comparison proanthocyanidins related compounds … · tions were made from ginkgo and ribes. abulk...

Plant Physiol. (1986) 82, 1132-11380032-0889/86/82/1132/07/$0 1.00/0

Comparison of Proanthocyanidins and Related Compounds inLeaves and Leaf-Derived Cell Cultures of Ginkgo bioloba L.,Pseudotsuga menziesii Franco, and Ribes sanguineum Pursh'

Received for publication July 2, 1986 and in revised form August 25, 1986

HELEN A. STAFFORD*, KELLY S. KREITLOW, AND HOPE H. LESTERBiology Department, Reed College, Portland, Oregon 97202

ABSTRACT

Proanthocyanidins, flavan-3-ols, and their flavanoid precursors inleaves and leaf-derived callus and cell suspension cultures have beenisolated and analyzed by high performance liquid chromatography withC,8 columns, paper chromatography, and by chemical and spectrophoto-metric methods. Cultures of Ginkgo biloba and Pseudotsuga menziesii(Douglas-fir) produced much greater amounts of proanthocyanidins thanleaves per milligram dry weight. In cultures, however, the prodelphinidincomponent relative to that of procyanidins decreased; this was mostpronounced in Pseudotsuga. In contrast, callus cultures of Ribes sangui-neum accumulated proanthocyanidins in amounts about equal to those inintact leaves per milligram dry weight and the prodelphinidin contentremained high. Although Ginkgo and Ribes leaves contained majoramounts of flavan-3-ols and dimers with the 2,3-cis-stereochemistry,their cultures tended to synthesize 2,3-trans-isomers instead. Glycosidesof flavanone and 3-hydroxyflavanone precursors accumulated in mediumto high amounts on a dry weight basis in leaves and cultures of Ribes andPseudotsuga, and the 3'-glycosidic linkage predominated when the latterspecies was cultured with 2,4-dichlorophenoxyacetic acid rather thannaphthaleneacetic acid.

Ellis (7) has recently reviewed the advantages and disadvan-tages of plant tisue and cell cultures in elucidating secondarymetabolism in plants. While some cultures do not synthesizesecondary compounds present in the tissue from which they werederived, others accumulate at least one ofthe secondary productsin high concentrations (5). We have demonstrated that cellcultures derived from cotyledons of Pseudotsuga menziesii werea major source of secondary products known as PA2 (21).The chemistry of PAs and their related flavan-3-ols has been

recently reviewed (10). Oligomeric PAs, also known as condensedtannins, generally consist of chains of 2,3-trans- and 2,3-cis-flavan-3-ol units, linked between carbons at positions 4 and 8(Fig. 1). Recent examples of 4,6-linkages have been found inPinus taeda that would permit branched polymers (11). Thehydroxylation pattern of the A-ring is usually 5,7-hydroxy, andthe B-ring is either 3'4'-dihydroxy (diphenolic) or 3'4'5'-trihy-droxy (triphenolic). Upon acid hydrolysis, the interflavanoidbonds are lysed and the 'upper' or extension units are oxidized

'Supported by National Science Foundation grant DMB-82 18301.2 Abbreviations: PA, proanthocyanidin; PC, procyanidin; PD, prodel-

phinidin; CSC, cell suspension cultures; S.P., Sep-Pak; DHQ, dihydro-quercetin; DHM, dihydromyricetin; ERIO, eriodictyol; BAW, bu-tanol:acetic acid:water (6:1:2).

to their respective anthocyanidins, i.e. cyanidins or delphinidins;this is the basis for both the name (proanthocyanidins) and thequantitative assay method used in this study.PAs are widely distributed in Gymnosperms (12), perhaps

even universally, and all cultures examined so far form significantamounts of these secondary products (1, 3, 4, 13, 17, 18). PAshave been identified only in a few angiosperm tissue cultures (5)and much of the data was based on nonspecific cytochemicalstains or spectrophotometric analyses of total phenolic com-pounds.The biosynthetic pathway to PAs and their related flavan-3-

ols is only partly known. Cell cultures derived from leaves ofPseudotsuga menziesii and Ginkgo biloba were vital to ourisotopic and enzymic studies of this pathway (21-26). The pres-ent study was designed to compare these Gymnosperm cellcultures, plus that from an Angiosperm, Ribes sanguineum, withthe leaves from which they were derived. Both the flavan-3-oland PA end products, as well as their flavanone and 3-hydroxy-flavanone precursors, were analyzed (Fig. 1).

MATERIALS AND METHODS

Standard Culture, Extraction, and Analytical Procedures. Cal-lus and CSC of Ginkgo biloba and Ribes sanguineum werederived from petioles or lamina of young leaves, respectively,from trees located on the Reed College Campus, and weremaintained at 25°C under 150 to 300 footcandles white fluores-cent light in a manner similar to that for Pseudotsuga menziesiicultured from cotyledons and young needles (20), but with thefollowing changes: 4 uM nicotinic acid and 0.5 AM pyridoxine-HCI were added to the nutrient medium, plus 1 mM arginine, inaddition for Ginkgo cultures. NAA was the auxin added in allcases, except where noted in Table VII. Before freezing, 100 mgculture samples from 3-week-old cultures were rinsed with H20on a nylon screen in a Buchner funnel and the surface H20 wasremoved by aspiration. The cells, weighed while still frozen, wereextracted in 70% methanol to give a soluble and insolublefraction that were analyzed for total PAs with the butanol-HClreagent. The methanol soluble fraction was further subdividedby chromatography on small C1,8 columns (Water Associates Sep-Pak) into the S.P. 20:80 (methanol:5% acetic acid, v/v) fractioncontaining flavan-3-ols and oligomers up to tetramers and theS.P. 70:30 (methanol:H20, v/v) fraction containing the higheroligomeric forms (21). In addition to the above PA assay, thesemethanol soluble fractions were analyzed by two-dimensionalpaper chromatography to estimate visually amounts of phenoliccompounds from the intensity of the Prussian blue positivereaction compared with standards, and by HPLC with C1,8 col-umns used previously to quantify amounts based on A at 280nm (20).

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PROANTHOCYANIDINS IN LEAVES AND CULTURES

OH OH

HO X R HOA 2,3- trans

o z ERIO:R=H 0 1 OH DHO: Rr-HH 0 5'-OH-ERIO:R-OH H 0 DHM: R =OH

jFLAVANONES 13-OH FLAVANONESI

NADPHReductases

OH

OH

HOR0 . R trans: R-H

HO OH 2,3-Cis R-HHI Rz OH

H OH R-OH

FFLAVAN -3,4-Cis- dio 'SI

L (leucocyanidins)

NADPH / rEXTENSION UNITSlReductoses L(quinone methidesgJ

initiating unit condensing enzyme

OHOH

Ho ,h R

H

2,3- trans COt: R H

gC: R =OH

2,3- cis epi: R- H

egc: R' OH

IFLAVAN- 3-OLS

'upper'extensionunits

1/

0

H

JOLIGOMERS

J n:o-8?OH~>OH

iBl

'R

Ilower'OH initiating

unit

FIG. 1. Diagram of the biosynthetic pathway leading to 2,3-cis- and2,3-trans-flavan-3-ols and oligomeric PAs, with both diphenolic andtriphenolic B-rings. A wavy line () indicates that a substituent is eitherabove (--) or below (-- -) the plane of ring C.

New Analytical Procedures. In addition to the Prussian bluespray, the following tests were done on a 0.5 cm2 section cut outfrom the center of a spot identified by its UV absorption onunsprayed, two-dimensional paper chromatograms: the Zn (2),and Mg (15) tests to distinguish between flavanones and 3-hydroxyflavanones, and the vanillin reaction (20) to identifyboth flavan-3-ols and PAs. With some glycosides in the Zn test,it was necessary to add the 6 N HCI first, dry with hot air, andthen add the Zn dust and more acid. The mechanism of the Znreaction has not been determined, but 3-hydroxyflavanones ap-pear to give rise to anthocyanidin-like colors; DHM gives apurple reaction typical of delphinidin while DHQ produces theredder hue of cyanidin. Flavanones give only a weak, delayedreaction. For the Mg test, the paper section was placed in a smalltest tube to which were added 50 Ml 70% methanol, a small pieceof Mg, and 2 drops of 6 N HCI. Both flavanones and 3-hydroxy-flavanones react with this reagent to give a reddish and purplishhue for the diphenolic and triphenolic B-ring types, respectively.The vanillin reaction was also done on a paper section in a smalltest tube by the addition of 50 each of a fresh solution of 4%

vanillin in 100% methanol and 12 N HCl.Ratios ofPC to PD were generally determined on the butanol-

HCl mixture produced in the PA assay ofthe methanol insolubleresidue. After one-dimensional chromatography on TLC-cellu-lose in acetic acid:HCl:H20 (3:0.3:1, v/v/v), the anthocyanidinbands were eluted in 70% methanol containing 1.0% (v/v) HClfor spectral analysis (21).HPLC profiles of the S.P. 20:80 fraction were difficult to

quantify because of the presence, in addition to discrete peaksindicating single compounds, of an 'underlay' due to a continu-ous series of PAs. This underlay was greatly lessened by extrac-tion of the flavan-3-ols and lower oligomers into ethyl acetate,after evaporation of the methanol. After addition of 0.1 ml H20,the extract was concentrated to an aqueous phase by evaporationof the ethyl acetate because this solvent interferes with HPLCelutions. This underlay was also observed as a continuous basalstreak of Prussian blue positive compounds at the bottom ofpaper chromatograms as these constituents did not migrate inthe BAW solvent, and just streaked in the 5% acetic acid solvent.Therefore, the quantitative estimates in Tables II and IV weresometimes based on visual comparisons of Prussian blue spotsafter paper chromatography, both before and after isolation ofappropriate peaks from HPLC analyses.

Purification of Precursors. To obtain enough precursor toanalyze its spectral changes upon addition of alkali etc. accordingto Markham (14), large scale (about 10 g fresh weight) prepara-tions were made from Ginkgo and Ribes. A bulk C,8 columnwas made by filling a 2.2 x 4.5 cm sintered glass funnel half-fullwith preparative C18 column packing material, 55-105 um(Waters Associates No. 51922). The column was washed andeluted with the same solvents as those for the small Sep-Pakcolumns (20); the precursors were generally eluted in the 70:30(methanol:H20) fraction. Subsequent purification was done bysequential one-dimensional strip chromatography (descending,on long Whatman No. 1 sheets) first in 5% acetic acid, and then,after elution in methanol:H20 (50:50, v/v) and concentration,in BAW. Column eluates and a small strip from the center ofthe chromatographed bands were monitored by UV, the Mg testto identify the flavanones and the Zn test for 3-hydroxyflava-nones. In some cases where there was too much overlap ofbandson paper chromatograms, large scale HPLC collections of theappropriate peaks were made and the compounds subsequentlyidentified by paper chromatography.

Standards and Conversion Factors. All standards used havebeen reported previously (21, 22, 26), except for 5'-hydroxyerio-dictyol that was kindly supplied by Dr. G. Forkmann, Universityof Tubingen, W. Germany. Factors used to convert to tig ofphenolic compound were calculated from the following E'%(absorption coefficient of a 1%, w/w, solution) values based oncommercial samples used previously or those reported in theliterature (20, 25): 120 at 280 nm for flavan-3-ols and dimerswith a diphenolic B-ring; 60 at 270 nm for similar compoundswith a triphenolic B-ring; 442, 346, and 306 at 290 nm formonoglucosides of ERIO, DHQ, and DHM, respectively (basedon molar absorption coefficients from H. Outtrup, CarlsbergLaboratories, personal communication). In addition, an E'% of150 was used to convert A at 550 nm to gg proanthocyanidinsfor both procyanidins and prodelphinidins (27). Since variationsin these values have been reported, they should only be consid-ered as approximations. The HPLC derived values were correctedfor analysis only at 280 nm when necessary and for percentrecovery. Visual estimates of the intensity of the Prussian bluereaction on paper chromatograms were based on chromato-graphed standards; this method was just as accurate as a stripdensitometer formerly used (24). Triphenolic B-ring compoundsgave approximately twice the intensity of blue as those with adiphenolic B-ring. Note that this 2-fold difference was in the

V)

0

U1)

a.

Uf)

0

z

Ul)

00.0zId

1133

? - -'

0 ..I,I'D.-

? OH

2,3- CiSL i

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Plant Physiol. Vol. 82, 1986

opposite direction of the difference between their absorptioncoefficients.

RESULTS AND DISCUSSIONGeneral Description of the Methanol Soluble and Insoluble

Fractions. The flavan-3-ols and smaller PA oligomers were foundin the S.P. 20:80 fraction. Higher oligomers and most of theother flavonoids were found in the S.P. 70:30 fraction. It is notclear whether the PAs in the methanol insoluble residue wereinsoluble in living cells of leaves or were artifacts of extraction.Upon paper chromatography, the major compounds that mi-

grated in both the BAW and 5% acetic acid solvents that are ofinterest in the PA pathway were the flavanone and 3-hydroxy-flavanone precursors such as ERIO and DHQ, present mainly asglycosides, the flavan-3-ol end-products, and dimeric and tri-meric PAs. Dimers were present in larger amounts than any ofthe other higher oligomers. The oligomers above trimers re-mained at the origin in the BAW solvent, but formed a basalstreak on the chromatograms up to an RF value of about 0.5 inthe 5% acetic acid solvent. Such a basal streak was found on thechromatograms derived from both the S.P. 20:80 and 70:30fractions. These higher oligomers appeared as a continuous seriesof ill-defined peaks on the HPLC profiles, and were presumablymade up of small amounts of oligomers of increasing chainlength and varying patterns of four types of flavan-3-ol units,consisting of 2,3-cis- or 2,3-trans-isomers with either di- or tri-phenolic B-rings. They accounted for over 50% of the cyanidinor delphinidin in the S.P. 20:80 and 100% in the S.P. 70:30fraction measured in the PA assay used in these studies, but werenot studied further. Except for determination of average mol wtand ratios of 2,3-trans:cis and PC:PD units (9), these higheroligomers have been poorly studied. They have generally notbeen considered to be associated with carbohydrates in livingcells, but recent evidence questions this (16). There was noevidence of any of the monophenolic PAs called propelargoni-dins in the species we have examined.PAs in Intact Leaves and Cultures of G. biloba (Table I). The

total PA content on a dry weight basis was much higher in thecultures than in the leaves from which they were derived. Thegreatest increase was in the S.P. 20:80 fraction of the methanolsoluble components. A similar increase in lower mol wt formswas obtained by Schrall and Becker (18) with twig-derived cul-tures of Ginkgo, while the molecular sizes of oligomers in calluscultures of Cryptomeria japonica were similar to those from theleaves from which they were derived (17).PC:PD Ratios. Intact leaves of Ginkgo contained up to 8 times

as much PD (triphenolic B-ring) as PC (diphenolic B-ring). Thiswas similar to ratios found in Metasequoia glyptostroboides, amember of the Taxodiaceae (27). The amount of PD relative toPC was much less in both callus and CSC, but was still present

Table I. PAs in Ginkgo Leaves (Lamina) and Tissue Cultures Derivedfrom Petioles plus the Ratio ofPC:PD

Values represent means + SE for 3 extracts (n = 3) and are based on

acid hydrolysis to anthocyanidins in butanol-HCI reagent.

Leaf Callus CSC

aqg/mg dry wta b

Methanol solubleS.P.20:80 35±3 407±7 358±68S.P. 70:30 7 ± 1 69 ± 3 85 ± 15

Methanol insoluble 28 ± 2 53 ± 4 122 ± 26

Total PA 70± 3 529 ± 11 565 ± 107

PC:PD ratios 1:8 1:1 1:1a Based on an average E'% at 550 nm of 150. b Dry/fresh weight:

lamina = 0.40, cultures = 0.10.

in at least equivalent amounts. This difference was not due justto the presence of younger cells in culture since even youngGinkgo leaves had very high PD contents.

Flavan-3-ols and Dimeric PAs (Table II). In general, the dis-tribution pattern of flavan-3-ols and dimers was similar to thatofthe total PAs, with much higher quantities found in the callusand CSC than in the intact leaf. The two major flavan-3-ols inthe leaf were the diphenolic 2,3-trans-isomer, catechin, and the2,3-cis-triphenolic flavan-3-ol, epigallocatechin. The cultures,however, showed a large increase not only in the 2,3-trans-catechin, but also in the triphenolic form ofthis 2,3-trans-isomer,gallocatechin; this is in contrast to the relatively constant amountof the 2,3-cis-isomer, epigallocatechin. The 2,3-cis-diphenolicflavan-3-ol, (-)-epicatechin, was generally either not detectableor was present only in trace amounts.The dimers of the intact leaf were present in quantities too

small relative to other types of phenolic compounds to be effec-tively quantified. The only major one was a PD tentativelyidentified as epigallocatechin-catechin. The two major dimers incultures were the all 2,3-trans-PC, catechin-catechin, and the all2,3-trans-PD, gallocatechin-catechin. The mixed isomeric PCdimer, epicatechin-catechin, was also detected in cultures, but inlesser amounts. Note that the designation of dimers and higheroligomers as PC or PD is determined by the 'upper' or extensionunits since these are the ones that are oxidized to an anthocyan-idin in the PA assay.PAs in Intact Leaves and Cultures of R. sanguineum (Table

III). This Angiosperm leaf was chosen for analysis because priorreports indicated that it contained a ratio ofPC to PD of 1:9 (9);

Table II. Flavan-3-ols and Major Dimeric PAs in Ginkgo Leaves andTissue Cultures

Estimates from both paper chromatography and HPLC from 3 ormore extracts.

Leaf Callus CSC

,g/mg dry wtFlavan-3-ols

Catechin 0.4a 8.0 4.2Epicatechin Trace 0.2 0Gallocatechin 0.08 5.0 4.0Epigallocatechin 0.3 0.2 0.2

DimersEpicatechin-catechin Trace 0.1 0.3Catechin-catechin Trace 0.3 0.8Gallocatechin-catechin Trace 0.3 0.5Epigallocatechin-catechin 0.15

aLate August collections. Leaves collected in June and late July hadvery small amounts of catechin (about 0.05 gg/mg dry weight); epigal-locatechin was the major flavan-3-ol.

Table III. PAs in Ribes Leaves (Lamina) and Tissute Clulthures Derivedfrom Them plus the Ratio ofPC:PD

Values represent means ± SE for 3 extracts (n = 3) and are based onacid hydrolysis to anthocyanidins in butanol-HCI reagent.

Leaf Callus CSC

alg/mg dry Utta'bMethanol soluble

S.P. 20:80 188 ± 8 229 ± 10 48 ± 13S.P.70:30 29±2 9±2 6± 1

Methanol insoluble 15 ± 8 53 ± 13 34 ± 4

Total PA 232 ± 8 291 ± 7 87 ± 13

PC:PD ratios 1:4 1:5 1:3

'Based on an average E'% at 550 nm of 150. bDry/fresh weight:lamina = 0.45, cultures 0.1.

1134 STAFFORD ET AL.




our ratio was 1:4. The leaf also contained total PAs equivalentto the highest amounts in the recently studied Taxodiaceae group(28). Cultures derived from either petioles or young leaves lessthan 1 cm in length retained high amounts ofPD relative to PC.In contrast to the cultures of the Gymnosperm species, the totalamount of PAs in the Ribes callus culture did not show a markedincrease, but was similar to that in intact leaves. PAs were very

low in CSC. The latter were very viscous in contrast to our othercultures, perhaps indicative of highly pectinaceous walls; dryweight to fresh weight ratios, however, were similar.

Flavan-3-ols and Dimers (Table IV). The leaves containedlarge amounts of the triphenolic 2,3-cis-flavan-3-ol, epigalloca-techin, and little or no detectable diphenolic catechin or epica-techin. However, upon culture, the catechin and gallocatechinpools (both 2,3-trans-isomers) increased as was the case inGinkgo. Two major PD dimers, found in approximately equalamounts in both leaves and culture, were tentatively identifiedas gallocatechin-catechin and gallocatechin-epigallocatechin; thelatter was reported previously as the major dimer in Ribes leavesby Foo and Porter (8).

Flavanoid Precursors of the PA Pathway in Tissue Cultures.None of the cultures contained detectable amounts of naringeninor dihydrokaempferol (monophenolic B-rings) as aglycones or

glycosides, but flavanones and 3-hydroxyflavanones with di- ortriphenolic B-rings were found. So far, we have been unable todetect any 5'-hydroxyeriodictyol as an aglycone or a glycoside;this flavanone with a triphenolic B-ring was recently demon-strated in flower petals and enzymic incubation mixtures ofmutant angiosperm species (6, 28).

Precursors in CSC from Pseudotsuga. Three major precursorshave now been detected in our Pseudotsuga CSC (strain V); twoof these were identified previously as (+)-dihydroquercetin-3'-glucoside and eriodictyol-7-glucoside. The former was dominantin needle extracts, while the latter was dominant in CSC (21). Athird precursor has now been tentatively identified as eriodictyol-3'-glucoside, based on the evidence summarized below and inTable V, with chromatographic information presented in TableVI.

Spectral peaks in 100% methanol and the bathochromic shiftswith alkali, A13+, and sodium acetate indicate that the 5 and 7hydroxyl groups on the A-ring of a flavonoid are free. Sinceflavanones and 3-hydroxyflavanones show no other diagnosticspectral shifts, we cannot differentiate between these two possi-bilities with spectral analyses alone ( 14). Two spot tests, however,can generally distinguish between these two types of flavonoids.The unknown glycoside isolated on paper chromatograms gavea bright cherry red color in the Mg test, a characteristic reactionof both types, but gave only a delayed light pink color with theZn test; this indicated that the unknown was a flavanone glyco-

Table IV. Flavan-3-ols and Major Dimeric PAs in Ribes Leaves andTissue Cultuires

Estimates from both paper chromatography and HPLC. Value of 0means less than 0.01 g/mg dry weight.

Leaf Callus CSC

/Lg/mg dry wt

Flavan-3-olsCatechin 0 1 0.3Epicatechin Trace 0 0Gallocatechin 0.4 7 0.8Epigallocatechin 2 2 0

DimersEpicatechin-catechin 0.6 0.5 0.5Catechin-catechin 0 0 0Gallocatechin-epigallocatechin 1 1 0.5Gallocatechin-catechin 1 1 0.5

Table V. Spectral Characteristics of Unknown Precursors inPseudotsuga and Ribes

Pseudotsuga Ribes

Methanol+NaOH+Al'++Na acetateZn testMg test

aglycone + glycoside285, 320a322b308c, 375324Delayed pinkRed

aglvcone + glycoside285, 320a324b314C, 375324PurpledPurpled

Tentative identification: ERIO-3'-glucoside DHM-3',5'-glycoside(rhamnoside?)

a Shoulder. b Intensification. c Major long wavelength peak.d After acid hydrolysis.

side. The aglycone, produced by acid hydrolysis of the glycosidein 2 N HCl at 95°C and extracted into ethyl acetate, gave spectralcharacteristics and spot test reactions similar to those of theglycoside. Paper chromatography of the ethyl acetate extract inseveral solvents and HPLC elution data indicated that the agly-cone was eriodictyol. The presumed sugar must be attached toeither the 3'- or 4'-hydroxyl of the B-ring. We have tentativelyidentified this unknown as eriodictyol-3'-glucoside, which has aglycosylation pattern similar to the DHQ glucoside that was alsopresent. Both compounds have also been reported in Pinusmassoniana (19).

Effects of Auxins on Precursors and PAs (Table VII). InPseudotsuga CSC, differences in the above precursor pools weredetected in cultures in which the auxin 2,4-D was substituted forNAA over a 2 year subculture period. The amounts of eriodic-tyol-7-glucoside were higher in the presence of NAA comparedwith 2,4-D, whereas eriodictyol-3'-glycoside and, in particular,DHQ-3'-glucoside were greatly increased.These two auxins caused no measurable difference in growth

rates (data not shown) or in total PA's and their distributionbetween the methanol soluble and insoluble fractions whenanalyzed 3 weeks after subculture (Table VII). This associationof growing cultures and high PA content is in contrast to manycultures, including ones derived from Pseudotsuga menziesii andfrom Pinus taeda (loblolly pine) (13), in which secondary prod-ucts such as flavonoids increased mainly when growth ratesdeclined and cell differentiation began (5). This may be due tothe fact that in order to study the PA pathway, we have routinelyselected cell lines that grow well, but accumulate large amountsof these compounds.

Precursors in Ginkgo. The only precursor found in eitherleaves or cultures of Ginkgo was a trace amount of the aglycone,DHM.

Precursors in Ribes. The major precursor found in cultures ofRibes was also found in large quantities in the leaf. Its spectralcharacteristics, summarized in Table V, were very similar tothose of eriodictyol-3'-glycoside in Pseudotsuga. The unhydro-lyzed compound did not give typical color reactions with eitherthe Mg or Zn tests. However, after acid hydrolysis of the Ribesunknown, the isolated aglycone showed the expected purple colorwith the Zn test for a 3-hydroxyflavanone. Spectral and HPLCanalyses and paper chromatography indicated that the aglyconewas DHM. Subsequently, we found that if the paper chromato-graphed area containing the unknown glycoside, detected by UVabsorption, was moistened first with HC1 and dried with heatprior to the regular Zn test, the typical purple hue for thetriphenolic DHM was observed. The unknown glycoside alsogave an initial black-purple color with the Prussian blue spray.So far, this unusual initial reaction has been observed only withglycosides containing just one free hydroxyl group on the B-ring,such as the 3'-glycoside of DHQ, but not its aglycone. Another

1135



Plant Physiol. Vol. 82, 1986

Table VI. Paper Chromatographic (RF values) and HPLC Elution Data (VF) for Precursors to theProanthocyanidin Pathway and Related Compounds

See Table IV in Ref. 27 for other data.VE (ml)

Methanol:5% Acetic Acid(v/v)

NAR-7-GERIO-7-GDHK-7-GDHQ-7-GDHQ-3'-GDHQ-3-rhamnosideNAR-7-glu-rhamnosidePsb-x: ERIO-3'-G?Ribes-x: DHM-di-rhamnosideNARERIO5'OH-ERIODHKDHQDHMCatechinGallocatechinEpigallocatechin

a Abbreviations: VE, elution volume;acid:water (25:23:5). b Pseudotstuga.

5%aceticacid0.50.470.680.660.60.59

0.380.510.250.220.160.380.40.40.420.40.3

0.560.470.50.380.480.630.720.550.460.90.850.680.90.80.70.550.360.28

5328107

0.43

0.850.720.440.540.440.210.850.6

6

24 12221431

20 1168392228

30 1614 88 5610

BAW, butanol:acetic acid:water (6:1:2);

107

7

136

321810128S

Table VII. Changes in Precursors and PAs and in Pseudotsugamenziesii Cell Suspension Cultures (Strain V) when 10J M 2,4-D wasSUtbstitiuted for 5 jAM NAA as the Auxin (plus 6-Benzylaminopurine at

0.5 gM in bothValues represent means + SE for 3 extracts (n = 3). Data from Tables

I and III in Ref. 21 for 1-year needles are shown for comparison.Cell Suspension Culture

NeedlesNAA 2,4-D

,ug/mg dry wtPrecursorsERIO-7-G 0.3 3.9 2.0ERIO-3'-G NDa 0.7 9.4DHQ-3'-G 0.9 2.9 91.0

ProanthocyanidinsMethanol soluble

S.P. 20:80 69 ± 7 351 ± 84 407 ± 41S.P.70:30 48±6 149±32 122± 16

Methanol insoluble 23 ± 3 75 ± 8 91 ± 18TotalPA 140± 12 575± 112 620±64

a Not determined.

peculiarity of this Ribes glycoside was that its aglycone elutedearlier than the glycoside on the HPLC C,8 column, whichindicated that the aglycone was more water soluble. Such areversal of the usual glycoside to aglycone elution sequence wassimilar to that obtained with astilbin, a DHQ-3-rhamnoside.While the aglycone of the Ribes glycoside was definitely DHM,its glycosidic linkages can only be tentatively postulated as thoseof a di-rhamnoside, with only one of its three hydroxyl groupsfree to explain the initial reaction with the Prussian blue spray.Comparative Summary of Data from Leaves and Cultures from

Pseudotsuga, Ginkgo, and Ribes (Table VIII; Fig. 1). Monomericflavan-3-ols and the oligomeric units from dimers up to at least

Table VIII. Summary ofComparisons ofLeafand Tissue Culturesfrom Pseudotsuga, Ginkgo, and Ribes

Values in parentheses estimates of flavan-3-ols and precursors as jug/mg dry weight from Tables I to V and from Ref. 21.

Total PC-D Major MajorPAS Flavan-3-ol(s) Precursor(s)

PseudotsugaLeaf' 140 1:1 Catc (2) ERIO-7-G (0.3)

DHQ-3'-G (2.0)Culturea-CSC 177 1:0 Cat (I 1) ERIO-7-G (2.0)

DHQ-3'-G (0.5)Culture"-CSC 575 1:0 Cat (8) ERIO-7-G (3.9)

ERIO-3'-G (0.7)DHQ-3'-G (2.9)

GinkgoLeaf 70 1:8 Cat (0.4) DHM (tr)

Egc (0.3)Culture-callus 529 1:1 Cat (8) NDc

Gc (5)Ribes

Leaf 232 1:4 Egc (2) DHM-di-Gly (13)DHQ-3'-G (tr)

Culture-callus 291 1:5 Gc (7) DHM-di-Gly (0.5)a Published data of Tables II and III of Ref. 21 for needles from 10

trees and for strain V CSC. b Current values for strain V after anadditional 5 years in culture. CAbbreviations: cat, catechin; egc,epigallocatechin; gc, gallocatechin; ND, none detected.

10 units are the two types of end product pools of the PApathway. Since 2,3-cis- and 2,3-trans-isomers with both di-phenolic and triphenolic B-rings are involved (Fig. 1), all possiblecombinations of oligomers may exist upon condensation. Thesepools may be even more complex if branching occurs at 4-.6linkage points in addition to the usual straight chains of 4-.8

RF

BAW CAW 20:80 30:70 40:60

CAW, chloroform:acetic

1136 STAFFORD ET AL.




interflavanoid bonds (1 1). The major precursors and end prod-ucts in cultures derived from leaves are summarized in TableVIII for Pseudotsuga, Ginkgo, and Ribes.

In the case of the two Gymnosperms studied, Ginkgo andPseudotsuga, there was a significant increase in total PAs incultures on a dry weight basis; the increase tended to be greatestin the more H20 soluble oligomers found in the S.P. 20:80fraction. PD relative to PC contents were lessened in both thesecultures when compared with intact leaves. No PDs were detectedin our present cell lines, but a PC:PD ratio of 1:0.25 was detectedin one of our earlier Pseudotsuga cultures (unpublished NMRdata of L. Porter, DSIR, Petone, New Zealand).The data for Pseudotsuga cultures in Table VIII are for strain

V studied previously (21). While 5 years ago this cell culture linecontained only medium amounts of PAs, at the time of thepresent study it contained higher amounts comparable to thoseof the no longer available NK strain studied earlier. In addition,precursor levels in strain V have now changed; the DHQ-3'-glucoside content was almost as high as that of ERIO-7-glucosideand low amounts of ERIO-3'-glucoside were now detected. Theincrease in the 3'-site for glycosylation compared with that atposition 7 is of special interest since it converts the B-ring froma diphenol to a functional 'monophenol' in terms of free o-phenolic groups.As these cultures contained no detectable DHM or its glycoside

or PDs, but did contain active reductases that converted DHMand its diol to gallocatechin (26), the block in PD synthesis mustbe at the level of synthesis of DHM. It is not known whetherDHM is synthesized in one step from DHQ or in two steps fromERIO with 5'-hydroxyeriodictyol as an intermediate. The accu-mulation of high concentrations of ERIO rather than DHQglycosides in cultures might be interpreted as supporting thelatter route, but neither 5'-hydroxyeriodictyol or its glycosidehave been detected in any of our cultures or leaves. Since bothERIO and DHQ glycosides were accumulated, however, theirconversion to flavan-3-ols or PAs might be considered the majorlimiting factor. Compared with the other species, leaves andcultures of Pseudotsuga contained the greatest variety of precur-sors.Ginkgo leaves and cultures did not accumulate any major

precursor, perhaps indicating a very tight enzyme complex anda highly regulated PA pathway. Ribes leaves accumulated highconcentrations of a DHM-glycoside relative to its PA content,indicating that a step leading to flavan-3-ols or PAs was limiting.In callus cultures, on the other hand, the amounts of the sameglycoside were low relative to a similar PA content, indicatingthat the production ofDHM might be the major limiting factorinstead.

In callus cultures of Ginkgo and Ribes, when compared withtheir intact leaves, the major flavan-3-ol with a triphenolic B-ring accumulated was the 2,3-trans-isomer, gallocatechin, ratherthan the 2,3-cis-isomer, epigallocatechin. In Ginkgo, this changewas accompanied by a 20-fold increase in the amount ofcatechin,the 2,3-trans-isomer with a diphenolic B-ring, while in Ribescallus this isomer was now found in detectable, but minoramounts. Such a change, of course, was not observed in ourPseudotsuga cultures since the 2,3-trans-flavan-3-ols were dom-inant in both leaves and cultures.The biosynthetic origin of the 2,3-cis-stereochemistry, or the

postulated enzymic condensation step in which an intermediatearising from a flavan-3,4-diol is 'added on' to a flavan-3-ol or analready existing oligomeric chain, are still unknown (23, 25).Furthermore, isotopic studies indicated that metabolic compart-mentation must be involved in order to account for the differ-ences in labeling patterns found in the flavan-3-ols, initiatingunits and extension units (22).

Since neither the major flavan-3-ols nor dimers detected in

culture may reflect the isomer pattern of the upper or extensionunits of the higher oligomers, the 2,3-cis-stereochemistry maystill be the major component of higher oligomers in our cultures.While the 2,3-cis-isomer is the most common one found inhigher oligomers, Ribes sanguineum leaves have been reportedto contain predominantly the 2,3-trans-isomer (9). The stereo-chemistry of the extension units of the higher oligomers in ourpresent Pseudotsuga cultures reported here have not been ex-amined, but those isolated from earlier cultures consisted pre-dominantly of the 2,3-cis-isomer (unpublished data of L. Porter,DSIR, Petone, New Zealand).

Little information concerning the regulation of this aspect ofthe PA pathway is available in the literature. Samejima et al.(17) reported briefly that the percent of 2,3-trans-isomers in theextension units of higher oligomers increased in light growncompared with dark grown cultures of another Gymnosperm,Cryptomeriajaponica.

CONCLUSIONS

We have examined PAs and related compounds in several cellcultures and compared them to the leaf tissues from which theywere derived. Each appears to have unique characteristics, butsome generalizations can be made.

(a) In the presence of NAA (or 2,4-D) and BAP in a ratio of10 (or 20): 1, callus or cell cultures from leaves of plants thataccumulate these secondary products appeared to favor the PApathway in contrast to other more highly oxidized flavonoids orto cinnamic acid derivatives. This may be due to the selection ofeither different cell types or to the two major factors known toincrease the accumulation of many flavonoids and other phe-nolics, i.e. higher light intensities and UV light present in naturalbut not in our growth chamber environments (5).

(b) In Gymnosperm cultures, there was a tendency towardgreater accumulation of PAs compared with leaves from whichthey were derived. This was not true for two Angiospermsstudied, Ribes sanguineum (Table III) and Leptarrhena pyroli-folia (unpublished data).

(c) In cultures with high PA contents, the PDs relative to PCsdecreased.

(d) The major flavan-3-ols and possibly dimers that accumu-lated in cultures were 2,3-trans rather than 2,3-cis-isomers. Priorstudies, however, indicated that the higher oligomers may stillconsist predominantly of 2,3-cis-units.

(e) Glycosidic flavanone and 3-hydroxyflavanone precursorsaccumulated in some, but not all cultures, and the type of auxinpresent during culture altered the relative amounts.Maintenance of cultures for this study of the PA pathway was

sometimes difficult, perhaps due to our constant selection forhigh PA content cultures. Because of their ability to precipitateproteins and carbohydrates, the higher oligomers of these sec-ondary products would be suicidal if not sequestered withinmembranes in the cell. Any leakiness of membranes will killcells, and some cultures started from the same parent tissueturned brown almost overnight, while others grew well. In addi-tion, some of our cultures show considerable morphologicalheterogeneity and may consist ofdifferent cell lines. The questionof the relationship of this to possible biochemical heterogeneityofPA accumulation will be discussed in a subsequent paper.

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3. CHAFE SC, DJ DURZAN 1973 Tannin inclusions in cell suspension cultures ofwhite spruce. Planta 113: 251-263

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13. JOHNSON MA, JA CARLSON 1982 Some redox considerations in conifer tissueculture. Proceedings of the 5th International Congress on Plant Tissue andCell Culture, pp 221-222

14. MARKHAM KR 1982 Techniques of Flavonoid Identification. Academic Press,New York

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17. SAMEJIMA M, T YOSHIMOTO 1983 The structural characteristics ofpolyflavanolsfrom the callus cells of Cryptomeriajaponica D. Don. Proceedings ofWoodand Pulping Chemistry Symposium, Tsukuba City, Japan, pp II 1-1 14

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19. SHEN Z, 0 THEANDER 1985 Flavonoid glycosides from needles of Pinusmassoniana. Phytochemistry 24: 155-158

20. STAFFORD HA, HH LESTER 1980 Procyanidins (condensed tannins) in greencell suspension cultures of Douglas fir compared with those in strawberryand avocado leaves by means of C,g-reversed phase chromatography. PlantPhysiol 66: 1085-1090

21. STAFFORD HA, HH LESTER 1981 Proanthocyanidins and potential precursorsin needles ofDouglas fir and in cell suspension cultures derived from seedlingshoot tissues. Plant Physiol 68: 1035-1040

22. STAFFORD HA, M SHIMAMOTO, HH LESTER 1982 Incorporation of ['4C]phen-ylalanine into flavan-3-ols and procyanidins in cell suspension cultures ofDouglas fir. Plant Physiol 69: 1055-1059.

23. STAFFORD HA 1983 Enzymic regulation of procyanidin biosynthesis: lack of aflav-3-en-3-ol intermediate. Phytochemistry 22: 2643-2646.

24. STAFFORD HA, HH LESTER 1984 Flavan-3-ol biosynthesis: The conversion of(+)-dihydroquercetin and flavan-3,4-cis-diol (leucocyanidin) to (+)-catechinby reductases extracted from cell suspension cultures of Douglas fir. PlantPhysiol 76: 184-186

25. STAFFORD HA, HH LESTER, U PORTER 1985 Further studies on the chemicaland enzymic synthesis of monomeric procyanidins (leucocyanidins or3'4'5,7-tetrahydroxyflavan-3,4-diols) from (2R,3R)-dihydroquercetin. Phy-tochemistry 24: 333-338

26. STAFFORD HA, HH LESTER 1985 Flavon-3-ol biosynthesis: the conversion of(+)-dihydromyricetin to its flavan-3-ol-diol (leucodelphinidin) and to (+)gallocatechin by reductases extracted from tissue cultures of Ginkgo bilobaand Pseudotsuga menziesii. Plant Physiol 78: 791-794

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28. STOTz G, P DE VLAMING, H WEIRING, AW SCHRAM, G FORKMANN 1985Genetic and biochemical studies on flavonoid 3'-hydroxylation in flowersof Petunia hybrida. Theor Appi Genet 70: 300-305

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