Persea americana, Mesocarp Structure, Light and Electron Microscope Study Author(s): Flora Murray Scott, Barbara G. Bystrom and E. Bowler Reviewed work(s): Source: Botanical Gazette, Vol. 124, No. 6 (Dec., 1963), pp. 423-428 Published by: The University of Chicago Press Stable URL: http://www.jstor.org/stable/2473211 . Accessed: 09/02/2013 21:55

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This content downloaded on Sat, 9 Feb 2013 21:55:02 PM All use subject to JSTOR Terms and Conditions 1963] HAIGH & GUARD-ROOT-TIP MITOSES 423

the rest of the quiescentarea, as delineatedby the boundaries of the quiescent zone on the side away CLOWES, only one division has been observed, from the root cap cannot be definedwith precision. whereasin the epidermalportion, a much smaller In the remainder of the apex behind this region, area, seven distinctdivisions are recorded.While there appears to be a fairlyuniform distribution of approximately1800 divisionshave been plotted,it divisions, with perhaps a rather higher density of would be desirableto studymany more sections in orderto clearlydefine the status of the epidermal division in the central, or "columella" area, than in region. the cortical and epidermal regions.

In thearea immediatelysurrounding the quiescent DEPARTMENT OF BIOLOGICAL SCIENCES centerthere is a gradualshading off of quiescence, PURDUE UNIVERSITY divisionsbecoming gradually more frequent, so that LAFAYETTE, INDIANA

LITERATURE CITED 1. CLOWES, F. A. The cytogenerativecentre in roots with 3. ---. Nucleic acid in root apical meristemsof Zea. New broad columellas.New Phytol. 52:48-57. 1953. Phytol. 55:29-35. 1956. 2. --. The promeristemand the minimalconstructional 4. JENSEN, W. A., and KAVALJIAN, L. G. An analysis of cell centrein grass root apices. New Phytol. 53:108-116. 1954. morphologyand periodicityof divisionin the root tips of Allium cepa. Amer. Jour.Bot. 45:365-372. 1958.

PERSEA AMERICANA, MESOCARP CELL STRUCTURE, LIGHT AND ELECTRON MICROSCOPE STUDY'

FLORA MURRAY SCOTT, BARBARA G. BYSTROM, AND E. BOWLER Introduction Ergasticsubstances, in particularoil and starch,were The mesocarpof the avocado fruitconsists pri- tested by stainingwith Sudan III, Sudan black, marilyof parenchymawith abundant oil and a net- osmicacid, and I2KI. Aqueousstains including neu- workof vascular strands(1, 19). The oil occursas tral red,Janus green, anilin blue, and thioninwere dropletsin the cytoplasmof unspecializedcells and usefulin stainingdifferentially but not definitively in oil sacs in scatteredidioblasts (3, 4). theoils in parenchymatouscells and oil-sac idioblasts. The structureof the oil-sac idioblasthas been For electronmicroscope study of cell walls,young describedin detail in the leaf of avocado, but the and maturetissues were cleared of the abundantoil interconnectionof idioblastswith surroundingcells and proteinaceousmaterials. Existing techniques is not indicatedin the originaldiagrams (4). The weremodified as required.The mostsuccessful meth- presentpaper deals with cell-wallstructure, inter- ods involvedthe use of acid maceration:1:1 10% cellularconnections, and theprotoplasts in thetissue chromicand 10% nitricacids; or acetylization:1:4 of the mesocarpof the fruitof Persea americana, glacial and concentratedsulfuric acids. These treat- var. Haas, as seen underlight and electronmicro- mentswere followed, when necessary, by removalof scopes. fattycomponents and by washingin 50% alcohol Material and methods followedby distilledwater. For cell-wallstudy cleared materialin distilled Fresh materialof youngand mature2fruits was waterwas fragmentedultrasonically for 25 secondsor sectionedand examinedunder the lightmicroscope, less in ultrasoniccell fragmentizerSF-50 (McKenna beforeand afterstaining with aqueous stains,also Laboratories,Santa Monica, California)and there- during and after treatmentwith standardmicro- aftermounted on Formvar-coatedgrids and shad- chemical reagents. owed withpalladium. Uncleared material was simi- The distributionof cellulose,pectic substances, larlyprocessed for comparison. lignin,and suberinwas determinedby I2KI-H2SO4, substancesin situ, ultrathin rutheniumred, phloroglucinol-HCl, and Sudan III. For thestudy of fatty sectioningwas effective.Segments of fruit were fixed 1 The authors thank the National Science Foundation in bufferedOS04 (7) or in KMnO4 (5) and wereim- for of this work. (NSF G23387) support bedded in Epon 812. Material was sectionedat 2 The term "mature" is used of fruitsat the "harvesting and 1000 on a Porter-Blummicro- ripe" stage, full grown and firmin texture.Softening of the between600 A A fruitoccurs after picking. tome. All sections were stained with Protargol.

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Observations were made on an RCA-EMU-2 micro- The polyhedralidioblasts in the same fruitsare scope. distinguishedfrom the parenchymaby largersize Observations and thickerwalls and by the presenceof oil sacs. Traces of ligninoccasionally occur in the . LIGHT MICROSCOPE.-In the youngestmesocarp examinedthe parenchymatouscells are polyhedral The patternof pittingon all cell facesis evidentin and approximatelyequal in size. The futureidio- youngerfruits before the wall is too heavilysuber- in blastsare alreadymarked by therefringence of their ized. In oldersuberized walls pittingis observed protoplast contents.Small air-filledintercellular spaces are ultrasonicallyshattered fragments. The defined ubiquitous. occurs during the growthof the is vacuolated,and thestalked oil sac is clearly of growth.The sac membrane fruit,and recentcell divisionis indicatedby varia- duringvarious stages but are impregnated tionin cell outline,diameter, and alignment,and by and stalk consistof cellulose fromthe earlieststages with a certainamount of thepresence of new thin cell walls between daughter The membraneap- nuclei.Idioblasts grow more rapidly than unspecial- -a suberin-likematerial. ized parenchymaand are distinguishedby their pearsvery finely punctate or ultraporous.From the sheaththat surroundsthe sac, cytoplasmicstrands radiatebetween toward the peripheral cyto- plasm, as in developingcystoliths and calcium oxalateidioblasts (9, 10). In thefull-grown idioblast, theoil sac fillsalmost the entire cell cavity and masks the nucleusand otherorganelles. In the tissuesof youngerfruits the cell walls and theprotoplasts of the idioblast react immediately by changein colorto standardmicrochemical reagents and to aqueousstains. In maturefruits the idioblasts generallyremain impervious indefinitely to the same reagentsand stains.It is not knownat what stage of developmentthis impermeabilitybecomes effec- tive. p 0 Pd OS ELECTRON MICROSCOPE.-The cellulose micro- FIG. 1. Diagramof avocado mesocarp cells showing pitted fibrillarpattern of the youngest cell walls examined cell wall and oil drops in protoplastsof parenchymacells and is a reticulum with pores and groups of pores that thickenedcell wall and oil sacs of idioblasts. In latter note mark the beginningof pits (15). Differentiationand and older oil at groupedpits, younger sacs, plasmodesmata wall thickeningof unspecialized parenchymatousand base, and radiatingprotoplasmic strands. P. pits; 0, oil; OSO oil sac; Pd, plasmodesmata.Actual diameterof idioblasts is idioblast cells is evident at an early stage of growth. 25 ,u. In the interparenchymatouscell faces, as already noted, the ubiquitous pits are large, elliptical or cir- thickerwalls and the presenceof youngstalked oil cular in outline, and become delimited by a macro- sacs. reticulum of microfibrils(fig. 2). In maturing cells, In maturefruits the distribution of thepolyhedral while deposition of microfibrilsthickens the wall as parenchymacells and the scatteredidioblasts is a whole, the pit membrane also thickens (fig. 3). clearlyseen. The cell faces of the parenchyma appear Near the rimof a cell face, some smallerpits, possibly netlike because of the very numerous pits (fig. 1). adjacent to an intercellular space, and others in The pits are elliptical,oval, or occasionally circular; random locations, are partially or completely oc- the major axis or diameteris from3 to 5 s0,except on cluded by microfibrils.Conspicuous pits remain un- cell faces contiguous with the idioblasts. On the obstructedand functional,however. latter,the pits, smaller, circular in outline,and more On cell faces contiguousto idioblasts,the pit numerous,match those of the idioblast wall. All pattern is similarto that of the idioblast. The meshes intercellularspaces throughoutthe tissue,including of the macroreticulum surround numerous small thosearound the idioblasts,are linedwith a suberin circularpits (fig.4). film. The protoplastof unspecializedparenchymatous The vacuolesof mature l parenchyma cells are filled cells includesnucleus and ,, withconspicuous oil dropsof varying size in addition elaioplasts (2), mitochondria,, and to chloroplasts,mitochondria, and numerousgran- such ergasticsubstances as oil, starch,and undeter- ules. All chloroplasts,are encircledby a corona of mined granules. All cells are interconnected by minutelipid droplets visible in freshmaterial before plasmodesmata (fig. 5). Osmiophilic drops are con- and afterstaining with Sudan black (13). spicuousin the cytoplasmof cells of all ages. They

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FIGS. 2-7.-Electron micrographsof various aspects of plasmodesmata, chloroplasts,fragment of nucleus, various avocado mesocarpparenchyma; P, pits; Pd, plasmodesmata; granules,and partof twoadjacent oil sacs; Luft fixation,ultra- E, elaioplast; SG, starch.Fig. 2, young; cell face with macro- thin section, X3150; fig.6, young; oil drops (black) in cyto- reticulumand pits, X2900; fig.3, older; pit with early stage plasm,elaioplasts (see fig.8); Palade fixation,ultrathin section, of membranethickening, X 15,500; fig.4, wall contiguousto X2800; fig.7, young;chloroplasts with lamellation and starch idioblast withcharacteristic smaller pits, X5900; fig.5, young; grain; Luft fixation,ultrathin section, X 10,000.

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vary in size and in older cells may mask nucleus, Duringthe markedelongation of theparenchym- otherorganelles, and cytoplasm(fig. 6). Chloroplasts atous cells in the onion root tip, the microfibrillar showtypical lamellation and containminute osmio- patternchanges from the characteristic meristematic philicdroplets and, frequently,starch grains (fig. 7). networkto parallelorientation (15). In the polyhe- Elaioplasts,in contrast,are unstratifiedin the pres- dral isodiametriccells of the avocado mesocarp, ent preparations.They occasionallyoccur in small whereall cell faces increasein area approximately groupsof two or threein randomlocations, and all evenly,no such changein patternoccurs. The cell possessa coronaof osmiophilicdroplets (fig. 8). wall thickensby depositionof successivelamellae, The walls of differentiatingidioblasts, in contrast but themicrofibrillar pattern remains reticular. Since to the parenchymatouswalls, are comparatively the cell wall beginsto thickenin the earlystages of dense fromthe earliestvisible stages of differentia-the growthof the fruit,long before any of the cells tion and remainso duringlater growth.A conspic- have reached their maximumvolume, the terms uous macroreticulumwith numeroussmall circular "primary"and "secondary,"in relation to wall pits is laid down at an earlystage (fig.9a). Subse- thickening,do not appear to be applicable. quentmicrofibrillar deposition may partially occlude Suberizationis the normalreaction of wounded someof these pits (fig.9b). Depositionof suberin-like livingtissue when exposed to air, as seen,for exam- materialin the idioblastwall beginsabout the time ple, in the cut tuberof a potato (8). Suberizationof thatthe suberin-pellicled intercellular spaces become intercellularspaces appears to be ubiquitousin the conspicuous.Isolated fragmentsof the suberized tissuesof higherplants and was evidentin the first layer of the wall resemblethe ultraporousor punc- intercellularspaces that arosein shootand roottips tate suberinlayer in theseed coat ofCercidium (14). (11, 12, 17). During the formationof intercellular Idioblast suberin,however, is distinguishedby the spaces, the plasmodesmata,ubiquitous on all faces ubiquitousperforations that markthe paths of the of thepolyhedral meristematic cells, are broken.In- interconnectingplasmodesmata (fig. 10). tercellularsuberization may, therefore,be regarded In the maturefruit ultrasonic treatment is effec- as a reactionof the protoplastto wounding.In the tive in isolatingcomplete idioblasts, in shattering protoxylemof Ricinus the suberizationof the spiral theirwalls, and in releasingthe enclosedsuberized vesselwall began at theintercellular spaces, extended oil sacs frequentlyintact. The oil sac, attachedto the along the middle lamella, and eventuallyimpreg- wall,distinguishes the idioblastat an earlystage of nated the cylindricalvessel wall (18). In the meso- development(fig. 11). The sac developsin a typical carp of Persea the patternof suberizationof the vacuolatedcell, and lipidmaterial is laid downin its idioblastwall may be similar.Suberin may be laid membrane.The latteris sheathedby cytoplasmfrom down firstin the intercellularspaces at the corners whichstrands radiate to the peripherallayer with of the polyhedralcells, may thenextend along the its interconnectingplasmodesmata (fig. 12). The zone of the middlelamella, and at the same time older,partially suberized sac is generallycoated with may impregnatethe celluloseframework. projectingremnants of the radiating strands (fig. 13). The plasmodesmataof the idioblasts occur in com- In occasionalsmall fragmentsof the membranea parativelythick strands,the paths of which are microfibrillarpitted structure is indicated. markedby theperforations of the suberin membrane. In the ultrathinsections of matureidioblasts the It is notknown at whatstage of growth, nor to what irregularoutline of the sac membranepresumably extent,if at all, suberizationblocks the plasmodes- indicatesa normallocalized variation in the consist- mata and the transportof materialsfrom chloro- encyof the living protoplasm (fig. 14). The sac mem- phyll-containingcells into the idioblast. In theyoung brane here is indistinguishablefrom the surfaceof mesocarp,the strainsand reagentsused penetrate the oil drop. rapidly;in themature mesocarp, on the otherhand, manyenter very slowly or possiblynot at all. Discussion The textureof the isolated suberinmembrane, The contentof theoil sacs in the leafof Perseais whetherdescribed as ultraporousor finelypunctate, stated to be terpene(4). The contentof the sacs in is strikinglysimilar in Persea and in Cercidium(14) thefruit is notnecessarily identical. A recentanalysis exceptfor the conspicuous perforations in theformer. (6) of the oils in the matureavocado fruit,variety Underthe electron microscope it is also indistinguish- Haas, indicateda mixtureof saturatedand unsatu- able fromthe cutin layer of the onion leaf and of the ratedfatty acids withslight traces of terpene:satu- epidermisof the root (16). It is evidentthat the ratedacids (palmitic,stearic), 23%7o and unsaturated physicalproperties of suberinand cutinrequire re- acids (palmitoleic,oleic, linoleic), 77%70. It is consid- considerationin relationto the functionsof absorp- ered probablethat tracesof terpenepresent in the tionin theroot and ofpenetration of fertilizer sprays oil sacs are responsiblefor differences in staining. intothe leaf.

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14f

FIGS. 8-14.-Electron micrographsof various aspects of desmata evident; Palade fixation,ultrathin section, X6250; avocado mesocarp parenchymaor oil-sac idioblasts; OS, oil fig. 12, young idioblast; fragmentof membraneof isolated sac; Pd, plasmodesmata; E, elaioplast. Fig. 8, young paren- sac with microfibrillarremains of wall and stalk and radiating chyma; elaioplasts with coronae of osmiophilicdrops; Palade cytoplasmicstrands, X10,000; fig. 13, older idioblast; frag- fixation,ultrathin section, X10,500; fig. 9a, young idioblast ment of membraneof isolated sac with remainingstumps of wall showingpit distribution,X 10,250; fig.9b, older idioblast projectingcytoplasmic strands, X20,000; fig.14, mature idio- wall with partial occlusion of pits by microfibrils,X 18,500; blast; oil sac with plasmodesmata at oil-sac base, peripheral fig.10, idioblast; isolated fragmentof suberinlayer with per- cytoplasm,and uneven surfaceof oil sac; Luft fixation,ultra- forationsand underlyingmicrofibrils, X52,500; fig.11, young thin section, X3150. idioblast with oil sac in cytoplasm,attached to wall; plasmo-

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The anatomicaldevelopment of the oil sac of interparenchymatouswalls are large,in contrastto Persea resemblesthat of the intracellularcystolith thoseof the parenchyma-idioblastcell faces. of Beloperone (10) and also that of the intracellular 4. In freshmaterial of the maturefruit the su- sheathof calciumoxalate druses (9). In the latter berized idioblasts are frequentlyimpermeable to structuresthe intracellular envelope consists initially aqueous stains and to standardmicrochemical re- ofcellulose and is sheathedby cytoplasmfrom which agentsthat react immediately with parenchymatous strandsradiate to the peripheralcytoplasm with its elements.At what stage of growththis impermea- plasmodesmata.In older cells a certainamount of bilitydevelops is notknown, nor is it knownto what suberinis depositedin the cystolithframework and extentwall suberizationslows down or blocks the in the calciumoxalate crystalenvelope. To what transportof materialsfrom chlorophyll-containing extentthis suberization blocks interconnection with parenchymato idioblast. thesurrounding cells is undetermined.In the case of 5. The details of interparenchymatousand of Persea it is evidentthat experimental work, possibly parenchyma-idioblastmicrofibrillar pit patternsare with radioactivetracers, is requiredto followthe evidentunder the electronmicroscope. path of oil precursorsfrom adjacent chlorophyll- 6. Chloroplastswith lamellae and starch and containingcells into the oil sacs of the idioblasts. elaioplastswith coronae of oil dropletsoccur in the parenchymatouscytoplasm. Summary 7. The oil sac is ensheathedby cytoplasmfrom 1. The bulk of the mesocarpof the fruitof the whichstrands radiate to the peripheralcytoplasm avocado (Persea americana) consistsof polyhedral and the cell wall withits plasmodesmata.Plasmo- parenchymatouscells and scatteredoil idioblasts. desmataalso occurat the base of the stalk. Oil is abundantin the cytoplasmof theparenchyma 8. Pores in the suberizedlayer of the cell wall of and also fillsthe oil sacs of the idioblasts. the idioblastmark the path of plasmodesmata. 2. In youngfruits all cell wallsconsist principally of cellulose.The thickwalls of idioblastsin the ma- DEPARTMENT OF turefruit are heavilysuberized. Intercellular spaces AND COLLEGE OF ENGINEERING are linedwith a suberinpellicle. UNIVERSITY OF CALIFORNIA 3. Plasmodesmataare ubiquitous;the pits on the Los ANGELES, CALIFORNIA

LITERATURE CITED

1. CUMMINGS, KATHERINE, and SCHROEDER, C. A. Anatomy in the apical meristemsof Ricinus and otherplants. BOT. of the avocado fruit.California Avocado Yearbook 1942: GAZ. 114:253-264. 1953. 56-64. 1942. 13. ---. Distributionand physical appearance of fats in 2. FAULL, ANNA F. Elaioplasts in Iris: a morphological living cells-introductorysurvey. Amer. Jour. Bot. 42: study. Jour.Arnold Arbor. 16:225-236. 1935. 475-480. 1955. 3. FREY-WYSSLING, A. Die Stoffausscheidungder hoheren 14. SCOTT, FLORA M.; BYSTROM3,BARBARA G.; and BOWLER, Pflanzen.Julius Springer, Berlin. 1935. E. Cercidiumfloridum seed coat, lightand electronmicro- 4. LEEMAN, A. Das Problemder Sekretzellen.Planta 6:216- scope study. Amer. Jour. Bot. 49:821-833. 1962. 233. 1928. 15. SCOTT, FLORA M.; HAMNER, K. C.; BAKER, ELIZABETH; and BOWLER, E. Electron microscopestudies of cell wall 5. LUFT, J. H. Permanganate-a new fixativefor electron microscopy.Jour. Biochem. Biophys. Cytol. 2:799-802. growthin the onion root. Amer. Jour. Bot. 43:313-324. 1956. 1956. 16. ---; - ; - ; and -. Electronmicroscope 6. NEVENZEL, J. Unpublisheddata, personalcommunication. study of the epidermisof Allium cepa. Amer. Jour. Bot. 1963. 45:449-461. 1958. 7. PALADE, G. E. Study of fixationfor electron microscopy. 17. SCOTT, FLORA M.; SCHROEDER, MARY R.; and TURRELL, Med. 1952. Jour.Exp. 95:285-297. F. M. Development,cell shape, suberizationof internal 8. PRIESTLEY, J. H., and WOFFENDEN, LETTICE M. Causal surfaceand abscission in the leaf of the Valencia orange, 1922. factorsin cork formation.New Phytol. 21:252-268. Citrussinensis. BOT. GAZ. 109:381-411. 1948. 9. SCOTT, FLORA M. Distributionof calcium oxalate crystals 18. SCOTT, FLORA M.; SJAHOLM,VIRGINIA; and BOWLER, E. in Ricinus. BOT. GAZ.103:225-246. 1941. Light and electron microscope studies of the primary 10. ---. Cystoliths and plasmodesmata in Beloperone, xylemof Ricinuscommunis. Amer. Jour. Bot. 47:162-173. Ficus, and Boehineria.BOT. GAZ. 107:372-378. 1946. 1960. 11. ---. Internal suberizationof tissues. BOT. GAZ. 111: 19. WINTON, A. L., and WINTON, KATE B. The Structureand 378-394. 1950. Compositionof Foods. Vol. 2. JohnWiley and Sons, New- 12. ---. Pits,intercellular spaces, and internalsuberization York. 1935.

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