~~ ~~ I n C, plants, serine synthesis is associated with photorespiratory glycine metabolism involving the tetrahydrofolate (THF)-dependent activities of the glycine decarboxylase complex (CDC) and serine hydroxymethyl transferase (SHMT). Alternatively, THF-dependent serine synthesis can occur via the C1 -THF synthase/SHMT pathway. We used 13C nuclear magnetic resonance to examine serine biosynthesis by these two pathways i n Arabidopsis fhaliana (L.) Heynh.Columbia wild type. We confirmed the tight coupling of the CDC/ SHMT system and observed directly i n a higher plant the flux of formate through the C1 -THF synthase/SHMT system. l h e accumulation of 13C-enriched serine over 24 h from the CDC/SHMT activities was 4-fold greater than that from C1 -THF synthase/SHMT activities. Our experiments strongly suggest that the two pathways operate independently in Arabidopsis. Plants exposed to methotrexate and sulfanilamide, powerful inhibitors of THF biosynthesis, reduced serine synthesis by both pathways. l h e results suggest that continuous supply of THF is essential to maintain high rates of serine metabolism. Nuclear magnetic resonance is a powerful tool for the examination of THF-mediated metabolism in its natural cellular environment.Folate coenzymes mediate single-carbon transfers in a variety of cellular processes such as purine biosynthesis, amino acid metabolism, thymidylate synthesis, and chloroplast and mitochondrial protein synthesis. In spite of the pivotal role THF plays in cellular metabolism, studies of THF biosynthesis and THF-dependent metabolism in plants remain sparse. We have recently begun wideranging studies of folate metabolism in Datuva cells and Arabidopsis plants (Wu et al., 1993(Wu et al., , 1994 Prabhu et al., 1994). In this study we addressed the THF-dependent biosynthesis of Ser in intact plants of Arabidopsis tkaliana (L.) Heynh. Columbia wild type using I3C NMR.THF-dependent Gly and Ser metabolism are closely linked in a variety of organisms (Schirch, 1984). The use of a common pool of THF allows transfer of the a-C of Gly

A procedure for cryogenic storage of alkaloid producing cell lines of periwinkle, Catharanthus roseus (L.) G. Don, has been developed. The procedure differs from established cryopreservation protocols in several aspects. Specifically, 4-day-old suspension subcultures of three cell lines were precultured in nutrient media supplemented with 1 molar sorbitol for 6 to 20 hours. The cells were then incubated in nutrient media with 1 molar sorbitol plus 5% DMSO in an ice bath for 1 hour and, thereafter, were frozen in this solution at a cooling rate of 0.5C per minute to -40°C prior to immersion in liquid nitrogen (LN). After rapid thawing in a 40°C water bath, the regrowth of LN stored cells was achieved by transferring them without washing onto filter paper discs over nutrient media solidified with agar for a period of 4 to 5 hours. The filter paper discs with the cells were then transferred to fresh media of the same composition for regrowth. The viability immediately after thawing as evaluated by the 2,3,5-triphenyl tetrazolium chloride method was about 60% of controls. Suspension cultures established from LN stored cells retained the capability for alkaloid synthesis and accumulation.Plant cells cultured in vitro are potential sources of useful chemicals such as alkaloids (1,5,16,23,31). The production of alkaloids by in vitro cultures can vary among cell lines and in individual lines be fairly stable (2, 4); in other cell lines, alkaloid content and spectra have been found subject to gradual change over years of subculture (6). Instead of repeated selection to maintain a high alkaloid level, cryopreservation would appear as an alternative to overcome stability problems (14, 31).Storage in LN2 followed by recovery of viable plant cells have been reported for many species (7,10,12,14,21,22,28,30) and cells of certain species retained the biosynthetic capacity for biotin (25), anthocyanin (8), and cardenolides (7). However, whether such a cryogenic technique could be applied to the preservation of cell cultures with capability for alkaloid synthesis and accumulation has not been determined and thus served as the primary objective of the present study. A procedure for prolonged cryogenic storage of periwinkle cell line no. 916, which did not produce alkaloids in detectable amounts, has been reported (14). We employed the procedure established for no. 916 (14) as well as other published protocols for the freeze-preservation of cultured plant cells (10,14,21,22,25,26,27,28,29); but none of them was found suitable for any of the alkaloid-producing periwinkle cell lines.In the present paper, we report a method for the successful cryopreservation of alkaloid-producing cell lines of periwinkle. It differs in several respects from previously established protocols.MATERIALS AND METHODS Plant Material. Alkaloid-producing periwinkle (Catharanthus roseus L., G. Don.) cell lines designated PRL nos. 200, 615, and 91601 were used in this study. These cell lines were initiated from callus of anther walls and filaments of p...

In C 3 plants large amounts of photorespiratory glycine (Gly) are converted to serine by the tetrahydrofolate (THF)-dependent activities of the Gly decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT). Using 13 C nuclear magnetic resonance, we monitored the flux of carbon through the GDC/SHMT enzyme system in Arabidopsis thaliana (L.) Heynh. Columbia exposed to inhibitors of THF-synthesizing enzymes. Plants exposed for 96 h to sulfanilamide, a dihydropteroate synthase inhibitor, showed little reduction in flux through GDC/SHMT. Two other sulfonamide analogs were tested with similar results, although all three analogs competitively inhibited the partially purified enzyme. However, methotrexate or aminopterin, which are confirmed inhibitors of Arabidopsis dihydrofolate reductase, decreased the flux through the GDC/SHMT system by 60% after 48 h and by 100% in 96 h. The uptake of [␣-13 C]Gly was not inhibited by either drug class. The specificity of methotrexate action was shown by the ability of 5-formyl-THF to restore flux through the GDC/SHMT pathway in methotrexate-inhibited plants. The experiments with sulfonamides strongly suggest that the mitochondrial THF pool has a long halflife. The studies with methotrexate support the additional, critical role of dihydrofolate reductase in recycling THF oxidized in thymidylate synthesis.

Clivorine (1) and ligularine (2), two hepatotoxic otonecine-type pyrrolizidine alkaloids isolated from Ligularia hodgsonii, an antitussive traditional Chinese medicine, were investigated in CDCl(3) and D(2)O by various NMR techniques to delineate why this type of alkaloid displays uncharacteristic solubility properties by dissolving in both nonpolar organic and aqueous solutions. The results demonstrated that both alkaloids exist in a non-ionized form in CDCl(3), but in an ionized form in D(2)O, suggesting that this unique dual solubility may play a role in the intoxication resultant from consumption of water extracts of herbs, including herbal teas, containing otonecine-type pyrrolizidine alkaloids.