The shikimic acid pathway is a major route for biosynthesis ofaromatic compounds in plants ... D; PEP, phos- phoenolpyruvate; E-4-P, erythrose-4-phosphate.
Received for publication January 17, 1990 Accepted May 7, 1990
Plant Physiol. (1990) 94, 507-510
0032-0889/90/94/0507/04/$01 .00/0
Induction of Shikimic Acid Pathway Enzymes by Light in Suspension Cultured Cells of Parsley (Petroselinum crispum) Kent F. McCue2 and Eric E. Conn* Department of Biochemistry and Biophysics, University Of California, Davis, California 95616 UV range of the spectrum (1), the plant can respond by converting phenylalanine to flavonoids which act to absorb the UV light and prevent damage to the plant's DNA. In suspension cultured cells of parsley (Petroselinum crispum), our system of choice, two sets of enzymes of flavonoid metabolism known as Group I and II can be coordinately induced by UV light (6, 12, 15). The group I enzymes, more recently termed the core enzymes of general phenylpropanoid metabolism (14), include PAL3; the group II enzymes are specific for the production of flavonoids (15). The enzymes of Group I can also be induced by the dilution of cell cultures (23) and by fungal elicitors (17). The induction of both groups I and II enzymes has been shown to occur at the level of gene transcription (3). Because of the extensive amount of work on the induction of flavonoid biosynthesis from phenylalanine, we chose to examine the effects of light on the enzyme activities of two enzymes of the shikimic acid pathway involved in the production of phenylalanine: DAHP synthase, which catalyzes the committed step in the shikimic acid pathway and CM, which catalyzes carbon flow into phenylalanine and tyrosine.
ABSTRACT Light treatment of suspension cultured cells of parsley (Petroselinum crispum) was shown to increase the activity of the shikimic acid pathway enzyme, 3-deoxy-o-arabino-heptulosonic acid-7-phosphate (DAHP) synthase (EC 4.1.2.15). DAHP synthase activity was assayed for two isoforms, DS-Mn and DS-Co (RJ Ganson, TA d'Amato, RA Jensen [1986] Plant Physiol 82: 203210). Light increased the enzymatic activity of the plastidic isoform DS-Mn as much as 2-fold, averaging 1.6-fold with >95% confidence. The cytosolic isoform DS-Co was unaffected. Cycloheximide and actinomycin D, translational and transcriptional inhibitors, respectively, both reversed induction of DS-Mn by light suggesting transcriptional regulation of the gene. Chonsmate mutase activity was assayed for the two isoforms CM I and CM II (BK Singh, JA Connelly, EE Conn [1985] Arch Biochem Biophys 243: 374-384). Treatment by light did not significantly affect either chorismate mutase isoform. The ratio of the two chonsmate mutase isoforms changed during the growth cycle, with an increase in the ratio of plastidic to cytosolic isoforms occurring towards the end of logarithmic growth.
MATERIALS AND METHODS The shikimic acid pathway is a major route for biosynthesis of aromatic compounds in plants and microorganisms including the proteinaceous amino acids phenylalanine, tyrosine, and tryptophan. The pathway is also important because of the volume of metabolites which traverse it in plants. The three aromatic amino acids mentioned are the primary metabolites which serve as precursors for many natural (secondary) products such as flavonoids, phenolic acids, coumarins, alkaloids, glucosinolates, and cyanogenic glycosides. In addition to these secondary compounds, the shikimic acid pathway provides precursors for many ubiquitous compounds important in the life ofthe plant such as the structural element lignin, the growth hormone indole acetic acid, quinones of the electron transport chain, and storage compounds like caffeoyl-quinate. Products of the shikimic acid pathway are also important in the response of plants to environmental stimuli. If a plant is subjected to high intensities of light (14), particularly in the
Cell Cultures
Suspension cultured cells of parsley (Petroselinum crispum) (Mill.) Nyman ex A. W. Hill [P. hortense Hoffm.]) were a gift of Dr. Joseph Chappell (University of Kentucky, Lexington), and were grown in the dark as previously described ( 19). Light Induction For light induction, several 9 d old cultures with conductivities between 0.50 and 0.60 mmho (10) were combined and 25 mL aliquots were transferred to 125 mL Erlenmeyer flasks for each treatment. This was done immediately prior to the induction treatment, or the addition of inhibitors, and ensured that cells in the different treatments were as homogeneous as possible. Light induction was carried out by placing flasks on a rotary shaker at 125 rpm under fluorescent lights for 16 h. The light source was a bank of 10 fluorescent lamps (8 foot, 'Abbreviations: PAL, phenylalanine ammonia-lyase; DAHP, 3deoxy-D-arabino-heptulosonic acid-7-phosphate; CM, chorismate
'This work supported in part by a McKnight Foundation Fellowship (K.F.M.) and by U.S. Public Health Service GM-05301-29. Present address: MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824-1312.
mutase; CHX, cycloheximide; act D, actinomycin D; PEP, phosphoenolpyruvate; E-4-P, erythrose-4-phosphate.
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MCCUE AND CONN
75 W, Westinghouse 96T12 Slimline) and 3 incandescent bulbs (40 W), at a distance of approximately 6 inches. The total flux at this distance was approximately 2000 ,E PAR. Control cultures were wrapped in foil and also placed under the light to minimize any variations due to temperature and shaker motion. Inhibitors
Inhibitors were added to the cell cultures 30 min before induction. CHX was added to culture flasks from a stock solution of 2 mg/mL to give a final concentration of 10 ,g/ mL; this concentration is reported (13) to be sufficient to prevent induction of group I and group II enzymes. Act D was added to cultures from a stock solution of 1.67 mg/mL in 95% ethanol to give a final concentration of 20 ,g/mL. Substrates
Analytical grade chemicals, other than substrates, were obtained from Fischer, Mallinckrodt, or Sigma. Substrates were obtained from Sigma and prepared as stock solutions as previously described ( 19). Enzyme Isolation
Cellular extracts were prepared by grinding cells to a fine powder in liquid nitrogen and homogenizing in 50 mm K2HPO4.HCl (pH 7.5), containing 0.1% (v/v) ,B-mercaptoethanol. Enzymes were assayed following removal of cellular debris by centrifugation and buffer exchange in Sephadex G25 medium (Pharmacia PD-10) as previously described (19). Chorismate mutase was assayed after exchange into 0.1 M Tris-HCl (pH 8.5). For DAHP synthase, 10 mM K2HPO4. HCI (pH 7.2) was employed. For PAL, 0.1 M borate NaOH (pH 8.8) was used. Enzyme Assay
DAHP synthase was assayed according to Ganson et al. (7) to discriminate between the isozymes DS-Mn and DS-Co in crude homogenates. Product was measured as previously described (19). DS-Mn was assayed in 50 mm K EPPS (pH 8.0) containing 0.5 mM MnC12, 3 mM PEP, 0.6 mm E-4-P, and 0.5 mM DT. DS-Co was assayed in 50 mM K.EPPS, (pH 8.6) containing 0.5 mM CoCl2, 2 mM PEP, and 6 mm E-4-P. CM isozymes were measured in crude homogenates according to Singh et al. (24). CM II was assayed in 0.1 M Tris. HCI (pH 8.5) with 0.3 mM chorismic acid. CM I + CM II were assayed by the addition of 1.0 mM tryptophan to the assay. The CM I of parsley has negligible activity in the absence of added tryptophan (
