Proc. Natl. Acad. Sci. USA Vol. 91, pp. 7608-7612, August 1994 Plant Biology Quantitative relationship between phenylalanine ammonia-lyase levels and phenylpropanoid accumulation in transgenic tobacco identifies a rate-determining step in natural product synthesis (cosuppresson/flux control/l n/metabolic engneerng/trasgenic plans) NICHOLAS J. BATE*, JOHN ORRt, WEITING Nit, AVRAHAM MEROMIt, TALIA NADLER-HASSAR*, PETER W. DOERNER*, RICHARD A. DIXONt, CHRIS J. LAMB*§, AND YONATAN ELKINDt *Plant Biology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037; tPlant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73402; and tFaculty of Agriculture, Hebrew University of Jerusalem, Rehovot, Israel Communicated by J. E. Varner, April 21, 1994 ABSTRACT Phenylalanine ammonia-lyase (PAL) cata- PAL, as the bridge between primary metabolism and natural lyzes the first step in phenylpropanold synthesis. The role of product biosynthesis, is a potential site for pathway regulation PAL In pathway regulation was investigated by measurement (9), and indeed PAL mRNA and enzyme levels are highly of product accumulation as a function of enzyme activity in a regulated spatially and temporally, associated with the tissue- coilection of near-isogenic transgenic tobacco plants exhibiting specific accumulation of phenylpropanoid products, exempli- a range of PAL levels from wild type to 0.2% of wild type. In fied by selective expression in differentiating xylem cells for leaf tissue, PAL level is the dominant factor regulating accu- production of lignin monomers at the onset of secondary wall mulation ofthe major product chlorogenic acid and overall flux deposition (10-13). However, genes encoding subsequent into the pathway. In stems, PAL at wild-type levels contributes, enzymes of phenylpropanoid biosynthesis are often coordi- together with downstream steps, in the regulation of lguin nately regulated with PAL (8), and hence these correlations do deposition and becomes the dominant, rate-determining step at not delineate whether perturbations in PAL enzyme levels levels 3- to 4-fold below wild type. The metabolic impact of contribute to the control ofpathway flux and accumulation of elevated PAL levels was investigated in transgenic leaf callus specific phenylpropanoid products. Moreover, while many that overexpressed PAL. Accumulation of the flavonoid rutin, reports demonstrate a correlation between changes in the the major product in wild-type callus, was not increased, but levels of phenylpropanoid biosynthetic enzymes and product several other products accumulated to similarly high levels. accumulation (3, 8, 14-17), there is little direct evidence that These data indicate that PAL is a key step in the regulation of product accumulation can be quantitatively accounted for by overall flux into the pathway and, hence, accumulation of changes in biosynthetic enzyme activity levels integrated over major phenylpropanoid products, with the regulatory archi- time, and it has been proposed that substrate control through tecture ofthe pathway poised so that downstream steps control changes in phenylalanine pool size might be the major site of partitioning into different branch pathways. phenylpropanoid regulation (18, 19). Generation of transgenic plants with altered levels of a Plants synthesize from phenylalanine a wide variety of nat- specific enzyme provides a new and powerful approach for ural products based on the phenylpropane skeleton (1). the analysis of pathway regulation (20). For example, mea- Phenylpropanoid products have important functions in plant surement of the rates of carbon fixation in a population of defense against pests and predators (1, 2), as UV protectants transgenic plants containing a ribulose-bisphosphate carbox- (3), and as signal molecules both internally and for commu- ylase antisense construct has allowed direct analysis of the nication with other organisms (4). Moreover, lignin, which is role of this enzyme in photosynthetic flux control (21). elaborated from cinnamyl alcohols by oxidative polymeriza- Transformation of tobacco with the bean PAL2 gene, mod- tion, is the major structural component of secondarily thick- ified by the inclusion of cauliflower mosaic virus 35S en- in hancer sequences in its promoter, generates transgenic plants ened cell walls of water-conducting xylem elements the with severely reduced PAL activity and correspondingly vascular system (5, 6). Lignin is the second most abundant lower levels of phenylpropanoid products (22). This para- biopolymer after cellulose and hence represents a major fate doxical suppression of PAL activity, which was one of the for fixed carbon in the biosphere. first examples of sense suppression, or cosuppression, fol- There is considerable interest in the regulation of phenyl- lowing introduction of transgene copies of plant genes (23, propanoid biosynthesis, both as a model for understanding 24), is progressively reversed in succeeding generations flux control in a complex biosynthetic pathway and also for homozygous for the PAL2 transgene. Thus PAL sense sup- the identification oftargets for biotechnological manipulation pression and subsequent recovery provide the equivalent of of product accumulation. Phenylpropanoid biosynthesis is an allelic series exhibiting a range ofPAL activities. We have initiated by the deamination of phenylalanine to give cin- used these plants to analyze the quantitative relationship namic acid, catalyzed by phenylalanine ammonia-lyase between PAL activity and phenylpropanoid product accu- (PAL, EC 4.3.1.5) (7). Successive hydroxylations of cin- mulation in a near-isogenic setting. namic acid to give p-coumaric acid and caffeic (3,4- dihydroxycinnamic) acid are followed by the formation of hydroxycinnamoyl CoA thioesters, which are the substrates MATERIALS AND METHODS for branch pathways for the production of lignin monomers, Plant Material. The generation of transgenic tobacco (Ni- flavonoids, coumarins, and simple esters (3, 8). cotiana tabacum cv. Xanthi) containing a bean PAL2 gene The publication costs ofthis article were defrayed in part by page charge Abbreviations: CGA, chlorogenic acid; CHS, chalcone synthase; payment. This article must therefore be hereby marked "advertisement" PAL, phenylalanine ammonia-lyase. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 7608 Downloaded by guest on September 27, 2021 Plant Biology: Bate et aL Proc. Natl. Acad. Sci. USA 91 (1994) 7609 with promoter sequences to -550 downstream of the cauli- alanine (485.7 mCi/mmol) was from Sigma. p-Coumaroyl flower mosaic virus 35S enhancer has been described (22). CoA was synthesized as described (27). All other chemicals The PAL2 primary transformant YE6-16 and progeny ho- were obtained from Sigma except where noted. mozygous for the transgene were selfed to generate pools of seed stocks for T1-T4 generations. Plants from these seed stocks, as well as wild-type plants from which the transgene RESULTS had been segregated out, were grown under greenhouse PAL Activity in PAL2 Transformant Progeny. The bean conditions and harvested when the plants were fully mature PAL2 primary transformant YE6-16 shows severely sup- (20-22 leaf nodes). Leaf tissue was harvested from the 11th pressed levels of PAL activity in leaf and stem tissue (22). nodal leafand stem sections were removed from the 10th and However, selfed progeny homozygous for the transgene 11th internodes and stored at -80"C. Small sections of stem show a progressive recovery in PAL activity. Fig. 1 shows were removed for immediate sectioning and staining, and the data for the recovery ofPAL activity in leaf tissue ofa single remainder was stored at -800C for the determination of pedigree of progeny from the To YE6-16 primary transform- enzyme activities, lignin, and soluble phenolics. Calli were ant, and a similar recovery in PAL levels was also observed generated from leaf tissue of wild-type plants and vegeta- in stems and other organs. By examining several different tively propagated PAL2 primary transformants YE10-6 and pedigrees arising from the YE6-16 primary transformant, we YE6-16 by excision of small tissue sections and culture on were able to obtain a large collection of near-isogenic plants Murashige and Skoog medium (25) with 1 /LM benzyladenine with different PAL and 2.5 pJM naphthylacetic acid in complete darkness. activity levels from wild type down to Phenolic Analysis. Leaf and callus samples were ground in 0.2% of wild type. liquid N2 and stored at -80oC for 3-4 weeks. One hundred PAL Activity and Phenylpropanoid Accumulation in Leaf nanomoles of isoferulic acid (Aldrich) in 100 p1 of methanol Tissue. CGA (3-caffeoylquinic acid) and rutin (quercitin 3-& plus 2 ml of acetonitrile was added to each sample, the D-rutinoside) are the major soluble phenylpropanoid prod- mixture was sonicated, 2 ml of ethyl acetate was added, and ucts in tobacco leaftissue, accounting for approximately 60% the mixture was vortexed and then centrifuged to separate and 10%1 oftotal phenolics, respectively (28). CGA is an ester the residue from the extracts. Two further extractions were conjugate of caffeic acid, which is an intermediate of the performed, once with 2 ml of hexane and 2 ml of acetonitrile central pathway of phenylpropanoid biosynthesis, whereas and once with 3 ml of water and 1 ml of acetonitrile. Extracts rutin is a disaccharide conjugate of quercitin and hence