Plant Physiol. (1984) 74, 901-9060032-0889/84/74/0901/06/$01.00/0

Endoplasmic Reticulum as a Site of Phenylpropanoid andFlavonoid Metabolism in Hippeastrum'

Received for publication September 15, 1983 and in revised form December 6, 1983

GEORGE J. WAGNER* AND GEZA HRAZDINAAgronomy Department, University ofKentucky, Lexington, Kentucky, 40546-0091 (G. J. W); and CornellUniversity, Department ofFood Science and Technology, New York State Agricultural Experiment Station,Geneva, New York 14456 (G. H.)

ABSTRACT

The nature of bound forms of enzymes of phenylpropanoid ad flavo-noid metabolism have been investigated in Hippeastrum CV Dutch RedHybrid. Particulate components of petal homogenates were fractionatedon sucrose gradients and the EDTA shift method was employed tocharacterize membranes of the endoplasmic reticulum. In magnesium-containing gradients, a portion of phenylaanine lyase, chakcnesynthase, glucosyl transferase, and all of the trans-_ciamate 4-mono-oxygenase and NADH Cytochrome c reductase (the last an endoplasmicreticulum marker) were associated with membranes equilibrating at 1.18specific gravity. In gradients lacking magnesium and coning EDTA,the above activities-except chalcone synthase, which was lost-andprotein were diminished at 1.18 specific gravity and enhanced at lowerdensities characteristic of membranes of the smooth endoplasmic reticu-lum. These results are consistent with the contention that endoplasmicreticulum is a site of phenylpropanoid and flavonoid metabolism inHippestrum.

with ER vesicles has not been ruled out (22). It is possible thatER-chloroplast associations which exist in vivo (2) may be main-tained in certain chloroplast preparations.

In earlier studies with Hippeastrum petal protoplasts (8), wefound that 5 to 15% ofthe activities ofkey phenylpropanoid andflavonoid pathway enzymes was associated with a particultatesubcellular fraction which was enriched in ER membranes. Thereis no published evidence to support the presence on or in thetonoplast ofenzymes ofthese pathways or ofenzymes leading toother secondary products. No evidence was found to suggest theoccurrence of UDPG-flavonoid glucosyltransferase (an enzymenear the end of the flavonoid biosynthetic sequence) on or inisolated tonoplast ofHippeastrum (24).To expand our understanding of the intracellular location of

secondary product formation in plants, we fractionated petalhomogenate-derived membranes on sucrose density gradientsand utilized the EDTA shift method to demonstrate that variouskey enzymes ofphenylpropanoid and flavonoid metabolism are,at least in part, ER-bound.

Biochemical and ultastructural studies have provided evidenceto support the contention that phenylpropanoid and flavonoidbiosynthesis occur on the ER (5, 11, 22, 25). A number ofinvestigators have shown that certain enzymes ofthese metabolicpathways are associated with microsomal membranes (11, 20,22) and Czichi and Kindl (3) demonstrated coupling betweenPAL2 and C4-H in microsomal preparations of potato andcucumber. Compelling micrographs of Douglas Fir callus cellssuggest that 'tannin'-containing vesicles can arise from dilatedregions of the rough RER, (16), and other ultrastructural studies(25) suggest that ER is the site of tannin synthesis in severaltannin accumulating plants. Chloroplasts have also been sug-gested as being sites of phenylpropanoid and flavonoid metabo-lism, but the evidence is contradictory. Various flavonoids, cer-tain cinnamic acid derivatives (9) and some phenylpropanoidand flavonoid biosynthetic enzymes were reported to occur inisolated plastids (14). Other reports conclude that plastids lackkey enzymes ofthese pathways (9, 22). The possibility that plastidpreparations which showed enzyme activities were contaminated

' The investigation reported in this paper (No. 83-3-147) is in connec-tion with a project ofthe Kentucky Agricultural Experiment Station andis published with approval of the Director.

2 Abbreviations: PAL, phenylalanine ammonia lyase; C4-H, trans-cinnamate-4-monooxygenase; CHS, chalcone synthase; CI, chalconeisomerase; GT, glucosyl transferase; RER, ribosome-bearing ER; SER,smooth ER; sp. gr., specific gravity.

MATERIALS AND METHODS

Plant Materials and Biochemicals. Hippeastrum cv Dutch RedHybrid was grown as previously described (23). Petals wereharvested from anthesis or 1- to 2-d postanthesis flowers andtheir bases were removed prior to homogenization. Cellulysinwas obtained from Calbiochem. Pectinase (P-5146), Cyt c (TypeIII), NADH (grade III), antimycin A, quercetin, and naringeninwere purchased from Sigma. L-[U-'4C]phenylalanine (450 mCi/mmol), UDP-D-[U-'4Clglucose (250 mCi/mmol), [2-'4C]malo-nyl CoA (32.1 mCi/mmol) were obtained from New EnglandNuclear Co. and [3-'4C]cinnamic acid (60 mCi/mmol) fromAmersham Co. p-Coumaryl-CoA was synthesized according toHrazdina, et al. (7).

Preparation of Extracts and Protoplast Fractions. Petals (ap-proximately 20 g fresh weight) were homogenized in a chilledmortar with 10 to 15 ml of 0.5 M K2HPO4/KH2PO4, pH 8.0, 4mM mercaptoethanol, 3.0 g ofPVP (Polyclar AT) and 1.5 g HCIwashed sand. Homogenates were centrifuged at 2000g for 10min and the supernatants filtered through Miracloth before beingapplied to sucrose gradients (see below). All manipulations weremade at 4°C. Protoplasts and protoplast fractions were isolatedfrom petals as peviously described (23, 26). Gentle osmotic shockof protoplasts in 0.2 M K2HPO4/KH2PO4, pH 8.0, resulted in 3protoplast fractions: intact vacuoles, particulate cytoplasm, anda fraction enriched in cytosol. The particulate cytoplasm con-sisted of aggregated-filterable (attached to stirrer and filtered byglass wool) materials produced during vacuole emergence fromprotoplasts (23 and references therein) and contained most ofthe cellular organelles and membranes (23) as well as unlysed

901

WAGNER AND HRAZDINA

protoplasts. The fraction enriched in cytosol consisted of nonfil-terable components which remained in the buffer after removalof particulate cytoplasm and intact vacuoles. The enriched cy-tosol fraction was separated into residual nonfiltered particulatematerials (membranes which did not aggregate with particulatecytoplasm) and soluble components by centrifugation at100,000g. Particulate cytoplasm, a fraction enriched in cytosol,and vacuole fractions of protoplasts were prepared for enzymeassay as previously described (8), except that the particulatefraction was homogenized and assayed directly for NADH Cyt creductase activity.

Sucrose Gradient Separations. Extracts of tissue (pH approxi-mately 6.0 due to the effect of tissue acids) were monitored forenzyme activities and divided into two equal parts. One portionwas made 10 mm with MgCl2 and applied to a 6 to 45% w/w(Fig. 1) or a 6 to 52% w/w (Figs. 2 and 3) linear sucrose gradient(32 ml) containing 40 mm, K-phosphate, pH 8.0, 10 mM MgCl2,5 mM mercaptoethanol, and was centrifuged in a swinging bucketrotor at l00,000g for 16 h (Figs. 1 and 3) or 7.5 h (Fig. 2) at 4°C.An equivalent portion was made 10 mm with Na2EDTA andcentrifuged in similar gradients which lacked Mg2" and contained10 mm Na2EDTA (EDTA shift method). Magnesium was addedjust prior to separation of membranes on gradients instead ofduring homogenization to ensure that samples applied to +Mg2++ -Mg2+ gradients were identical prior to addition of Mg24 orEDTA. Gradients were prepared over a 60% w/w sucrose cush-ion. After centrifugation, gradients were fractionated into 1.2 mlaliquots by pumping from below with 65% w/w sucrose. Aliquots(100-200 IAI) of fractions were used directly for assay (see below).Enzyme Assays. PAL (EC 4.3.1.5) was monitored as described

in (10) with L-[U-'4C]phenylalanine and C4-H (EC 1.15.13.11)as described in (10) with [3-'4C]cinnamic acid. CHS, CL (EC5.5.1.6), and UDPG flavonoid GT (EC 2.4.1.9 1) were assayed asdescribed in (8). Quercetin (recrystallized 3x from hot aqueousethanol was used as substrate for the latter. Cyt c oxidase andNADH Cyt c reductase were assayed essentially as summarizedby Mathews (13) and protein according to Bradford (1). Specificgravity was determined at 20°C by refractive index measurementand anthocyanin was monitored as previously described (8).UDP glucose hydrolase was monitored as the release of ['4C]glucose from UDP-D-[U-'4C]glucose. ['4C]glucose migrated withthe solvent front in the chromatographic system described (8).

RESULTSEnzyme Analysis of Protoplast Fractions. Data of Table I

summarize the distribution of enzyme activities in fractions ofHippeastrum protoplasts. Results for CHS, CI and UDPG-fla-vonoid GT were reported earlier (8). A portion (5-15%) of therecovered activities of these enzymes was present in particulatecomponents recovered after lysis of protoplasts. Only a smallportion of certain activities which occurred in the fraction en-

riched in cytosol was sedimented at 100,000g. PAL and C4-Hwere not detected in Hippeastrum protoplasts; however, the latterwas found in the particulate cytoplasm fraction from Tulipaprotoplasts (not shown). Particulate cytoplasm and 100,000gsedimented fractions were enriched in ER membranes as indi-cated by the occurrence of antimycin A insensitive NADH Cytc reductase (Table I). The distribution patterns for Cyt c reductaseand Cyt c oxidase were similar to those observed earlier in similarexperiments using Tulipa petal and leaf protoplasts (23).

Gradient Analysis of Homogenate-Derived Membranes. At-tempts to separate and identify components of the particulatecytoplasm fraction ofprotoplasts were unsuccessful. We thereforerecovered membranes directly from petal tissue. Results obtainedafter separation of 2000g soluble components of a petal extracton 6 to 45% w/w linear sucrose gradients containing magnesium(+Mg2+), or lacking magnesium (-Mg2+, +EDTA) are shown inFigure 1. In the presence of Mg2+, Cyt c oxidase and NADH Cytc reductase equilibrated at 1.18 to 1.2 sp. gr. the expected regionofequilibration ofinner mitochondrial membrane and RER (18)in this gradient (Figs. 1, a and c, respectively). Activities at > 1.2sp. gr. represent components which accumulated at the gradient-sucrose cushion interface. In the absence of Mg2", NADH Cyt creductase activity completely shifted to a lower density (1.08 to1.16 sp. gr.) characteristic of SER (18). This result is consistentwith the partial or complete loss of ribosomes from RER (19).Protein profiles (Fig. Id) reflect the solubilization of ribosomalprotein which occurs when Mg2` is absent and endogenous Mg2`is chelated by EDTA. As expected, inner mitochondrial mem-brane (Cyt c oxidase, Fig. la) was unaffected by the absence ofMg2e. PAL activity occurred primarily as a peak centered at 1.07sp. gr. but a small peak of activity was observed at 1.16 to 1.19sp. gr. in the presence of Mg2+. The latter peak was diminishedin the absence of Mg2" with a concomitant increase in PALactivity in the region of SER, at 1.1 to 1.15 sp. gr. The samplezone gradient interface was at about 1.03, and as indicated byresidual anthocyanin (not shown), mixing ofsample zone micro-solutes occurred to about 1.07 sp. gr. The nature of the largePAL peak at 1.07 sp. gr. is considered in the discussion. C4-Hprofiles were very similar to those of the ER marker NADH Cytc reductase, indicating association of this enzyme with RER.Results obtained with 6 to 52% gradients are shown in Figure 2.Since RER membrane was completely retained within thesegradients (as compared with 6 to 45% gradients in Fig. 1) theeffects of depleting Mg2e (EDTA shift) on protein (Fig. 2d) andNADH Cyt c reductase (Fig. 2b) were clear and RER was definedas having a specific gravity of 1.16 to 1.21.As expected for ER membrane, NADH Cyt c reductase was

largely antimycin A insensitive (Fig. 2b). Anthocyanin (Fig. 2a)and protein (Fig. 2d) marked the sample zone-gradient interface(about 1.03 sp. gr.) and the region of microsolute diffusion into

Table 1. Summary ofthe Distribution ofEnzyme Activities in Fractions ofHippeastrum Petal ProtoplastsProtoplast Fractions

Enzyme Particulate Fraction enriched in cytosolCytoplasm ~~~~~~~~VacuoleCytoplasm l00,OOOgsoluble lOO,OOOg pellet

% ofrecovered activityChalcone synthase 5-15 80-95 1-4 obChalcone isomerase 5-6 94 0 obUDPG-flavonoid GT 6-12 88-96 1 1bNADH Cytcreductase 70 2 27 1.6Cyt c oxidase 72 0 28 0.2

* For method of preparation see -Materials and Methods" and (8).b Only soluble vacuole contents monitored.

902 Plant Physiol. Vol. 74, 1984

ENDOPLASMIC RETICULUM AS A SITE OF FLAVONOID METABOLISMSPECIFIC GRAVITY

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FIG. 1. Sucrose gradient (6 to 45% w/w sucrose) profiles of 2000gsoluble components of Hippeastrum petal homogenates centrifuged atI00,OOOg for 16 h at 4°C. 0 and A, the gradient which contained 10 mM

MgCI2; * and A, an otherwise identical gradient which lacked Mg2" andcontained 10 mM Na2EDTA. The experiment utilized the EDTA-shiftmethod to monitor ER membranes before and after removal of ribo-somes.

1 5 9 13 17 21 25FRACTION NUMBER

29

FIG. 2. Sucrose gradient (6 to 52% w/w sucrose) profiles of 2000gsoluble components of Hippeastrum petal homogenates centrifuged at100,000g for 7.5 h at 4°C. 0 and A, a gradient which contained 10 mMMgCl2; * and A, an otherwise identical gradient which lacked Mg2' andcontained 10 mM Na2EDTA. Only the -Mg2+ gradient was monitoredfor C4-H (X-X in 2a). Anthocyanin profile ofthe +Mg2' gradient (notshown) was identical to that of the -Mg2' gradient shown in Fig. la.NADH Cyt c reductase (2b) was measured in the presence (A, A) andabsence (0, *) of I t/ml antimycin A.

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this gradient (to about 1.08 sp. gr.). As expected, Chl, a markerfor thylakoid membrane, equilibrated at about 1.19 sp. gr. in-thepresence and absence of Mg2" (Fig. 2a). Similar to the resultshown in Figure lb, C4-H equilibrated in the -Mg2' gradientin the region expected for SER (Fig. 2a). UDPG-flavonoid GTactivity occurred in the gradients at low levels, but in the presenceof Mg2e, a small peak was observed in the region of RER. Thisactivity was diminished and perhaps partially solubilized in theabsence of Mg2e and increased somewhat at lower density (Fig.2c). CHS activity was not detected in +Mg2' gradient experi-ments shown in Figures 1 and 2. An alternative procedure wasutilized which resulted in the detection of low levels of this keyenzyme in similar gradients. In this procedure, the homogenatewas centrifuged at 5000g for 5 min, the resulting supernatantstirred with 1 g Dowex-l (P04 form, pH 8.0) to remove residualand inhibitory phenolic compounds and the mixture centrifugedat 10OOg for 5 min. The supernatant was divided among +Mg2"and -Mg2+, +EDTA gradients which were subsequently centri-fuged at 100,000g for 16 h at 4°C. Results (Fig. 3b) indicated theoccurrence of low levels of CHS activity throughout the +Mg2+gradient, but the highest activity occurred in the region of RERas characterized by the protein profile (Fig. 3c). The only activityobserved in the -Mg2+ gradient was in the sample zone. GT andUDP-glucose hydrolase activity (Fig. 3a) and protein (Fig. 3c)were only monitored in the +Mg2" gradient. Maximal GT activ-ity occurred at about 1.06 sp. gr. (qualitatively similar to thatobserved in Fig. 2c) but substantial activity also occurred in theregion of RER. Interestingly, and in contrast to that observed inFigure 2c, little GT activity was observed near the surface of thesample zone. UDP-glucose hydrolase, measured as the release of['4C]glucose from UDP-D-[U-'4C]glucose, occurred only in thedensity range expected for dictyosomes (1.06 to 1.14 sp. gr.),(18).

DISCUSSION

We have sought to determine if various enzymes of phenyl-propanoid and flavonoid metabolism are associated with intra-cellular membranes. Experiments with Hippeastrum protoplasts(Table I; Ref. 8) indicated that a small but significant portion ofthe total activities of CHS, CI, and GT recovered in fractions ofHippeastrum protoplasts occurred in the particulate cytoplasm(aggregated, glass-wool-filtered) fraction recovered after osmoticshock of protoplasts. ER and mitochondrial markers were pri-marily associated with this fraction, but about one third of thetotal activity of these marker enzymes occurred as sedimentablecomponents of the nonfilterable fraction which is enriched incytosol, indicating the presence of ER and inner mitochondrialmembrane in the latter fraction. In contrast, CHS, CI, and GT,which we have considered to be ER-associated, were not foundin significant amounts in this fraction. It is possible that ER(bearing flavonoid enzymes), which escapes entrapment in theparticulate cytoplasm fraction and therefore occurs in the frac-tion enriched in cytosol, may be stripped of weakly associatedenzymes during or after osmotic shock of protoplasts. Alterna-tively, ER in the particulate cytoplasm may represent a differentsize or compositional class of ER than that in the fractionenriched in cytosol. Phenylpropanoid and flavonoid biosyntheticenzymes (with the exception ofC4-H) are generally found to besoluble in homogenates of various tissues which contain them(22). The first enzyme of phenylpropanoid metabolism, PAL,was not observed in protoplast fractions of either Hippeastrumor Tulipa. Others have been unable to detect this enzyme inenzymically isolated protoplasts (22).C4-H, a particulate oxidoreductase which is considered to be

part of the core sequence in phenylpropanoid metabolism (6)was not detected in Hippeastrum protoplasts or protoplast frac-tions-reasons unknown-but was observed as a particulate

Plant Physiol. Vol. 74, 1984

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FIG. 3. Sucrose gradient (6 to 52% w/w) profiles of 5,000g soluble,Dowex 1-phosphate treated components of Hippeastrum petal homoge-nate centrifuged at 100,OOOg for 16 h at 4C. 0, X-X (3a), a gradientcontaining 10 mM MgC2; 0 (3b), an otherwise identical gradient whichlacked Mg2e and contained 10 mM Na2EDTA.

component (93% in the particulate cytoplasm fraction) of Tulipaprotoplasts (data not shown). This activity was only found inbound form in gradients used here to separate membranes oftissue homogenates (see below). CA-H has been shown in differ-ential centrifugation experiments to be bound to microsomes inSorghum (20), Helianthus (13), potato, and cucumber (3, 1 1). Invery important work which suggests the functioning of multien-zyme complexes in phenylpropanoid metabolism, Czichi andKindl (3, 1 1) observed cooperation or channeling between PALand C4-H in microsomal fractions of cucumber and potato.

Since only low levels of phenylpropanoid and flavonoid en-zyme activities occurred in the particulate fraction of protoplastsand PAL, and CA-H were not observed in Hippeastrum proto-plasts, we isolated membranes directly from Hippeastrum petaltissue and fractionated them on linear sucrose gradients. ERmembranes were characterized using the EDTA-shift method,

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LIXIII

ENDOPLASMIC RETICULUM AS A SITE OF FLAVONOID METABOLISM

first introduced by Sabatini et al. (19), first used by Lord et aL.(12) to characterize plant ER, and used by Saunders et al. (20)to demonstrate ER association of C4H in Sorghum. Thismethod is based on the ability of EDTA to strip ribosomes fromRER causing the membrane (SER) to equilibrate to a lowerdensity in a density gradient. Comparison of the equilibriumdensity ofmembrane on Mg2" versus EDTA-containing gradientscan serve to identify ER-bound components. Results reportedhere (Figs. 1-3) using this method suggest that a portion of PAL,CHS, GT, and all of the C4-H in Hippeastrum petal extracts isbound to ER. Since PAL, CHS, and GT are very soluble enzymes(21, 22), it is possible that in vivo a large portion or perhaps allof these enzymes are bound to ER.EDTA-shift experiments reported on here indicate that bound

forms do not represent nonspecific binding artifacts. We knowof no report which shows that nonspecific binding of proteins tomembranes occurs principally with ER membranes. Additionalevidence against nonspecific binding was obtained from studiesof Tulipa petal homogenates (data not shown) where we founda fraction of the observed activities of PAL, C4-H, CHS, CI,and GT, associated with components soluble at 20,000g butsedimented at I00,000g, would contain both RER and SER. Thedistribution of activities between the I00,000g soluble and insol-uble fractions was not greatly affected by addition of 10 mMEDTA before centrifugation of the homogenate at 20,000g.These results suggest that bound activities of Tulipa like those ofHippeastrum are not a result of divalent, cation-dependent ag-gregation phenomena. In addition, homogenization of tulippetals in a medium of high ionic strength (0.8 M K-phosphate,pH 8.0, 4 mm mercaptoethanol) also resulted in 20,000g soluble-l00,000g insoluble activity of the above enzymes. This resultsuggests that nonspecific ionic binding is not responsible for theoccurrence of bound activities (15, 17).While the ER-flavonoid enzyme association was the most

interesting point revealed by the gradient studies reported, severaladditional and interesting observations were made. The bulk ofPAL activity recovered in both +Mg2+ and -Mg2" gradientsshown in Figure la occurred at 1.05 to 1.08 sp. gr. after 16 h ofcentrifugation. This indicated sedimentation of PAL beyond theregion of sample zone-gradient mixing. No PAL occurred nearthe top of the sample zone. In contrast, soluble protein wasdistributed throughout the sample zone (fractions 1 throughabout 1 1, Fig. ld). This result suggests that PAL (monomer molwt -300,000) was either in aggregated form, was associated withcarbohydrate to form complexes of high mol wt, or was associ-ated with a multienzyme complex which survived dissociation.Hanson and Havir (6 and personal communication) have notedthe occurrence ofPAL-carbohydrate complexes in certain tissues.The possibility that the prominent PAL peak represents a mul-tienzyme aggregate is the most intriguing of the above. Similarexperiments with immature Hippeastrum petals resulted in pri-marily soluble PAL and less but significant activity at 1.05 to1.08 sp. gr. (data not shown).While the bulk of C4-H, the enzyme most likely to be asso-

ciated with PAL, equilibrated at a somewhat higher density thanPAL in the gradient described in Figure la, some C4-H activitywas observed at 1.04 to 1.07 sp. gr. in gradients shown in Figuresla and 2a. Also, C4-H activity has been observed at this densityin other gradients of Hippeastrum and Tulipa (data not shown).As noted above, 20,000g soluble-l00,000g insoluble activitieshave been observed in homogenates of Hippeastrum prepared in0.8 M K-phosphate buffer. Multienzyme complexes of the gly-colytic sequence are preserved in high ionic strength medium(4). These results are consistent with, but not proof of, theoccurrence ofmultienzyme complexes containing these enzymes.Stafford (21, 22) has discussed the evidence for, and logic of, theexpectation that multienzyme complexes function in secondary

product biosynthesis.In the gradients shown in Figure 2c, GT was observed through-

out the sample zone as well as in the regions of RER and SER.In contrast, in the gradient shown in Figure 3a, no GT occurrednear the top of the sample zone while a prominent peak ofactivity occurred at 1.05 to 1.08 sp. gr. It is possible that theconditions of sample preparation (or tissue condition) prior tothe gradient separation shown in Figure 3 preserved multienzymecomplexes of which GT was a part. Alternatively, these condi-tions may have led to artifactual aggregate formation in thesample zone. The later explanation is considered less likely sincethe most substantial activity observed in the experiment de-scribed by Figure 2c also occurred at about 1.04 to 1.08 sp. gr.The occurrence of PAL, GT (possibly CHS, Fig. 3b), and aportion of C4-H at about 1.04 to 1.08 sp. gr. (a density belowthat of membranes which are well characterized in higher plants,[18]), may indicate partial survival of multienzyme complexesin these experiments. Efforts are underway to determine the molwt and rate sedimentation characteristics of activities occurringin this region and to devise separation protocols which betterpreserve labile enzyme (CHS and C4-H) activities.

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3. CZICHI U, H KINDL 1977 Phenylalanine ammonia lyase and cinnamic acidhydroxylases as assembled consecutive enzymes on microsomal membranesof cucumber cotyledons: cooperation and subcellular distribution. Planta134: 133-143

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10. HRAZDINA G, GF PARSONS 1982 Induction of flavonoid synthesizing enzymesby light in etiolated pea (Pisum sativum cv Midfreeze) seedlings. PlantPhysiol 70: 506-510

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23. WAGNER G 1979 Content and vacuole/extravacuole distribution of neutral 26. WAGNER G 1983 Higher plant vacuoles and tonoplasts. In JL Hall, AL Moore,sugars, free amino acids and anthocyanin in protoplasts. Plant Physiol 64: eds, Isolation of Membranes and Organelles from Plant Cells. Academic88-93 Press, New York, pp 83-118