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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.

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.

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.

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.

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.

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.

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.

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 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 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.

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.

Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are the major purine alkaloids in plants. To investigate the diversity of N-methyltransferases involved in purine alkaloid biosynthesis, we isolated the genes homologous for caffeine synthase from theobromine-accumulating plants. The predicted amino acid sequences of N-methyltransferases in theobromine-accumulating species in Camellia were more than 80% identical to caffeine synthase in C. sinensis. However, there was a little homology among the N-methyltransferases between Camellia and Theobroma. The recombinant enzymes derived from theobromine-accumulating plants had only 3-N-methyltransferase activity. The accumulation of purine alkaloids was, therefore, dependent on the substrate specificity of N-methyltransferase determined by one amino acid residue in the central part of the protein.

To estimate the operation of the de novo and salvage pathways of pyridine nucleotide synthesis, [3H]quinolinic acid, an intermediate of the de novo synthesis, and [14C]nicotinamide and [14C]nicotinic acid, substrates of the salvage pathways, were administered to cultured white spruce (Picea glauca) and Madagascar periwinkle (Catharanthus roseus) cells and overall metabolism of these radioactive compounds was examined over the culture period. In P. glauca cells, all three precursors were able to generate pyridine nucleotides (mainly NAD and NADP) and trigonelline. Supplied [3H]quinolinic acid was efficiently converted to pyridine nucleotides and trigonelline in both at the logarithmic and the stationary stages of cell growth, although uptake of [3H]quinolinic acid by P. glauca cells was very low. [14C]Nicotinic acid and [14C]nicotinamide were taken up by the cells in a relatively facile manner, and 15–20 and 32–58% of total radioactivity from these compounds was found in pyridine nucleotides (mainly NAD and NADP) and trigonelline, respectively, after 18 h incubation. In C. roseus cells, these three precursors were utilised for pyridine nucleotides, but in contrast to P. glauca, nicotinic acid glucoside, but not trigonelline, was heavily labeled. Nicotinic acid and nicotinamide were better precursors for pyridine nucleotide synthesis than quinolinic acid. In Pi-starved cells, the uptake of quinolinic acid, nicotinic acid and nicotinamide was markedly decreased. Pyridine nucleotide synthesis de novo was greatly reduced in Pi-starved C. roseus cells, while little effect was found in the salvage pathway of nicotinic acid. Pi-deficiency slightly increased the rate of nicotinic acid-glucoside synthesis from nicotinic acid and nicotinamide. From the in vitro determination of enzyme activity, it is concluded that quinolinic acid and nicotinic acid are converted to nucleotides by quinolinate phosphoribosyltransferase (2.4.2.19) and nicotinate phosphoribosyltransferase (2.4.1.11), respectively. High activity of nicotinamidase (3.5.1.19) but no detectable activity of nicotinamide phosphoribosyltransferase (2.4.2.12) suggests that nicotinamide is converted to nicotinic acid, and then salvaged by nicotinate phosphoribosyltransferase.

Pyrimidine deoxyribonucleotide metabolism was investigated during maturation and germination of white spruce somatic embryos by following the metabolic fate of [2- 14 C]cytidine, [2- 14 C]deoxycytidine and [2- 14 C]thymidine. The de-novo pathway of deoxyribonucleotides was estimated indirectly, by the ability of the tissue to incorporate cytidine into DNA after conversion to dCTP. The salvage pathway was estimated by the utilization of labelled cytidine, deoxycytidine and thymidine for synthesis of deoxyribonucleotides and nucleic acids. Utilization of cytidine for DNA synthesis, via the de novo pathway, was always lower than that observed for RNA throughout the course of the experiment. Incorporation of cytidine into RNA was found to occur either directly, after conversion to CTP, mediated by the enzymes cytidine kinase, nucleoside monophosphate kinase and nucleoside diphosphate kinase, or indirectly, after conversion to UTP via uridine and UMP. Active incorporation of uridine into RNA of white spruce-cultured cells was demonstrated previously. Salvage of deoxycytidine and thymidine was operative in maturing and germinating white spruce somatic embryos, as label from both compounds was recovered in nucleotides and DNA. However, the utilization of these precursors by the cells was different. Salvage of deoxycytidine was always higher than that observed for thymidine, which was extensively catabolized to CO 2 at all stages of embryo development.

Pyrimidine nucleotide synthesis was investigated in isolated germinating zygotic embryos and separated megagametophytes of white spruce by following the metabolic fate of 14C-labelled orotic acid, uridine, and uracil, as well as by measuring the activities of the major enzymes participating in nucleotide synthesis. The rate of nucleic acid synthesis in these tissues was also examined by tracer experiments and autoradiographic studies conducted with labelled thymidine, and by conventional light microscopy. From our results, it emerges that changes in the contribution of the de novo and salvage pathways of pyrimidines play an important role during the initial stages of zygotic embryo germination. Preferential utilization of uridine for nucleic acid synthesis, via the salvage pathway, was observed at the onset of germination, before the restoration of a fully functional de novo pathway. Similar metabolic changes, not observed in the gametophytic tissue, were also documented in somatic embryos previously. These alterations of the overall pyrimidine metabolism may represent a strategy for ensuring the germinating embryos with a large nucleotide pool. Utilization of 14C-thymidine for nucleic acid synthesis increased in both dissected embryos and megagametophytes during germination. Autoradiographic and light microscopic studies indicated that soon after imbibition, DNA synthesis was preferentially initiated along the embryonic axis, especially in the cortical cells. Apical meristem reactivation was a later event, and the root meristem became activated before the shoot meristem. Taken together, these results indicate that precise changes in nucleotide and nucleic acid metabolism occur during the early phases of embryo germination.

Changes in pyrimidine metabolism were investigated in germinating white spruce somatic embryos by following the metabolic fate of [2-14C]uracil and [2-14C]uridine, intermediate metabolites of the salvage pathway and [6-14C]orotic acid, a central metabolite of the de novo. nucleotide biosynthesis. An active uridine salvage was found to be responsible for the enlargement of the nucleotide pool at the inception of germination. Uridine kinase, which catalyzes the conversion of uridine to uridine monophosphate (UMP), was found to be very active in partially dried embryos and during the early phases of imbibition. The contribution of uracil to the nucleotide pool was negligible since a large amount of radioactivity from [2-14C]uracil was recovered in degradation products. As germination progressed, the decline of the uridine salvage pathway was concomitant with an increase of the de novo biosynthetic pathway. The central enzyme of the de novo pathway, orotate phosphoribosyltransferase, showed increased activity and contributed to the larger amount of orotate being anabolized. These results suggest that although both the salvage and de novo pathways operate in germinating white spruce somatic embryos, their contribution to the enlargement of the nucleotide pool appears tightly regulated as germination progresses.

The imposition of a partial drying treatment (PDT) on mature white spruce somatic embryos is a necessary step for successful germination and embryo conversion into plantlets. Purine and pyrimidine metabolism was investigated during the PDT of white spruce somatic embryos by following the metabolic fate of 14 C-labeled adenine, adenosine, and inosine, as purine intermediates, and orotic acid, uridine, and uracil, as pyrimidine intermediates, as well as examining the activities of key enzymes. Both the salvage and the degradation pathways of purines were operative in partially dried embryos. Adenine and adenosine were extensively salvaged by the enzymes adenine phosphoribosyltransferase and adenosine kinase, respectively. The activity of the former enzyme increased during the PDT. In both mature and partially dried embryos, a large proportion of inosine was recovered as degradation products. The de novo pathway of pyrimidine nucleotide biosynthesis, estimated by the incorporation of orotic acid into the nucleotides and nucleic acids, was high at the end of the maturation period and declined during the PDT. Uridine was the main substrate for the pyrimidine salvage pathway, since a large proportion of uracil was recovered as degradation products, i.e. CO 2 and β-ureidopropionic acid in both mature and partially dried embryos. Uridine was mainly salvaged by uridine kinase, whose activity was found to increase during the PDT. Taken together these results indicate that the PDT might be required for increasing the activity of adenine and uridine salvage enzymes, which could contribute to the enlargement of the nucleotide pool required at the onset of germination.

In order to examine the biosynthesis, interconversion, and degradation of purine and pyrimidine nucleotides in white spruce cells, radiolabeled adenine, adenosine, inosine, uracil, uridine, and orotic acid were supplied exogenously to the cells and the overall metabolism of these compounds was monitored. [8- 14 C]adenine and [8- 14 C]adenosine were metabolized to adenylates and part of the adenylates were converted to guanylates and incorporated into both adenine and guanine bases of nucleic acids. A small amount of [8- 14 C]inosine was converted into nucleotides and incorporated into both adenine and guanine bases of nucleic acids. High adenosine kinase and adenine phosphoribosyltransferase activities in the extract suggested that adenosine and adenine were converted to AMP by these enzymes. No adenosine nucleosidase activity was detected. Inosine was apparently converted to AMP by inosine kinase and/or a non-specific nucleoside phosphotransferase. The radioactivity of [8- 14 C]adenosine, [8- 14 C]adenine, and [8- 14 C]inosine was also detected in ureide, especially allantoic acid, and CO 2 . Among these 3 precursors, the radioactivity from [8- 14 C]inosine was predominantly incorporated into CO 2 . These results suggest the operation of a conventional degradation pathway. Both [2- 14 C]uracil and [2- 14 C]uridine were converted to uridine nucleotides and incorporated into uracil and cytosine bases of nucleic acids. The salvage enzymes, uridine kinase and uracil phosphoribosyltransferase, were detected in white spruce extracts. [6- 14 C]orotic acid, an intermediate of the de novo pyrimidine biosynthesis, was efficiently converted into uridine nucleotides and also incorporated into uracil and cytosine bases of nucleic acids. High activity of orotate phosphoribosyltransferase was observed in the extracts. A large proportion of radioactivity from [2- 14 C]uracil was recovered as CO 2 and β-ureidopropionate. Thus, a reductive pathway of uracil degradation is functional in these cells. Therefore, white spruce cells in culture demonstrate both the de novo and salvage pathways of purine and pyrimidine metabolism, as well as some degradation of the substrates into CO 2 .

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.

The aim of the present study was to investigate the metabolic fate of guanine nucleotides in higher plants. The rate of uptake of [8- 14 C]guanosine by suspension-cultured Catharanthus roseus cells was more than 20 times higher than that of [8- 14 C]guanine. The rate of uptake of [8- 14 C]guanosine increased with the age of the culture. Pulse-chase experiments with [8- 14 C]guanosine revealed that some of the guanosine that had been taken up by the cells was converted to guanine nucleotides and incorporated into nucleic acids. A significant amount of [8- 14 C]guanosine was degraded directly to xanthine, allantoin and allantoic acid, with the generation of l4 CO 2 as the final product. The rate of salvage of [8- 14 C]guanosine for the synthesis of nucleic acids was highest in young cells, while the rate of degradation increased with the age of the cells. In segments of roots from Vigna mungo seedlings, nearly 50% of the [8- 14 C]guanosine that had been absorbed over the course of 15 min was recovered in guanine nucleotides. A significant amount of the radioactivity in nucleotides became associated with nucleic acids and ureides during 'chase' periods. In segments of young leaves of Camellia sinensis, [8- 14 C]guanosine was initially incorporated into guanine nucleotides, nucleic acids, theobromine and ureides, and the radioactivity in these compounds was transferred to caffeine and CO 2 during a 24-h incubation. Our results suggest that guanosine is an intermediate in the catabolism of guanine nucleotides and that it is re-utilised for nucleotide synthesis by 'salvage' reactions. Guanosine was catabolised by the conventional degradation pathway via xanthine and allantoin. In some plants, guanosine is also utilised for the formation of ureide or the biosynthesis of caffeine.

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.

The aim of this study was to investigate the S-adenosylmethionine dependent N-methyltransferase(s) (NMT) associated with the three methylation steps in the caffeine biosynthesis pathway in tea (Camellia sinensis L.). NMT activity in cell-free preparations from young leaves was purified by anion-exchange and gel-filtration column chromatography. In both systems, a single zone of NMT activity, with broad substrate specificity was detected. The N-3 position of dimethylxanthine and monomethylxanthines was methylated more readily than N-1 while comparatively little substitution occurred at the N-7 locus. When xanthosine was used as a substrate only the N-7 position was methylated. These results indicate that a single NMT may participate in the conversion of xanthosine to caffeine. The apparent M r of the NMT, estimated by gel filtration chromatography, was 61 000. The substrate specificity of the NMT is compatible with the operation of a xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine pathway as the main biosynthetic route to caffeine in young tea leaves. The data also indicate that the conversion of 7-methylxanthine → paraxanthine → caffeine may function as one of a number of minor pathways that also contribute to the production of caffeine.

Summary A tissue culture system was established from pistils of flower buds of a mangrove plant, Sonneratia alba . The optimum growth rate of callus was observed in Murashige and Skoog's medium that contained 50 mM NaCl. In this tissue culture system, we examined the effects of short-term salt stress on the size of the pool of purine nucleotides, as well as the capacity for the salvage and the degradation of purines. At 100 mM NaCl, the size of the total adenylate pool was reduced, but the «adenylate energy charge» was, by contrast, increased. No marked change in the size of guanylate pool occurred in response to NaCl. The rate of conversion of [8- 14 C] adenosine to adenylate was decreased by NaCl, but that to nucleic acids was not significandy affected. In contrast to the results obtained with maize root tips (Peterson et al., 1988), salt stress did not increase the catabolism of [8- 14 C] hypoxanthine. Most of the radioactivity from [8- 14 C] adenosine was accumulated in adenine while that in [8- 14 C] hypoxanthine remained unaffected in Sonneratia alba . The low capacity of the purine-degradation system might ensure a supply of purine bases and nucleosides that can be utilized as substrates for the rapid and efficient biosynthesis of nucleotides by the salvage pathways.

A limited number of plant species accumulate purine alkaloids, such as caffeine and theobromine, which are synthesized from xanthosine, a catabolite of purine nucleotides. The main biosynthetic pathway is a sequence consisting of xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine. This review summarizes the occurrence of purine alkaloids in the plant kingdom, the caffeine biosynthesis routes from purine precursors, the enzymes and genes of N-methyltransferases, key enzymes of caffeine biosynthesis, caffeine catabolism and the possible ecological role of caffeine. Finally, we introduce transgenic plants in which caffeine production is either suppressed or induced by the introduction of caffeine encoded genes. Such plants have the potential to be used for the production of decaffeinated coffee and tea or as natural pesticides in agriculturally important crops.

Besides regulating meristem formation and maintenance in vivo, SHOOTMERISTEMLESS (STM) has been shown to affect embryogenesis. While the over-expression of Brassica napus (Bn)STM enhances the number of microspore-derived embryos produced in culture and their ability to regenerate viable plants, a down-regulation of this gene represses the embryogenic process (Elhiti et al., J Exp Bot, 61:4069-4085, 2010). Synthesis and degradation of pyrimidine and purine nucleotides were measured in developing microspore-derived embryos (MDEs) generated from B. napus lines ectopically expressing or down-regulating BnSTM. Pyrimidine metabolism was investigated by following the metabolic fate of exogenously supplied (14)C-uridine, uracil and orotic acid, whereas purine metabolism was estimated by using (14)C-adenine, adenosine and inosine. The improvement in embryo number and quality affected by the ectopic expression of BnSTM was linked to the increased pyrimidine and purine salvage activity during the early phases of embryogenesis and the enlargement of the adenylate pool (ATP + ADP) required for the active growth of the embryos. This was due to an increase in transcriptional and enzymatic activity of several salvage enzymes, including adenine phosphoribosyltransferase (APRT) and adenosine kinase (ADK). The highly operative salvage pathway induced by the ectopic expression of BnSTM was associated with a slow catabolism of nucleotides, suggesting the presence of an antagonist mechanism controlling the rate of salvage and degradation pathways. During the second half of embryogenesis utilization of uridine for UTP + UDPglucose (UDPG) synthesis increased in the embryos over-expressing BnSTM, and this coincided with a better post-germination performance. All these events were precluded by the down-regulation of BnSTM which repressed the formation of the embryos and their post-embryonic performance. Overall, this work provides evidence that precise metabolic changes are associated with proper embryo development in culture.

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.

Purine metabolism and the biosynthesis of purine alkaloids in leaves of mate plants were investigated by administering [8−14C]adenine,[8−14C]guanosine and [8−14C]hypoxanthine to mate plants of different ages. Each precursor was incorporated into theobromine and caffeine in young leaves. No biosynthesis of these purine alkaloids took place in mature dark-green leaves. Pulse-chase experiments and the inhibitory effects of coformycin suggest that the biosynthesis of caffeine from adenine nucleotides is initiated by the reaction catalysed by AMP deaminase. The conventional pathway for the degradation of purines via allantoin and allantoic acid appeared to be operative in both young and mature leaves. No significant degradation of caffeine was detected in the tracer experiments. The levels of adenine nucleotides in young mate leaves were about five times higher than those of guanine nucleotides. Other possible pathways of purine metabolism in mate leaves are discussed.

The biosynthesis of purine alkaloids in flower buds of tea plants was investigated. More than 25% of total radioactivity of [8-14C]adenine taken up by stamens isolated from tea flower buds was found to have been incorporated into purine alkaloids, namely, theobromine and caffeine, 24 h after administration of the labelled compound. Pulse-chase experiments indicated that [8-14C]adenine taken up by the stamens was converted to adenine nucleotides and subsequently incorporated into theobromine and caffeine. Since 5 μM coformycin, an inhibitor of AMP deaminase, inhibited the incorporation of radioactivity into the purine alkaloids, synthesis of caffeine from adenine nucleotides seems to be initiated by the reaction of AMP deaminase. Although most of the radioactivity from [8-14C]inosine was recovered as CO2 and ureides, considerable amounts of radioactivity were recovered as purine alkaloids. The incorporation of radioactivity from [8-14C]inosine into the purine alkaloids was not affected by coformycin. The five enzymes involved in synthesis of 5-phosphoribosyl-1-pyrophosphate from glucose were present in the stamens and petals of tea flower buds. From present and previous results, the pathway for the biosynthesis of caffeine from adenine nucleotides in flower buds of tea is discussed.

Methylxanthines and methyluric acids are purine alkaloids that are synthesized in quantity in a limited number of plant species, including tea, coffee and cacao. This review summarizes the pathways, enzymes and related genes of caffeine biosynthesis. The main biosynthetic pathway is a sequence consisting of xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine. Catabolism of caffeine starts with its conversion to theophylline. Typically, this reaction is very slow in caffeine-accumulating plants. Finally, the ecological roles of caffeine and the production of decaffeinated coffee plants are discussed.

Salt stress improves the quality of tomato fruits. To clarify the mechanism(s) underlying this phenomenon, we investigated metabolic alterations in tomato fruits exposed to 160 mM salt, focusing on metabolism of organic acids related to the tricarboxylic acid (TCA) cycle and gamma-aminobutyric acid (GABA). Quantitative analyses revealed that most amino acids increased in response to salt stress throughout fruit development, and the effect of the stress was greater in the pericarp than in the columella, whereas organic acids did not show a remarkable tendency to salt stress. The transcript levels of 20 genes encoding enzymes of the TCA cycle and peripheral pathways were also analyzed in salt-stressed fruit. Genes responsive to salt stress could be categorized into two types, which were expressed during early development or ripening stages. During fruit development, phosphoenolpyruvate carboxylase 2 and phosphoenolpyruvate carboxykinase displayed contrasting expression patterns between early development and ripening, suggesting a switch of carbohydrate metabolism after the turning stage. Our results revealed a new metabolic pathway for GABA during the development of tomato fruits. At the start of ripening, GABA is first converted to malate via succinate semialdehyde, and it passes into a shunt through pyruvate. Then, it flows back to the TCA cycle and is stored as citrate, which contributes as a substrate for respiration during fruit maturation.

Cellular brassinolide (BL) levels regulate the development of Brassica napus microspore-derived embryos (MDEs). Synthesis and degradation of nucleotides were measured on developing MDEs treated with BL or brassinazole (BrZ), a biosynthetic inhibitor of BL. Purine metabolism was investigated by following the metabolic fate of (14)C-labelled adenine and adenosine, substrates of the salvage pathway, and inosine, an intermediate of both salvage and degradation pathways. For pyrimidine, orotic acid, uridine and uracil were employed as markers for the de novo (orotic acid), salvage (uridine and uracil), and degradation (uracil) pathways. Our results indicate that utilization of adenine, adenosine, and uridine for nucleotides and nucleic acids increased significantly in BL-treated embryos at day 15 and remained high throughout the culture period. These metabolic changes were ascribed to the activities of the respective salvage enzymes: adenine phosphoribosyltransferase (EC 2.4.2.7), adenosine kinase (EC 2.7.1.20), and uridine kinase (EC 2.7.1.48), which were induced by BL applications. The BL promotion of salvage synthesis was accompanied by a reduction in the activities of the degradation pathways, suggesting the presence of competitive anabolic and catabolic mechanisms utilizing the labelled precursors. In BrZ-treated embryos, with depleted BL levels, the salvage activity of both purine and pyrimidine nucleotides was reduced and this was associated to structural abnormalities and poor embryonic performance. In these embryos, the activities of major salvage enzymes were consistently lower to those measured in their control (untreated) counterparts.

The metabolism of purine nucleotides and purine alkaloids (e.g. caffeine and theobromine) are reviewed in tea and coffee plants. Purine metabolism in these plants is similar to that in other plants, which do not contain caffeine; however, tea and coffee plants have purine nucleotides, including those produced directly by the pathway of purine biosynthesis de novo , as effective precursors of caffeine. Xanthosine is the first methyl acceptor from S -adenosylmethionine in caffeine biosynthesis, and is also metabolized by a purine degradation pathway via xanthine. The regulation of purine alkaloid biosynthesis remains elusive, but the activity of the three N -methyltransferases is considered. The production and accumulation of the alkaloids are associated with the developmental stage of tissues (i.e. leaves, flowers, fruits and seeds) and with seasonal changes, especially in tea grown in temperate climates. The metabolism (especially biosynthesis) of purine alkaloids differs among Camellia species. In Coffea plants and in cultured cells, the rate of caffeine synthesis and turnover (i.e. biodegradation and/or biotransformation to xanthine or to methyluric acids) differs markedly among species. Ecological roles of the alkaloids have been reported, but their physiological significance in tea and coffee plants remains uncertain.

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 distribution of phenolic compounds in young and developing leaves, stems, main and lateral roots and cotyledons of 8-week-old tea (Camellia sinensis) seedlings was investigated using HPLC-MS(2). Fourteen compounds, flavan-3-ols, chlorogenic acids, and kaempferol-O-glycosides, were identified on the basis of their retention time, absorbance spectrum, and MS fragmentation pattern. The major phenolics were (-)-epigallocatechin-3-O-gallate and (-)-epicatechin-3-O-gallate, located principally in the green parts of the seedlings. Considerable amounts of radioactivity from [ring-(14)C]phenylalanine were incorporated in (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin-3-O-gallate and (-)-epigallocatechin-3-O-gallate, by tissues of young and developing leaves and stems. Expression of genes encoding enzymes involved in flavan-3-ol biosynthesis, CHS, CHI, F3H, F3'5'H, DFR, ANS, ANR and LAR was investigated. Transcripts of all genes, except LAR, were more abundant in leaves and stems than in roots and cotyledons. No significant difference was found in the amount of transcript of LAR. These findings indicate that in tea seedlings flavan-3-ols are produced by a naringenin-chalcone-->naringenin-->dihydrokaempferol pathway. Dihydrokaempferol is a branch point in the synthesis of (-)-epigallocatechin-3-O-gallate and other flavan-3-ols which can be formed by routes beginning with either a flavonoid 3'-hydroxylase mediated conversion of the flavonol to dihydroquercetin or a flavonoid 3',5'-hydroxylase-catalysed conversion to dihydromyricetin with subsequent steps involving sequential reactions catalysed by dihydroflavanol 4-reductase, anthocyanidin synthase, anthocyanidin reductase and flavan-3-ol gallate synthase.

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.

We examined the distribution of ethylamine, glutamic acid and alanine, which are utilized in theanine biosynthesis, and other major amino acids in leaves, stems, cotyledons and roots of 6-week-old tea seedlings. Ethylamine and glutamic acid, which are substrates of theanine synthetase, were distributed almost uniformly in all parts of the seedlings; the contents in μmol/g fresh wt varied from 0.44 - 0.88 (ethylamine) and 1.6 - 2.4 (glutamic acid). The content of alanine, a possible precursor of ethylamine synthesis, was significantly higher in roots (3.1 μmol/g fresh wt) than in other parts. Incorporation of radioactivity from [U-14C]- alanine into theanine was also higher in roots than in other organs. In 10-week-old seedlings, [1-14C]ethylamine was converted to theanine in young and developed leaves, stems, main and lateral roots; the highest rates of conversion were detected in the main and lateral roots. These results suggest that the theanine synthesis preferentially takes place in roots but is not restricted to them; substrates and the enzymatic machinery for theanine synthesis are available in all parts of tea seedlings

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.

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.

To determine the biosynthetic pathway to trigonelline, the metabolism of [carboxyl-(14)C]nicotinate mononucleotide (NaMN) and [carboxyl-(14)C]nicotinate riboside (NaR) in protein extracts and tissues of embryonic axes from germinating mungbeans (Phaseolus aureus) was investigated. In crude cell-free protein extracts, in the presence of S-adenosyl-L-methionine, radioactivity from [(14)C]NaMN was incorporated into NaR, nicotinate and trigonelline. Activities of NaMN nucleotidase, NaR nucleosidase and trigonelline synthase were also observed in the extracts. Exogenously supplied [(14)C]NaR, taken up by embryonic axes segments, was readily converted to nicotinate and trigonelline. It is concluded that the NaMN-->NaR-->nicotinate-->trigonelline pathway is operative in the embryonic axes of mungbean seedlings. This result suggests that trigonelline is synthesised not only from NAD but also via the de novo biosynthetic pathway of pyridine nucleotides.

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.

Growth of suspension-cultured Catharanthus roseus cells ceased during phosphate starvation, but the cells grew again upon addition of Pi even after long-term starvation. The metabolic fate of [ 33 P]Pi was studied in 1-week-old stationary phase cells in ordinary culture and in 1- or 2-week-old Pi-starved cells. Immediately after administration, the most heavily labelled organic compounds are nucleotides, followed by sugar phosphates. Two weeks Pi starvation slowed down the speed of incorporation of 33 P into nucleotides. The RNA, protein, and free nucleotide content all decreased gradually during Pi starvation; however, these compounds, especially nucleotides, increased markedly in the 24 h after addition of Pi. These responses are found in all cells examined, although the total amounts of these compounds were lower in the long-term Pi-deficient cells. Of the nucleotides, a marked increase was observed in nucleoside triphosphates and UDP-glucose. The transcript level of phosphate transporter and the activities of acid phosphatase, 5′- and 3′-nucleotidase, and adenosine nucleosidase were all reduced by the addition of Pi. In contrast, the activities of adenine phosphoribosyltransferase, nicotinate phosphoribosyltransferase, and nicotinamidase, which are salvage enzymes of purine and pyridine nucleotides, were markedly increased in the Pi-fed cells. Little or no increase was observed in adenosine kinase. In the light of these results, the possible involvement of net nucleotide synthesis in the initial metabolic events of recovery from Pi deficiency are discussed.

Nucleotide metabolism was investigated during white spruce organogenesis by following the metabolic fate of 14C-labeled adenine, adenosine and inosine, as purine precursors, and orotic acid, uridine, and uracil, as pyrimidine intermediates. Key enzymes of purine and pyrimidine metabolism were also assayed during the organogenic process. White spruce epicotyl explants cultured on shoot-forming (SF) medium had a better ability to utilize adenine and adenosine for nucleotide and nucleic acid synthesis, compared to tissue cultured on non-shoot forming (NSF) medium. High levels of salvage products were observed in SF tissue after 10 days in culture, when shoot formation was initiated along the epicotyl axis of the explants. Such a differential utilization of purine precursors was mainly due to the higher specific activity of the two adenine and adenosine salvage enzymes, adenine phosphoribosyltransferase (APRT) and adenosine kinase (AK), measured in SF tissue. Similar catabolism of inosine was observed in both SF and NSF conditions during the 30 days of culture. For pyrimidines, the higher activities of the de novo, salvage, and degradation pathways observed in SF tissue, compared to NSF tissue throughout the course of the experiment, clearly denote a faster turnover of pyrimidine nucleotides in the former. Taken together, these results suggest that a better utilization of purine bases and nucleosides for nucleotide and nucleic acid synthesis, as well as a more rapid turnover of pyrimidine nucleotides, represent a physiological switch, which occurs during the initiation and continuation of the organogenic process in white spruce.

In anti-sense and RNA interference transgenic plants of Coffea canephora in which the expression of CaMXMT1 was suppressed, caffeine biosynthesis from [8-(14)C]adenine was investigated, together with the overall metabolism of [8-(14)C]adenine. Compared with wild type control plants, total purine alkaloid biosynthesis from adenine and conversion of theobromine to caffeine were both reduced in the transgenic plants. As found previously, [8-(14)C]adenine was metabolised to salvage products (nucleotides and RNA), to degradation products (ureides and CO(2)) and to purine alkaloids (theobromine and caffeine). In the transgenic plants, metabolism of [8-(14)C]adenine shifted from purine alkaloid synthesis to purine catabolism or salvage for nucleotides. HPLC analysis revealed a significantly reduced caffeine content in the transgenic plants. A small quantity (less than 20 nmol g(-1) fresh weight) of xanthosine had accumulated in at least one of the transgenic plants.