Transgenic plants for therapeutic proteins: linking upstream and downstream strategies.

We have described two very different and innovative plant-based production systems--postharvest production and recovery of recombinant product from tobacco leaves using an inducible promoter and oleosin-mediated recovery of recombinant product from oilseeds using a seed-specific promoter. Both base technologies are broadly applicable to numerous classes of pharmaceutical and industrial proteins. As with any emerging technology, the key to success may lie in identifying those products and applications that would most benefit from the unique advantages offered by each system. The postharvest tobacco leaf system appears effective for proteins requiring complex posttranslational processing and endomembrane targeting. Because of the remarkable fecundity and biomass production capacity of tobacco, biomass scale-up is very rapid and production costs are low. Clearly the development of equally cost-effective extraction and purification technologies will be critical for full realization of the commercial opportunities afforded by transgenic plant-based bioproduction. The recovery of protein from tobacco leaves or oleosin-partitioned proteins by oil-body separations represent significant break-throughs for cost-effective commercialization strategies. Additional low-cost, high-affinity separation technologies need to be developed for effective scale-up purification of plant-synthesized recombinant proteins. Clearly successful commercialization of plant-synthesized biopharmaceuticals must effectively link upstream strategies involving gene and protein design with downstream strategies for reproducible GMP-level recovery of bioactive recombinant protein. Both the tobacco and oilseed systems are uniquely designed to address issues of biomass storage, product recovery, quality assurance, and regulatory scrutiny in addition to issues of transgene expression and protein processing.

[S]-AR-R 15896AR-A novel anticonvulsant: acute safety, pharmacokinetic and pharmacodynamic properties.

A rational, chemical, synthetic effort to identify promising low-affinity uncompetitive N-methyl-D-aspartic acid receptor antagonists for use as antiepileptic drugs led to the discovery of AR-R 15035AR, or [RS]-alpha-phenyl-2-pyridine-ethanamine.2HCl. Chiral separation followed by intensive in vivo screening resulted in the selection of the [S] enantiomer, AR-R 15896AR, as the best compound for further preclinical development. AR-R 15896AR prevented tonic seizures in rodents for up to 6 to 8 h in response to maximal electroshock (MES), 4-aminopyridine, bicuculline, or strychnine, as well as characteristic seizures following injections of N-methyl-DL-aspartic or kainic acids. AR-R 15896AR was ineffective in two kindling models of epilepsy, did not produce tolerance to MES, and was devoid of proconvulsant and phencyclidine-like properties in mice and rats, respectively. Therapeutic indices for AR-R 15896AR were comparable to or exceeded those for standard anticonvulsants. Orally administered AR-R 15896AR rapidly entered the rat brain and was eliminated in parallel from the plasma and plasma-free compartment. A dose-response relationship between plasma and brain levels after p.o. or i.v. administration of AR-R 15896AR and protection against MES was highly correlative. The time course for loss of protection against MES mirrored the elimination of the compound from brain and plasma. The total brain concentration (25 microM) of drug at the ED50 value (approximately 3 mg/kg) for protection against MES seizures was consistent with the reported affinity of AR-R 15896AR at the N-methyl-D- aspartic acid binding site (IC50 value = 1.3 microM). The present findings demonstrated the attractiveness of AR-R 15896AR as a candidate for further development to treat epilepsy.

Expression of a defense-related 3-hydroxy-3-methylglutaryl CoA reductase gene in response to parasitization by Orobanche spp.

Orobanche spp. are angiosperms that live parasitically on the roots of other plants, and are capable of significantly reducing the yield and quality of their crop hosts. We have demonstrated that parasitization by Orobanche induces expression of hmg2, a defense-related isogene of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) in tobacco. Transgenic tobacco plants expressing a construct containing 2.3 kb of the tomato hmg2 gene promoter fused to the beta-glucuronidase (GUS) reporter gene were parasitized by O. aegyptiaca. Expression of the hmg2:GUS construct was detected within 1 day following penetration of the host root by the O. aegyptiaca radicle and was localized to the region immediately around the site of parasite invasion. This expression continued and intensified over the course of O. aegyptiaca development. In addition, the hmg2:GUS expression was induced by secondary parasitization, where secondary roots of O. aegyptiaca contacted the host root at a distance from the primary attachment site. This GUS expression was specific to plants containing the hmg2:GUS construct, and was not observed in control plants transformed with a construct of the cauliflower mosaic virus 35S promoter fused to the GUS gene. These results indicate that Orobanche parasitization initiates rapid and sustained induction of a defense-related gene in the host root.

Responses of Antioxidants to Paraquat in Pea Leaves (Relationships to Resistance).

Differnential sensitivity to the oxidant paraquat was observed in pea (Pisum sativum L.) based on cultivar and leaf age. To assess contributions of inductive responses of the antioxidant enzymes in short-term resistance to oxidative damage, activities of glutathione reductase (GR), superoxide dismutase (SOD), and ascorbate peroxidase (APX) and transcript levels for plastidic GR, Cu,Zn SOD, and cytosolic APX were determined. Responses to paraquat exposure from three different leaf age classes of pea were studied. Resistance was correlated with leaf age, photosynthetic rates, enzyme activities, and pretreatment levels of plastid GR and plastid Cu,Zn SOD transcripts. In response to paraquat, small increases in activities of GR and APX were observed in the more resistant leaves. These changes were not reflected at the mRNA level for the plastidic GR or Cu,Zn SOD. Paraquat-mediated increases in cytosolic APX mRNA occurred in all leaf types, irrespective of resistance. Developmentally controlled mechanisms determining basal antioxidant enzyme activities, and not inductive responses, appear to be critical factors mediating short-term oxidative stress resistance.

Developmental and environmental regulation of tissue- and cell-specific expression for a pea protein farnesyltransferase gene in transgenic plants.

Farnesylation mediates membrane targeting and in vivo activities of several key regulatory proteins such as Ras and Ras-related GTPases and protein kinases in yeast and mammals, and is implicated in cell cycle control and abscisic acid (ABA) signaling in plants. In this study, the developmental expression of a pea protein farnesyltransferase (FTase) gene was examined using transgenic expression of the beta-glucuronidase (GUS) gene fused to a 3.2 kb 5' upstream sequence of the gene encoding the pea FTase beta subunit. Coordinate expression of the GUS transgene and endogenous tobacco FTase beta subunit gene in tobacco cell lines suggests that the 3.2 kb region contains the key FTase promoter elements. In transgenic tobacco plants, GUS expression is most prominent in meristematic tissues such as root tips, lateral root primordia and the shoot apex, supporting a role for FTase in the control of the cell cycle in plants. GUS activity was also detected in mature embryos and imbibed embryos, in accordance with a role for FTase in ABA signaling that modulates seed dormancy and germination. In addition, GUS activity was detected in regions that border two organs, e.g. junctions between stems and leaf petioles, cotyledons and hypocotyls, roots and hypocotyls, and primary and secondary roots. GUS is expressed in phloem complexes that are adjacent to actively growing tissues such as young leaves, roots of light-grown seedlings, and hypocotyls of dark-grown seedlings. Both light and sugar (e.g. sucrose) treatments repressed GUS expression in dark-grown seedlings. These expression patterns suggest a potential involvement of FTase in the regulation of nutrient allocation into actively growing tissues.

Protein farnesyltransferase in plants: molecular characterization and involvement in cell cycle control.

Farnesylation is required for membrane targeting, protein-protein interactions, and the biological activity of key regulatory proteins, such as Ras small GTPases and protein kinases in a wide range of eukaryotes. In this report, we describe the molecular identification of a plant protein farnesyltransferase (FTase) and evidence for its role in the control of the cell cycle in plants. A pea gene encoding a homolog of the FTase beta subunit was previously cloned using a polymerase chain reaction-based strategy. A similar approach was used to clone a pea gene encoding a homolog of the FTase alpha subunit. The biochemical function of the pea FTase homologs was demonstrated by the reconstitution of FTase enzyme activity using FTase fusion proteins coexpressed in Escherichia coll. RNA gel blot analyses showed that levels of FTase mRNAs are generally higher in tissues, such as those of nodules, that are active in cell division. The relationship of FTase to cell division was further analyzed during the growth of suspension-cultured tobacco BY-2 cells. A biphasic fluctuation of FTase enzyme activity preceded corresponding changes in mitotic activity at the early log phase of cell growth. Moreover, manumycin, a specific inhibitor of FTase, was effective in inhibiting mitosis and growth in these cells. Using synchronized BY-2 cells, manumycin completely blocked mitosis when added at the early S phase but not when added at the G2 phase. These data suggest that FTase is required for the plant cell cycle, perhaps by modulating the progression through the S phase and the transition from G1 to the S phase.

Bioproduction of human enzymes in transgenic tobacco.

Transgenic plants have significant potential in the bioproduction of complex human therapeutic proteins due to ease of genetic manipulation, lack of potential contamination with human pathogens, conservation of eukaryotic cell machinery mediating protein modification, and low cost of biomass production. Tobacco has been used as our initial transgenic system because Agrobacterium-mediated transformation is highly efficient, prolific seed production greatly facilitates biomass scale-up, and development of new "health-positive" uses for tobacco has significant regional support. We have targeted bioproduction of complex recombinant human proteins with commercial potential as human pharmaceuticals. Human protein C (hPC), a highly processed serum protease of the coagulation/anticoagulation cascade, was produced at low levels in transgenic tobacco leaves. Analogous to its processing in mammalian systems, tobacco-synthesized hPC appears to undergo multiple proteolytic cleavages, disulfide bond formation, and N-linked glycosylation. Although tobacco-derived hPC has not yet been tested for all posttranslational modifications or for enzymatic (anticlotting) activity, these results are promising and suggest considerable conservation of protein processing machinery between plants and animals. CropTech researchers have also produced the human lysosomal enzyme glucocerebrosidase (hGC) in transgenic tobacco. This glycoprotein has significant commercial potential as replacement therapy in patients with Gaucher's disease. Regular intravenous administration of modified glucocerebrosidase, derived from human placentae or CHO cells, has proven highly effective in reducing disease manifestations in patients with Gaucher's disease. However, the enzyme is expensive (dubbed the "world's most expensive drug" by the media), making it a dramatic model for evaluating the potential of plants to provide a safe, low-cost source of bioactive human enzymes. Transgenic tobacco plants were generated that contained the human glucocerebrosidase cDNA under the control of an inducible plant promoter. hGC expression was demonstrated in plant extracts by enzyme activity assay and immunologic cross-reactivity with anti-hGC antibodies. Tobacco-synthesized hGC comigrates with human placental-derived hGC during electrophoretic separations, is glycosylated, and, most significantly, is enzymatically active. Although expression levels vary depending on transformant and induction protocol, hGC production of > 1 mg/g fresh weight of leaf tissue has been attained in crude extracts. Our studies provide strong support for the utilization of tobacco for high-level production of active hGC for purification and eventual therapeutic use at potentially much reduced costs. Furthermore, this technology should be directly adaptable to the production of a variety of other complex human proteins of biologic and pharmaceutical interest.

The N-terminal domain of tomato 3-hydroxy-3-methylglutaryl-CoA reductases. Sequence, microsomal targeting, and glycosylation.

The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of 3-hydroxy-3-methylglutaryl-CoA to mevalonic acid, considered the rate-limiting step in isoprenoid biosynthesis. In plants, isoprenoid compounds play important roles in mediating plant growth and development, electron transport, photosynthesis, and disease resistance. Sequence comparisons of plant HMGR proteins with those from yeast and mammalian systems reveal high levels of sequence identity within the catalytic domain but significant divergence in the membrane domain. Mammalian HMGRs are integral membrane proteins of the endoplasmic reticulum with eight membrane-spanning regions. In contrast, the membrane domain of plant HMGRs is predicted to contain only one to two transmembrane spans. We have isolated and sequenced a clone (pCD4) encoding exon 1 of tomato hmg1. The membrane domain structures of two differentially regulated tomato HMGR isoforms, HMG1 and HMG2, were analyzed using in vitro transcription and translation systems. Microsomal membrane insertion of the tomato HMGRs is co-translational and does not involve cleavage of an N-terminal targeting peptide. HMGR membrane topography was established by protease protection studies of the HMG1 membrane domain and an analogous region of HMG2 engineered to contain a c-myc epitope tag. The data indicate that both tomato HMGRs span the membrane two times with both the C and N termini located in the cytosol. Lumenal localization of the short peptide predicted to lie within the endoplasmic reticulum was further confirmed by in vitro glycosylation of an asparagine-linked glycosylation site present in HMG2.

Differential response of Cu,Zn superoxide dismutases in two pea cultivars during a short-term exposure to sulfur dioxide.

Pea cultivars Progress and Nugget have been shown previously to be differentially sensitive with respect to apparent photosynthesis in a short-term exposure to 0.8 microliters/l SO2. One possible contributing factor to the relative insensitivity of apparent photosynthesis of Progress to SO2 is an increase in superoxide dismutase (SOD) activities. We show here that both chloroplastic and cytoplastic Cu,Zn-SOD proteins increased in Progress on exposure to sulfur dioxide whereas both proteins decreased in Nugget. The increase in cytosolic Cu,Zn-SOD protein was greater than that of chloroplastic Cu,Zn-SOD protein. Using a gene-specific probe for the plastid SOD, northern blot analysis revealed an initial decrease in transcript abundance of the chloroplastic Cu,Zn-SOD gene in Progress on exposure to SO2 with an eventual recovery to pre-exposure levels. The transcript levels of the chloroplastic Cu,Zn-SOD decreased in Nugget over the time period of the exposure. These results suggest that a combination of translational and post-translational mechanisms may be involved in SO2-induced changes in cytosolic and plastidic Cu,Zn-SODs in pea.

Kainic acid and 4-aminopyridine seizure models in mice: evaluation of efficacy of anti-epileptic agents and calcium antagonists.

Seizures may be induced in mice in response to stimulation of subtypes of glutamate receptors by kainic acid or inhibition of certain voltage-dependent potassium channels by 4-aminopyridine (4-AP). The anti-seizure efficacy of intraperitoneally administered anticonvulsants and Ca++ antagonists to CF-1 mice was tested using these models. The order of potency for prevention of kainate convulsions and the subsequent lethality was: dihydropyridine Ca++ antagonists (nicardipine, nisoldipine > nitrendipine > nifedipine > nimodipine) followed by verapamil > prenylamine > diltiazem > flunarizine > remacemide HCl > ethosuximide > valproate. In the 4-AP model the order of potency to prevent hind limb tonic extension was: MK801(+/-) > lamotrigine > phenytoin, phenobarbital > carbamazepine > FPL 12495AA (the desglycine metabolite of remacemide HCl), remacemide HCl > flunarizine > prenylamine >>> valproate. Therefore, compounds that limit activation of kainate receptors and voltage-operated linked calcium channels are active in the kainate model. Agents effective against maximal electroshock appear to be effective in the 4-AP model.

Protein farnesyltransferase in plants. Molecular cloning and expression of a homolog of the beta subunit from the garden pea.

Protein farnesyltransferase is a heterodimeric enzyme that attaches a farnesyl moiety to C-terminal cysteine residues. Both the alpha and beta subunits have recently been cloned and sequenced from yeast and rat. Degenerate oligonucleotides, corresponding to conserved regions of the beta subunit, were used as primers for the polymerase chain reaction to amplify cDNA synthesized from total cellular RNA from the apical buds of pea (Pisum sativum L.) seedlings. The 171-bp fragment obtained encodes an open reading frame of 57 amino acids showing 65% identity to the rat protein farnesyltransferase beta subunit. Using this fragment to screen a pea cDNA library, one full-length cDNA clone, designated PsFTb, was obtained that contains an open reading frame encoding a polypeptide of 419 amino acids. The predicted amino acid sequence exhibits 48 and 40% identity to the rat and yeast beta subunits, respectively, indicating that this cDNA encodes a pea homolog of the beta subunit of farnesyltransferase. Gel blot hybridizations show that PsFTb is likely to be encoded by a single-copy gene and is expressed as a transcript of approximately 1.7 kb. During photoregulated leaf development in continuous white light, PsFTb transcript levels within apical buds decline by approximately 5-fold.

Regulation of Defense-related Gene Expression during Plant-Pathogen Interactions.

Plants have evolved a broad array of defense mechanisms involved in disease resistance. These include synthesis of phytoalexin antibiotics and proteinase inhibitors, deposition of cell wall materials, and accumulation of hydrolytic enzymes such as chitinases. Resistance appears to depend on the ability of the host to recognize the pathogen rapidly and induce these defense responses in order to limit pathogen spread. Application of molecular technologies has yielded significant new information on mechanisms involved in pathogen recognition, signal transduction, and defense-related gene activation, and is leading to novel strategies for engineering enhanced disease resistance. We are using these approaches to analyze regulation of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), a key enzyme mediating the production of terpenoid defense compounds. This enzyme is encoded by four genes in tomato; hmg2 gene expression is specifically associated with responses to pathogen or defense elicitors. Transgenic plants containing DNA constructs that fuse the hmg2 promoter to a reporter gene have been used to analyze both tissue specificity and patterns of defense-related expression. Because this gene is rapidly induced in tissues directly surrounding the site of ingress by a variety of pathogens, it may serve as a valuable tool in engineering new disease-resistance mechanisms.

Purification of Multiple Forms of Glutathione Reductase from Pea (Pisum sativum L.) Seedlings and Enzyme Levels in Ozone-Fumigated Pea Leaves.

Glutathione reductase was purified from pea seedlings using a procedure that included 2',5'-ADP Sepharose, fast protein liquid chromatography (FPLC)-anion exchange, and FPLC-hydrophobic interaction chromatography. The purified glutathione reductase was resolved into six isoforms by chromatofocusing. The isoform eluting with an isoelectric point of 4.9 accounted for 18% of the total activity. The five isoforms with isoelectric points between 4.1 and 4.8 accounted for 82% of the activity. Purified glutathione reductase from isolated, intact chloroplasts also resolved into six isoforms after chromatofocusing. The isoform eluting at pH 4.9 constituted a minor fraction of the total activity. By comparing the chromatofocusing profile of the seedling extract with that of the chloroplast extract, we inferred that the least acidic isoform was extraplastidic and that the five isoforms eluting from pH 4.1 to 4.8 were plastidic. Both the plastidic (five isoforms were pooled) and extraplastidic glutathione reductases had a native molecular mass of 114 kD. The plastidic glutathione reductase is a homodimer with a subunit molecular mass of 55 kD. Both glutathione reductases had optimum activity at pH 7.8. The K(m) for the oxidized form of glutathione (GSSG) was 56.0 and 33.8 mum for plastidic and extraplastidic glutathione reductase, respectively, at 25 degrees C. The K(m) for NADPH was 4.8 and 4.0 mum for plastidic and extraplastidic isoforms, respectively. Antiserum raised against the plastidic glutathione reductase recognized a 55-kD polypeptide from purified antigen on western blots. In addition to the 55-kD polypeptide, another 36-kD polypeptide appeared on western blots of leaf crude extracts and the purified extraplastidic isoform. The lower molecular mass polypeptide might represent GSSG-independent enzyme activity observed on activity-staining gels of crude extracts or a protein that has an epitope similar to that in glutathione reductase. Fumigation with 75 nL L(-1) ozone for 4 h on 2 consecutive days had no significant effect on glutathione reductase activity in peas (Pisum sativum L.). However, immunoblotting showed a greater level of glutathione reductase protein in extracts from ozone-fumigated plants compared with that in control plants at the time when the target concentration was first reached, approximately 40 min from the start of the fumigation, and 4 h on the first day of fumigation.

Erwinia carotovora subsp. carotovora extracellular protease: characterization and nucleotide sequence of the gene.

The prt1 gene encoding extracellular protease from Erwinia carotovora subsp. carotovora EC14 in cosmid pCA7 was subcloned to create plasmid pSK1. The partial nucleotide sequence of the insert in pSK1 (1,878 bp) revealed a 1,041-bp open reading frame (ORF1) that correlated with protease activity in deletion mutants. ORF1 encodes a polypeptide of 347 amino acids with a calculated molecular mass of 38,826 Da. Escherichia coli transformed with pSK1 or pSK23, a subclone of pSK1, produces a protease (Prt1) intracellularly with a molecular mass of 38 kDa and a pI of 4.8. Prt1 activity was inhibited by phenanthroline, suggesting that it is a metalloprotease. The prt1 promoter was localized between 173 and 1,173 bp upstream of ORF1 by constructing transcriptional lacZ fusions. Primer extension identified the prt1 transcription start site 205 bp upstream of ORF1. The deduced amino acid sequence of ORF1 showed significant sequence identity to metalloproteases from Bacillus thermoproteolyticus (thermolysin), B. subtilis (neutral protease), Legionella pneumophila (metalloprotease), and Pseudomonas aeruginosa (elastase). It has less sequence similarity to metalloproteases from Serratia marcescens and Erwinia chrysanthemi. Locations for three zinc ligands and the active site for E. carotovora subsp. carotovora protease were predicted from thermolysin.

Differential activation of potato 3-hydroxy-3-methylglutaryl coenzyme A reductase genes by wounding and pathogen challenge.

Potato genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) were expressed in response to pathogen, elicitor, and wounding. HMGR catalyzes the rate-limiting step in isoprenoid biosynthesis leading to accumulation of phytoalexins and steroid glycoalkaloids. Wounding caused increases in HMGR mRNA levels. A rapid and transient peak occurred 30 minutes after wounding, followed by a slower peak at 14 hours; both were correlated with increased enzyme activity. Induction of HMGR mRNA by the soft rot pathogen Erwinia carotovora subsp carotovora or arachidonic acid began 8 hours after challenge and continued through 22 hours. Potato HMGR is encoded by a gene family. An HMGR gene-specific probe was used to demonstrate that one isogene of the HMGR family is pathogen activated and is distinct from isogene(s) that are wound activated. This provides evidence that defense-related increases in HMGR activity are due to mRNA level increases and that HMGR isogenes are activated differentially by wounding or pathogen challenge.

Microbiological and pharmacokinetic studies of acyl demycinosyltylosin and related tylosin derivatives.

A series of tylosins and acyl derivatives of 23-O-demycinosyltylosin (DMT) were initially tested for in vitro antibacterial activity and serum levels in squirrel monkeys (po) and mice (iv). Overall, the DMT compounds were more active in vitro than the tylosins. Two tetraacylated DMTs, Sch 37644 and Sch 38646, were selected from the initial studies for further evaluation and compared to erythromycin and A-56268 (6-O-methyl erythromycin). Sch 37644 and Sch 38646 were 2 to 8-fold less potent in vitro against Gram-positive bacteria than erythromycin and A-56268. In squirrel monkeys, Sch 37644 (AUC, 19.7 micrograms.hour ml) and A-56268 (21.6 micrograms.hour/ml) had similar serum levels following po administration of 20 mg/kg, while Sch 38646 (11.8 micrograms.hour/ml) and erythromycin (1.5 micrograms.hour/ml) had lower levels. In mice administered 200 mg/kg orally, Sch 37644 (AUC, 19.4 micrograms.hour/ml) and Sch 38646 (15.4 micrograms.hour/ml) had higher serum levels than erythromycin (5.7 micrograms.hour/ml). A-56268 was the most active po macrolide in mouse protection studies (PD50S) against Staphylococci and Streptococci, while Sch 37644 and Sch 38646 were similar to erythromycin.

Differential regulation of phenylalanine ammonia-lyase genes during plant development and by environmental cues.

Phenylalanine ammonia-lyase (PAL) catalyzes the first reaction in the biosynthesis from phenylalanine of a wide variety of phenylpropanoid natural products including lignin, flavonoid pigments, and phytoalexins. In bean (Phaseolus vulgaris L.), PAL is encoded by a family of three genes. We show here by RNase protection with gene-specific probes that these genes are expressed differentially during development and in response to different environmental cues. While all three genes are expressed at high levels in roots, only PAL1 and PAL2 are expressed in shoots and only PAL1 is expressed in leaves. Strikingly, PAL2 is expressed at very high levels in petals, where PAL1 is only very weakly expressed and PAL3 is not expressed. All three genes are induced by mechanical wounding of hypocotyls, but fungal infection only activates PAL1 and PAL3. Illumination of etiolated hypocotyls activates PAL1 and PAL2 but not PAL3. Corresponding differential patterns of synthesis of specific PAL polypeptide isoforms were observed by two-dimensional gel electrophoretic analysis of in vitro translation products encoded by RNA isolated from hypocotyls stimulated by light, wounding, or infection. The specific isoforms encoded by transcripts of the three PAL genes were identified by inhibition of synthesis in vitro with gene-specific anti-sense transcripts followed by comparative two-dimensional gel electrophoretic analysis of the pattern of translation products. These data indicate that selective expression of PAL genes encoding functional variants is governed by a complex set of regulatory networks for developmental and environmental control of phenylpropanoid biosynthesis.

Phenylalanine ammonia-lyase gene organization and structure.

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) genomic sequences were isolated from bean (Phaseolus vulgaris L.) genomic libraries using elicitor-induced bean PAL cDNA sequences as a probe. Southern blot hybridization of genomic DNA fragments revealed three divergent classes of PAL genes in the bean genome. Polymorphic forms were observed within each class. The nucleotide sequences of two PAL genes, gPAL2 (class II) and gPAL3 (class III), were determined. gPAL2 contains an open reading frame encoding a polypeptide of 712 amino acids, interrupted by a 1720 bp intron in the codon for amino acid 130. gPAL3 encodes a polypeptide of 710 amino acids showing 72% similarity with that encoded by gPAL2, and contains a 447 bp intron at the same location. At the nucleotide level, gPAL2 and gPAL3 show 59% sequence similarity in exon I, 74% similarity in exon II, and extensive sequence divergence in the intron, 5' and 3' flanking regions. S1 nuclease protection identified transcription start sites of gPAL2 and gPAL3 respectively 99 bp and 35 bp upstream from the initiation codon ATG, and showed that gPAL2 but not gPAL3 was activated by elicitor, whereas both were activated by wounding of hypocotyls. The 5' flanking region of both genes contain TATA and CAAT boxes, and sequences resembling the SV40 enhancer core. gPAL2 contains a 40 bp palindromic sequence and a 22 bp motif that are also found at similar positions relative to the TATA box in 5' flanking regions of other elicitor-induced bean genes.

L-Phenylalanine ammonia-lyase fromPhaseolus vulgaris: Modulation of the levels of active enzyme bytrans-cinnamic acid.

The extractable activity ofL-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in cell suspension cultures of bean (Phaseolus vulgaris) is greatly induced following exposure to an elicitor preparation from the cell walls of the phytopathogenic fungusColletotrichum lindemuthianum. Following exogenous application oftrans-cinnamic acid (the product of the PAL reaction) to elicitor-induced cells, the activity of the enzyme rapidly declines. Loss of enzyme activity is accompanied by inhibition of the rate of synthesis of PAL subunits, as determined by [(35)S]methionine pulse-labelling followed by specific immunoprecipitation; this is insufficient to account for the rapid loss of PAL enzyme activity. Pulse-chase and immune blotting experiments indicate that cinnamic acid does not affect the rate of degradation of enzyme subunits, but rather mediates inactivation of the enzyme. A non-dialysable factor from cinnamicacid-treated bean cells stimulates removal of PAL activity from enzyme extracts in vitro; this effect is dependent on the presence of cinnamic acid. Such loss of enzyme activity in vitro is accompanied by an apparent loss or reduction of the dehydroalanine residue of the enzyme's active site, as detected by active-site-specific tritiation, although levels of immunoprecipitable enzyme subunits do not decrease. Furthermore, cinnamic-acid-mediated loss of enzyme activity in vivo is accompanied, in pulse-chase experiments, by a greater relative loss of(35)S-labelled enzyme subunits precipitated by an immobilised active-site affinity ligand than of subunits precipitated with anti-immunoglobulin G. It is therefore suggested that a possible mechanism for cinnamic-acid-mediated removal of PAL activity may involve modification of the dehydroalanine residue of the enzyme's active site.