Search

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.

Primary and secondary metabolism in Coffea plants is reviewed. After a brief introduction of carbohydrate and nitrogen metabolism, the biosyntheses of three major secondary metabolites in coffee, caffeine, trigonelline and chlorogenic acids, are reviewed. Caffeine is a purine alkaloid and produced from purine nucleotides with the four-step biosynthetic pathway from xanthosine being catalyzed by three enzymes belonging to the SABATH family of N-methyltransferases. Catabolism of caffeine occurs in only a limited number of Coffea species. Trigonelline is a pyridine alkaloid produced from a pyridine nucleotide, NAD. Nicotinic acid, an intermediate of the pyridine cycle, serves as the substrate for trigonelline synthase. This enzyme is also a SABATH family N-methyltransferase and very similar to the enzymes operating in the caffeine biosynthetic pathway. Biosynthesis of chlorogenic acids begins with the core phenylpropanoid pathway. The route to 5-O-caffeoylquinic acid (5-CQA) from p-coumaroyl-CoA in Coffea plants is p-coumaroyl-CoA → 5-O-p-coumaroylshikimic acid → 5-O-caffeoylshikimic acid → caffeoyl-CoA → 5-CQA. Occurrence and plant physiological aspects of the metabolism of the three secondary metabolites are also described.

Caffeine (1,3,7- N-trimethylxanthine) and trigonelline (1 N-methylnicotinic acid) are major alkaloids in coffee plants. The key enzymes involved in the biosynthesis 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.

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.

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.

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.

This review describes the occurrence and biosynthesis of trigonelline (N-methylnicotinic acid) and related nicotinic acid metabolites. High concentrations of trigonelline are found in seeds of coffee, and some members of the Fabaceae, while trace amounts occur in many other species. In contrast, the occurrence of other pyridine alkaloids derived from nicotinic acid is limited. Nicotinic acid, a precursor of the secondary pyridine metabolites, is derived from pyridine nucleotides. In planta, pyridine nucleotide biosynthesis de novo is initiated from aspartic acid. The degradation of NAD and its regeneration from the catabolites is called the pyridine nucleotide cycle (PNC). Isotopic labelling and enzymatic studies indicate a seven-component PNC VII pathway is the major route. All plants examined convert exogenous nicotinic acid to trigonelline and/or nicotinic acid N-glucoside (NaG). In general, NaG formation is restricted to ferns and selected orders of angiosperms, whereas other plants produce trigonelline. The biosynthesis of other pyridine alkaloids, of which many details remain to be resolved, is discussed briefly. The potential in planta roles of trigonelline, including detoxification, nyctinasty and host selection are discussed. Coffee beverage is the major food containing trigonelline and some vegetables also contain trigonelline. The possible effects of trigonelline on health mediated via hypoglycaemic, neuroprotective, anti-cancer, estrogenic, and antibacterial activities are reviewed. Finally, potential genetic manipulation of biosynthetic pathways to create trigonelline- and vitamin B3-rich plants and agricultural uses of trigonelline-rich genetically modified crops and trees are discussed.

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.

To investigate the physiological role of trigonelline in plant cells, the effects of nicotinic acid, nicotinamide and trigonelline on the division and colony formation of lettuce cells were investigated. Four days after treatment with 0.1–1.0 mM nicotinic acid, division of isolated protoplasts was significantly inhibited. In contrast, a little or no inhibition was found in protoplasts treated with nicotinamide or trigonelline. Nine days after treatment there was a marked inhibitory effect on the colony formation of cells derived from the protoplasts treated with nicotinic acid or nicotinamide, but no effect or even a stimulatory effect was observed in trigonelline-treated protoplasts. These observations imply that nicotinic acid is toxic in high concentration for cell division of plant cells. Trigonelline formation from nicotinic acid and nicotinamide appears to be a result of detoxification of nicotinic acid which is produced by the pyridine nucleotide cycle in the cells or supplied exogenously to the cells.

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 β-carotene. γ-Oryzanol (279 μg/g) was also present as a mixture of 24-methylenecycloartenol ferulate, campesterol ferulate, cycloartenol ferulate and β-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.

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.

Pyridine compounds, including nicotinic acid and nicotinamide, are key metabolites of both the salvage pathway for NAD and the biosynthesis of related secondary compounds. We examined the in situ metabolic fate of [carbonyl-(14)C]nicotinamide, [2-(14)C]nicotinic acid and [carboxyl-(14)C]nicotinic acid riboside in tissue segments of tea (Camellia sinensis) plants, and determined the activity of enzymes involved in pyridine metabolism in protein extracts from young tea leaves. Exogenously supplied (14)C-labelled nicotinamide was readily converted to nicotinic acid, and some nicotinic acid was salvaged to nicotinic acid mononucleotide and then utilized for the synthesis of NAD and NADP. The nicotinic acid riboside salvage pathway discovered recently in mungbean cotyledons is also operative in tea leaves. Nicotinic acid was converted to nicotinic acid N-glucoside, but not to trigonelline (N-methylnicotinic acid), in any part of tea seedlings. Active catabolism of nicotinic acid was observed in tea leaves. The fate of [2-(14)C]nicotinic acid indicates that glutaric acid is a major catabolite of nicotinic acid; it was further metabolised, and carbon atoms were finally released as CO(2). The catabolic pathway observed in tea leaves appears to start with the nicotinic acid N-glucoside formation; this pathway differs from catabolic pathways observed in microorganisms. Profiles of pyridine metabolism in tea plants are discussed.

Xanthosine is a catabolite of purine nucleotides. Our studies using excised tissues of various plant species indicate that xanthosine salvage is negligible and that xanthosine is catabolised predominantly via xanthine. A recent report using intact Arabidopsis thaliana seedlings (Riegler et al., 2011. New Phytol. 191, 349–359) showed that significant amounts of xanthosine were utilised for RNA synthesis. We report here similar, more detailed 14C-feeding experiments of xanthosine and xanthine using intact mungbean seedlings. Less than 3% of radioactivity from [8-14C]xanthosine and 1% from [8-14C]xanthine was incorporated into the RNA fraction; the rest of the radioactivity was incorporated into purine catabolites, including ureides, urea and CO2. Allopurinol, which is a xanthine oxidoreductase inhibitor, markedly inhibited purine catabolism, and radioactivity from these two precursors was retained in xanthine. Even then, no significant salvage of xanthosine and xanthine was observed. Rapid catabolism and slow salvage of xanthosine and xanthine appear to be inherent properties of many plant species.

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.

Silica/poly(vinyl alcohol)(PVA) organic-inorganic hybrid gas barrier films were prepared by sol-gel method with microwave irradiation using tetraethoxysilane (TEOS), methyltriethoxysilane (MeTEOS) and poly(vinyl alcohol) on polypropylene (PP) substrates. Oxygen permeation through the films was measured by a variable-pressure method. Water vapor transmission rate of the films was also evaluated by dish method. Oxygen permeability coefficients of the hybrid layer was small and about one-sixth of those of poly(vinylidene chloride) (PVDC) and water vapor transmission rate of the hybrid layer was the same order of PVDC. Pencil hardnesses (50 g load) of the PP with the hybrid layers were more than HB. These values were considerably higher than that of PP (6B). These properties were thought to be due to well dispersion of inorganic segments (silica) and organic segments (PVA) at molecular level in the hybrid. From the results, it was found that the organic-inorganic hybrids could be applicable to gas barrier films.

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.

As part of a research program on nucleotide metabolism in potato tubers (Solanum tuberosum L.), profiles of pyridine (nicotinamide) metabolism were examined based on the in situ metabolic fate of radio-labelled precursors and the in vitro activities of enzymes. In potato tubers, [(3)H]quinolinic acid, which is an intermediate of de novo pyridine nucleotide synthesis, and [(14)C]nicotinamide, a catabolite of NAD, were utilised for pyridine nucleotide synthesis. The in situ tracer experiments and in vitro enzyme assays suggest the operation of multiple pyridine nucleotide cycles. In addition to the previously proposed cycle consisting of seven metabolites, we found a new cycle that includes newly discovered nicotinamide riboside deaminase which is also functional in potato tubers. This cycle bypasses nicotinamide and nicotinic acid; it is NAD --nicotinamide mononucleotide --nicotinamide riboside --nicotinic acid riboside --nicotinic acid mononucleotide --nicotinic acid adenine dinucleotide --NAD. Degradation of the pyridine ring was extremely low in potato tubers. Nicotinic acid glucoside is formed from nicotinic acid in potato tubers. Comparative studies of [carboxyl-(14)C]nicotinic acid metabolism indicate that nicotinic acid is converted to nicotinic acid glucoside in all organs of potato plants. Trigonelline synthesis from [carboxyl-(14)C]nicotinic acid was also found. Conversion was greater in green parts of plants, such as leaves and stem, than in underground parts of potato plants. Nicotinic acid utilised for the biosynthesis of these conjugates seems to be derived not only from the pyridine nucleotide cycle, but also from the de novo synthesis of nicotinic acid mononucleotide.

The aim of this study was to investigate the antitumor efficacy of treatment, identify prognostic factors, and construct a prognostic index in patients with hepatocellular carcinoma treated by transcatheter arterial infusion chemotherapy (TAI) using cisplatin suspended in lipiodol.We analyzed the outcomes in a total of 94 consecutive patients with previously untreated hepatocellular carcinoma who were treated by TAI using cisplatin suspended in lipiodol.Twenty-seven patients (29%) showed complete response and 21 patients (22%) showed partial response, with an overall response rate of 51% (95% confidence interval, 41-61%). The median survival time was 2.5 years and the proportions of survivors at 1, 2, and 5 years were 81.6, 65.2, and 18.3%, respectively. The results of multivariate analysis indicated a significant association of serum albuminor =3.0 g/dL, maximum tumor sizeor =3.0 cm, absence of ascites, and unilateral distribution of the tumors with a favorable survival. For clinical application, we also propose a prognostic index based on a combination of these prognostic factors. Based on this index, the patients were classified into three groups: those with good, intermediate, and poor prognosis. The median survival times in these three groups were 4.3, 2.7, and 1.1 years, respectively (p0.01).TAI with cisplatin suspended in lipiodol exhibited favorable tumor efficacy and survival in patients with hepatocellular carcinoma. The prognostic factors identified and the index proposed based on these factors may be useful for predicting life expectancy, determining treatment strategies, and designing future clinical trials.

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.

To determine whether caffeine biosynthesis is controlled by the availability of purine precursors and/or methyl-donors, we examined the effect of some purine compounds on purine alkaloid accumulation, using tea callus cultures. No stimulation of caffeine biosynthesis was observed when the calli were cultured with 0.5 mM adenosine, guanosine or hypoxanthine for 3 weeks. However, 0.5 mM paraxanthine doubled the caffeine level relative to controls. Adenosine stimulated the growth of callus and reduced the caffeine concentration 3 months after inoculation. These results indicate that methylation of xanthosine by 7-methylxanthosine synthase is the most plausible rate-limiting step of caffeine biosynthesis; the supply of non-methylated purine precursors or availability of S-adenosyl- l -methionine are not the principal controlling factors of caffeine biosynthesis. Adenosine salvage to adenine nucleotide synthesis may contribute to the growth of tea calli, but not to caffeine biosynthesis.

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.

A large amount of gamma-aminobutyric acid (GABA) was found to accumulate in tomato (Solanum lycopersicum) fruits before the breaker stage. Shortly thereafter, GABA was rapidly catabolized after the breaker stage. We screened the GABA-rich tomato cultivar 'DG03-9' which did not show rapid GABA catabolism after the breaker stage. Although GABA hyperaccumulation and rapid catabolism in fruits is well known, the mechanisms are not clearly understood. In order to clarify these mechanisms, we performed comparative studies of 'Micro-Tom' and 'DG03-9' fruits for the analysis of gene expression levels, protein levels and enzymatic activity levels of GABA biosynthesis- and catabolism-related enzymes. During GABA accumulation, we found positive correlations among GABA contents and expression levels of SlGAD2 and SlGAD3. Both of these genes encode glutamate decarboxylase (GAD) which is a key enzyme of GABA biosynthesis. During GABA catabolism, we found a strong correlation between GABA contents and enzyme activity of alpha-ketoglutarate-dependent GABA transaminase (GABA-TK). The contents of glutamate and aspartate, which are synthesized from GABA and glutamate, respectively, increased with elevation of GABA-TK enzymatic activity. GABA-TK is the major GABA transaminase form in animals and appears to be a minor form in plants. In 'DG03-9' fruits, GAD enzymatic activity was prolonged until the ripening stage, and GABA-TK activity was significantly low. Taken together, our results suggest that GAD and GABA-TK play crucial roles in GABA accumulation and catabolism, respectively, in tomato fruits.

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 metabolic fate of [carbonyl-(14)C]nicotinamide, [8-(14)C]adenine and [8-(14)C]inosine was examined in microspore-derived canola (Brassica napus) embryos at different developmental stages: globular stage (day 10, stage 1), early cotyledonary stage (day 20, stage 2), late cotyledonary stage (day 25, stage 3), and fully developed stage (day 35, stage 4). Uptake of [8-(14)C]nicotinamide by the embryos was always rapid. A lower uptake rate was found for [8-(14)C]adenine and [8-(14)C]inosine, especially at stages 1 and 2. [Carbonyl-(14)C]nicotinamide was converted to nicotinic acid and further metabolized to pyridine nucleotides (NAD/NADP). Some radioactivity was also associated to nicotinic acid glucoside. [8-(14)C]adenine was efficiently utilized for the synthesis of adenine nucleotides and RNA. A small fraction of adenine was degraded to CO(2) via ureides. Up to 40% of [8-(14)C]inosine was salvaged to nucleotides and RNA, although degradation of [8-(14)C]inosine to CO(2) was pronounced. At stage 1, highest salvage activities of nicotinamide, adenine and inosine were observed. In contrast, the lowest purine salvage and highest purine catabolism were found in stage 3 embryos. These results suggest that both nicotinamide and purine salvage for NAD/NADP and purine nucleotides synthesis are extremely high in the globular stage (stage 1). These activities decrease gradually until the late cotyledonary stage (stage 3), before increasing again in the fully developed embryos (stage 4). Overall it appears that nicotinamide and purine salvage are required in support of active growth during the initial phases of embryogenesis and at the end of the maturation period, in preparation for post-embryonic growth.

Using semi-quantitative reverse transcription-PCR, we studied the expression of genes encoding caffeine synthase (TCS1), inosine-5′-monophosphate dehydrogenase (TIDH), Sadenosyl- l-methionine synthase (sAMS), phenylalanine ammonia-lyase (PAL) and α-tubulin (Tua1) in young and mature leaves, stems and roots of 4-month-old tea seedlings and young and old tea tissue cultures. The amounts of transcripts of TCS1 were much higher in young leaves than in other parts of the plant. Expression of TIDH was greater in leaves than in other parts. Little difference in the amounts of transcripts of PAL, sAMS and Tua1 was found between various organs of tea seedlings. Larger amounts of transcripts of TCS1 and PAL were found in young callus tissues than in old tissues. These results support our conclusion deriving from previous enzymatic and metabolic studies that caffeine is synthesized mainly in young leaf tissues. We propose that marked caffeine biosynthesis in young leaves is dependent on a greater expression of the TCS1 gene in the organ

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.

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.

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.

MIM capacitors using one of the standard back-end of line (BEOL) metal layers as bottom electrode have emerged as key passive components for analog/mixedsignal (AMS) and radio frequency (RF) integrated circuits, due to advantages of reduced series resistance and lower substrate coupling effect at low cost. Upon the MIM characteristics, the emphasis is on the capacitors second-order voltage linearity since it is critical for the dynamic range of analog circuit, where the firstorder component of voltage linearity can be cancelled out by differential techniques. The dispersion phenomenon with varying applied voltage observed for capacitor with plasma enhanced (PE) CVD nitride as the dielectric were explained by bulknitride traps, though reduction of the capacitance dispersion phenomenon were extensively investigated. In this work, nitride films, prepared by the ALD method, are demonstrated the precise properties for AMS and RF integrated circuits, adapted to the dielectric of MIM capacitors.

Changes in the pattern of pyrimidine nucleotide metabolism were investigated in Pinus radiata cotyledons cultured under shoot-forming (SF; +N(6)-benzyladenine) and non-shoot-forming (NSF, -N(6)-benzyladenine) conditions, as well as in cotyledons unresponsive (OLD) to N(6)-benzyladenine. This was carried out by following the metabolic fate of externally supplied (14)C-labeled orotic acid, intermediate of the de novo pathway, and (14)C-labeled uridine and uracil, substrates of the salvage pathway. Nucleic acid synthesis was also investigated by following the metabolic fate of (14)C-labeled thymidine during shoot bud formation and development. The de novo synthesis of pyrimidine nucleotides was operative under both SF and NSF conditions, and the activity of orotate phosphoribosyltransferase (OPRT), a key enzyme of the de novo pathway, was higher in SF tissue. Utilization of both uridine and uracil for nucleotide and nucleic acid synthesis clearly indicated that the salvage pathway of pyrimidine metabolism is also operative during shoot organogenesis. In general, uridine was a better substrate for the synthesis of salvage products than uracil, possibly due to the higher activity of uridine kinase (UK), compared to uracil phosphoribosyltransferase (UPRT). Incorporation of uridine into the nucleic acid fraction of OLD cotyledons was lower than that observed for their responsive (day 0) counterparts. Similarly, uracil utilization for nucleic acid synthesis was lower in NSF cotyledons, compared to that observed for SF tissue after 10 days in culture. This difference was ascribed to higher UPRT activity measured in the latter. Thus, there was an apparent difference in the utilization of nucleotides derived from uracil and uridine for nucleotide synthesis. The increased ability to produce pyrimidine nucleotides via the salvage pathway during shoot bud formation may be required in support of nucleic acid synthesis occurring during the process. Studies on thymidine metabolism confirmed this notion.

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.

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.

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.

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.

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.

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.

To investigate the short-term (3 h) effect of salt on the metabolism of purine, pyrimidine and pyridine nucleotides in mangrove (Bruguiera sexangula) cells, we examined the uptake and overall metabolism of radiolabelled intermediates involved in the de novo pathways and substrates of salvage pathways for nucleotide biosynthesis in the presence and absence of 100 mM NaCl. Uptake by the cells of substrates for the salvage pathways was much faster than uptake of intermediates of the de novo pathways. The activity of the de novo pyrimidine biosynthesis estimated by [2-14C]orotate metabolism was not significantly affected by the salt. About 20–30% of [2-14C]uridine, [2-14C]uracil and more than 50% of [2-14C]cytidine were salvaged for pyrimidine nucleotide biosynthesis. However, substantial quantities of these compounds were degraded to 14CO2 via β-ureidopropionate (β-UP), and degradation of β-UP was increased by the salt. The activities of the de novo pathway, estimated by [2-14C] 5-aminoimidazole-4-carboxamide ribonucleoside, and the salvage pathways from [8-14C]adenosine and [8-14C]guanosine for the purine nucleotide biosynthesis were not influenced by the salt. Most [8-14C]hypoxanthine was catabolised to 14CO2, and other purine compounds are also catabolised via xanthine. Purine catabolism was stimulated by the salt. [3H]Quinolinate, [carbonyl-14C]nicotinamide and [carboxyl-14C]nicotinic acid were utilised for the biosynthesis of pyridine nucleotides. The salvage pathways for pyridine nucleotides were significantly stimulated by the salt. Trigonelline was synthesised from all pyridine precursors that were examined; its synthesis was also stimulated by the salt. We discuss the physiological role of the salt-stimulated reactions of nucleotide metabolism.

Caffeine is a key component of many popular drinks. The caffeine biosynthetic pathway has been elucidated, several of the genes encoding key N-methyltransferases have been cloned, and transgenic coffee plants with a reduced caffeine content have been produced.

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.

Uridine and cytidine are major nucleosides and are produced as catabolites of pyrimidine nucleotides. To study the metabolic fates and role of these nucleosides in plants, we have performed pulse (2 h) and chase (12 h) experiments with [2-14C]uridine and [2-14C]cytidine and determined the activities of some related enzymes using tubers and fully expanded leaves from 10-week-old potato plants (Solanum tuberosum L.). In tubers, more than 94% of exogenously supplied [2-14C]uridine and [2-14C]cytidine was converted to pyrimidine nucleotides and RNA during 2-h pulse, and radioactivity in these salvage products still remained at 12 h after the chase. Little degradation of pyrimidine was found. A similar pyrimidine salvage was operative in leaves, although more than 20% of the radioactivity from [2-14C]uridine and [2-14C]cytidine was released as 14CO2 during the chase. Enzyme profile data show that uridine/cytidine kinase (EC 2.7.1.48) activity is higher in tubers than in leaves, but uridine nucleosidase (EC 3.2.2.3) activity was higher in leaves. In leaves, radioactivity from [U-14C]uracil was incorporated into β-ureidopropionic acid, CO2, β-alanine, pantothenic acid and several common amino acids. Our results suggest two functions of uridine and cytidine metabolism in leaves; these nucleosides are not only substrates for the classical pyrimidine salvage pathways but also starting materials for the biosynthesis of β-alanine. Subsequently, some β-alanine units are utilized for the synthesis of pantothenic acid in potato leaves.