Plant Physiol. (1982) 70, 745-748 0032-0889/82/70/0745/04/$00.50/0 Distribution of Secondary Plant Metabolites and Their Biosynthetic Enzymes in Pea (Pisum sativum L.) Leaves' ANTHOCYANINS AND FLAVONOL GLYCOSIDES Received for publication March 4, 1982 and in revised form April 19, 1982 GEZA HRAZDINA, GERALD A. MARX, AND HARVEY C. HOCH Departments of Food Science and Technology (G. H.), Seed and Vegetable Sciences (G. A. M.), and Plant Pathology (H. C. H.), Cornell University, Geneva, New York 14456 ABSTRACT Leaves of a novel strain of peas (Pisum sativum L.) were used to determine the distribution of secondary metabolites and their biosynthetic enzymes. Leaf epidermal layers in this strain are easily separated from the parenchyma. Anthocyanins and flavonol glycosides were localized in epidermal vacuoles only. Among the biosynthetic enzymes studied, phenyl- alanine ammonia-lyase (PAL, EC 4.3.1.5), S-adenosyl-1-methionine (SAM):caffeic acid and SAM:quercetin methyltransferases (o-dihydric phenol methyltransferase, EC 2.1.1.42) and a flavonoid 7-0-glucosyltrans- ferase (EC 2.4.1.91) were chiefly localized in the parenchyma, whereas trans-cinnamate 4-monooxygenase (EC 1.14.13.11), hydroxycinna- mate:CoA ligases (EC 6.2.1.12) and a flavonoid 3-"Oglucosyltransferase (EC 2.4.1.91) were found mainly in the epidermis. Flavanone (chalcone) synthase activity was found only in the epidermis, whereas chalcone isomerase (EC 5.5.1.6) was evenly distributed in epidermal and parenchyma tissues. The function of secondary metabolites in living tissues is still obscure. Many of the attempts made in the past to resolve this problem included studies at the subcellular level (3, 8, 10, 13, 22, 27, 29). Flavonoids and their biosynthetic enzymes have often been the target of these investigations, but the reports were con- tradictory. For example, tonoplasts (7), plastids (21, 22, 24, 29, 30) and cytosol (8, 15) have been suggested as the sites of phenylpro- panoid and flavonoid biosynthesis. Hence, it became evident that our understanding of the location of the biosynthetic sites might be improved by investigating specific tissues. To investigate the tissue-specific distribution of secondary plant metabolites, we have used, as a model system, anthocyanins, flavonol glycosides, and their biosynthetic enzymes in pea (Pisum sativum L.) leaves. Properties of a new pea mutant Argenteum (14, 19) allowed us to separate the abaxial and adaxial epidermal layers from the parenchyma. This genetic line also has the capacity to produce anthocyanins in the leaves when exposed to low temperatures. Anthocyanin production in peas is dependent on a dominant ground factor A. Ordinarily, the pigments are localized at specific sites (leaf axils, flowers, fruits, and seeds) via a controlling genetic system that is well-established (2). The anthocyanin and flavonol glycoside composition of the genus Pisum is described in detail in (9, 27). ' Supported by State and University funds. MATERIALS AND METHODS All experiments were performed at least in triplicate. Data presented here were obtained from four independent experiments. Plant Material. Pea seedlings of a line designated A 681-230 were grown in a growth chamber (EY8VH Controlled Environ- ment) under 18°C daylight (6 AM to 6 PM) and 15°C nighttime (6 PM to 6 AM) temperatures in sand flats under 200 ,uE/m2 illumi- nation with twelve 120 cm VHO CW, four 60 cm CW HO fluorescent tubes and eight GO w incandescent bulbs at 80% RH. To induce anthocyanin production, the nighttime temperature was reduced to 10°C after seedling emergence. Chemicals. CoASH, glucose 6-P, glucose 6-P dehydrogenase, NADP+, ATP, naringenin and p-coumaric acid were obtained from Sigma; [3-14CJ cinnamic acid (57 mCi/mmol) from Amer- sham Radiochemicals; S-adenosyl-l-methionine (SAM, methyl- "C, 58.6 mCi/mmol), uridine diphosphoglucose (glucose UL-" C) (223 mCi/mmol) from International Chemical and Nuclear Corp. (Irvine, CA); [U-_4CJphenylalanine (420 mCi/mmol) and [2-14C] malonyl-CoA (45.6 mCi/mmol) from New England Nuclear; 4- hydroxycinnamyl-CoA was synthesized as reported previously (16). Naringenin chalcone was synthesized according to Moustafa and Wong (20), quercetin 3-glucoside as previously described (4). Anthocyanin and Flavonol Glycoside Content was determined as follows: Ten leaves were fractionated into epidermal (upper and lower) and parenchyma layers. Each layer was extracted with 1.0 ml methanol-0. 1 N HCI for 10 min and the extract decanted. Twenty pl of the extract was added to 980 pl methanol-0. 1 N HCI and the absorption spectrum recorded in a spectrophotometer. The concentration of anthocyanins was determined from the A at 530 nm using a molar extinction coefficient (e) of 38,000 l/M-cm, that of the flavonol glycosides at 360 nm (e = 20000 l/M cm, determined from a pure sample of quercetin 3-glucoside). Homogenization of the lissues. Fifteen pea leaves were frac- tionated into epidermal (upper and lower) and parenchyma layers. Each tissue fraction was homogenized at 4°C in 4.0 ml 0.2 M K2HPO4/KH2PO4 buffer (pH 8.0), containing 4 mm 2-mercapto- ethanol with 200 mg PVP and 150 mg granular silica in a mortar. The homogenate was centrifuged for 2 min at 11,500g, percolated through a Dowex 1X2/ Amberlite XAD-4 column (2:1 ratio, 5 mm x 30 mm) to remove phenolic material. The percolate was used directly for the determination of enzyme activities. Enzyme Assays. All incubations were performed at 30°C. Phenylalanine Ammonia-Lyase. Activity was determined by a radiotracer method modified from Amrhein and Zenk (1). The incubation mixture contained 100 td plant extract and 5 il [U- 14CJ phenylalanine and was incubated 20 min. The reaction was stopped by adding 20 il concentrated acetic acid, extracted for 5 min with 200 Al ethyl acetate, centrifuged 1 min at 11,500g and 745 Downloaded from https://academic.oup.com/plphys/article/70/3/745/6078744 by guest on 23 September 2021
Distribution of Secondary Plant Metabolites and TheirBiosynthetic Enzymes in Pea (Pisum sativum L.) Leaves'ANTHOCYANINS AND FLAVONOL GLYCOSIDES
Received for publication March 4, 1982 and in revised form April 19, 1982
GEZA HRAZDINA, GERALD A. MARX, AND HARVEY C. HOCHDepartments ofFood Science and Technology (G. H.), Seed and Vegetable Sciences (G. A. M.), and PlantPathology (H. C. H.), Cornell University, Geneva, New York 14456
ABSTRACT
Leaves of a novel strain of peas (Pisum sativum L.) were used todetermine the distribution of secondary metabolites and their biosyntheticenzymes. Leaf epidermal layers in this strain are easily separated from theparenchyma. Anthocyanins and flavonol glycosides were localized inepidermal vacuoles only. Among the biosynthetic enzymes studied, phenyl-alanine ammonia-lyase (PAL, EC 4.3.1.5), S-adenosyl-1-methionine(SAM):caffeic acid and SAM:quercetin methyltransferases (o-dihydricphenol methyltransferase, EC 2.1.1.42) and a flavonoid 7-0-glucosyltrans-ferase (EC 2.4.1.91) were chiefly localized in the parenchyma, whereastrans-cinnamate 4-monooxygenase (EC 1.14.13.11), hydroxycinna-mate:CoA ligases (EC 6.2.1.12) and a flavonoid 3-"Oglucosyltransferase(EC 2.4.1.91) were found mainly in the epidermis. Flavanone (chalcone)synthase activity was found only in the epidermis, whereas chalconeisomerase (EC 5.5.1.6) was evenly distributed in epidermal and parenchymatissues.
The function of secondary metabolites in living tissues is stillobscure. Many of the attempts made in the past to resolve thisproblem included studies at the subcellular level (3, 8, 10, 13, 22,27, 29). Flavonoids and their biosynthetic enzymes have oftenbeen the target of these investigations, but the reports were con-tradictory. For example, tonoplasts (7), plastids (21, 22, 24, 29, 30)and cytosol (8, 15) have been suggested as the sites of phenylpro-panoid and flavonoid biosynthesis. Hence, it became evident thatour understanding of the location of the biosynthetic sites mightbe improved by investigating specific tissues. To investigate thetissue-specific distribution ofsecondary plant metabolites, we haveused, as a model system, anthocyanins, flavonol glycosides, andtheir biosynthetic enzymes in pea (Pisum sativum L.) leaves.
Properties of a new pea mutant Argenteum (14, 19) allowed usto separate the abaxial and adaxial epidermal layers from theparenchyma. This genetic line also has the capacity to produceanthocyanins in the leaves when exposed to low temperatures.Anthocyanin production in peas is dependent on a dominantground factor A. Ordinarily, the pigments are localized at specificsites (leaf axils, flowers, fruits, and seeds) via a controlling geneticsystem that is well-established (2). The anthocyanin and flavonolglycoside composition of the genus Pisum is described in detail in(9, 27).
' Supported by State and University funds.
MATERIALS AND METHODSAll experiments were performed at least in triplicate. Data
presented here were obtained from four independent experiments.Plant Material. Pea seedlings of a line designated A 681-230
were grown in a growth chamber (EY8VH Controlled Environ-ment) under 18°C daylight (6 AM to 6 PM) and 15°C nighttime (6PM to 6 AM) temperatures in sand flats under 200 ,uE/m2 illumi-nation with twelve 120 cm VHO CW, four 60 cm CW HOfluorescent tubes and eight GO w incandescent bulbs at 80% RH.To induce anthocyanin production, the nighttime temperaturewas reduced to 10°C after seedling emergence.
Chemicals. CoASH, glucose 6-P, glucose 6-P dehydrogenase,NADP+, ATP, naringenin and p-coumaric acid were obtainedfrom Sigma; [3-14CJ cinnamic acid (57 mCi/mmol) from Amer-sham Radiochemicals; S-adenosyl-l-methionine (SAM, methyl-"C, 58.6 mCi/mmol), uridine diphosphoglucose (glucose UL-" C)(223 mCi/mmol) from International Chemical and Nuclear Corp.(Irvine, CA); [U-_4CJphenylalanine (420 mCi/mmol) and [2-14C]malonyl-CoA (45.6 mCi/mmol) from New England Nuclear; 4-hydroxycinnamyl-CoA was synthesized as reported previously(16). Naringenin chalcone was synthesized according to Moustafaand Wong (20), quercetin 3-glucoside as previously described (4).
Anthocyanin and Flavonol Glycoside Content was determinedas follows: Ten leaves were fractionated into epidermal (upperand lower) and parenchyma layers. Each layer was extracted with1.0 ml methanol-0. 1 N HCI for 10 min and the extract decanted.Twenty pl of the extract was added to 980 pl methanol-0. 1 N HCIand the absorption spectrum recorded in a spectrophotometer.The concentration of anthocyanins was determined from the A at530 nm using a molar extinction coefficient (e) of 38,000 l/M-cm,that of the flavonol glycosides at 360 nm (e = 20000 l/M cm,determined from a pure sample of quercetin 3-glucoside).Homogenization of the lissues. Fifteen pea leaves were frac-
tionated into epidermal (upper and lower) and parenchyma layers.Each tissue fraction was homogenized at 4°C in 4.0 ml 0.2 MK2HPO4/KH2PO4 buffer (pH 8.0), containing 4 mm 2-mercapto-ethanol with 200 mg PVP and 150 mg granular silica in a mortar.The homogenate was centrifuged for 2 min at 11,500g, percolatedthrough a Dowex 1X2/ Amberlite XAD-4 column (2:1 ratio, 5mm x 30 mm) to remove phenolic material. The percolate wasused directly for the determination of enzyme activities.Enzyme Assays. All incubations were performed at 30°C.Phenylalanine Ammonia-Lyase. Activity was determined by a
radiotracer method modified from Amrhein and Zenk (1). Theincubation mixture contained 100 td plant extract and 5 il [U-14CJ phenylalanine and was incubated 20 min. The reaction wasstopped by adding 20 il concentrated acetic acid, extracted for 5min with 200 Al ethyl acetate, centrifuged 1 min at 11,500g and
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Plant Physiol. Vol. 70, 1982
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FIG. 1. A, Cross-section of a pea leaf (Pisum sativum L., mutant A 681-230) (x200). B, Removal of the upper epidermal layer of pea leaf (mutantA 681-230). C, Scanning electron micrograph (SEM) of the palisade sideof the upper epidermal layer (x580). D, Palisade cells of a normal pea leafafter removal of the upper epidermis (SEM, x700). Electron micrographswere taken as described in Hoch et al. (14).
100 Al of the ethyl acetate extract was used directly in 5 ml toluenecocktail (2.5 g PPO/l) for activity determination by liquid scintil-lation spectrometry.
trans-Cinnamate 4-Monooxygenase. Activity was determinedby a modified method of Hahlbrock et al. (11). The reactionmixture contained 250 nmol NADP', 50 nmol glucose 6-P, 1.0unit glucose 6-P dehydrogenase, 30 nmol [3-14C]cinnamic acid,and 200 Al plant extract in a total volume of 505 [LI. The reactionmixture was incubated 40 min, 500 ,ug p-coumaric acid in 50 tlconcentrated acetic acid was added and the reaction productextracted into 300 Al ethyl acetate as above. One hundred tdaliquots of the extract were chromatographed on 4-cm wideWhatman 3MM chromatography paper strips for 2.5 h with theupper phase of benzene:acetic acid:H20 (2:2:1), the p-coumaricacid area cut out and activity determined by liquid scintillationspectrometry in 17 ml toluene cocktail of above.
Hydroxycinnamate:CoA Ligase. Activity was determined by thehydroxamic acid assay as described in Knobloch and Hahlbrock(18).
SAM2:Caffeic Acid and SAM:Quercetin Methyltransferase.Activities were determined according to Hrazdina et al. (15).
Flavanone (Chalcone) Synthase. Activity was determined as inHrazdina et al. (16).
Chalcone Isomerase. Activity was determined according to (12).The reaction mixture contained 36 nmol naringenin chalcone (in10 p1 ethylene glycol monomethyl ether), 5 ,umol KCN and 10 ,ultissue extract in 1.0 ml 0.2 M K2KPO4/KH2PO4 buffer (pH 8.0).Chemical isomerization of the chalcone was determined by omit-ting the tissue extract.
UDPglucose:Flavonoid 3-0- and 7-0-Glucosyltransferase. Ac-tivities were determined as described in (17) with quercetin assubstrate.
Protein. Protein was determined according to Schaffner andWeissman (25).
RESULTS
Pea leaves are of a relatively simple construction, consisting ofmonocellular epidermal layers bounding a monocellular layer oftightly packed palisade cells and a 3 to 5 cell layer deep spongymesophyll. Vascular tissue is present in both palisade and meso-phyll layers (Fig. IA). The loosely attached epidermal layers ofthe Arg mutant can be easily removed from the parenchyma (Fig.1B). The tissues (epidermal and parenchyma) so obtained arerelatively undamaged, only a small amount of torn vascular tissueadheres to the epidermal layers (Fig. IC). The parenchyma wasvirtually free from epidermal contamination and the tissues re-mained largely intact. In pea cultivars not containing the Argmutant, removal of the epidermis is accompanied by destructionof the adjacent cell layers (Fig. ID).
Table I summarizes the data for the tissues, flavonoid com-pounds and enzymes. Anthocyanins and flavonol glycosides werefound in the epidermal tissues exclusively. The distribution of theflavonoid biosynthetic enzymes was not so clear. The majority ofPAL activity, the first enzyme in the phenylpropanoid path, waslocated in the parenchyma, whereas the activity of the nextenzyme, trans-cinnamate 4-monooxygenase was found mainly inthe epidermal layers. p-Coumarate and caffeate:CoA ligase activ-ities were found mainly in the epidermis, ferulate-CoA:ligase waschiefly of parenchymal origin. Caffeic acid and quercetin meth-yltransferase activities were limited to the parenchyma. Amongthe specific anthocyanin and flavonol glycoside pathway enzymesflavanone (chalcone) synthase could be found in epidermal tissuesonly, whereas the activities of chalcone isomerase and flavonol 3-0-glucosyltransferase were approximately evenly distributed be-
2Abbreviation: SAM, S-adenosyl-l-methionine.
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SECONDARY METABOLITES AND ENZYMES IN PISUM
Table I. Tissue-Specific Distribution of Flavonoids and Enzymes Involved in Their Biosynthesis in Pisum sativum Leaves
Flavonoids and Enzymes Upper Epidermis Parenchyma Lower Epidermis
Enzymes kat kg-' pro- kat kg-' pro- kat kg-' pro-kat/leaf tein kat/leaf tein kat/leaf tein
Phenylalanine ammonialyase 1.1 x l0-,- 16.1 2.4 x 108 5.2 x i0-'5 79.4 2.5 x 10-9 2.9 x 10-9 4.5 7.0 x 10-9
Cinnamicacid-4-hydroxylase 5.1 x i0-'5 51.4 1.1 x 10-7 2.7 x 10-15 27.8 1.3 x 10-9 2.0 x 1i-`5 20.7 4.9 x 10-8p-Coumarate:CoA ligase 2.5 x 10-12 28.3 5.5 x 10-5 3.8 x 10-12 43.3 1.8 x 10-6 2.5 x 10-12 28.3 5.9 x 10-5Caffeate:CoA ligase 13.8 x 10-12 33.0 3.0 x 10-4 15.7 x 10-12 38.0 7.4 x 10-6 11.8 x 10-12 29.0 2.8 x 10-4Ferulate:CoA ligase 2.3 x 10-12 20.2 5.1 x 10-5 6.7 x 10-12 58.2 3.2 x 10-6 2.5 x 10-12 21.5 5.9 x 10-5SAM:caffeic acid methyl
transferase 6.3 x 10`0 4.7 4.1 x 10-3 1.3 x 10-8 93.6 4.7 x 10-3 2.3 x 10-10 1.7 7.6 x 10-3Flavanone synthase 3.8 x 10-" 88.7 1.8 x 10-5 0 0 0 4.8 x 10-12 11.3 8.5 x 10-6Chalcone isomerase 11.3 x 10-12 31.6 2.6 x 10-4 17.7 x 101-2 49.7 8.4 x 10-6 6.6 x 10-12 18.6 1.6 x 10-4SAM:quercetin methyl trans-
ferase 3.2 x 10`0 6.2 7.1 X 10-3 4.8 x 10-8 93.8 1.8 x 10-3 0 0 0UDPG:flavonol-3-O-gluco-
syltransferase 8.2 x 10-13 34.5 1.0 x 10-6 1.3 x 10-12 54.7 3.3 x 10-8 2.5 x 10-'3 10.5 3.8 x 10-7UDPG:Flavonol-7-O-gluco-
syltransferase 1.3 x 10-14 17.5 2.9 x 10-7 4.6 x 10-'4 62.1 1.7 x 10-8 1.5 x 10-'4 20.4 5.0 x 10-7
tween epidermal and parenchyma tissues. The majority of aflavonol 7-0-glucosyltransferase activity was found in the paren-chyma.
DISCUSSION
Distribution of the first six enzyme activities shown in Table I(e.g. PAL, trans-cinnamate 4-monooxygenase, the ligases andcaffeic acid methyltransferase) is not decisive in establishing thesite of flavonoid biosynthesis in pea leaves, inasmuch as theseenzymes also participate in the lignin biosynthetic pathway. Therewas, however, no consistency in the localization of the specificflavonoid pathway enzymes either. Flavanone (chalcone) syn-thase, the first enzyme of this path is epidermal only, whilechalcone isomerase and the flavonol 3-0-glycosyltransferase seemto be almost evenly distributed between the epidermis and par-enchyma. The role of quercetin methyl transferase and the fla-vonol 7-0-glucosyltransferase is not understood presently, inas-much as methylated flavonols and flavonol 7-0-glycosides havenot been detected in this plant genus (9). Methylated quercetinderivatives may be produced by an unspecific O-dihydric phenolmethyltransferase and quercetin 7-0-glucoside can be an in vitroproduct of an unspecific glucosyltransferase.
Because the anthocyanins and flavonol glycosides were foundonly in epidermal vacuoles, and biosynthetic enzymes in bothepidermal and parenchyma tissues, the data permit two differentinterpretations. One is that the biosynthetic sequence of thesecompounds is closely coordinated between cells and tissues, andthe precursors and end products are specifically transported fromone tissue to another. There is ample evidence in plants forintertissue transport of primary metabolites.The other interpretation is that anthocyanins and flavonol
glycosides in the genus Pisum are synthesized in the epidermallayers only. One would expect that the parenchyma is the majorsite of synthesis for lignin precursors. Because the first fourenzymes mentioned above are members of both the lignin and thespecific flavonol glycoside pathway, their activities are expectedto be found in both epidermis and parenchyma. Chalcone isom-erase activity in the parenchyma could also be caused by oxidasesnot related to the flavonoid pathway.There is suggestive evidence for the synthesis of flavonoids in
the epidermal layers only. Association ofphenylpropanoid (5) andspecific flavonoid pathway enzymes (29) with the ER in variousplants suggest that flavonoid biosynthesis, like that of other sec-ondary metabolites (6), takes place on a tightly spaced multien-zyme complex (26). In such a complex, the intermediates betweenthe first and last step would be highly channeled and the endproducts ofthe reaction, e.g. the glycosylated compounds, releasedfrom the last enzyme in the sequence. Such multienzyme com-plexes would not permit the shuttle of intermediary metabolicpath products across tissues, and end products released wouldaccumulate in the tissues where they are synthesized.
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