cell isoperoxidases in sweet potatoplants in relation to mechanical

Plant Physiol. (1976) 57, 74-79

Cell Isoperoxidases in Sweet Potato Plants in Relation to

Mechanical Injury and EthyleneReceived for publication July 9, 1975 and in revised form September 16, 1975

HELENA BIRECKA AND JAMES CATALFAMODepartment of Biological Sciences, Union College, Schenectady, New York 12308

PAUL URBANElectron Microscopy Laboratory, Veterans Administrations Hospital, Albany, New York 12208

ABSTRACT

Leaves and storage roots of sweet potato plants (Ipomea batatas)showed the same qualitative isoperoxidase patterns and a similar distribu-tion of distinctive isoperoxidases between the cell protoplast and cell wallfree, ionically bound, and covalently bound fractions. No changes in thequalitative isoenzyme spectrum were found in relation to age, mechanicalinjury, or ethylene action. Thus, as in tobacco plants, the cell isoperoxi-dases in sweet potato did not reflect the possible differential mRNAsynthesis in relation to organ, age, or injury. Transcription does not seemto be a limiting factor in injury- and ethylene-dependent peroxidaseenhancement during the first 24 hr.

The contribution of the wall ionically bound and protoplast fractionswas highest in young and old leaves, respectively. In the protoplast andwall ionically and covalently bound fractions, 14, 6, and 5 isoenzymebands were found; in addition, 4 bands, not detected in the protoplast,were also revealed in the covalently bound fraction. The distinctive"juvenile" and, developing with age, "mature" isoforms were mainlyfound in the ionically bound and protoplast fractions, respectively.

The injury-enhanced and/or ethylene-enhanced peroxidase develop-ment was most pronounced in young leaves. Ethylene suppressed someinjury-enhanced, had no effect on some other injury-enhanced, andgreatly promoted some of the injury-unaffected or enhanced isoperoxi-dases. After ethylene removal, an increase in the "mature" isoforms wasfound in the protoplast of intact leaves.

Electron microscopy of leaves revealed peroxidase in membrane-boundvesicles located mainly in the vacuole; a thin layer of reaction productswas also found on the wall's outer surface. No Golgi apparatus were seenin the ceUs of control or ethylene-treated intact leaves. In ethylene-treated intact or injured leaves accumulations of reaction products be-tween the plasmalemma and wall were also found. Numerous Golgiapparatus with dark stained vesicles were seen in injured, and especiallyin injured and ethylene-treated leaves; the vacuolar bodies seemed tooccur in very great number.

Previous investigation (2-4) has shown that sweet potatostorage roots differ significantly from tobacco pith and leavesand carrot roots in the peroxidase reaction to mechanical in-jury, C2H4, IAA, and actinomycin D. However, like tobaccotissues, sweet potato root isoperoxidases were distinctive intheir distribution between cell wall and protoplast fractions as

well as in their reaction to cut injury. The root isoperoxidaseswere also distinctive in their reaction to C2H4. This report dealsmainly with age-, mechanical injury-, and C2H4-related changesin cell peroxidase of sweet potato leaves.

MATERIALS AND METHODS

Healthy storage roots of Ipomea batatas were purchasedlocally. Plants grown from several buds of single roots, with 2 to3 or 7 to 8 shoots, were used in each experiment, especially inexperiments in which leaf isoperoxidases were compared to theroot isoenzymes.

Mechanical injury was induced in the 3rd to 4th (lower) and9th to 12th (upper) leaves in two ways: (a) by excising discs 5 to7 mm in diameter; the discs were exposed to air in the presenceor absence of 250 mm Hg2 (C104)2 or to ethylene (50 ,ul/l) in 750-ml air-tight Plexiglas boxes for 24 hr in shade; or (b) by gentlyrubbing half laminae of leaves, attached to the plants, withCarborundum which then was removed with water; the adja-cent half laminae were left untreated. The whole plants or theirshoots were placed in 50-liter Plexiglas boxes. Sections ofstorage roots 10 mm in diameter and 3 mm thick were treated asthe leaf discs.

Procedures related to isolation of the protoplast and wall free,ionically bound, and covalently bound peroxidase fractions,were similar to those previously used (3). The protoplast frac-tion was extracted with 20 mm phosphate buffer, pH 6. TritonX-100 extracts showed little or no peroxidase activity. The walldebris was treated twice with I M NaCl to isolate the ionicallybound fraction. Peroxidase determination, electrophoresis, andisoperoxidase scanning were carried out as previously. In orderto prevent gel-overloading with dominant isoperoxidases and todetect minor ones, several dilutions of particular samples wereused.The procedures used in electron microscopy studies were

similar to those previously applied to tobacco leaves (4). Thediscs were incubated with 0.2% diaminobenzoic acid and 0.001,0.01, or 0.1% H202 for 60 min in a 0.05 M tris buffer (pH 7.6).

RESULTS

Two and five experiments with roots and leaves, respec-tively, were carried out; all yielded similar results. Some of theresults are here presented. In storage roots, the contribution ofthe protoplast and wall fractions to the total cell peroxidaseactivity, and the enzyme reactions to cut injury and C2H4 (TableI) were similar to those observed in previous experiments (2, 3).The activities found in leaves of plants with shoots of various

ages were significantly higher than those in storage roots. Theolder the shoots the higher was the enzyme activity in theirleaves; in all cases the total activity in the lower leaves washigher than that in upper ones (Fig. 1). In young leaves with alow total peroxidase activity, the contribution of the protoplastfraction ranged between 19 and 32%, whereas that of the wallionically bound fraction ranged between 63 and 59%. With an

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ISOPEROXIDASES IN SWEET POTATO

increase in total activity the share of the protoplast fractionincreased to 70 to 80%. A significant increase in the activity ofthe wall covalently bound fraction was also observed withsenescence. The share of the extracted free peroxidase fractionin the walls was not greater than 1% of the protoplast activity.

Mechanical injury caused a significant increase in peroxidaseactivity, especially in young leaves (Table II). The increase wasobserved mainly in the protoplast and ionically bound fractions.In experiments with older plants, the peroxidase reaction toinjury was relatively weaker, but its character was similar.The enzyme reaction to C2H4 or to C2H4 in combination with

injury was much stronger than its reaction to injury alone. Thelargest increases occurred in the protoplast. The enhancementof the protoplast and wall ionically bound fractions in upperleaves, especially in young plants, was significantly greater thanthat in lower leaves. The wall covalently bound fraction alsoshowed a marked reaction to C2H4 and to C2H4 combined withinjury, this reaction being very pronounced in lower leaves.

Electrophoresis revealed the presence of 3 cathodic and 11

anodic bands in the protoplast fraction of the roots and leaves(Fig. 2). No qualitative differences in the isoperoxidase patternwere found in relation to leaf age or organ. In the wall ionicallybound fraction of leaves and roots, cathodic C and five anodic

Table I. Effect of Cut Injury and Ethylene on Peroxidase Activity inStorage Roots

Sections 10 mm in diameter and 3 mm thick were exposed for 24 hr toair in the presence or absence of 0.25 M Hg2(CI0j)2 or to air with50 ,ul/I C2H4 added.

Immediately Exposure after ExcisionPeroxidase after Exci- Air +

sion Hg2(CIO.)2 Air Air + C2H4

lAA/min g fresh Wt

Protoplast 64 848 1036 4402Wall

Free 8 34 51 56lonically bound 10 161 160 188Covalently bound 16 72 93 298

Total 98 1115 1340 4944

isoenzymes Al, A2, A5-A7, present in the protoplast, weredetected. In roots A5-A7 occurred in traces only. In bothorgans, the wall covalently bound fraction, 75 to 80% of which

Protoplast

10*

90

so

4*

3*

20

I,

466

1,752

M Wall ionically bound

Wall covalently bound

7

1

1764,040

1,3046600

3,600£6000

-I~I~ I - ~ I ~ L E- on=I PL.1 I % -I IU L U L

Plantswith 2-3 shoots

U L U L U Lp~~~~~~~~~~~~~~~

Plants with 7-8 shoots

FIG. 1. Peroxidase distribution in wall of upper and lower leaves ofplants with shoots of various ages. Lower and upper leaves correspondto the 2nd and 3rd and 7th to 10th (expanding) leaves, respectively, onthe shoot. The contribution of the free peroxidase fraction in the cellwall of lower leaves was cl1% of the total cell peroxidase activity. Traceactivities were found in the free fraction of upper leaves; values abovethe bars indicate total cell peroxidase activities (100%) - AA/min-gfresh weight.

Table II. Effect of Mechanical Injury and C2H,4 on Peroxidase Activity in LeavesHalf laminae of the 3rd to 4th (lower) or 9th to 12th (upper) leaves, attached to young plants with 2 to 3 shoots, were treated with Carborundum

or left intact. From the adjacent half laminae samples were taken immediately before treatment. The whole plants were placed in Plexiglas boxesand exposed for 24 hr to air in the presence of Hg2(CI04)2 or to air with 50 ,1A/I C21H4 added. Laminae discs 6 mm in diameter were cut out with acork borer from lower and upper leaves of old plants with 7 to 8 shoots and exposed for 24 hr to air in the presence or absence of Hg2(CI04)2 or to airwith 50 Ad/I C2H4 added.

Attached Leaves Leaf Discs

Peroxidase Fractions' Activity be- Increase in activity after treatment Increase in activity after treatmentfore treat- Injur1air Intact air + Injury air + Activity before

ment2 nuyar+ Itctar+ Ijr i treatment Air + Ar Ar+CHHg2(CIO.)2 C2H. C2H. Hg2(C10)2 Ar Ar+CH

AAImin-g fresh Xi

Upper leavesProtoplast 194 1,004 7,578 7,486 802 944 1,4% 6,404Cell wall

Free trace 6 7 38Ionically bound 355 936 1,206 1,828 1,000 757 830 1,096Covalently bound 56 40 204 396 204 44 56 108

Lower leavesProtoplast 610 419 2,174 2,078 10,102 1,068 1,240 5,857Cell wall

Free 16 33 46 62lonically bound 507 126 244 608 2,512 312 506 618Covalently bound 336 63 348 460 1,152 164 344 544

Trace activities were found in the wall free fraction in the attached upper leaves. In the lower ones, this fraction was <1% of the protoplastactivity.

2 Average from control half laminae of four plants. Differences in peroxidase activities between plants ranged from S to 10%.

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was released by cellulase and pectinase, contained C I and fouranodic bands (Al-A4) also present in the protoplast. In addi-tion, three slowly moving cathodic bands (C,l2) and one anodic(A,5) band were also revealed. Like the C2-C3 of the protoplastfraction, A,5 of the wall covalently bound one could be de-

Protoplast

l l \ I I I

3 2 11 2 3 4 S 6 7 a 9 10 11

Wall ionically bound

IL. . .

Wall covalently bound

-ziiiiiiiit11 I11C1-

c A

Origin

FIG. 2. Isoperoxidase patterns in leaf laminae and storage roots ofsweet potato plants. Starch gel electrophoresis at pH 8.3, 10 v x cm-'for 3 to 4 hr. Anodic (A) and cathodic (C) isoenzymes revealed usingguaiacol-H202 as substrates. A8 and A9 were detected in the wallcovalently bound fraction only in older leaves.

tected only using large root samples. The wall free fraction inroots contained A3 and A4; in old leaves, A9 (dominant) wasalso detected.

In the protoplast fraction of roots and young and old leaves,A4, Al, and A9 were dominant, respectively (Table III). Inroots as well as leaves of both ages, Al in the wall ionicallybound fraction and A3-A4 in the covalently bound one weredominant (Tables IV and V). The wall ionically bound fractionin leaves showed a significantly higher activity of CI, and espe-

cially of Al when compared to the protoplast fraction. Theactivity of A3-A4 in the wall covalently bound fraction washigher than or similar to that in the protoplast fraction.The greatest age-dependent increase in activity was shown by

A9 in the protoplast; smaller increases were also shown by Alin the wall ionically bound fraction and A3-A4 in the wallcovalently bound one. No significant age-dependent increaseswere found in the activity of Cl-C3, A5-A7, and A0O-AI 1.The isoperoxidases were distinctive in their reaction to me-

chanical injury and C2H4. In the protoplast of all tissues, C2showed the greatest increase in activity in response to injury;this increase being partially suppressed by C2H4, especially inleaves. Relatively low activities of C2 were also found in thewall free and ionically bound fractions of injured tissues. Injurystrongly enhanced the activity of CI and especially Al in theprotoplast fraction of young leaves and storage roots. Thegreatest increase in these isoforms was found in the wall ioni-

Table III. Effect of Mechanical Injury and C2H,4 on Protoplast Isoperoxidases in Storage Roots and Attached Leaves

Root Sections Attached Leaves

Treatment Upper LowerIsoperoxi- Treatment Treatment

dases Immediately Immediately Immedi-after excision Air + Air + Immediately bedi-Hg2(C10 , 2 before treat Intur+ Intact air + C2H Injured air + ately before Injry air Intact air Injured air

H2(CoI042 ntcai+c1 C,H4 Hg2(Clo4)2 + C2H, + C2H4

AA/min-g fresh stt

CathodicC3 trace 20 17 2 62 8 46 1 19 2 11C2 trace 266 235 2 589 10 221 1 321 3 76Ci 1 94 201 28 73 1176 818 32 31 189 204

AnodicAl 7 128 388 75 182 1672 1102 122 131 708 389A2 4 29 48 12 60 372 244 28 24 136 121A3 8 65 101 18 41 80 83 43 44 110 82A4 23 24 35 8 12 21 20 34 40 53 59A5 2 8 241 4 8 27 11 4 10 11 8A6 11 189 160 23 56 64 44 25 64 82 64A7 3 10 44 8 12 192 92 20 33 106 40A8 1 11 992 4 40 2288 2220 52 56 400 374A9 3 49 10 43 1174 821 245 252 772 512

AlIt 1 5 1938 trace trace 252 1200 3 4 92 306All 436 1200 ~~~~~~~~~~~~~~~~~~~~120442

Table IV. Effect of Mechanical Injury and C2H4 on the Wall lonically Bound Isoperoxidases in Attached Leaves

Upper Leaves Lower Leaves

*soperoxidases Increase after treatment Increase after treatmentActivity before Activity before

treatment Injury air + Intact air + C214 Injury air + C2H4 treatment Injury air + Intact air + C2H4 Injury air + C2H4Hg2(CI04)2 nuyar+~H Hg2(CI04)2

AAiming fresh vt

C2 56 4 41 2C 1 77 222 286 408 84 10 41 112Al 227 533 743 1230 386 53 152 447A5-A72 51 125 140 161 35 22 40 38A8-A 11 37 25 11 9

' Including the activity of A2 amounting to 4 to 6% that of Al.2 Injury- or C2H4-enhanced A6 or A7, respectively.

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Table V. Effect of Mechanical Injury and C2H4 on Activity of WallCovalently Bound Isoperoxidases in Attached Leaves

Activity before Treatment Increase after Injury and Exposureto C2,H

Isoperoxidases' Leaves

Upper Lower Upper Lower

AA lminig fresh wt

CathodicC 1-2 trace 39 3Cl 3 21 4

AnodicAl 4 30 2A2 13 46 2 9A3-A42 30 117 27 129A15 3 32 2A8-A93 40 84 133AIO-All 286 189

' About 75 to 80% of the covalently bound isoperoxidases werereleased after treatment with cellulase and pectinase. The calculationswere made taking into account the total activity of the fraction.

2 Injury- or C2H4-enhanced A3 or A4, respectively.3 A9 was the prevailing isoenzyme.

cally bound fraction, and no increase was found in the cova-lently bound one. In the young leaves as well as the roots,injury also enhanced slightly some other isoperoxidases in par-ticular fractions. However, A4, A5, A7 and AIO-AI I did notreact to mechanical injury.

Six isoperoxidases, Cl, Al, and A8-All reacted to C2H4most strongly, their increase being found mainly in the proto-plast. A large increase of Cl and Al was also observed in theionically bound fraction; small amounts of A8-AI I in this frac-tion may be due to artifacts. A8-AI I were also found in thecovalently bound one. C2H4 also enhanced the activity of A7 inthe protoplast and wall ionically bound fractions and of A4 inthe wall covalently bound one.C2H, in combination with injury induced a smaller increase in

the activity of A l and A9 and a greater increase in that of AI1-All in the protoplast. The activity of Al in the ionically boundfraction was higher than in the corresponding fraction of intactethylene-treated leaves. The activity of the covalently boundAIO-A 1 amounted to 14 to 20% of that in the protoplast. Thesetwo isoforms, released by cellulase and pectinase, showed aslightly greater electrophoretic mobility than did the corre-sponding bands present in the protoplast. A8-A 11 were de-tected in the wall free fraction of C2H4-treated root sections andleaves.

Intact C2H4-treated leaves did not show significant changes inthe activity of the wall peroxidase fractions I and 5 days afterthe plants were flushed with C2H4-free air and removed fromthe Plexiglas boxes. The activity of the protoplast fraction inlower and upper leaves increased in this period by 32 and 23%,respectively. In both, a significant increase in the activity of A9was found. In upper leaves, this increase was accompanied bysome decrease in the activities of A8 and A 10-A 1 1; in the lowerleaves a slight increase in the activity of C2 was found 5 daysafter C2H4 removal.

Electron microscopy of upper and lower control leaves re-vealed reaction product in membrane-bound vesicles locatedprimarily in the vacuole and occasionally in the ground cyto-plasm (Fig. 3a). The vesicles contained both spheres of densereaction product and a more electron transparent background.There was no apparent spatial relationship between the vesiclesand Golgi apparatus. In fact, no Golgi apparatus could be seenin control leaf cells. The cells also had a thin layer of reactionproduct on the wall outer surface (Fig. 3a). At the junction

between cells, this layer was not in the position of the middlelamella. No specifically localized reaction products could bedetected in the walls. The peroxidase layer on the wall outersurface was seen in all treatments.

In mechanically injured upper leaves exposed to air in thepresence of mercuric perchlorate, the extracellular peroxidaselayer was stained more heavily and in areas of large intercellularspaces there were some aggregations of reaction products. Thevacuolar bodies were again present and showed both deepstaining and the more electron transparent matrix. In these cellsGolgi apparatus were often found, and some of the vesiclescontained reaction product.

Like the control, intact leaves treated with C2H4 possessedvacuolar bodies which seemed to occur in a greater number. Inaddition, spherical or oval accumulations of reaction productwere found between the plasmalemma and the cell wall (Fig.3b). These aggregations did not have the electron transparentmatrix. Golgi apparatus were not seen in the cells of intactC2H4-treated leaves in any experiment.Numerous Golgi apparatus showing dark reaction product

were found in mechanically injured leaves treated with C2H4(Fig. 3c). No photographs showed unequivocal fusion of theGolgi vesicles with either the tonoplast or the plasmalemma.The proximity of the Golgi vesicles to either of these membranesand the presence of reaction product nearby in the vacuole orthe space between the plasmalemma and the cell wall is sugges-tive of a possible secretory function of the stained vesicles. Theinjured C2H4-treated tissues also contained spherical or ovalaccumulations of reaction product between the plasmalemmaand cell walls. The vacuolar bodies seemed to occur in a verygreat number in this tissue.When incubated in the presence of 3-amino- 1,2,4-triazole, the

vacuolar bodies showed a slight decrease in staining, indicatingthat catalase could have contributed to the formation of thereaction product. In neither treatment did the endoplasmicreticulum show any significant staining.The described accumulations of reaction products were seen

in cells incubated not only with 0.001% but also with 0.01 and0.1% H202. However, high concentration of H202 affected theground cytoplasm.

DISCUSSION

The great attention paid to isoforms of various enzymes,including peroxidase, in recent literature is due partially to thefact that isoenzyme expression has been linked very often todifferential gene action during cellular differentiation, organismdevelopment, injury, or infection (15).

In a previous study (4) on three tobacco varieties no differ-ences in the qualitative pattern of cell isoperoxidases werefound between the stem pith and leaves. Neither were anyqualitative differences found in relation to age, mechanicalinjury, or leaf infection. In this study on sweet potato plants noqualitative differences in the cell isoperoxidase pattern could befound between storage roots and leaves. Neither were anyqualitative differences detected in relation to the stem leaf posi-tion, age, mechanical injury or C2H4 effects. The two fast mov-ing cathodic isoenzymes (C2-C3) which were not previously de-tected in roots immediately after excision (2, 4) could be de-tected in large samples of intact roots; these two isoforms wereeasily detectable in intact leaves. Thus, at least in the two plantspecies under consideration, the cell peroxidase patterns do notreflect the possible differential mRNA synthesis in relation toorgan, age, or injury. In sweet potato root sections actinomycinD stimulated the activity of injury- and C2H4-dependent peroxi-dase development (2), indicating that in both cases the processof transcription was not a limiting factor in the enzyme enhance-ment.

In tobacco pith or sweet potato roots, cycloheximide had an

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BIRECKA, URBAN, AND CATALFAMO

V

C

3bM

-:.r;T,*..

-.i i9fS~0 ' s +

g; 5 ,., <' t/ffit < 9...

FIG. 3. a: Control mesophyll cell. Peroxidase product is seen indense bodies in vacuole, large arrow, along cell wall, white arrow,between plasmalemma and cell wall, small arrow. C, chloroplast; M,mitochondria; Mi, microbody; V, vacuole. x 16,000. b: Intact meso-

inhibitory effect on the injury- or injury and C2H4-dependent,respectively, isoperoxidase enhancement, thus indicating thatde novo synthesis of the isoenzymes is involved.

Cycloheximide has been considered as a nonspecific inhibitorof protein synthesis in vivo, also affecting other processes in thecells (12). Thus, the observed inhibitory effect of cycloheximidecould be an indirect one related to processes which are trig-gered by injury, perhaps including the formation of a woundhormone and proteinase inhibitor (8, 14). A more direct proof ofsynthesis de novo of isoperoxidases, enhanced by injury andC2H4, was given for sweet potato roots using blasticidin S and'4C-leucine (16). In injured horseradish roots, cycloheximideinhibited '4C-leucine incorporation into the peptide portion ofisoperoxidases, but had little effect on the incorporation of

V

phyll cell treated with ethylene. Note reaction product between plasma-lemma and cell wall. x 20,000. c: Injured mesophyll cell treated withethylene. Golgi apparatus (G) contains peroxidase reaction product. x55,000.

neutral sugars (10). On the other hand, cycloheximide did notaffect the injury-dependent increase of peroxidase activity incarrot roots (2). Thus, one may assume that in the sweet potatoroots and leaves under study synthesis de novo could havetaken place.

In TMV-infected and/or injured tobacco leaves, peroxidasewas detected in the endoplasmic reticulum, Golgi apparatus,vacuole, cell walls, and intercellular spaces. Secretions of thereaction product by Golgi vesicles into the vacuole and cell wallwere also observed, indicating a sequence of glycoenzymesynthesis and transport similar to that observed in mammaliancells as well as recently observed in plant cells (7). However,neither pith nor leaf isoperoxidases reacted to C2H4 in tobaccoplants. Electron microscopy of sweet potato leaves showed a

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somewhat different picture. There is not doubt that a greatportion of the peroxidase of intact and especially injured and/orC2H4-treated cells is located in the vacuole. However, thevacuolar membrane-bound vesicles with reaction product andspherical or oval-shaped accumulations of reaction productbetween the plasmalemma and the wall of ethylene treated cellswere detected in sweet potato leaves, but were not found intobacco leaves. In C2H4-treated intact leaves, the great enhance-ment of peroxidase activity was not accompanied by activationof the Golgi apparatus. The latter was observed only in mechani-cally injured tissues, and especially in tissues which were in-injured and treated with C2H4. If ethylene induced in intactleaves de novo. synthesis of C2H4-sensitive isoperoxidases,Golgi vesicles do not seem to take part in synthesis of thecarbohydrate moiety of these glycoenzymes. Chrispeels (5) hasproposed that glycosylation of hydroxyproline residues in exten-sion occurs within the cytoplasm, and that the glycoprotein issubsequently transferred to the cell wall by a smooth surfacedmembrane, probably the smooth endoplasmic reticulum. It ispossible that a similar process took place with regard to some

C2H4-enhanced isoperoxidases and their transport into the vac-uole and/or cell wall in sweet potato leaves. There is some

evidence that peroxidase, at least in horse radish, does notcontain hydroxyproline (I1, 17). The great accumulation ofperoxidase in the vacuole in C2H4-treated leaves would indicatethat this hormone could cause an increase in the permeability ofthe tonoplast as observed in morning glory flowers (9), althoughthe effect of C2H4 on membrane permeability is generally con-

sidered insignificant (1).The electron micrographs as well as the analytical data con-

firmed our postulation that peroxidase reactions to C2H4 isdifferent from its reaction to injury. In the roots and leaves ofsweet potato, C2H4 suppressed some injury-enhanced, had noeffect on some other injury-enhanced, and greatly promotedsome of the injury-unaffected or enhanced isoperoxidases.The isoperoxidase distribution among various cell fractions

does not seem to be significantly affected by possible extractionartifacts. As in tobacco plants, young leaves of sweet potatoshowed a relatively low peroxidase activity in the protoplastfraction and a high activity of the wall bound fractions as

compared to old leaves. One could also distinguish to someextent "'juvenile'" from "mature" isoperoxidases, as in to-bacco. Mechanical injury most enhanced the "juvenile" iso-form, moving it to the wall ionically bound fraction, and also aminor cathodic isoenzyme remaining mainly in the protoplast.The mode of enhancement of these isoforms in the cell mayperhaps be different. C2H4 affected the "'juvenile," "mature,"and some of the minor anodic isoperoxidases. Thus, its effect

seems to be less specific than that of injury. However, bothinjury and C2H4 had their greatest effect in young leaves. Asimilar age-dependent response to injury was observed in to-bacco leaves and a similar response to C2H4 was found in peainternodes (13). C2H4 increased the wall covalently bound frac-tion to a much greater extent than did injury. Neither injury northe hormone affected isoperoxidases which seem to be charac-teristic only of this fraction, unless they are artifacts of theanalytic procedure. Although the isoperoxidases were quitedistinctive in their distribution between the protoplast and cellwall fractions, any speculation as to their possible role in thewalls or protoplast cannot be substantiated by the data ob-tained. The significant increase in the vacuolar peroxidase ininjured and/or C2H4-treated tissues indicates an important elimi-nation (temporary?) of the enzyme from metabolic processes inthe cytoplasm.

LITERATURE CITED

1. ADELES, F. B. 1973. Ethylene in Plant Biology. Academic Press, New York. pp. 237-279.2. BIRECKA, H. AND A. MILLER. 1974. Cell wall and protoplast isoperoxidases in relation to

injury, indoleacetic acid, and ethylene effect. Plant Physiol. 53: 569-574.3. BIRECKA, H.. K. A. BRIBER. AND J. L. CATALFAMO. 1973. Comparative studies on tobacco

pith and sweet potato root isoperoxidases in relation to injury, indoleacetic acid, andethylene effects. Plant Physiol. 52: 43-49.

4. BIRECKA, H., J. L. CATALFAMO, AND P. URBAN. 1975. Cell wall and protoplast isoperoxi-dases in tobacco pith in relation to mechanical injury and infection with tobacco mosaicvirus. Plant Physiol. 55: 611-618.

5. CHRISPEELS, M. J. 1969. Synthesis and secretion of hydroxyproline containing macromole-cules in carrot. I. Kinetic analysis. Plant Physiol. 44: 1187-1193.

6. DASHEK, W. V. 1970. Synthesis and transport of hydroxyproline-rich components insuspension cultures of sycamore-maple cells. Plant Physiol. 46: 831-838.

7. GARDINER, M. AND M. J. CHRISPEELS. 1975. Involvement of Golgi apparatus in thesynthesis and secretion of hydroxyproline-rich cell wall glycoprotein. Plant Physiol. 55:536-541.

8. GREEN, T. R. AND C. A. RYAN. 1972. Wound-induced proteinase inhibitor in plant leaves: a

possible defense mechanism against insects. Science, 175: 776-777.9. HANSON, A. D. AND H. KENDE. 1975. Ethylene-enhanced ion and sucrose efflux in

morning glory flower tissue. Plant Physiol. 55: 663-669.10. LEW, J. Y. AND L. M. SHANNON. 1973. Incorporation of carbohydrate residues into

peroxidase isoenzymes in horse radish. Plant Physiol. 52: 462-465.11. LiN, E. H. AND D. T. A. LAMPORT. 1974. An accounting of horse radish peroxidase

isoenzymes associated with the cell wall and evidence that peroxidase does not containhydroxyproline. Plant Physiol. 54: 870-876.

12. MCMAHAN, D. 1975. Cycloheximide is not a specific inhibitor of protein synthesis in vivo.Plant Physiol. 55: 815-821.

13. RIDGE, I. AND D. J. OSBORNE. 1970. Regulation of peroxidase activity by ethylene inPisumsativum. Requirements for protein and RNA synthesis. J. Exp. Bot. 21: 720-734.

14. RYAN, C. A. 1974. Assay and biochemical properties of the proteinase inhibitor-inducingfactor, a wound hormone. Plant Physiol. 54: 328-332.

15. SCANDOLIOS, J. G. 1974. Isoenzymes in development and differentiation. Annu. Rev.Plant. Physiol. 25: 225-258.

16. SHANNON, L. M., I. URITAINI, AND H. IMASEKI. 1971. De novo synthesis of peroxidaseisozymes in sweet potato tubers. Plant Physiol. 47: 493-498.

17. SHIH, J. H. C., L. M. SHANNON, E. KAY, AND J. Y. LEW. 1971. Peroxidase isoenzymesfrom horse radish roots. IV. Structural relationship. J. Biol. Chem. 246: 4546-4551.

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cell isoperoxidases in sweet potatoplants in relation to mechanical

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