Ultrastructure of Tomato Fruit Ripening and the Role ofPolygalacturonase Isoenzymes in Cell Wall Degradation
Received for publication November 16, 1982 and in revised form March 17, 1983
PHILIP R. CROOKES1 AND DONALD GRIERSONDepartment ofPhysiology and Environmental Science, University ofNottingham, School ofAgriculture, SuttonBonington, Loughborough, Leicestershire LE12 SRD United Kingdom
ABSTRACT
Ultrastructural changes in the pericarp of tomato (Lycpekon escuden-tun Mill) fruit were followed during ripening. Ethylene production wasmonitored by gas chromatography and samples analyzed at successivestages of the ripening process.
Changes in the cytoplasm}c uktrastructure were not consistent with thesuggestion that ripening is a 'senescence' pbenomenon. A large degree ofultrastructural organization, especially of the mitochondria, chromoplasts,and rough endoplasmic reticulum, was retained by ripe fruit.
Striking changes in the structure of the cell wall were noted, begnningwith dissolution of the middle lamella and eventual disruption of theprimary cell wall. These changes were correlated with appearance ofpolygalacturonase (EC 3.2.1.15) isoenzymes. Application of purifli tomatopolygalacturonase lsoenzymes to mature green fruit tissue duplicated thechanges in the cell wall noted duing normal ripening. Possible roles of thepolygalacturonase isoenzymes in cell wall disorganizadon are discussed.
One of the most characteristic changes during the ripening offleshy fruits is a decrease in firmness. This has been shown to beassociated with an increase in the activities of cell wall degradingenzymes, particularly PG2 (5, 7), although a role has been sug-gested for cellulase (4, 11).Tomato fruit contains at least two isoenzyme forms of PG (13,
22, 25). It has been suggested that some interconversion of the twoforms can occur (23) and that the increase in PG activity andprotein noted during ripening is due to de novo synthesis of PG(24). Changes in the mRNA populations of ripening avocado (3)and tomato (14) have been reported, and it has been suggestedthat at least some of the control of ripening occurs at the level oftranscription and translation (3, 6).
Conversely, it has been suggested that ripening is a 'senescence'phenomenon (18) during which there is an increase in permeabilityleading to a loss of compartmentalization which regulates theactivity of enzymes involved in ripening (19). Evidence for thissuggestion has come from work showing a loss of cellular orga-nization at the ultrastructural level during ripening (1). Morerecently, it has been suggested that this interpretation is question-able and observed changes in permeability and cellular organiza-tion may be due to osmotic damage to the delicate cells of ripefruit (12).The aim of this work was to examine the degree of structural
' Recipient ofa Science and Engineering Research Council postgraduatestudentship.
2Abbreviations: PG, polygalacturonase; EM, electron microscopy;PAGE, polyacrylamide gel electrophoresis.
organization retained by ripening tomato fruit. It was hoped thata combined study of the changes in isoenzymes, the pattern ofwall dissolution, and the effect of applied purified enzymes wouldhelp to clarify the role of the PG isoenzymes and cellulase in fruitsoftening.
MATERIALS AND METHODS
Plant Material. Tomato plants (Lycopersicon esculentum MiWlcv Ailsa Craig) were grown in a glasshouse and fruit was harvestedat the mature green stage just before being used for the experi-ments.Measurement of Ethylene Production. Fruit were kept in glass
jars in a growth room (daylength 16 h, 24°C day, 14WC night).The jars were sealed daily and the amount of ethylene accumu-lating over 1 h was measured by GC using an Alumina Fl column.
Electron Microscopy. Small pieces of pericarp were cut andfixed in 3% (v/v) glutaraldehyde in 20 mm phosphate buffer (pH7.0) for 2 h at room temperature, rinsed in buffer, and postfixedin 1% (w/v) aqueous osmium tetroxide for 2 h at room tempera-ture. Fixed tissue was dehydrated through a graded alcohol seriesincluding 1% (w/v) phosphotungstic acid in the first absolutealcohol step and embedded in acrylic resin (London Resin Co.,Basingstoke, England). Sections were cut using a Reichart OM U2ultra micro-tome, contrasted with Reynolds lead citrate and ex-amined in a Phillips EM300 electron microscope.Enzyme digestions were carried out prior to fixation using 75
mg fungal cellulase (BDH Chemicals Ltd., Poole, England) in 2ml 0.1 M acetate buffer (pH 5.0) for 2 h at 40°C, 100 ,ug purifiedPGl in 2 ml of 0.15 M NaCi, 0.05 M sodium acetate (pH 3.8)overnight at 25 °C or 250 ,ug purified PG2 in the same buffer andconditions. Purified tomato isoenzymes were prepared as de-scribed previously (22). One g of pericarp tissue from a maturegreen fruit was used in each experiment. Tissue was also incubatedin buffer solutions alone prior to fixation.Enzyme Extraction. Total cell wall bound proteins were pre-
pared as described previously (22). Tomato pericarp was slicedinto water (1:1 w/v) and homogenized using a Polytron homoge-nizer (Kinematica Gmbh, Luzern, Switzerland). The homogenatewas centrifuged at 2,400g for 10 min and the pellet resuspendedin 1 M NaCl. The pH was adjusted to 6 with 1 M NaOH and thewhole stirred for 3 h at 4°C. Debris was sedimented for 10 min at2,400g and the supernatant filtered and made to 75% saturationwith (NH4)2SO4. The precipitate was sedimented for 20 min at10,000g and resuspended in 0.15 M NaCl (pH 6). The extract wasdialyzed against 0.15 M NaCl (pH 6) and used as a crude cell wallprotein preparation.PG was assayed in 1 ml of 0.5% (w/v) polygalacturonic acid,
0.15 M NaCl, 0.05 M sodium acetate (pH 3.8) at 25°C. Reducinggroups were detected by the arsenomolybdate method of Nelson(10).PG isoenzymes were estimated by heat stability. Samples of
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POLYGALACTURONASE AND THE ULTRASTRUCTURE OF RIPENING
800
700
;C7 600CE 500YEcC
I 400
( 3000.
200
100
00 1 2 3 4 5 6 7 8 9 10 11
DAYS FROM ONSET OF ETHYLENEPRODUCTION
FIG. 1. Changes in PG isoenzymes during ripening. (0), Total PGactivity; (0), PGl activity according to heat stability.
crude extract were heated (650C, 5 min), cooled, and assayed asdescribed. Heating removes 100%1o of isoenzyme 2 activity but only10%o of isoenzyme 1 activity (22).Polyacrylamide Gel Electrophoresis. Proteins were fractionated
in a 10%o acrylamide gel (15). Enzyme activity was detected asdescribed by Lisker and Retig (9). The gel was washed in waterand incubated in 20 mm acetate buffer (pH 4.5) for 10 min. Thiswas replaced by 1% (w/v) polygalacturonic acid in the same bufferfor 15 min at 370C and, after washing, the gel was stained with0.05% (w/v) ruthenium red and destained in water.
RESULTS
Changes in PG Isoenzymes during Ripening. PG activity wasfirst detectable 2 or 3 d after the onset of ethylene production androse to a level of about 750 nmol galacturonic acid produced g-'fresh weight min- after about 11 d. Initially, all the activity wasascribable to PGl according to heat stability. As ripening pro-ceeded, PG2 activity was detected and increased at a greater ratethan PG1 until the latter accounted for only 27% of total PGactivity (Fig. 1).The estimates of PG isoenzyme activity were confirmed by
fractionation by PAGE under nondenaturing conditions. Isoen-zymes 1 and 2 were clearly separated and PG2 was resolved intotwo bands of activity (Fig. 2). The pattern of isoenzyme changesfound by the heat-stability and nondenaturing PAGE methodswere similar.
Ultrastructural Changes during Ripening. The pericarp of ma-ture green tomato fruit was found to be composed of largeisodiametric parenchymous cells of approximately 300 ,um diam-eter. Seen at the EM level, the cytoplasm was confined to a narrowlayer about 10 pm thick adjacent to the cell wall. The cytoplasmwas vacuolate but contained a number of common organellesincluding chloroplasts containing numerous grana and plastoglob-uli (Fig. 3A) and often starch grains, ER (Fig. 3B), and mitochon-
A B C D E F
FIG. 2. Polyacrylamide gel of tomato cell wall proteins stained for PGactivity. Regions of activity appear light on the dark background. Lane A,purified PGl; lane B, purified PG2; lanes C-F, 4, 5, 7, and 12 d from theonset of ethylene production, respectively; lane G, cell wall proteins froma Never-ripe fruit at the same stage as that represented in Figure 6D.
dria (Fig. 3B). The cell wall was seen to be composed of denselypacked fibrils in an electron-translucent matrix (Fig. 3, A and B).The middle lamella was visible as a more electron-dense regionbetween the walls of adjacent cells (Fig. 3, A and B).One or 2 d after the onset of ethylene production, few changes
were noted in the cells (Fig. 3, C and D). Fewer and smaller starchgrains were seen in the chloroplasts, crystalloid microbodies werepresent, and ER was visible which, at higher magnifications, wasseen to have ribosomes attached (Fig. 3D).Three or 4 d after the onset of ethylene production, a change in
the appearance ofthe cell wall was noted along the middle lamella(Fig. 3, E and F). This was coincident with the first detectable PGactivity, which was almost entirely in the form of isoenzyme 1according to heat stability (Fig. 1) and mobility during PAGE(Fig. 2). Chloroplasts, although still present (Fig. 3F), were fewerin number and the appearance of immature chromoplasts wasnoted (Fig. 3E).Four to 5 d after the onset of ethylene production, cell wall
disruption was much more advanced (Fig. 4A), and the transitionof chloroplasts to chromoplasts complete (Fig. 4, A and B).Chromoplasts were much as described previously (17). Lycopenewas largely present in the form of crystalloid remnants (Fig. 4B),the angular lycopene crystals being lost during dehydration leav-ing undulating membranes transversing electron-translucent areas.Rod-shaped crystalloids were also seen. Plastoglobuli remained inthe plastids. These are thought to contain carotenoid and xantho-phyll pigments (8).The pattern and extent of cell wall disruption is shown in Figure
4, A and B. At this stage, PG activity was about 200 nmolgalacturonic acid produced g-l fresh weight min-, approximately80%o of which was in the form of PG I (Figs. 1 and 2). PG clearlycaused dissolution of the middle lamella and the release of fibrouscomponents of the primary wall into the resulting electron-trans-
e illF t biRs :-: .:- iSS&'8 oaW gRig,&:ql!Ux c.: , k:*!, . xS \ R. t; .:.g }e. es 2 ........ ::2u,' e ; Ri.s. r . \ /.e s : :. eR b r°, f swi...: !ai.;tt........ A,, <6. ,. :*: .'. w: ... ^. .. : .
FIG. 3. Changes in ultrastructure during normal ripening. A and B, Mature green fruit. Cytoplasm contains many normal organelles includingmitochondria (m), chloroplasts (chl), endoplasmic reticulum (er), and crystalloid microbodies (cm). The cell wall (cw) consists of fibrils in an electron-translucent matrix. Middle lamella (ml) is visible as an electron-dense region between walls of adjacent cells. C and D, One to 2 d from the onset ofethylene production. There has been an increase in endoplasmic reticulum (er) which clearly has ribosomes attached (rer). Cell wall (cw) structure isunchanged. E and F, Cell wall dissolution has been initiated along the middle lamella (ml). The chloroplast (chl) to chromoplast (chmi) transition hasbegun. A (x 9,600), B (x 12,200), C (X 24,200), D (x 52,500), E (x 12,700), F (x 22,500); bar = 0.5 Pm.
FIG. 4. Changes in ultrastructure during normal ripening. A and B, Four to 5 d after the onset of ethylene production. Dissolution of the middlelamella (ml) is more advanced and 'bow-shaped' regions (bs) visible in the primary cell wall. Plastids are chromoplasts containing angular lycopenecrystalloid remnants (cr). C and D, Seven or more d from the onset of ethylene production. Cell wall (cw) degradation is complete. The cytoplasmremains relatively intact with normal mitochondria (m), chromoplasts (chm), and endoplasmic reticulum (er). A (x 24,200), B (x 24,200), C (x 7,800),D (x 16,300); bar = 0.5 ,sm.
FIG. 5. Effect of applied cellulose. A, Incubation in buffer has no noticeable effect on cell wall appearance. B, Incubation with cellulose results in ahigh lighting of the middle lamella (ml), swelling of the cell wall, and the appearance of electron-dense regions. A (x 24,500), B (x 7,980); bar = 0.5Am.
FIG. 6. Effect of applied purified PG isoenzymes. A, Incubation with buffer has no noticeable effect on cell wall appearance. B and C, The effectsof isoenzymes I and 2, respectively, are indistinguishable. The middle lamella (ml) region has been removed and there is evidence of dissolution of thefibrils of the primary wall. D, Ultrastructure of a Never-ripe fruit containing only PGI activity. Dissolution of the middle lamella (ml) region of the cellwall has occurred. Plastid structure has been altered with angular lycopene crystalloid remnants being replaced by an intraplastid membrane system(ims). A (x 13,700), B (x 10,200), C (x 21,000), D (x 8,760); bar = 0.5 ,m.
lucent area (Fig. 4B). The outer regions of the wall were seen tobe composed of parallel arrays of fibres arranged in a patternsimilar to the 'bow shapes' described in mung bean hypocotyl cellwalls partially degraded by purified fungal PG (16).At later stages (7 or more d after the onset of ethylene produc-
tion), extensive wall disintegration was noted (Fig. 4C). Thepattern of disruption was essentially similar to that describedearlier, although much more dissolution of the primary cell wallwas evident. Despite the difficulties of processing such delicatetissue for EM, a large degree of organization was retained in thecytoplasm of fully red, soft fruit. Chromoplasts, mitochondria,and ER are clearly visible in Figure 4D.
Effect of Applied Cell Wail Degrading Enzymes. The effect ofincubating mature green pericarp tissue with a commercial fungalcellulase preparation is shown in Figure 5. Incubation in pH 5buffer had little noticeable effect on cell wall structure (compareFig. 5A with 3B), but the inclusion of cellulase in the buffercaused the middle lamella and other regions of the wall to appearmore electron-dense (Fig. SB). Considerable swelling of the cell
wall also occurred. The degradative effect of cellulase did notresemble that seen during normal ripening or after adding purifiedPG.The pattern of disruption produced by added tomato PGl (Fig.
6B) was essentially the same as that noted in the early stages ofripening (compare Fig. 6B with 3E). Cell wall disruption occurredmainly in the middle lamella region leading to the separation ofthe two primary cell walls. Little dissolution of the fibrillar regionsofthe primary walls was noted. Incubation ofmature green tomatopericarp with purified tomato PG2 produced the pattern of walldisruption shown in Figure 6C. There was no discernible differ-ence between the effects of PG I and PG2 on the appearance ofthe cell wall.The same pattern of wall disruption was observed in a Never-
ripe mutant fruit which was allowed to change color and softennaturally (Fig. 6D). This specimen had only PGl according toheat stability (data not shown) and mobility ofPG during PAGE(Fig. 2). Plastid structure was also altered by this mutation.Plastoglobuli remained, but an intraplastid membrane system
POLYGALACTURONASE AND THE ULTRASTRUCTURE OF RIPENING
replaced the lycopene crystalloids characteristic of normal fruit atthis stage.
DISCUSSION
PG activity was first detectable 2 or 3 d after the onset ofethylene production as described previously (24), at about thesame time as the plastid transformation and pigment changesbecome apparent (Fig. 1). This is further evidence that the hy-pothesis of Tigchelaar et aL (21) that PG is the initial trigger offruit ripening should be rejected.
During the early stages of ripening, the most abundant PGisoenzyme was PGI, based on heat stability and mobility duringPAGE. The results are in good agreement (Figs. 1 and 2) andshow that PG2 accumulates later in ripening. Figure 2 shows thatPG2 can be resolved into two separate polypeptides by PAGE.These two polypeptides, which have been designated PG2A andPG2B, are immunologically related (25) and copurify after gelfiltration and ion-exchange chromatography. It is thought theymay differ only in their carbohydrate moieties (25).The electron micrographs presented here show ripening to
consist of a series of specific changes in the ultrastructure of thefruit which occur during the period when PG (24), and possiblyother enzymes, are synthesized. There is no evidence from thisstudy of the loss of ultrastructural organization during ripeningdescribed by Bain and Mercer (1). Even the cytoplasm of a fullyripe, soft red tomato retained a large degree of structural integrity(Fig. 4D). These observations are in agreement with those madeon ripening avocado (12).Red tomato fruit have an active protein synthesizing machinery
with translatable poly A-containing mRNA (14) and polyribo-somes (Speirs, Brady, Grierson, Ali, in preparation). There havebeen many reports of an increase in protein and RNA synthesisduring ripening (6) and the increase in PG activity has been shownto be due to de novo synthesis (24). Since specific changes in polyA-containing mRNA have been noted during the ripening oftomato (14) and avocado (3), it seems likely that at least some ofthe control of ripening occurs at the level of transcription andtranslation. The retention of a high degree of cytoplasmic orga-nization in ripening fruit is consistent with these observations. Itis possible that PG is synthesized on the RER noted immediatelyprior to the appearance of PG activity (Fig. 3D).The most obvious changes in cell structure that occur during
ripening are the transition from chloroplast to chromoplast, whichhas been described in more detail previously (17), and the disrup-tion of the cell wall. During ripening, extensive dissolution of thecell wall becomes apparent which can be closely correlated withthe increase in PG activity. The pattern of cell wall disruption issimilar to that described during the ripening of apples and pears(2). A similar pattern of wall disorganization has been noted incell wall material treated with purified fungal PG (16). Purifiedtomato PG2 has been shown to degrade tomato fruit cell wallmaterial in vitro (20) and so there can be little doubt that PG isthe major cell wall degrading enzyme in ripening tomato fruit.The role of the isoenzymes of PG in wall degradation is less clearinasmuch as both PGI and PG2 are able to disorganize cell wallstructure when added to mature green tissue (Fig. 6). Duringripening, the first detectable dissolution of the middle lamellaoccurs very early in ripening, when fruit contain mainly PGI(Figs. 1 and 2). This suggests that in vivo PGI is responsible for
initiating wall disruption, by attacking the middle lamella. Laterstages of disorganization, in which the primary wall is attacked,may be due to the action of PG2. Further evidence for this comesfrom studies with Never-ripe fruit (Fig. 6D). This mutant softensmore slowly than wild type and the specimens examined in thisstudy, which were about the same age as a fully ripe normal fruit,had only PGl activity. PG2 activity is detectable in very oldNever-ripe fruit although it rarely accounts for more than 50%Yo oftotal PG activity (unpublished data). The slow softening of thismutant is probably related to the reduced amount of total PGproduced (22).
Acknowledgment-Purified PG isoenzymes were a kind gift from Dr. G. A.Tucker.
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