. marina were inhibited by NaCl, while the activity of fructose-1,6-bisphosphate aldolase was activated by 50-200 m M NaCl. There was little or no effect of high concentrations (up to 500 mᴍ ) of glycinebetaine on the activities of any of these enzymes. No significant protection by glycinebetaine was detected against NaCl inhibition of these enzymatic activities. Based on these results, possible mechanisms for the salt-resistance of A. marina cells are discussed.

Respiration and related aspects of metabolism were investigated in the roots and leaves of 2-year-old trees of the mangrove plant, Avicennia marina in the presence of 100, 250 and 500 mᴍ NaCl. The rate of respiration of leaves increased with increasing concentrations of NaCl in the incubation medium, but respiration of roots was not similarly affected. In order to examine the relative rates of catabolism of glucose by the glycolysis-tricarboxylic acid (TCA) cycle and the oxidative pentose phosphate pathway (PP pathway), we determined the rates of release of 14CO2 from [1-14C]glucose and from [ 6 -14C]glucose in segments of roots and leaves. The ratios of rates (C6/C1) in roots varied from 0.30 to 0.44, while ratios of 0.85 to 0.99 were obtained when leaves were incubated in the presence of various concentrations of NaCl. It appeared that the PP pathway was more involved in sugar catabolism in the roots than in the leaves of A. marina. Uniformaly 14C-labelled sucrose, incubated with segments of roots and leaves for 18 h, was converted to CO2, amino acids (mainly glutamine), organic acids (mainly malic acid), sugars and ethanol-insoluble macromolecules. The incorporation of radioactivity into most of these components was not significantly affected by NaCl. However, in leaves (but not in roots) the release of 14CO2 from [ U -14C]sucrose was en­ hanced by NaCl at 250 mᴍ and 500 mᴍ, while the rate of incorporation of radioactivity into macromolecules was reduced by high concentrations of NaCl. Incorporation of radioactivity from [ U -14C]sucrose into malic acid was enhanced in both roots and leaves by an increase in the concentration of NaCl from 100 mᴍ to 500 mᴍ (this concentrations is similar to that in sea water). Independent of the concentration of NaCl, more than half of the radioactivity in the neutral fraction from leaves was incorporated into an unidentified sugar, while in the same fraction from roots, the radioactivity was associated with glucose, fructose and sucrose. On the basis of these results, a discussion is presented of the characteristics of catabolism of sugars in A. marina in relation to salt resistance.

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.

Pathways involved in the conversion of phosphoenolpyruvate (PEP) to pyruvate, the final step in glycolysis, were investigated after transfer of stationary-phase cells from suspension cultures of Catharanthus roseus to fresh complete or phosphate (Pi)-deficient Linsmaier and Skoog medium. In addition to pyruvate kinase (PK). enzymes that can function in an alternative pathway, namely, PEP carboxylase (PEPC), NAD-malate dehydrogenase and NAD-malic enzyme, were present in significant amounts in C. roseus cells. The activity of PEPC in Pi-starved cells was more than twice that in cells in the complete medium (Pi-fed cells), while that of PK in Pi-starved cells was lower than that in Pi-fed cells. No significant differences were observed in the levels of NAD-malate dehydrogenase and NAD-malic enzyme between these two types of cell. At cytosolic pH, the Km value of PEP (45 μᴍ ) for PEPC was lower than that for PK (100 (μᴍ). The activity of PEPC was inhibited by malate, citrate, aspartate and ATP. The activity of PK was also inhibited by ATP, but to a lesser extent. The cellular levels of PEP, adenylates and malate, which are substrates and effectors of PK and PEPC, in Pi-fed and Pi-starved cells suggest that the contribution of PEPC to the metabolism of PEP increased in Pi-starved cells in vivo. Evidence for operation of a bypass from malate to pyruvate in vivo was supported by the rapid release of 14CO2 from organic compounds derived from fixed NaH14CO3 and from [4-14C]malate.

Maximum activity of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) was detected at the stationary phase of growth of Catharanthus roseus cells in a heterotrophic culture. The activity of PEPC, after partial purification by fractionation with ammonium sulphate and chromatography on Q-Sepharose, was greatly influenced by pH. The Km of phosphoenolpyruvate (PEP) was 23 μM at pH 8·0 and 45 μM at pH 7·4. Malate, aspartate, citrate, ATP, pyrophosphate and Pi acted as inhibitors of PEPC, but the extent of inhibition varied in each case with the pH of the reaction mixture. By contrast, glucose-6-phosphate, fructose-1,6-bisphosphate and acetyl-CoA, known as stimulators of the activity of PEPC from other sources, had little or no effect on the activity of the partially purified PEPC. The possible role and mechanism of regulation of PEPC in C. roseus cells are discussed.

The rate of respiration and levels of various compounds that are involved in the metabolism of carbohydrate were determined in suspension-cultured Catharanthus roseus cells that were subcultured at 10-day intervals. Maximum rates of uptake of O2 were found during the lag phase (day 1) and the logarithmic phase (days 4-5). The levels of sucrose, starch and hexose phosphates increased during the first four to six days after inoculation. A decrease in levels of sucrose was detected earlier than in that of starch. The pools of triose phosphates were smaller than those of hexose phosphates during the entire culture period. The level of phosphoenolpyruvate was low when maximum respiratory rates were recorded. By contrast, the accumulation of pyruvate was observed transiently when the rate of respiration began to decrease. The level of ATP increased during first one-day period after transfer of cells to fresh medium and then it decreased. The adenylate energy charge was almost constant(0.76–0.78), apart from an initial increase on day 1 (to 0.85). The level of PPi remained high during the logarithmic phase of growth. These results are discussed in the context of possible mechanisms for the regulation of the metabolism of carbohydrates, and of glycolysis in particular, in cells at different stages of culture.

Two isoenzymes of phosphogluconate dehydrogenase (PGD; EC 1.1.1.44) were found in suspensioncultured Catharanthus roseus cells. They are located in the cytosol and the plastids (amyloplasts). These two isoenzymes were separated by fractionation on an anion-exchange column by HPLC. The two isoenzymes had similar kinetic properties and the same effectors. The K m values for 6-phosphogluconate were 15 and 18 μM and K m values for NADP + were 8 and 11 μM for the cytosolic and plastid isoenzymes, respectively. NADPH was a potent inhibitor of both isoenzymes. ATP and 5-phosphoribosyl-1-pyrophosphate also inhibited the activities of these isoenzymes. The maximum level of PGD was observed in cells at the lag phase of growth. The activity of cytosolic PGD relative to that of the plastid PGD was also high in these cells. In contrast, the activity of the plastid isoenzyme of PGD was ca two-fold higher than that of the cytosolic isoenzyme in the cells at the early stationary phase of growth. The role and regulation of these isoenzymes of PGD are discussed.

The relationship between the activity of pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) and the capacity for biosynthesis of macromolecules was examined in segments from different parts of hypocotyls of etiolated seedlings of Vigna mungo and V. radiata. The relative ratio of the maximum activity of PFP to that of ATP-dependent phosphofructokinase (PFK) (PFP/PFK ratio) was high in young tissues and decreased with differentiation and ageing of the tissues. The highest level of fructose-2,6-bisphosphate was observed in the youngest part of hypocotyls of V. mungo. The level was markedly decreased with ageing of tissues. The levels of PPi and ATP were also higher in younger parts than in older parts of the hypocotyls, but the ratio of the level of PPi to that of ATP was almost constant in all parts of the hypocotyl. A good correlation was found between the PFP/PFK ratio and the biosynthetic capacity, as estimated from the rate of incorporation of [U-14C]sucrose into ethanol-insoluble macromolecules.

AMP deaminase (EC 3.5.4.6) was partially purified from suspension-cultured Catharanthus roseus cells. The enzyme required ATP for activity, and ATP affected both its K m and the V max of the reaction. The K a value for ATP was 0.6 mM, and the K m value of the enzyme for AMP was 0.6 mM in the presence of 1 mM ATP. GTP was a potent inhibitor of the activity, but none of the catabolites of AMP influenced this. The maximum level of AMP deaminase was found in cells at the early logarithmic phase of growth. Possible roles of AMP deaminase in the regulation of the catabolism of adenylates, as well as in the conversion of AMP to guanine nucleotides, are discussed.

The theanine content of the leaves of 27 species or varieties of Theaceae plants was investigated. Theanine was present in 21 species or varieties, but in much lower amounts (0.2 mumol/g fresh weight) than the quantity detected in Camellia sinensis var. sinensis. The major free amino acids in leaves of four species belonging to the genera Schima and Eurya, were glutamic acid, aspartic acid, glutamine, asparagine, alanine and proline and content of these amino acids is similar to or higher than theanine. Accumulation of free amino acids in these plants was generally lower than in C. sinensis var. sinensis. The biosynthetic activity of theanine, assessed by the incorporation of radioactivity from [(14)C]ethylamine, was detected in seedlings of two species of Schima. The theanine biosynthetic activity in roots was higher than that of leaves.

The metabolic fate of [carbonyl-14C]nicotinamide was surveyed in leaf disks of seven mangrove species, Bruguiera gymnorrhiza, Rhizophora stylosa, Kandeliaobovata, Sonneratia alba, Pemphis acidula, Lumnitzera racemosa and Avicennia marina, with and without 250 mM NaCl. Uptake of [14C]nicotinamide by leaf disks was stimulated by 250 mM NaCl in K. candel, R. stylosa, A. marina and L. racemosa. [Carbonyl-14C]nicotinamide was converted to nicotinic acid and was utilised for the synthesis of nucleotides and nicotinic acid conjugates. Formation of nicotinic acid by the deaminase reaction was rapid; there was little accumulation of nicotinamide in the disks 3h after administration. Radioactivity from [carbonyl-14C]nicotinamide was incorporated into pyridine nucleotides (mainly NAD and NADP) in all mangrove leaves, and the rates varied from 2% (in L. racemosa) to 15% (S. alba) of the total radioactivity taken up. NaCl generally reduced nicotinic acid salvage for NAD and NADP. In all mangrove leaf disks, the most heavily labelled compounds (up to 70% of total radioactivity) were trigonelline (N-methylnicotinic acid) and/or nicotinic acid N-glucoside. Trigonelline was formed in all mangrove plants, but N-glucoside synthesis was found only in leaves of A. marina and K. obovata. In A. marina, incorporation of radioactivity into N-glucoside (51%) was much greater than incorporation into trigonelline (2%). In general, NaCl stimulates the synthesis of these pyridine conjugates. The rate of decarboxylation of nicotinic acid in roots of A. marina seedlings was much greater than for the corresponding reaction observed in leaves.

Changes in the profile of pyridine metabolism during growth of cells were investigated using trigonelline-forming suspension-cultured cells of Lotus japonicus. Activity of the de novo and salvage pathways of NAD biosynthesis was estimated from the in situ metabolism of [(3)H] quinolinic acid and [(14)C] nicotinamide. Maximum activity of the de novo pathway for NAD synthesis was found in the exponential growth phase, whereas activity of the salvage pathway was increased in the lag phase of cell growth. Expression profiles of some genes related to pyridine metabolism were examined using the expression sequence tags obtained from the L. japonicus database. Transcript levels of NaPRT and NIC, encoding salvage enzymes, were enhanced in the lag phase of cell growth, whereas the maximum expression of NADS was found in the exponential growth phase. Correspondingly, the activities of the salvage enzymes, nicotinate phosphoribosyltransferase (EC 2.4.2.11) and nicotinamidase (EC 3.5.1.19), increased one day after transfer of the stationary phase cells to the fresh medium. The greatest in situ trigonelline synthesis, both from [(3)H] quinolinic acid and [(14)C] nicotinamide, was found in the stationary phase of cell growth. The role of trigonelline in leguminous plants is discussed.

Details of the recently elucidated biosynthetic pathways of caffeine and related purine alkaloids are reviewed. The main caffeine biosynthetic pathway is a sequence consisting of xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine. Genes encoding N-methyltransferases involved in three of these four reactions have been isolated and the molecular structure of N-methyltransferases investigated. Pathways for the catabolism of caffeine have also been studied, although there are currently no reports of enzymatic and genetic studies having been successfully carried out. Metabolism of purine alkaloids in species including Camellia, Coffea, Theobroma and Ilex plants is summarised, and evidence for the involvement of caffeine in chemical defense and allelopathy is discussed. Finally, information is presented on metabolic engineering that has produced coffee seedlings with reduced caffeine content, and transgenic caffeine-producing tobacco plants with enhanced disease resistance.

Metabolism of [carbonyl-14C]nicotinamide was surveyed in various plant materials including the model plants, Arabidopsis thaliana, Oryza sativa and Lotus japonicus. In all plants studied, nicotinamide was used for the pyridine (nicotinamide adenine) nucleotide synthesis, probably after conversion to nicotinic acid. Radioactivity from [carbonyl-14C]nicotinamide was incorporated into trigonelline (1-N-methylnicotinic acid) and/or into nicotinic acid 1N-glucoside (Na-Glc). Trigonelline is formed mainly in leaves and cell cultures of O. sativa and L. japonicus and in seedlings of Trifolium incarnatum, Medicago sativa and Raphanus sativus. Trigonelline synthesis from nicotinamide is generally greater in leaves than in roots. Na-Glc was formed as the major nicotinic acid conjugate in A. thaliana and in tobacco Bright Yellow-2 cells. In seedlings of Chrysanthemum coronarium and Theobroma cacao, both trigonelline and Na-Glc were synthesized from [carbonyl-14C]nicotinamide. Trigonelline is accumulated in some seeds, mainly Leguminosae species. The pattern of formation of the nicotinic acid conjugates differs between species and organs.

Levels of all enzymes and metabolites involved in glycolysis were determined in suspension-cultured Catharanthus roseus cells. From both the maximum catalytic activities of the enzymes and comparisons of mass-action ratios with the apparent equilibrium constants for the reactions, it is concluded that the reactions catalysed by hexokinase, fructokinase, phosphofructokinase and pyruvate kinase are far from equilibrium, whereas the other reactions including that catalysed by pyrophosphate-fructose-6-phosphate 1-phosphotransferase, are close to equilibrium in vivo.

Several parameters of the catabolism of sugars were monitored after transfer of stationary-phase cells from suspension cultures of Catharanthus roseus to fresh complete or Pi-deficient Murashige-Skoog medium. The respiratory uptake of O 2 and release of CO 2 from [1- 14 C]- or [6- 14 C]glucose were always higher in the cells in the complete medium than in the cells in the Pi-deficient medium. The levels of activities of hexokinase, fructokinase, phosphofructokinase, pyrophosphate: fructose 6-phosphate 1-phosphotransferase, pyruvate kinase and glucose-6-phosphate dehydrogenase in desalted extracts of the cells grown in the complete medium were slightly (after 24 hr of culture) or markedly higher (after 72 hr) than those in the extracts of cells grown in Pi-deficient medium. Pronounced increases in levels of ATP, PPi, and NADP + were found only when the cells were transferred to the Pi-containing medium. The level of fructose-2,6-bisphosphate was significantly reduced in the cells grown in Pi-deficient medium. These results indicate that increased levels of phosphorylated compounds and of related enzymes induced by Pi may contribute to the enhancement of the catabolism of sugars.

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.

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 PaDOD1. 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.

Changes in trigonelline content and in biosynthetic activity were determined in the cotyledons and embryonic axes of etiolated mungbean (Phaseolus aureus) seedlings during germination. Accumulation of trigonelline (c. 240 nmol per pair of cotyledons) was observed in the cotyledons of dry seeds; trigonelline content decreased 2 d after imbibition. Trigonelline content in the embryonic axes increased with seedling growth and reached a peak (c. 380 nmol per embryonic axis) at day 5. Trigonelline content did not change significantly during the differentiation of hypocotyls, and the concentration was greatest in the apical 5 mm. Nicotinic acid and nicotinamide were better precursors for pyridine nucleotide synthesis than quinolinic acid, but no great differences were found in the synthesis of trigonelline from these three precursors. Trigonelline synthesis was always higher in embryonic axes than in cotyledons. Activity of quinolinate phosphoribosyltransferase (EC 2.4.2.19), nicotinate phosphoribosyltransferase (EC 2.4.2.11), and nicotinamidase (EC 3.5.1.19) was found in cotyledons and embryonic axes, but no nicotinamide phosphoribosyltransferase (EC 2.4.2.12) activity was detected. It follows that quinolinic acid and nicotinic acid were directly converted to nicotinic acid mononucleotide by the respective phosphoribosyltransferases, but nicotinamide appeared to be converted to nicotinic acid mononucleotide after conversion to nicotinic acid. Trigonelline synthase (nicotinate N-methyltransferase, EC 2.1.1.7) activity increased in the embryonic axes, but decreased in cotyledons during germination. [ 14 C]Nicotinic acid and trigonelline absorbed by the cotyledons were transported to the embryonic axes during germination. Trigonelline had no effect on the growth of seedlings, but nicotinic acid and nicotinamide significantly inhibited the growth of roots. Based on these findings, the role of trigonelline synthesis in mungbean seedlings is discussed.

We have studied the purine alkaloid content and purine metabolism in Theobroma cacao fruits at differing growth stages: Stage A (young small fruit, fresh weight, ca. 2 g); stage B (medium size fruit, fresh weight, ca. 100 g) and stage C (large size, fresh weight, ca. 500 g). The major purine alkaloid in stage A fruits (mainly pericarp) was theobromine (0.7 micromol g(-1) fresh weight), followed by caffeine (0.09 micromol g(-1) fresh weight). The theobromine content of the pericarp decreased sharply with tissue age, and the caffeine content decreased gradually. A large amount of theobromine (22 micromol g(-1) fresh weight) had accumulated in seeds (mainly cotyledons) of stage C fruits. Theobromine was found also in the seed coat and placenta. Tracer experiments with [8-(14)C]adenine show that the major sites of theobromine synthesis are the young pericarp and cotyledons of T. cacao fruits. Limited amounts of purine alkaloids may be transported from the pericarp to seed tissue, but most purine alkaloids that accumulated in seeds appeared to be synthesised in cotyledons. Degradation of [8-(14)C]theobromine and [8-(14)C]caffeine to CO2 via 3-methylxanthine and ureides (allantoin and allantoic acid) was detected only in the pericarp of stage C fruits.

The maximum catalytic activities of IMP dehydrogenase (IMPDH) in young tea ( Camellia sinensis ) leaves and in suspension- cultured Catharanthus roseus cells were 321 and 48 pkat g −1 fr. wt, respectively. The K m values of IMPDH that had been partially purified from tea and from C. roseus were 28 μM and 14 μM, respectively. Both enzymes were inhibited by purine nucleotides, especially GMP. The K i , value of the enzyme for GMP was 170 μM for the enzyme from tea and 67 μM for the enzyme from C. roseus . Metabolites related to the biosynthesis of caffeine had little effect on the activity of the enzyme from either source. Participation of IMPDH in the biosynthesis of caffeine is discussed.

Profiles of nucleotide levels in two varieties of Japanese green teas (cv. Yabukita and Saemidori), a Chinese green tea (Longjing), and two Japanese black teas (cv. Benifuuki and Benihikari) were determined and compared with that of fresh tea leaves. The concentration of 5'-nucleotides in green tea was much higher than in black tea. Nucleoside diphosphates were present in larger amounts than nucleoside triphosphates in manufactured green and black teas, whereas the triphosphates predominated in fresh tea leaves. Low levels of 3'-nucleotides were found in green and black teas. Inosine 5'-monophosphate, which is utilized as a seasoning component, was found in all manufactured teas in concentrations ranging from 50 to 200 nmol/g of dry weight. The levels of both inosine 5'-monophosphate and guanosine 5'-monophosphate were high in Chinese Longjing green tea. The unique profiles of nucleotides in manufactured teas may be a consequence of the action of degradation enzymes, such as ribonuclease, apyrase, phosphatase, nucleotidase, and adenosine 5'-monophsphate deaminase during the commercial processing of the young leaves.

Caffeine synthase is an enzyme that catalyses the final two steps in the caffeine biosynthesis pathway. We have cloned the gene encoding caffeine synthase from young leaves of tea (Camellia sinensis), opening up the possibility of creating tea and coffee (Coffea arabica) plants that are naturally deficient in caffeine. Consumers concerned about the possible adverse effects of caffeine consumption will welcome this development towards caffeine-free drinks that retain their flavour.

Changes in cytoplasmic pH of suspension-cultured cells of Catharanthus roseus under extreme acid conditions were measured with the pH-dependent fluorescence dye; 2',7'-bis-(2-carboxyethyl)-5 (and-6) carboxyfluorescein (-acetoxymethylester) (BCECF). When cells were treated with 1 mM HC1 (pH 3 solution), the cytoplasmic pH first decreased then returned to the original level. Treatment with 10 mM HC1 (pH 2 solution) acidified the cytoplasm to a greater extent, and the acidification continued at a constant level throughout the measurement. Treatment with a pH 2 solution resulted in a gradual decrease of the malate content, indicating the operation of biochemical pH regulation mechanism. The pH 2 treatment also caused a sudden decrease of the intracellular level of Pi. The cellular content of total phosphorus did not change during the acidification. The Pi was converted to the organic phosphate form. The ATP level was not increased by the pH 2 treatment, but slightly decreased. The role of Pi, which might be functioning as a regulatory factor of cytoplasmic pH, a non-competitive inhibitor of the H+ -pumps of both the plasma membrane and tonoplast is discussed.

Floret elongation and levels of precarthamin were investigated in freshly collected flowers of Carthamus tinctorius. Accumulation of precarthamin was found to be induced at the early stages of floret elongation. [U-14C]Acetate and [U-14C]phenylalanine were incorporated into precarthamin in the detached florets from the flower bud. The results suggest that precarthamin is synthesized via the acetate-shikimate pathway.

The aim of this study was to reveal the pyridine metabolism in leaves of two mangrove legumes, Derris indica (= Millettia pinnata or Pongamia pinnata) and Caesalpinia crista. Radioactivity from [carbonyl-14C]nicotinamide supplied exogenously to young leaf disks was recovered in nicotinic acid, nicotinic acid mononucleotide, NAD, NADP, nicotinamide mononucleotide and trigonelline. These mangrove species, especially D. indica, have strong ability to convert nicotinamide to trigonelline, but not to nicotinic acid glucoside. The endogenous trigonelline content in leaves of D. indica was more than 830 μg/g dry weight. This value is 5-12 times greater than that in leaves of Glycine max. There was little short-term effect of 250 and 500 mM NaCl (equivalent to ca. 50% and 100% sea water) on nicotinamide metabolism.

Trigonelline is synthesized from nicotinic acid in many plant species. This compound accumulates in seeds of leguminous plants and Coffea sp. The present article reviews the distribution of nicotinic acid conjugates and biosynthetic pathways of trigonelline, including the pyridine nucleotide cycle and de novo synthesis. The function of trigonelline in plants is also discussed.