Biosynthesis of Abscisic Acid by Cercospora rosicola Passerini

Transcription

1 APPLID AND NVIRONMNTAL MICROBIOLOGY, Apr. 1981, p /81/ $02.00/0 Vol. 41, No. 4 Influence of Nitrogen Source, Thiamine, and Light on Biosynthesis of Abscisic Acid by Cercospora rosicola Passerini SHIRLY M. NORMAN,* VINCNT P. MAIR, AND LINDA C. CHOLS Fruit and Vegetable Chemistry Laboratory, U.S. Department ofagriculture, Science and ducation Administration, Agricultural Research, Pasadena, California 1981 Received 20 October 1980/Accepted 7 February 1981 Abscisic acid production by Cercospora rosicola Passerini in liquid shake culture was measured with different amino acids in combination and singly as nitrogen sources and with different amounts of thiamine in the media. Production of abscisic acid was highest with aspartic acid-glutamic acid and aspartic acidglutamic acid-serine mixtures as nitrogen sources. Single amino acids that supported the highest production of abscisic acid were asparagine and monosodium glutamate. Thiamine was important for abscisic acid production. Leucine inhibited abscisic acid production. C. rosicola produced abscisic acid in the dark, but production more than doubled in the presence of light. Cercospora rosicola Passerini is the only mold reported to produce the secondary metabolite (+)-abscisic acid (ABA) (2). The biochemistry and physiology of this important plant hormone was recently reviewed by Walton (5). In comparison with plants, C. rosicola provides a simple system for the study of ABA biosynthesis and regulation. Little information is available on the factors affecting ABA production by this fungus. Assante et al. (2) reported that C. rosicola produced ABA when grown on agar containing extracts of potato, yeast, malt, or oatmeal but did not produce ABA when grown on agar containing peptone or on Sabouraud, Czapek, and nutrient formulas. We developed a chemically defined liquid medium for cultivating C. rosicola (4). We now report the effects of nitrogen source, thiamine, and light on ABA production by C. rosicola. MATRLALS AND MTHODS Organisms. The C. rosicola Passerini culture (strain ) used was obtained from the Centraalbureau voor Schimmelcultures, Baarn, The Netherlands. Primary slants and working petri dish cultures were maintained on potato dextrose agar (Difco Laboratories, Detroit, Mich.). Chemicals. All L-amino acids and other chemicals were from commercial sources. Reverse osmosisdeionized water (Milli-Q System, Millipore Corp., Bedford, Mass.) was used for all procedures. (+)-ABA, a 50% mixture of cis,trans (ABA) and trans,trans (t-aba) isomers (R. J. Reynolds Tobacco Co., Winston-Salem, N.C.), was used as a standard for quantitation. Liquid culture. ach milliliter of test medium contained the following: one or more amino acids (mixtures of amino acids were of equal amounts) or 981 other nitrogen source equivalent to 0.25 mg of nitrogen, 20 mg of glucose, 0.2 mg of MgSO4, 0.5 mg of KCI, 0.1 mg of CaCl2.2H20, 0.8 mg of KH2PO4, and mg of thiamine (except where stated otherwise). Also, each liter of test medium contained 1 ml of a mixture of trace metals (0.05 g of FeSO4.7H20, g of MnCl2, 0.25 g of ZnSO4, 0.4 g of CuSO4.4H20, and g of H3BO3 in 100 ml of water). Autoclaved test media (100 ml) in 500-ml rlenmeyer flasks were inoculated with 2 ml of mycelia blended in sterile water (Waring blender). All treatments in duplicate were incubated on reciprocating shakers at 24 to 26 C for 1 to 3 weeks under continuous fluorescent lighting (Verda-A-Ray north white, 32 in. [ca. 81 cm] above cultures). Cultures were filtered, and mycelia were freeze-dried and weighed. Details of culture and media are described elsewhere (4). ABA quantitation. ABA production was quantitated by high-pressure liquid chromatography. A sample of the liquid growth medium (ph 2.5) was passed through a C18 reversed-phase cartridge (Sep-Pak, Waters Associates, Milford, Mass.) for cleanup, and the ABA fraction was eluted from the cartridge with 75% methanol in M H3PO4. The fraction was separated on a Partisil PXS 5/25 octyldecylsilane column (Whatman Inc., Clifton, N.J.) eluted with 50% methanol in M H3PO4 at a rate of 1.0 ml/min, and ABA was detected at 268 nm. The lower limit of detectable ABA by the high-pressure liquid chromatographic method was 0.01 pg/mg of mycelium. The reversed-phase cleanup and high-pressure liquid chromatographic quantitation of ABA are described in detail elsewhere (S. M. Norman, V. P. Maier, and L. C. chols, submitted for publication). All ABA values represent the sum of ABA and t-aba unless stated otherwise. RSULTS AND DISCUSSION ABA content of mycelium and culture medium. ABA was produced and exuded by C.

2 982 NORMAN, MAIR, AND CHOLS rosicola in both surface agar and liquid shake cultures. C. rosicola began to exude droplets of liquid on the mycelial surface about 1 to 2 weeks after inoculation of agar. This exudation continued until the cultures were 4 to 6 weeks old and then stopped, and the droplets evaporated. Up to 200 lil of exudate could be collected with a microliter syringe from one petri dish culture. The amount of ABA in the exudate ranged from 42 to 130 ug/ml. C. rosicola formed mycelial pellets under our conditions of liquid shake culture in the defined media. When mycelium from a liquid shake culture was filtered, washed thoroughly with water, and subsequently extracted with ethyl acetate, no measurable ABA was found in the ethyl acetate extract of the mycelium. The ABA content of broth from 1-week-old cultures ranged from 5 to 30 jig/ml. ffects of light on ABA production. Both ABA and t-aba were found in the culture media of C. rosicola. The ratio of ABA/t-ABA averaged 17.3 ± 4.1 in 35 cultures after 7 days in the light. The identies of ABA and t-aba were confirmed by combined gas chromatographymass spectrometry of the methyl esters. ABA isomerizes to t-aba, giving an equilibrium mixture of both isomers on exposure to light (3). To determine whether t-aba is produced by C. rosicola or is a result of light-catalyzed isomerization, we grew the cultures in light and in darkness. The fungus produced about twice as much ABA in light as in the dark in liquid and surface cultures (Table 1). The t-aba content in light-grown cultures was about 40 and 5 times higher in liquid and surface cultures, respectively, than in the corresponding dark-grown cultures. Also, a test with the liquid shake culture showed that the ratio of ABA/t-ABA decreased with time (81, 23, 14, 10, and 8 for 3, 8, 10, 15, and 19 days, respectively). These results suggest that most of the t-aba was formed via light-catalyzed isomerization of ABA. However, this suggestion does not rule out the possibility that t-aba is produced in small quantities by the fungus. TABL 1. ffect of light on the production ofaba and t-aba by C. rosicola Culture ag/mg of mycelium Mycelium Type Age Treat- (days) ment t-aba ABA Liquid shake 7 Light mg/ml Liquid shake 7 Dark mg/mi Potato dextrose 14 Light mg/dish agar Potato dextrose 14 Dark mg/dish agari APPL. NVIRON. MICROBIOL. ffect of thiamine on ABA production. Thiamine was an important factor for growth of C. rosicola. Growth increased with increasing amounts of thiamine, up to about 0.05 ytg/ml, in the media. ABA production per gram of mycelium also increased with increasing thiamine content up to about 0.05,ug/ml (Fig. 1). Additional amounts of thiamine in the media did not affect ABA production. ffect of carbon source on fungus growth and on ABA production. Four sugars were tested for growth and ABA production. Mycelial weights in liquid shake culture ranged from 5 to 6 mg/ml after 1 week. ABA production was 4.0, 2.5, 1.7, or 3.4 mg/g of mycelium with glucose, lactose, sucrose, or galactose as the carbon source, respectively. ffect of nitrogen source on fungus growth and on ABA production. Mycelial production of C. rosicola was from 3 to 5 mg/ml in 14 days with certain amino acids as the sole nitrogen source, namely, asparagine, monosodium glutamate, aspartic acid, glutamic acid, glycine, and glutamine. Mycelial production was 1 to 3 mg/ml with serine, leucine, potato extract, or ammonium nitrate as the sole nitrogen source. ABA production did not follow the same pattern as growth (Table 2). ABA production was highest with asparagine and monosodium glutamate, intermediate with aspartic acid, glutamic acid, and potato extract, and lowest with glutamine, glycine, and serine. Leucine and ammonium nitrate did not support significant ABA production. Other nitrogen sources tested were arginine, valine, lysine, and ammonium acetate; these compounds supported mycelial production of <1 mg/ml and ABA production of <2,tg/ml. Results of a time study of growth and ABA production of C. rosicola with monosodium glutamate as the sole nitrogen source are shown in Fig. 2. Growth paralleled ABA content of the liquid medium until day 6; then growth slowed, whereas ABA production continued at the same rate. ABA content of the medium reached a maximum on about day 13 and then began to decrease. ssential nutrients were probably depleted by day 17, for growth decreased. Production of the secondary metabolite ABA in the early states of growth may have been related to the internal microenvironment of the pellet. The extent of fungal pelletting has been reported to depend on the species, individual strain, size of inoculum, growth medium, or the physical environment within the culture vessel (6). xcept for the mixture of arginine, lysine, and valine, mixtures of amino acids did not support growth as well as most of the single amino acids (cf. Tables 2 and 3). ABA production was higher with the aspartic acid-glutamic acid and aspartic acid-glutamic acid-serine combinations than

3 VOL. 41, 1981 ABSCISIC ACID BIOSYNTHSIS BY C. ROSICOLA 983 * 5 X T I Thiamine (pg/mi) FIG. 1. ffect of thiamine content in liquid shake culture on ABA production of C. rosicola. TABL 2. Growth and ABA production of C. rosicola after 14 days of culture in liquid media containing different single nitrogen sourcesa ABA Myce- Nitrogen source (pg/mg lium dry of myce- wt lium) (mg/ml) Monosodium glutamate Asparagine Aspartic acid Glutamic acid Potato extract Serine Glycine Glutamine Ammonium nitrate Leucine a Values represent means of duplicate flasks from three to five tests. with any single amino acid. ABA production was the same whether aspartic acid was used with or without serine and whether glutamic acid was used with or without urea. Mixtures of other amino acids did not enhance ABA production. Leucine inhibited by 85 and 77%, respectively, ABA production with glutamic acid or aspartic acid plus glutamic acid. Four mixtures of amino acids tested but not listed in Table 3 were (i) arginine and leucine, (ii) arginine and lysine, (iii) glutamic acid, leucine, and arginine, and (iv) leucine, arginine, and lysine; these mixtures supported growth of<1 mg/ml and ABA production of <2,ug/ml. Because leucine inhibited ABA production of C. rosicola, glutamic acid and urea were tested separately with and without leucine (Table 4). Leucine inhibited ABA production 83 and 44% when combined with glutamic acid and urea, respectively. Assante et al. (2) found that C. rosicola did not produce ABA when grown on agar containing peptone or on Sabouraud, nutrient, and Czapek formulas. Sabouraud and nutrient formulas contain neopeptone and peptone, respectively. We found that C. rosicola grew rapidly (6.8 mg/ ml) in a peptone-glucose liquid shake broth but did not produce ABA. The fungus grows in extremely fine, oblong pellets in peptone broth in contrast to larger, spherical pellets in the chemically defined media. Peptone contains 15 amino acids, of which glycine, glutamic acid, arginine, aspartic acid, lysine, leucine, tyrosine, and valine are the major components (1). The leucine may inhibit ABA production or the type of growth may be responsible for lack of ABA production, or both. Czapek medium contains sodium nitrate, sucrose, potassium phosphate, magnesium sulfate, and iron sulfate but does not contain thiamine or trace metals. We were unable to grow the fungus in Czapek liquid shake culture with added thiamine. C. rosicola produces large amounts of ABA, which are exuded into the medium. We defined

4 984 NORMAN, MAIR, AND CHOLS APPL. NVIRON. MICROBIOL. a.5i Ac Ti;m. (days) FIG. 2. ffect of time on growth and ABA production of C. rosicola in liquid shake culture containing monosodium glutamate. TABL 3. Growth and ABA production of C. rosicola after 14 days of culture in liquid media containing mixtures of amino acids and urea" ABA Myce- Nitrogen source (jig/mg lium dry of myce- wt lium) (mg/ml) Serine, aspartic acid, glutamic acid Aspartic acid, glutamic acid Serine, aspartic acid Urea, glutamic acid Aspartic acid, glutamic acid, leucine, valine, serine, lysine, arginine Valine, serine Valine, serine, aspartic acid Lysine, valine, serine Arginine, lysine, valine Aspartic acid, glutamic acid, leucine Lysine, valine Glutamic acid, leucine " Values represent means of duplicate cultures from three to five tests. some of the requirements for ABA biosynthesis by liquid shake cultures in chemically defined media. C. rosicola produced ABA in the dark, but light stimulated additional production. Thiamine was required for both growth and 10 TABL 4. L-Leucine inhibition ofaba production of C. rosicola after 14 days of liquid shake culture ABA Mycelium Nitrogen source (jig/mg of mycedry wt (mg/ml) lium) (m/i Glutamic acid Glutamic acid, leucine Urea Urea, leucine ABA biosynthesis. When mixed nitrogen sources were tested, production of ABA was highest with mixtures of (i) aspartic acid and glutamic acid and (ii) aspartic acid, glutamic acid, and serine. Single amino acids that supported the highest production of ABA were asparagine and monosodium glutamate. Leucine and ammonium nitrate supported growth but little ABA production. The ABA normally produced with either urea or glutamic acid as the nitrogen source was inhibited 50 to 80% by the addition of leucine. The inhibitory effect of leucine is being investigated further. Other nutritional and environmental requirements of C. rosicola for maximum production of ABA are also being studied. Defining these requirements will further improve the usefulness of C. rosicola as a tool for studying the biosynthesis, metabolism, and regulation of ABA and as a route for commerical produc- I

5 VOL. 41, 1981 ABSCISIC ACID BIOSYNTHSIS BY C. ROSICOLA 985 tion of the natural (+)-enantiomer. At the present time, the synthetic ABA available commercially is a racemic mixture of (-)- and (+)-ABA. LITRATUR CITD 1. Ardetti, J Clonal propagation of orchids by means of tissue culture, p In J. Ardetti (ed.), Orchid biology: reviews and perspectives. Cornell University Press, Ithaca, N.Y. 2. Assante, G., L. Merlini, and G. Nasini (+)-Abscisic acid, a metabolite of the fungus Cercospora rosicola. xperimentia 33: Milborrow, B. V Abscisic acid, p In D. S. Letham, P. B. Goodwin, and T. J. V. Higgins (ed.), Phytohormones and related compounds: a comprehensive treatise, vol. 1. lsevier, Amsterdam. 4. Norman, S. M., V. P. Maier, and L. C. chols Development of a defined medium for growth of Cercospora rosicola Passerini. Appl. nviron. Microbiol. 41: Walton, D. C Biochemistry and physiology of abscisic acid. Annu. Rev. Plant. Physiol. 31: Whitaker, A., and P. A. Long Fungal pelleting. Process Biochem. 8: Downloaded from on November 3, 2018 by guest