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    TH E JO UR NA L O F H IST OC H EM IST RY A ND C YT OC HE MIST RYCopyr ight 1973 by The H istoch em ical So cie ty , In c.

    PLAN T M ICROBOD IE S EUG EN E L . V IG IL

    V ol. 21 . No . 11 . pp . 9 58-962 , 1 973Pr in te d in U .S .A .

    D epartm en t o f B io logy , M arque tte U n ivers ity, M ilw aukee , W isconsin 5 3 2 3 3R ece ived fo r pu b lica tion Ju ly 20 , 1973

    P lan t m ic ro bod ies com pr ise a d is tinc t class o fo rg an e lles d is tr ib u ted ub iqu itous ly in ce lls.T hese s in g le m em brane-bo unded organe llesrange from 0.2 to 1 .5 in d iam ete r and arecharac te ris tica lly asso c ia ted w ith o ne or m orec is te rnae o f rou gh (F ig . 1 ) o r sm ooth end op las-m ic re ticu lum . T he coarse to fin e ly granu la rm atrix w hich ex ten ds to the lim iting m em braneoften con ta ins a cen tra l am o rphou s or parac rys -ta lline core (F ig . 3 ).

    F in e struc tu ra l s tu d ies hav e linked deve lop-m en t o f m icrob od ies w ith th e endop lasm ic re -ticu lum , since it is possib le to find d irec tco n tin u itie s be tw een rough endop lasm ic re ticu -lum and m icro body m em branes (35 , 36 ) (seea lso F ig . 2 ). B io ch em ica lly , iso la ted m icrob odym em bran es from sev era l p lan t tissues and ra tlive r a re s im ila r to the end op lasm ic re ticu lum inphosp ho lip id com po sition an d co n ta in low lev-e ls o f the m icro som al enzym es reduced n ico tin -am ide ad en in e d inuc leo tide cy tochnom e c re -du ctase and redu ced n ico tinam ide aden ine d i-nu cleo tide phospha te cy tochrom e c reduc tase(6 ). In add ition , pu lse -lab eled slices o f cas to rbean endo sperm w ith C -ch o lin e incorpora tedlabe led carbo n in to lec ith in firs t in a m icroso -m al f raction and then in m icrobod ies (1 9).T aken toge ther, the above da ta p rov ide ev i-dence fo r the d irec t fo rm ation of p lan t m icro -bod ies from endop lasm ic re ticu lum .

    Parac rys ta lline co res a re p rom inen t in m anym icrobod ies (7 -9 , 17 , 2 2 , 3 4-36) and deve lop byreorg an iza tion an d com paction of m atrix m ate -ria l (F ig . 3 ) (3 5 , 36 ). En zym e cy tochem is try fo rca ta lase us in g the m od if ied d iam ino benz id inem etho d of N ov ik off an d G old fisch er (24) d em -ons tra ted the presence o f cata lase in th e crys tal-line co re (F ig . 5 ) (7 , 8 , 17 , 34-36). C a ta laseactiv ity w as eithe r un ifo rm ly d istribu ted in them atrix o f core less m icrobod ies (F ig . 4 ) o r w as

    1 This stud y w as su ppor ted by N atio na l Ins titu teso f H ealth T ra in in g G ran t H D 0017405S1 to D r. H ew -so n Sw if t and M arque tte U nivers ity C omm ittee onR esearch G ran t 5641 .

    presen t in variou s fib n illa r s truc tu res in m icro -bod ies o f d iffe ren t tissues (8 , 1 7).

    T he sen sitiv ity o f the d iam inobenz id inem etho d fo r ca ta lase ac tiv ity w as im portan t inestab lish in g the iden tity o f m icrobod ies inp lan ts. T h is is because som e plan t cells con ta ino ther sing le m em brane -bou nded org ane lleslack in g ca ta lase w h ich are easily m is taken fo rm icro bod ies. A lso , deve lop in g vacuo les con ta ina very ac tive perox idase (25 , 36) . H ow ever, theoccurrence of ca ta lase -con ta in ing org ane lles ina w ide range of p lan t ce lls in various tissues (36)su pports ea rlie r fin e stru ctu ral find ings tha tm ic robo d ies w ere organe lles o f p lan t ce lls (9 ,2 3). W ith the excep tion of ce rta in a lgae (12 , 13 ,36) and Eug lena grac ilis (14), ca ta lase is gen-e ra lly foun d in m orp ho log ica lly iden tif iab lem icrob od ies. T he absence of cata lase in m icro -bod ies o f som e org an ism s sugges ts the absen ceof enzym es tha t g en erate hy drogen perox ideas h as b een show n recen tly fo r E . g ra cilis (15) .

    T he fun c tion o f p lan t m ic ro bod ies can besep arated in to tw o m ain b iochem ica l pa thw ays,invo lv ing (a ) m etabo lism o f g ly co la te p rod ucedduring pho tosyn thes is by ribu lose d iphospha teca rb oxy lase (1 , 3 , 22 ) and (b ) conv ersion ofsto red lip id in to sucrose d uring seed germ ina-tion . Iso la tion of m icrobo d ies from sp inachleaves in T o lberts lab ora to ry p rov ided the f irs tev id ence fo r the presence o f sev eral enzym esinv o lved in g lyco la te ox id ation in a d is tin c tp ar tic le a long w ith a la rge am ount o f ca ta lase(31 ). S ince g lyco la te ox id ation had been show np rev ious ly as part o f a ligh t-d ep en den t resp ira -tion (ph o to resp ira tion ) tha t w as sep arate fromm ito ch ondn ia l resp ira tion (18 , 29 , 38), loca liza -tion of severa l ox idases and ca ta lase in p ero x-isom es sugges ted th at a spec ific m etabo licpa thw ay ex isted in p lan t m ic robo d ies (3 0). T heim portance of perox isom al enzym es in in te r-m ed ia ry m etabo lism of lea f ce lls and thu s in thegrow th of the en tire p lan t w as ev idenced bythe loss o f ca rb on d iox ide in th e course of g ly -co la te ox ida tion (18 , 28). T h is loss o f ca rbon

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    PLA NT M ICRO BO DIES 95 9d iox ide grea tly a ffec ts the pho tosyn the tic e f-f ic iency o f p lan ts w ith h igh leve ls o f pero x i-som al enzym es w hich fav or h igher ra tes o f pho -to resp ira tion . T hus , it is no t su rp risin g tha tsy n thesis and ox ida tio n o f g lyco la te in ligh t, asp art o f p ho to resp ira tion , sev erely lim its g row thand crop pro duc tiv ity (3 7).

    N o t a ll c ro p p lan ts exh ib it h igh lev e ls o fpho to resp ira tion . In fac t m os t tro p ica l p lan tssu ch as sugar can e and corn are e ffic ien t p ro -ducers o f d ry m atte r. In con trast to tem pera tep lan ts like tobacco , trop ica l p lan ts do n o t f ixca rbon d io x ide in itia lly in to th ree -ca rbon sugarsv ia n ibu lo se d iph osp ha te ca rbo xy lase . In stead ,fou r-ca rbon ac ids a re fo rm ed (usua lly m ala te )a fte r fixa tion of CO 2 in to ph osp hoeno lpy ruva t.e .T hen as m ala te is m etabo lized in to carbohy -d ra te it m oves from the s ite o f syn thes is in thecy top lasm of m esophy ll ce lls to bund le shea thcell ch lo rop lasts acco rd ing to the H atch-S lackpa thw ay (16 ). W hile it has been show n tha tp ho to resp ira tio n d oes o ccu r in bund le shea thce lls (5 ), th e lev el o f p erox isom al enzym es (2 1 ,26) and th e n um ber of m icro bod ies w ere g en er-a lly low (8 , 17 , 21) .

    D uring g erm in ation of seeds co n tain in g la rgeam ounts o f s to red lip id , conv ersion o f lip id in tosucro se invo lv es th e g lyoxy la te cyc le (2 ) . W henendosperm tissue of g erm in a ting casto r beanswa s hom ogen ized gen tly in a h igh su crose m e-d ium , B re idenbach and B eevers w ere ab le toob ta in a m itocho ndn ia l frac tio n w hich con-tained partic les h av in g g lyox y la te cyc le en -zym es bound to them (4) . T hese partic les ,w h ich they ca lled g lyo xysom es, w ere boundedby a sing le m em brane and a lso con ta ined ca ta-lase an d en zym es fo r fl-ox id ation in the g ranu la rm atrix (2 ) . F in e s truc tu ra l exam ina tion of thesam e tissue revea led num ero us m icrob od ies(3 5). In add ition , m ic rob od ies w ere app osed tolip id inc lus ions and undoub ted ly fac ilitatedefficien t tians fe r o f fa tty ac ids from the lip idbod ies to m icrobod ies (F ig . 2 ). T he localiza tio nof ca talase by enzym e cy tochem istry in m icro -bod ies o f cas to r bean endosp erm (F ig . 4 ) (34)co nfirm ed the id en tity o f g lyoxy som es as aspec ia l ty pe of m icrobo dy .

    A ltho ugh g lyoxysom es and leaf perox isom esdo no t g en era lly app ear in the sam e tissue , th eyh ave been fo und in co ty ledons du ring g reen ingw hen lip ids a re nearly dep le ted and pho to syn -thesis beg ins in the deve lop ing ch lo rop lasts (20 ,

    27 , 3 3). S ince th ere is a para lle l d ram aticchange in enzym e ac tiv itie s fo r g lyoxy la te andg lyco la te cyc le enzym es, it is im po rtan t to knoww hether th e re is m o re th an on e type o f m icro -body based o n enzym e com plem ent and buoy -an t dens ity . C lea rly , th e re is no new syn the-sis o f ca ta lase (1 0) to sugges t d e novo fo rmat ionof perox isom es as g lyo xysom al enzym es de-c rease . A ltho ugh th ere is a sh ift in buoy an tdens ity to a low er va lue fo r p erox isom es fromtha t o f th e o rig ina l g lyoxy som es , e lec tron m i-c roscopy h as no t uncovered ev idence fo r fo rm a-tio n of new m icrob od ies (33 , 36 ). D uring dark tolig h t trans ition som e m icrob od ies in co ty ledonsof cucum ber (3 3) and w ate rm elon (36) becom em odified by invag ina tions of cy top lasm to fo rmsm all v acu o les. T he vacu o le -con ta in in g m i-c robo d ies w ere on ly p resen t dur in g th e tran si-tion in enzym e com p lem ent from g ly oxysom esto p erox isom es (3 3).

    S ig n ifican tly , m icrobod ies in p lan t ce lls d iffe rfrom those in an im al ce lls w hich tu rn ov erregu la rly by the ir app aren t lo ngev ity and thelack o f a w ell de fined lysosom al sys tem fo ro rg an elle rem ova l during cell d iffe ren tia tion inp lan ts. I f a s in g le p opu la tion of m icrobo d ies ischarac te ris tic o f p lan t ce lls genera lly , th en anychanges in enzym e com plem ent during ce llg row th and d iffe ren tia tion cou ld possib ly beaccom plished by passage th rough th e en dop las-m ic re ticu lum . H en ce, it w ou ld be ra re to f in dm icrobod ies tu rn ing over during early stages ofce ll d iffe ren tia tion . Th is app ears to b e tru e fo rp lan t ce lls and loss o f m icrob od ies from thecy top lasm prob ab ly co inc ides w ith onse t o fsenescence as has been show n fo r casto r beanendosp erm (11 , 3 5). D isappearance of m icro -bod ies from th is tis su e occurs by seques tra tionin to au tophag ic vacu o les (3 5).

    Th e pro b lem of tu rn over o f w hole m icrobod ieso r ind iv idua l p ro te in s th erein , pa rticu la rly ing reen in g co ty led ons , has n o t y et been c lear lyreso lved e ithe r by b iochem is try or e lec tron m i-c roscopy . T he answ er w ill m o st like ly com efrom enzym e cy tochem istry , esp ec ia lly w ithn ew tech n iques fo r loca liz ing the g ly oxysom alen zym e m ala te syn th ase (32 ) and the perox iso -m al en zym e glyco la te ox idase (28).

    ACKNOWLEDGMENT SI th an k D r. H ew son Sw ift fo r th e pos tdoc to ra l

    tra ineesh ip d uring w hich m ost o f th e w ork cited

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    PLA NT M ICR OB OD IES 96 1here w as perfo rm ed and a lso D r. Z . H ruban fo rencouragem ent an d in te rest in p lan t m ic ro -bod ies .

    REFERENCES1. A ndrew s T J , L orim er G H , T olbert N E : R ibu lo se

    d ip hospha te o xygenase . I. S yn thes is o f phospho-g lyco la te b y frac tion -I p ro te in of leaves. B io ch em -is try 12 :11 , 1973

    2 . B eevers H : G lyoxysom es o f casto r b ean endo-sperm and th e ir rela tion to g lu co neogenes is . A nn

    N Y A cad S c i 168:313 , 1 96 93 . B ow es G , O gren W L : O xygen in h ib itio n and o ther

    p ropertie s o f soy bean n ibu lose 1 , 5 -d ipho sph ateca rbo xy lase . J B io l C h em 247:2171 , 19 72

    4. Breiden bach R W, Beev ers H: A sso cia tion of th eg ly oxy la te cyc le enzym es in a no ve l subce llu la rp ar tic le from casto r bean endosperm . B io ch emB iophys R es C omm un 27:462 , 1967

    5 . C ho lle t R , Og r en WL: O xygen in h ib its m aizeb und le shea th ph o tosy n thesis. B io ch em B iophysR es C omm un 46 :2062 , 19726 . D ona ldso n RP , T o lbert N E , S chnarrenb erger C :A com parison of m icro body m em bran es w ithm icrosom es and m itochondria f rom plan t andan im al tis sue . A rch B iochem B iophy s 152:199 ,1972

    7 . F rederick SE , N ew com b EH : C ytochem ica l lo ca l-ization o f ca ta lase in leaf m ic rob od ies (perox i-som es). J C e ll B io l 43 :3 43 , 1969

    8 . F rederick SE , N ew com b EH : U ltras truc tu re an dd is tribu tio n of m icrobo d ies in leaves of g rassesw ith an d w ithou t C O ,-ph o to resp ira tion . P lan ta96:152 , 1971

    9 . F rederick SE , N ew com b EH , V ig il EL , W erg inW P: F ine struc tu ra l charac te ris tic s o f p lan t m i-crobodies. Pla nta 81:229, 1968

    10 . G erhard t B : U n te rsuchungen zur Funk tionsan-derun g der M icrobo d ies in d en K eim bla tte rn vonHe lianthus a nnus L. Planta 110:15, 1973

    11 . G erhard t B , B eevers H : D eve lopm enta l s tud ies ong lyox ysom es in R ic inus endosperm . J C e ll B io l44 :94 , 19 70

    12 . G erhard t B , B erg er C : M icrobo d ies u nd D iam in o-b en z id in R eak tion in den A ceta t-F lag ella tenPo ly tom ella caeca un d Chlo rog on ium elong atum .P lan ta 100:155 , 1971

    13 . G irau d G , C zan insk i Y : L o ca lisa tions u ltrastruc -tu ra les d activ it#{2 33}s o xydas iq ues chez le C hlam y-

    dom onas re inhard i. C R A cad S c i [D I (P ar is )27 3 :2500 , 19 71

    14 . G rav es LB Jr, T re lease RN , B ecker WM : P articu -la te na tu re o f g ly co late dehy drogen ase in E u-g lena : p oss ib le loca lization in m icrobo d ies . B io -chem B iophys Re s C ommu n 44:280, 197115 . G raves LB Jr, T re lease RN , G rill A , B ecker WM :Localiza tio n of g lyoxy la te cyc le enzym es in g ly ox-ysom es in E ug lena . J P ro tozoo l 19 :5 27 , 197 2

    16 . H atch M D , S lack CR : P ho tosyn the tic CO 2-fixa -tion pa thw ays . A nnu R ev P lan t Ph ysio l 21:141,1970

    17 . H illia rd JH , G racen V E , W est SH : L eaf m icro -bod ies (p erox isom es) an d cata lase loca liza tion inp lan ts d iffe ring in the ir ph o tosy n the tic ca rbo npa thw ays. P lan ta 97 :93 , 1971

    18 . Jackson W A , V olk R i: Pho to resp ira tion . A nnuR ev P lan t Phy sio l 21 :385 , 1970

    19 . K agaw a T , L ord JM , B eev ers H : T h e orig in andtu rn over o f o rgan elle m em branes in cas to r b eanen dosp enm . P lan t Ph ysio l 51:61, 1973

    20 . K ag aw a T , M cG regor D I, B eevers H : D eve lop -m en t o f enzym es in the co ty ledon s o f w ate rm elonseed lin gs. P lan t P hys io l 51:66 , 1973

    21 . L iu AY , B lack C C Jr: G lyco la te m etabo lism inm eso phy ll ce lls an d bund le shea th ce lls iso la tedfrom crabgrass, D ig ita ria san gu ina lis L . Scopleaves. A rch B iochem B iophys 149 :26 9 , 1972

    2 2. L or im er G H , A nd rew s T J, T o lbert N E : R ib u losed ip hospha te o xygenase . II. Fu rthe r p ro of o f reac -tion pro duc ts and m ech an ism of ac tion . B iochem -is try 12 :18 , 1973

    23 . M ollenhauer H H , M orr# {233 }D J, K elley AG : Thew idespread occurrence of p lan t cy tosom es resem -b lin g an im al m icrob od ies. P ro top lasm a 62 :44 ,1966

    24 . N ov ikoff A B , G old fischer 5 : Visualization of per -ox isom es (m icrobo d ies ) and m itochondria w ithd iaminobenzidin e. J Histochem Cytoche m17:675, 1969

    25. Po ux N: Loc alisation dactivi t#{233}snzymatiquesd an s le m # {2 33 }r is t# {2 32 }m ead icu la ire d e cucum is sativusL . IV . R eac tions ayes la d iam inob en zid ine m iseen ev idence de peroxy som es . J M icro sc 14:183 ,1972

    26 . R ehfe ld DW , R and all D D , T o lbert N E : E nzym esof the g lyco la te p athw ay in p lan ts w ithou t C 02 -pho to resp ira tion . C an J Bo t 48 :1219 , 197 0

    27 . Schn arren berger C , O eser A , To lb ert N E : D eve l-o pm ent o f m icrobo d ies in sun flow er co ty ledo ns

    FIG. 1. Microbody (M b ) in a parenchym al ce ll o f a tob acco ro o t. N o te tha t the endop lasm ic re ticu lum (ER )ad jacen t to the m icrobody m em brane lacks r ib osom es . x 102 ,0 00 .

    FIG. 2 . Por tion of a ce ll f rom cas to r bean endo sperm show ing num erou s m icrobod ies assoc ia ted w ith lip idbodies (LB). Tw o m icrobo d ies app ear to b e a ttach ed to segm ents o f rou gh en dop lasm ic re ticu lum (arrow s).x25,000.

    FIG. 3 . M icrobo dy w ith a w ell de fined cry sta l. O a t co leop tile . x74 ,000 .F IG . 4 . M icrobod ies in a portion of a casto r bean endosperm cell sta ined fo r ca ta lase w ith d iam inobenz id ine .

    T he dark ly sta ined m icrobo d ies stand o u t aga inst the o ther o rgane lles. S ec tion co un te rsta ined w ith lead c itra teand urany l aceta te . x 3 ,200 .

    FIG. 5 . S ec tion of a m esophy ll cell in y oung tobacco lea f p rev ious ly reac ted w ith d iam inob enz id in e fo rca ta lase ac tiv ity . T he crys ta llo id is the site o f m os t ac tiv ity . T he on ly o ther de tectab le perox id atic ac tiv ity is onthe vacuo la r m em b rane (a rrow ). x3 2 ,0 00 .

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    96 2 VIGILand cas to r bean endosp erm during g erm in a tion .P lan t P hysio l 42:566 , 1971

    28 . S hn itka TK , T a lib i G G : Cytoch em ica l loca liza -tion by fe rricyan ide reduc tio n of a -hyd roxy ac ido x idase ac tiv ity in pero x isom es of ra t k idney .H isto chem is try 2 7:13 7, 19 71

    29 . T o lbert N E : P ho tosyn the tic M echan ism s inGr een Plants. Nationa l Academy of Sciences-Natio nal Research Council Public ation 1145.1963 , p 648

    30 . T o lbert N E : M icrob od ies-p erox isom es an dg lyox ysom es. A nnu R ev P lan t Phy sio l 22 :4 5 , 1971

    31 . Tolbert N E , O eser A , K isak i T , H agem an RH ,Y am azak i RK : P erox isom es from sp in ach leavescon ta in ing enzym es re lated to g lyco la te m etab o-lism . J B io l C hem 243:5179 , 1968

    32 . T re lease RN , B ecker WM : Cytochem ica l loca liza-tion of m ala te syn thase in g lyo xysom es. J C e llB io l 5 5 :262a , 1 972

    33 . T re lease RN , B eck er WM , G rub er PJ , N ew com b

    EH : M icrob od ies (g lyoxysom es and pero x isom es)in cucum ber co ty ledo ns. C orre la tive b ioch em ica land u ltras truc tu ra l s tu dy in lig h t- and dark -g row n seed lings. P lan t P hys io l 48 :4 61 , 19 71

    34 . V ig il EL : C ytochem ica l lo ca liza tion o f c ata la se(pe rox ida tic ) ac tiv ity in p lan t m ic rob od ies. JH is to ch em C ytochem 17 :4 25 , 196 9

    35 . V ig il EL : C ytochem ica l and deve lopm enta lch anges in m icro bod ies (g lyox ysom es) and re -la ted organe lles o f casto r bean endosperm . J C e llB io l 4 6 :435 , 19 70

    36 . V ig il EL : S truc tu re and fu nc tion of p lan t m ic ro -b od ies. S ub-C ell B iochem 2:237 , 1973

    37 . Z e litch I : T he re lationsh ip of g lyco lic ac id toresp ira tion an d pho to syn th es is in to bacco leaves.J B io l C hem 234 :3 077 , 1 959

    38 . Z elitch I: P lan t p rodu ctiv ity and th e con tro l o fp ho to resp ira tion . P roc N atl A cad Sc i (U SA )70 :579 , 1973