The effects of infection by Phytophthora infestans on the control of phenylpropanoid metabolism in wounded potato tissue

During the first 24 h of in vitro incubation of excised potato tuber (Solanum tuberosum L.) discs, the appearance of phenylalanine ammonia-lyase (PAL; EC 3.4.1.5) and the accumulation of chlorogenic acid are both stimulated by infection with Phytophthora infestans (Mont.) de Bary. Whereas in control tissue the level of PAL reached a stable plateau value after 40 h, in infected tissue it subsequently rose again, in one experiment, as the fungal mycelium developed. In the infected but not the control tissue, the level of chlorogenic acid subsequently fell to about to about 20% of its maximum after 50 h. The time courses of increases in cinnamic acid 4-hydroxylase (CA4H; EC 1.14.13.11; 0–60 h) and of caffeic acid acid o-methyltransferase (COMT; EC 2.1.1.42; 0–160 h) are not altered by fungal infection. If the discs are restored to the tuber environment immediately after excision, by placing them inside a host tuber, the activity of PAL as well as those of CA4H and COMT remained at the constant low endogenous level for at least 60 h, irrespective of whether the discs had first been inoculated with P. infestans. The increase in PAL may not be an obligatory feature of the P. infestans/potato compatible interaction but dependent on an underlying wound response. The experiments provide further evidence that PAL is the rate limiting step of chlorogenic acid biosynthesis in potato tuber discs.Abbreviations PAL phenylalanine ammonia-lyase - CA4H cinnamic acid 4-hydroxylase - COMT caffeic acid o-methyltransferase - CGA chlrogenic acid (5-o-caffeoylquinic acid) - gfwt gram fresh weight     

Partial Male Sterility in Transgenic Tobacco Carrying Antisense and Sense PAL cDNA under the Control of a Tapetum-Specific Promoter

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5 [EC] ) catalyzes thefirst step in the synthesis of phenylpropanoids. An antisenseor sense PAL cDNA of sweet potato under the control of a tapetum-specificpromoter of rice was introduced into tobacco. A reduction inpollen fertility was observed in two out of seventeen antisensePAL transformants and in six out of nineteen sense PAL transformants.The pollen fertility of these plants ranged from 8% to 60%.The distorted pollen grains that did not germinate lacked starchand flavonols. PAL activity in anthers at the microspore stagewas also reduced to some extent and the level of PAL activitywas positively correlated with the number of fertile pollengrains at the flowering stage. Although it was unclear how theantisense or sense transgene affected the PAL activity in anthers,our results clearly demonstrate that the PAL activity in theanther tapetum has a significant effect on the development ofmicrospores. (Received October 9, 1995; Accepted March 10, 1996)     

Optimization of reaction conditions and stabilization of phenylalanine ammonia lyase-containing Rhodotorula glutinis cells during bioconversion of trans-cinnamic acid to L-phenylalanine

Studies were performed to elucidate the optimal reaction conditions (pH, temperature, ammonia concentration and biocatalyst loading) for bioconversion of trans-cinnamic acid (t-CA) to L-phenylalanine (L-Phe) by L-phenylalanine ammonia lyase (PAL) containing Rhodotorula glutinis cells. All treatments with permeabilizing agents stimulated L-Phe production and also enhanced instability of the catalyst, except Triton X-100 which gave a superior (56%) increase in conversion as compared to the control and a significant stabilization of PAL enzyme. Inclusion of several activity modifiers and stabilizer additives in reaction mixtures were shown to enhance the yield of L-Phe and maintained PAL stability over several successive incubations during the bioconversion process. Maximum stabilization of PAL and enhancement of L-Phe production was achieved with addition of 20% polyhydric alcohol (glycerol). The production of L-Phe continued to the fifth cycle and the total yield increased 2.3 times compared to the yield produced by the control (without glycerol addition) during the repeated batch process. Reducing agents such as 2-mercaptoethanol and thioglycolic acid were added to the bioconversion mixture in order to reduce the effects of oxygen on PAL catalyst life. Production of L-Phe by addition of 400 mgL(-1) of thioglycolic acid was maximized over the control by 55%. When both 20% glycerol and 400 mgL(-1) thioglycolic acid were simultaneously present in the reaction mixture, reuseability and stability of biocatalyst (PAL) were extended to eight consecutive cycles and conversion rate and overall productivity of L-Phe were higher than that of the control. These results may lead to improvements in the production of the essential amino acid L-Phe.     

Colocalization of L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase for metabolic channeling in phenylpropanoid biosynthesis

Metabolic channeling has been proposed to occur at the entry point into plant phenylpropanoid biosynthesis. To determine whether isoforms of L-Phe ammonia-lyase (PAL), the first enzyme in the pathway, can associate with the next enzyme, the endomembrane-bound cinnamate 4-hydroxylase (C4H), to facilitate channeling, we generated transgenic tobacco (Nicotiana tabacum) plants independently expressing epitope-tagged versions of two PAL isoforms (PAL1 and PAL2) and C4H. Subcellular fractionation and protein gel blot analysis using epitope- and PAL isoform-specific antibodies indicated both microsomal and cytosolic locations of PAL1 but only cytosolic localization of PAL2. However, both PAL isoforms were microsomally localized in plants overexpressing C4H. These results, which suggest that C4H itself may organize the complex for membrane association of PAL, were confirmed using PAL-green fluorescent protein (GFP) fusions with localization by confocal microscopy. Coexpression of unlabeled PAL1 with PAL2-GFP resulted in a shift of fluorescence localization from endomembranes to cytosol in C4H overexpressing plants, whereas coexpression of unlabeled PAL2 with PAL1-GFP did not affect PAL1-GFP localization, indicating that PAL1 has a higher affinity for its membrane localization site than does PAL2. Dual-labeling immunofluorescence and fluorescence energy resonance transfer (FRET) studies confirmed colocalization of PAL and C4H. However, FRET analysis with acceptor photobleaching suggested that the colocalization was not tight.     

Incorporation of 15N from ammonium into the N-linked oligosaccharides of an immunoadhesin glycoprotein expressed in Chinese hamster ovary cells

Elevated ammonium concentrations in the medium of cultivated cells have been shown to increase the intracellular levels of uridine-5'-diphospho- N-acetylglucosamine (UDP-GlcNAc) and uridine-5'-diphospho-N- acetylgalactosamine (UDP-GalNAc; Ryll et al., 1994). These sugar nucleotides are substrates for glycosyltransferases in the glycosylation pathway. In our experiments, recombinant Chinese hamster ovary cells producing an immunoadhesin glycoprotein (GP1-IgG) have been cultivated under controlled cell culture conditions in the presence of different ammonium concentrations.15N-Labeled ammonium chloride (15NH4Cl) was added exogenously to the cell culture media to determine if ammonium was incorporated into UDP-GlcNAc and cytidine-5'- monophospho-N-acetylneuraminic acid (CMP-NANA) pools, and subsequently incorporated into GP1-IgG as N-linked glycans. The intracellular pools of UDP-activated hexosamines (UDP-GNAc) were followed during the time course of the experiment. To assess the extent of15NH4+incorporation into the glycans of GP1-IgG, the glycoprotein was first purified to homogeneity by protein A chromatography. Enzymatically released N- glycans were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. N-Glycans synthesized in the presence of15NH4Cl revealed an N-glycan-dependent increase in mass-to-charge of 2.5-4.8 Da. These results indicate that 60-70% of the total nitrogen containing monosaccharides had incorporated15N. Presumably,15NH4+was incorporated into GlcNAc and N- acetylneuraminic acid as proposed earlier (Ryll et al., 1994). This might be a universal and previously not described reaction in mammalian cells when exposed to nonphysiological but in cell culture commonly found concentrations of ammonium. The data presented here are of significance for glycoprotein production in mammalian cell culture, since it has been shown previously that elevated levels of UDP- activated hexosamines affect N-glycan characteristics such as branching and degree of amino sugar incorporation. In addition, our results demonstrate that isotope labeling in combination with MALDI-TOF-MS can be used as an alternate tool to radioactive labeling of sugar substrates in metabolic studies.      

Continuous production of L-phenylalanine by Rhodotorula glutinis immobilized cells using a column reactor

Studies have been conducted on L-phenylalanine (L-Phe) production and phenylalanine ammonia lyase (PAL) stabilization in the presence of several optimum effectors and reducing agents under bioconversion of transcinnamic acid (t-CA) conditions during repeated batch operations. L-Phe production was maximized and reuseability of PAL catalyst was extended to eight consecutive cycles (repeated batches) in the presence of optimum effectors (glutamic acid, polyethylene glycol and glycerol), thioglycolic acid and sparging with nitrogen gas. These best optimum bioconversion conditions desensitize the PAL catalyst to substantially elevated higher substrate t-CA concentrations and inhibit inactivation of PAL enzyme over longer reaction periods compared to the control. The fed batch mode operation of bioconversion of total t-CA (300 mM) to L-Phe was superior (65.2%, conversion), comparing with conventional batch and repeated batch (58.4%, conversion) operations after 120 h. Gamma irradiation process was employed to polymerize and crosslink polyvinyl alcohol (PVA) with N,N'-methylene-bisacrylamide (BIS) agent. The use of immobilized PAL biocatalyst containing cells in PVA-BIS copolymer gel carrier produced by radiation polymerization is obviously advantageous with regards to the yield of L-Phe which was increased in average 1.2-fold when compare to those obtained with free cells during optimum bioconversion process. When comparing the magnitudes of gamma irradiation effects on immobilized entrapped yeast cells in PVA-BIS copolymer gel carrier using scanning electron microscopy it was show that yeast cells were protected and capable to overcome these conditions and had normal shape and other features as free (unirradiated) intact yeast cells. Optimum conditions for continuous production of L-Phe by PVA-BIS copolymer carrier entrapped yeast cells in a packed bed column reactor in recycle fed-batch mode were investigated. Under these optimum conditions L-Phe accumulated to concentration 240.1 mM represts a total conversion yield of 80% (w/w) from (300 mM) t-CA after 84 h of reaction process, which was higher than that obtained after 120 h of reaction, 65.2% (w/w) from (300 mM) t-CA with free cells in fed-batch mode. The results also demonstrated that during about 4 weeks of repeated continuous recycle fed batch mode experiments (using immobilized cells in packed bed reactor), the final production of L-Phe concentrations decreased gradually in eight consecutive runs with no sign of breakage or disintegration of the carrier gel beads.     

Cloning, expression, site-directed mutagenesis and immunolocalization of phenylalanine ammonia-lyase in Bambusa oldhamii

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) from green bamboo was isolated and cloned from the shell of Bambusa oldhamii. The K(m) of bamboo shell PAL for L-Phe was 476 μM, and the molecular mass of native PAL was estimated as 275 kDa and the molecular mass of a subunit was about 76 kDa, indicating that PAL from bamboo also exists as a tetramer. The optimum temperature for PAL activity was 50°C and the optimal pH 9.0. The identity of the purified bamboo shell PAL was confirmed using Q-TOF tandem MS/MS de novo sequencing. Four PAL genes, designated as BoPAL1 to BoPAL4, were cloned from B. oldhamii. The open reading frames of BoPAL3 and BoPAL4 were 2142 and 2106 bp in size, respectively: BoPAL2-4 contained one intron and two exons, but no intron was found in BoPAL1. BoPAL4 expressed in Escherichia coli possessed both PAL and tyrosine ammonia-lyase activities. While recombinant wild-type PAL proteins had similar biochemical properties to the native bamboo shell PAL, both site-directed mutagenesis of BoPAL1 F133H and BoPAL2 F134H, respectively, showed decreased k(cat)/K(m) values toward L-Phe, whereas BoPAL2 F134H showed a slightly increased k(cat)/K(m) value toward L-Tyr. These data suggest other residues largely control Phe/Tyr substrate specificity. An antibody raised against the purified shell PAL was generated for histochemical studies. In bamboo shell and branch shoots, PAL was localized primarily in sclerenchyma cells.     

Inhibition of the gene expression for granule-bound starch synthase I by RNA interference in sweet potato plants

Granule-bound starch synthase I (GBSSI) is one of the key enzymes catalyzing the formation of amylose, a linear α(1,4)D-glucan polymer, from ADP-glucose. Amylose-free transgenic sweet potato plants were produced by inhibiting sweet potato GBSSI gene expression through RNA interference. The gene construct consisting of an inverted repeat of the first exon separated by intron 1 of GBSSI driven by the CaMV 35S promoter was integrated into the sweet potato genome by Agrobacterium tumefaciens-mediated transformation. In over 70% of the regenerated transgenic plants, the expression of GBSSI was inactivated giving rise to storage roots containing amylopectin but not amylose. Electrophoresis analysis failed to detect the GBSSI protein, suggesting that gene silencing of the GBSSI gene had occurred. These results clearly demonstrate that amylose synthesis is completely inhibited in storage roots of sweet potato plants by the constitutive production of the double-stranded RNA of GBSSI fragments. We conclude that RNA interference is an effective method for inhibiting gene expression in the starch metabolic pathway.     

Effects of ethephon and norbornadiene on sterol and glycoalkaloid biosynthesis in potato tuber discs

The accumulation of glycoalkaloids that normally takes place in aerobically incubated potato ( Solanum tuberosum L.) tuber discs has been found to be inhibited by the ethylene-releasing substance ethephon. Using ethephon and the ethylene action inhibitor norborna-2,5-diene, the effect of ethylene on the synthesis of sterols and glycoalkaloids, which partly share their biosynthetic pathway, was investigated.
Control discs showed incorporation of (2-¹⁴C)mevalonic acid into free sterols, steryl esters, steryl glycosides and acylated steryl glycosides at 24 h, thereafter the radioactivity decreased in free sterols and steryl esters concomitant with the appearance of radioactivity in glycoalkaloids. Discs with ethephon additions contained more radioactivity in all sterol classes at all time-points, but no glycoalkaloids were formed.
The enzyme S-adenosyl- l -methionine:sterol C24 methyltransferase (SMT, EC 2. 1. 1. 41), located at one presumed branching point in the sterol and glycoalkaloid pathway, was characterized and found to exhibit similar characteristics as in other plants, but a lower specific activity. The activity of SMT increased in ageing tuber discs and this increase was further stimulated by ethephon, but inhibited by norborna-2,5-diene. The activity of the glycoalkaloid-specific enzyme UDP-glucose:solanidine glucosyltransferase (EC 2. 4. 1) also increased after slicing, but here ethephon additions counteracted the induction. The activity of the sterol-specific UDP-glucose:sterol glucosyltransferase (EC 2. 4. 1) was unaffected by either tuber slicing or ethephon additions.
The results indicate that ethylene stimulates sterol synthesis in wounded potato discs, and that the wound-induction of SMT is regulated by ethylene.     

Structure and characterization of a cDNA clone for phenylalanine ammonia-lyase from cut-injured roots of sweet potato

A cDNA clone for phenylalanine ammonia-lyase (PAL) induced in wounded sweet potato (Ipomoea batatas Lam.) root was obtained by immunoscreening a cDNA library. The protein produced in Escherichia coli cells containing the plasmid pPAL02 was indistinguishable from sweet potato PAL as judged by Ouchterlony double diffusion assays. The M_r of its subunit was 77,000. The cells converted [¹⁴C]-l-phenylalanine into [¹⁴C]-t-cinnamic acid and PAL activity was detected in the homogenate of the cells. The activity was dependent on the presence of the pPAL02 plasmid DNA. The nucleotide sequence of the cDNA contained a 2121-base pair (bp) open-reading frame capable of coding for a polypeptide with 707 amino acids (M_r 77, 137), a 22-bp 5′-noncoding region and a 207-bp 3′-noncoding region. The results suggest that the insert DNA fully encoded the amino acid sequence for sweet potato PAL that is induced by wounding. Comparison of the deduced amino acid sequence with that of a PAL cDNA fragment from Phaseolus vulgaris revealed 78.9% homology. The sequence from amino acid residues 258 to 494 was highly conserved, showing 90.7% homology.     

Wounding-induced Enhancement of the Activity of Chromatin-bound DNA-Dependent RNA Polymerases in Sweet Potato Root

Chromatin fractions were isolated from intact and wounded sweet potato root tissues. The synthesis of RNA by the chromatin fractions was dependent on four ribonucleoside triphosphates and a divalent cation such as Mg²⁺ and Mn²⁺, Mn²⁺ being most effective. Whereas phosphate did not interfere with the polymerase reaction, it was totally blocked by pyrophosphate. The reaction was inhibited by DNase and actinomycin D as well as RNase and trypsin. The RNA polymerases of sweet potato root needed SH-groups for catalysis. Activity of chromatin-bound RNA polymerases (EC 2.7.7.6) promptly increased in the 6 hr after wounding and then decreased gradually up to 24 hr. Under the present experimental conditions it was mostly due to the activity of RNA polymerase I. RNA polymerase II contributed only about 5 to 15% to the total activity. The increase in the activity after wounding was completely inhibited by cycloheximide. Plant hormones such as 2,4-dichlorophenoxyacetic acid, gibberellic acid and dibutyryl cyclic adenosine 3′,5′-monophosphate stimulated the increase in RNA polymerases three to four times after wounding. Ethylene partially suppressed the wound-induced increase of RNA polymerases.     

Production of ethylene by sweet potato roots infected by black rot fungus

Ethylene production by sweet potato roots infected by the blackrot fungus, Ceratocystis fimbriata, increased strikingly afterinfection. The fungus grown on potato extract containing 1%sucrose or steamed sweet potato produced no ethylene. Thus,ethylene was proven to be produced from the host tissue affectedby fungus invasion. The ethylene production seemed to be stimulatedby carbon dioxide. Oxygen was essential for production, butexcess oxygen, probably over 80%, was found to be inhibitory.Apparent fungus growth on sweet potato was reduced under a hightension of oxygen, but this was not a cause of reduced ethyleneproduction in excess oxygen. When tissue plugs of infected sweet potato which were activelyproducing ethylene were sliced into thin discs, ethylene productionwas abolished with the exception that the first 1 mm discs atthe 1st and 2nd day stages produced a significant amount ofethylene. Similarly, plugs which were removed from fungus-invadedparts did not produce an appreciable amount of ethylene. Theproduction of ethylene was observed only by tissue plugs whichconsisted of both fungus invaded and noninvaded parts. Infected sweet potato tissue produced ethylene at a rate comparableto that in apples and may provide a goodsystem for the studyof ethylene biosynthesis. ¹Part 72 of the Phytopathological Chemistry of Sweet Potatowith Black Rot and Injury.     

Beta-1,3-glucooligosaccharide induced activation of four enzymes responsible for N-p-coumaroyloctopamine biosynthesis in potato (Solanum tuberosum cv.) tuber tissue

Potato tuber disks, when treated with laminarin, a beta-1,3-glucooligosaccharide from Laminaria digitata, accumulate a hydroxycinnamoyl amide compound, N-p-coumaroyloctopamine (p-CO). The biosynthesis of p-CO was investigated by feeding experiments, in order to show that the precursors of N-p-coumaroyl and octopamine moieties of p-CO are L-phenylalanine and L-tyrosine, respectively. The treatment of potato tuber tissue with laminarin resulted in elevated activities of four enzymes which are putatively involved in p-CO biosynthesis: phenylalanine ammonia lyase (PAL; EC 4.3.1.5), 4-hydroxycinnamic acid:CoA ligase (4CL; EC 6.2.1.12), hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110) and tyrosine decarboxylase (TyrDC; EC 4.1.1.25). Among these, the response of TyrDC was specific to laminarin treatment, thus indicating that the regulation of TyrDC activity is critical for the accumulation of p-CO in potato tuber tissue.     

IAA Oxidase preparations from fresh and aged Ipomoea batatas tuber discs

IAA oxidase preparations from fresh sweet potato tuber discs oxidized IAA only in the presence of added phenolic cofactors, and the pH optimum for enzyme activity depended on the cofactor used. Ageing of tuber discs, either by aeration in distilled water or by incubation on moist filter paper, resulted in increased peroxidase and phenol-stimulated IAA oxidase activities, as well as the development of IAA oxidase activity in the absence of added cofactors. High phenolase activity of fresh tuber discs decreased considerably with ageing. Phenol-stimulated IAA oxidase activity reached maximal levels before IAA oxidase activity in the absence of added cofactors. Enzyme preparations from aged tuber discs had double pH optima, similar to those previously described for sweet potato root IAA oxidase preparations. IAA in the concentration range 10^?4 to 10^?2 M inhibited the increase in peroxidase and IAA oxidase activities with ageing. DCP-stimulated IAA oxidase activities in preparations from both fresh and aged sweet potato tuber discs were inhibited by manganous ion.     

A new isopentenyl diphosphate isomerase gene from sweet potato: cloning,characterization and color complementation

Isopentenyl diphosphate isomerase (IDI, EC 5.3.3.2) catalyzes the revisable conversion of 5-carbon isopentenyl diphosphate and its isomer dimethylallyl diphosphate, which are the essential precursors for isoprenoids, including carotenoids. Here we report on the cloning and characterization of a novel cDNA encoding IDI from sweet potato. The full-length cDNA is 1155 bp with an ORF of 892 bp encoding a polypeptide of 296 amino acids, which was designated as IbIDI (GenBank Acc. No: DQ150100). The computational molecular weight is 33.8 kDa and the theoretical isoelectric point is 5.76. The deduced amino acid sequence of IbIDI is similar to the known plant IDIs. The tissue expression analysis revealed that IbIDI expressed at higher level in sweet-potato’s mature leaves and tender leaves than that in tubers, meanwhile, no expression signal could be detected in veins. Recombinant IbIDI was heterologously expressed in engineered Escherichia coli which led to the reconstruction of the carotenoid pathway. In the engineered E. coli, IbIDI could take the role of Arabidopsis IDI gene to produce the orange β-carotene. In summary, cloning and characterization of the novel IDI gene from sweet potato will facilitate our understanding of the molecular genetical mechanism of carotenoid biosynthesis and promote the metabolic engineering studies of carotenoid in sweet potato.     

Some investigations on inactivation of phenylalanine ammonia-lyase in cut-injured sweet potato root tissue

In sweet potato roots, activity of the phenylalanine ammonia-lyase(PAL)-inactivating system in crude enzyme solution increasedmarkedly in response to cut injury after a lag period of about10 hr and reached a maximum after 24 hr of incubation. The resultscoincided with previous results from experiments using a proteinsynthetic inhibitor. The inactivating system could be precipitatedby centrifugation and was distributed in a different patternfrom mitochondrial and microsomal marker enzymes, accordingto data from cellular fractionation by differential and sucrosedensity gradient centrifugation. The optimum pH of the inactivationwas 6.0. Previous studies showed that PAL content changed inparallel with PAL activity in vivo. However, immunochemicalstudies indicated that the inactivation was not due to proteolysis.Furthermore, proteinase activity in sweet potato tissue didnot change in response to cut injury. These results suggestedthat PAL was first inactivated by the inactivating system, thenthe inactivated PAL was rapidly decomposed by the proteinase. ¹ This paper constitutes Part 130 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. This work was supportedin part by a grant from the Ministry of Education. ² Present address: Faculty of Agriculture, Yamaguchi University,Yamaguchi 753, Japan. (Received May 14, 1977; )     

Ethylene-controlled Induction of Phenylalanine Ammonia-lyase in Citrus Fruit Peel

l-Phenylalanine ammonia-lyase (PAL) activity is low in the external layers (flavedo) of intact mature grapefruit peel. Flavedo discs evince upon incubation increasing PAL activity and ethylene production. Light has no effect in enhancing PAL activity in discs. Exogenous ethylene stimulates PAL activity in the flavedo of intact mature grapefruits (half maximum stimulation at 15 ppm); such activity rapidly decreases when fruit is removed from the ethylene containing atmosphere. Carbon dioxide inhibits both ethylene production and PAL activity of discs; exogenous ethylene only partly relieves PAL inhibition. Cycloheximide inhibits both PAL activity and ethylene production by flavedo discs. The same concentration of cycloheximide also inhibits PAL activity of discs in the presence of exogenous ethylene. Protein synthesis seems therefore to be needed at both levels of ethylene evolution and enhancement of PAL activity.     

Immunolocalization of phenylalanine ammonia-lyase and cinnamate-4-hydroxylase in differentiating xylem of poplar

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) and cinnamate-4-hydroxylase (C4H; EC 1.14.13.11) are pivotal enzymes involved in lignification. We synthesized peptides as the epitopes according to the amino acid sequences of these enzymes, coupled them with hemocyanin, and injected them into mice. The antiserums against peptides of PAL and C4H specifically detected PAL and C4H in the crude enzymes extracted from differentiating xylem of poplar, respectively. PAL and C4H were localized in differentiating xylem of poplar. PAL labeling was mainly localized in the cytosol, and somewhat localized on the rough-endoplasmic reticulum (r-ER) and the Golgi apparatus. In contrast, C4H was mainly observed on r-ER and the Golgi apparatus. These findings suggest that conversion of phenylalanine to cinnamic acid occurs in the cytosol and the following reaction occurs near the membrane of r-ER and the Golgi apparatus. The possibility of coordinated localization of PAL and C4H is discussed.