Modes of Action of Selected Herbicides

FAlt~d by P. (; RAH'IELS

and H K NISIIIMUI'O

Pdm P.C. Barf&mdR . K . NWimota.. . . . . . . . . . . . . . . . . . Ctu* l . Hormonal Gnlml ad Herbicidal ldlvmr m D1prptld.r

Synthnu In Squh Cdykdrm . F . M . Ahion o d A . Tmy

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lihnlun rrvirw . . . . . . . . . . . . . . . . . . . . . . . . Wed d h@rbic&on dippcidpu adivity . . . . . . . . . . . . . . . Hnmmsl qul~tiunddipptdvr . . . . . . . . . . . . . . . ldlwnrrd hrrbi~4dman hannmnlmntr~l d d~prptidnr . . . Dcmmlynthnh t i diprpt~dnr . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . Gmrrsl dwuoiun : Summary . . . . . . . . . . . . . . . . . . . . . . . , . . . . Litrraturrr~ted . . . . . . . . . . . . . . . . . .

Chopr 2 . Effn3s of Hrhirldm an Bud S p r d l q d Purplr Nutdlp . R . K . NlrMrnto

Introduction . . . . . . . . ... . . . . . . . . . . . . . . . . . . . I. iteratun rcvw . . . . . ... . . . . . . . . . . . . . . . . . Budrprmtinp, d purple nul- Iuhcr) . . . . . . . . . . . . . . . . Rcspcmed purple nutssdg to qmtnl@pplicat~unr d b11)iruln S u m r y . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . Literatun cited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chpcer 3 . Adbn d SmQ2 6706 m ClmtmoM Synthah and ChlumpM. P . C . Borldc

I n t d u d b n a d h a t w miew . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F&bn d nmtd in phnb

Clrohnqpnalr: inhibnkm by Snrdm 0706 . . . . . . . . . . . . . . . . . . . . . . . . . . . GarnldhcuwkmdmodedrdhodSDnda67Mi . . . . . . . . . . Chloc.oplut.devslqnnmt: inhibition bySDndar6706

SuMOuy ............................................ Litcnturr citrd . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Cbpter4 . Crimih.ncdiatul Rapaa d MethyknoxMdc with a P t q m d k i c d d m d Actim for 2.41). V . A . McMahon ad C . L . vilkn*r

lnbDduabn ......................................... 1 'Dtrtun review ......................................

Chrplrr 5. CyliJ~irnl r ~ d Blochrm~cal W e i r d Trdluralin on Mitun. P C. Barf&. F D. Hm, a d D. E. Bayn

THE AUTHORS

F. M Ashlon u Prdctar, Dcprrtmnt dBdmy, Uniwnity dGllfomia. Davis.

P. C. &Wll i s Prdm)r. lkpertmnt d Plud Scimm. University d A r i m . Tue M.

D. E h)*r u Prdmnr, Drpmrtmmt d Botany. Univmity dQlifornia. Davis

F. D. k ww Anirt.nt Pmfa*w. Dcpartmlnt d Botany and Plant Palhohgy. C a b rm& Stab Univcnity, Fort Collins. He b mw with thc Departmnt d many and Plad Pothohgy, Pu& l lnlmjty, hfaymt..

R. Tmy u r r C h t t Sbdmf. DQ.macntdB&ny* UaivcmitydC.tilmrilDavir

Western Regional Carpcr~tive Rersrch Projects on wmd (W.52. W 4 3 , W-77) havebeen mnitrnedwith a wide ranged herbicidE.u**dnmtrul prob lem over the yean. Thw projects ranged from purrly phy~iolqical invnl i gations on translocation and mo& uf action d herbicidm to practical matten such as environmental lactors that alter herbicidr dficiol~cy. In the Wr, be c a w of mounting public cwwm over i r n d i r t c hrulth hazards and lonp term ecvlogical mnrsyucnirs of pediridrs in thr mvironment, herbicidal march projects shifted in part away Imm d i e s d better crop protection toward assessment d the dfect d herbicidal pollutantr in the mvirtmmmt. Project W.108 was initiated to determinr the d c t s d rqetitiw application or persistent pollution kvels of herbicides on plant communities and indi. vidual species d the plant mmmunity. This bulktin dralr with the effects or action d herbicides on the rtmcture. chemical comporitian, and function d plant species.

Because many different herbicides were u d in the project, the range d studin was mrrespndingly broad. Experiments were designed to rtudy: (a) mechanism d adion d rveral herbicides on dipptidar synthesis in quash cdyledonsc (b) effects d herbicide and growth regulatorr on bud sprouting d purple nutsedge; Ic) cytological and bhhemial effects d trifluralin on rwt tip; (d) inhibition d carotmoid synthesis by Whz 67MI; and (el mode of at. tion of 2,4-D in l m tissue. Additional rxperbKnts dealt with the dfeds d growth regulaton and other additives upon herbicide action.

The findingr of this pmjnt may be used to drvelop principkr that may m b k man to avoid environmental pollution with hcrbicida whik rctalnina their advsnt~gn. It is,espmially important tohove an undentnndingd t h e i leas d htrbicids on plant biochemical procum so that this information may be related to the plant mmmunity and io ecda(lira1 mnmqumm. For exah pk, a slight r l t i t i o n d thc phot&ynthetic effiekney d r hant specin could have e n o m conrqurnca on a plant community and the whok wsydem in which the plant Amuni ty is found. Furthtr, morphdogil a d b b chemical EbDnlpr c a d by herbicides my prow to br voluabk i n d i c a b d thcprrrmccd specific herbieidnl pdlutants in tirmvimnmmt.

P C ' r ' . I R W N ' ' ,

COMMITTEE MEMBERS. J. L. Andem. Drp.rtmnt d Plant p i rnn , Utah Slate Univcnity, Lqm. Utah: F. M. Ashton and D. E. Bayer. I)rpsgmnl d Bofany. Univrnity d California. Davis, Crlilomir: P. C . Bnnrls and K. C.~mil ton. Depart men1 d Plant S r k . Univonity d Ar i rw. T u r n . Anmu: 8.;. Day. Californu Aqicukural Lpcrimnt Station. Berkeley, Cllifornia: L. C Erirbbn and C . A. Le. Plan1 and Sod K k n n Departmnt. Un~vrnity d Idaho. M-. Idaho; 1. L. Fulls md F. D. Hm. Drpnrtmmt d Many and P L t Pmtholw. G~brsdu Yetr University. Fort Cdlinr. Cakmdw C. H. Hriskr, Bioehcmirtry Divis~on. University d Nevada, Rm). Nwada: H. H. Hull. U. 5. Department d Agrirulfun. Tucum. Ariwna: J M. Hodgon. Plant and Soil Scimw DpM, Mmtnw State Unlrrnity. Bozrman. Urntam; L S. Jordan, Plant ScknrnDrpnrhncnf. Univrnity dCllifornia. Riverside. Clllfimb: V. A. McMahon. Divisiond Biodrmistry, Univenity d Wyoming. Lam- mb, Wpmiw. R. ]. Milbr. Cd!qe d Agricuhn. Univtnity d Idaho. Mosow. Ida- ho; R. 0. M~uiu. Drpnrtmmt d Agr~cultural Chemistry. 0- Slate University. G w a l l i Omgon: T. J. Muzik, Depart- d @mamy and Soils, Wuhiqtm Shte Univrnity. Pullmn. Wuhiqpn: R. K. Niimola Ceparhncnt d Horticulture, UnC ~ n i l y d Hawaii. Hudulu. Hawaii; J . W. Whihvorth. Deprbnmt d Agrmumy, Nrw k i m S h k Univmity, Unimrity Park. New Mexico.

m T M C AGENCIES Tiu ~grkuhural Expriment 3 8 t h d Arironn. Calif&, Cdmdo. Hawaii. 1d.h M d n r New&. New Merim. Utah. Wuhit@m, ud WyanlnS. and rht Crmpntlw St** R-W~ S w h , UUnflal Wte Wrtnntd AMItum.

Chapter I . Hormonal Control and HehicidPI Inf lwna

on Dipcptidase Synthesis in Squash Cotykdom

F. M. Ashton and H. Tsay

Dipeptidax, onr d the prutrulylic rnzymn d quash (Cururblta morimu Duch. 'Hubbard') cotyledons, has been isolated ad pur i fd (A$l<m and Dahmm. 1967). Thu rnzyme has been skwn In iwrruse rvcralfold during x d germination. Its drvelnprnrnt was inhibikd by protein synthmk inhibi. ton. N.6bcruyladminr (BA) ltimulatrd&velqnnrnt d thr mayme aclivity in half-ssedc of quash (Sze and Arhtm. 1971 J. Howcvrr, thnc studln did n d unequivocally ahow whelhrr the dipeptidar wm synthnized & nooo or artw through activation d a zymogrn. Thr inhibit* studin s u w c d thnt it was synthesized de notlo. Specificity of control d the dipcptidar by plant b r . mones, effect d herbicides on its develupment, and the herbicidal action on h o m l mntml proasses have nnt brm studid.

Theobjcctivesd this study wrre to investigak (a) the effect d Mclder on diprptidaa activity. (b) the hormonal qu latbn d dipcptidax. (c) the in. fluena d herbicides on hormonal control d dfpCPtidaa. and Id) the dr n w o synthesis d dipptidar. Some d the results reported in this p p r hnvr been previously publimed(Tsay and Ashton. 1971. 19740, 1974b).

The 1 e c t d herbicides on variau enzymes bas ken mimsd many t i m in the lprt two dsldn ( F d 1953; Weintraub, 1953: Wort, 1954, 1964: Woodlord d aL. 1958: H i b d PI.. 1863: MwrLnd. 1967: Rokr tan and K i r k a d 1970, and Ahton and Craftr. 19731. Hem the subject h limited to thoc mocn mnceminn herbicides u d in tk d v . namelv: auxin. cbchlo&r;il (2.-itrik), &ball 17aabt&dd2.2.i~heptme 2.3diarbmylic a 4 . and naptatam 1N.I-napthylphhlamk add. 7hc term

"auxin." d as a generic term, includm synthetic and naturally m r r i n g impounds having thr growth-promolion prupertics of indnl-3.acdir acid (IAA). The auxin iwnpwndr disruled herein are: 2.4.D 1(2,4dichlornphc- m y ) a&ir add], dicamba (3.6dichlorcbo-aniric acid), and IAA. h a w 2.4- D has alur often hrrn used at low ivmnntratlons as a h o r n , it and dher auxin compuunb u r d as hormonn nrrdiacurrd as one topic, auxin. Dicam- 110 u a l s1r herbicide in mat d the cam is d i r u d sepamtely.

Auxin

Thr influrncm ol auxln on dilfemnt enzymes havr k ~ n t e n s i v e l y studied rinw 1950, but t l w invrltiptims hrvr ncr( r h w n a 4 in i t e effect m the wnu rnzym Irnm diflcrmt plant mnteridls. Th diffrknt rvgponrn may mlak lo plant material itself, roncmtrstion d auxin u4 , t ime d treatmmt. and other lactors.

Scvrral invrstigators rrportrd the effect of auxin on ri&nuc'leasc IRNam). Ln, and Suchrr 11970, I9711 r v r t r d that NAA (naptha a i r t~c acid) at a rate ul 10 pdpl inhibited inm-as of HNam in leaf w t i i ~ ? of Hhovo discolor and &.arp t i s w wtionr d b a n plants. In corn 4esucotyl sections, hnwrvrr. 2.4.D i n c n a d thr HNas aclivity at lcnver co~centrations and in- hibitrd ewzyme actnilv m l higher cwmntrations (Shanyn et ill.. 1964). A ranking d qwics b a d on HNam IS c lmly correlated with the ranking bawd un nlativr rrsislanw to 2.4.D. according to Burkhalter end Carter (1969). Thrir mults Jhmvd lhnl changes in RNA and protein lrvcls bllowing 2,4-D trratments epvr inmnclus~ve results, howcver. RNase levels drclined lollnw- ing trratmenl d rndl ivr rperies but r a t in more mistant plants.

Manv nll wall degradrtbnenzymcs have been fwnd to increase in activity in mponr to auxin lnatment. IAA treatment at thr apices of ckapitated r t ida td pa, bean. and wrn wedlings increased thr amwnt and ~ i l i c ac. tivity d wllularr in adjacent li~sw(Maclachlan r t PI., 1968). When applied to epirotyls of ywng pas. 10 ppm d auxin i n d u d formtion d allulax, and this activity continued to i n c m x with increasing auxin conantrations up to SOW ppm lFan and Marlachlan. 19661. Hnwever, in barley mlcopliks and p a cpicdyl internode -1% cellulase was not i n d u d by auxin; but Ma 1.3- nnd/or M a I .ti-glucanrre md hemicellular activity incnan l after a %hour IAA treatment (Tanimnto and Madn . 1968).

I n slices d som plant tisue, invert- can be i n d u d by washing. When d i m d bart-mot t ime were incubated ins solution d IM or 2.4-D, invertan adivity dcercnrd (Palmer. 1967). Howwer. in t i d e d i m d sugarcane. LM and NAA initially m h a d invert- dmlopmmt. but later were inhibitory l G W i e( 41.. 1968. 1W1. Enzyma other than invert- in tiaruc dim were n b w e d to & infiumcal by auxins N M inhibited permidue syw tbni in diasdsignrcane(Palrner. 1888). and a short-term apo~un d auxin

to bda-mot t i f W I and Jerusalem artichdre disks stimulated activitb d arid phocphntar and ATPar(Cluniou rt 01.. 1868).

The infiumrn d auxin on crthrr cnzymc aydmr &vr a l u heen rtudtsd. Auxin affected Ihr activity d nitrate redudau in p a rrdlingt (Sprivtrv st al.. 19701, glutathionc mludnr, and glu&phnrphptc drhydrqrnar in nrcird dton cwlyl& (Baslrr and Wills. 1W8). aunrbase in bean (PanEr and Franke. 1970). mcorbonylar in p u ruot t i p (Kim and Bidwrll, i(m7l. and aldolar, aminapeptidasc, nr wll ma glutamate: orulardstr truluminas in Atropa belhdonna (Simla and Sqmwn. 187 11.

I n an in uivo system. 2.4-D i n d u d addit i~ml rhmmml~ri.hrund HNA polymrrasc formatian in r)ybran hyprcdyb (O'lrirn, 19871. At hrrhiridvl concmtrations, hmwrr . 2.4.D and IAA inhibitd DNA rynthrsis by Esch* richta roli, DNA polymerase supp)rtd by frw DNA chronlntin, or nur lw h i d m prepad from ptw emhryos(Srhwimmrr. 19611). Auxin was alm bund to be involved in thr activation d enrvmr udivity. Cilratr syathetur isulstcd from corn rculrlla, cauliflower. and Iran plants muld hr nrlivilted hy IAA in tdtro and activation imu rm l at the crtcrnal St1 Itulfhydryll group d thr rn- zyme(Serkiuian. 1W8. 1972).

Dicamha

Mowland el al. (1969) reported that tlicamha inhibitd (;A.inducrJ lgilr tKwllir arid) alpha.amylaw syntkls in distal.hslvn of barlry d r . In cotyledons ul germinating quash m l r , dirsmtvs at 1t)"M inhibit4 ckvrlup ment dpruleinnsactivily slightly (Ashtl~n&nl.. I(Hi81.

I n barley d, dichlobmil was rqx~rted to inhibit dcvelapment d (;A- i n d u d alpha-amylase (Pennrr. 1968: Dcvlh and Cunningham, 1970) and phytare (Penner. 1968): whm applied to germinating quash &. it rvcwly inhibited &ling ~ row th and devclopmcnt d protrinaw activity (Ashtun rl a].. 1968). I f BA is added to the dichlobenil tmatment. some of thr prctteinase activity inhibition muld lx nullifkd (Pmnrr and Ashton. 1968).

Endothall inhibited development d alpha-amylase in thr m d a p r m of pr . minating barley Jmb (Penner. 1968). I t also depresxd protmlytk e n z y m in geminating qua& rah (AJlton rt al., I-), and this deprmion was nd dimd by additiond BA(Penner and Ashton 1W8J.

Napt8JIm

Naptalam h d link df& on dcvdevdopmmt d prdeiclpr adivlty In gcrmi- nating & mxb (Ashton et al., 19681. Itr influence on other enzymc ry* tcmsholnotbmnrrputcd.

HAWAII A((:W(:ULIURALt.A, c.MMuCI SlAllON

Protcin in a mppr d~nagc r r r r v c in many plant d. This dorage protein is hydrolyd during d germination by prdmlytic mzymsr, and the hydm lytic pmlurts a n uad by thc developing d l i n g for synthesis d new dm& tural and functional proteins and for energy, Inhibition d t k rnzyma by hrrhirida rmld haw a profound d f rd on &ling [~rautb and development. Protrlnar activity in the cotyledon ob quash seeds incredm sweralfold dur. ing d grrmination (Wily and Aghton, 19671. One or miwe proteinam pro hbly a n involved in this increar (Pmner and Ashton. I $), and some herbi- ridm inhibit it (Ashtonct al., I(W8).

Ashtt~1,1W,l967) are probably synthesized dc novo. The purpar ol thir experiment was to survey the herbic d i n the pro

kin*. study (Ashton el al. I!%&) for thrir effect upon L i d a a activity and to cumpare thrir dfcct on the development of these tw&zytnes.

lkrulh and D ' i 1)ipptidua activity of squash cotyledons incream dur iq gemtination (Szc

and Ashton. 197 11. Htnvcvcr, when pQdr were germinatad in various herbi. cidr solutions, thr drvrlopmrnt of dipeptidaa activity was altered by several U( the hrrbicides (Tablr 1 -I I. Herbicides that inhibited dnnlopment d dipep tidaa activity by 70 percent or mure were 2.4-D, diamba, fenac I(2.3.6- trichlanphrnyl)awtic acid), and naptalarn. T h w inhibiting by 63 to 34 per- m t were picloram (4.arnim3.S.6-triAlompicolinic acid), dichlobmil. bromowynil (3.5dibro~hydruxybcnzonitrile). amitmlc (3.arnimtria- zde), ulrarirw ~2~rhlomc(ethylamim)-Mi~r~lami1~)~btriazi, & tbII, and bmmacil15-hromo3-rrr-butyMmcthyluracil). T h e causing only slight inhibition. 24 to 16 percent, werr diphenamid (N.Ndimethy1-2,2- diphcnyluwtamidc), ehlorpropharn (impropy1 m-chlorocarbanilitc), and tri- fluralin ~eap.trifl-2,6dinitr~~,N.N~dipmpyCptoIuidine. Monumn 13. ~prhlomphmyltl.ldidhylum] waa saenthlly the same as the mntrol. Hwrvrr. dalapon (2.2dichlompropiimir acid), bmoulidr [O,~ isnpmpyl phoaphomdithioatr Sester with N~2-mrcaptoithyl)bmmwsuK~mi~, CDM (N.N-diallyl-2.fhlompwtam~. and CDU: (2-chloroallyl diethyl. dithkcarbinald were somewhat stimubtory.

HNbidm t h t inhibited d i m i d u s activity do not -rily .ffat p teinase activity (Ahton et rl.. lW) to the am &gree, In gmer~i. herbicide

T * I - t . ~ i d u d C r * r . a i * Y v h a v l d a J q u h & ~ d i *.hrkrbiriWp*(*r

- -- .... - -. .

1HprlUkbl.r PNXFMV Hnblchit rttlvtl\. l*nr*lI *rltrl l$ ~ r r t w d l m t m d ( i r m l c r d m d n n l n d d t m l m l ' -. -- - - -- -. --- . . . . . . 1.44 1n"M H 11 I>n.amb 10"M I 7 )Ih Fmar 10" hl 21 il Nsyralrn~ IIP'M H I 43 PrLlrvrn lV IM 1: :(I

n w h k h ~ l 1 (I* M 1U .Ill Bnmnrrnl IU"M 41 lZl7hl AmttnC IO.',M 4~ &:I A1111llw 10-M hl UI Elukdhull IU"M M 70 Rnmr~l . lIl.'M (UI HI) 1)ptnurnacl I U ~ M :h 11:

(:hloq,ryh.rn 5 x M 14 I riil(0l 'I rdlursl8n 1 S r ID-M 81 $11 Mcmum ll14M 1111 O ' I

Hnrul,#& Sr lU"M 112 'l%H21 (I IAA 10.' M I IH 56 <:l)FL: I04M 120 I l lh l l l D~lrpw$ IW'M I!) Wk (imtrol 111) llUl . . .. -. . . . . . - . . .. . . . -. - . . .-

'l;rumAsl1tcmnrl.ll9hU1 K t ~ u r n t , p m ~ l l r * l u ~ l r u ~ ~ A 1 l l c t ~ ~ ~ 1 u l I ) 1 1 W 1 l l 1 1 4 i I 1

inhibition of development d dipeplidar activity was greater than i~~hihition d protrinax activity. This was true with 10 d thr I l ) herbicide# rvuluatd: arnitrtde. naptalarn. utrazine, diearnba, brolauxynil, bromcil, diphumid. 2.4-D, fenac. and p~clorarn. A notable nct.ptlom wax CDAA, which inhibited pruteinase activity but did nd inhibit dippUdav activity. Tlw t h r e e t i i n hibition of the develqment of these two types d activity wal similar for w v m l herbicides: bmuiide, chlorpropham, dichklhil. endothall, snd monwon. Thedata d Ashtom and DeWitt 11971) lor prvteitmu adivity wrre used for the above & m p n r h for bromoxynil. bmmlide, CDFZ. and chlor- propham b u r they d water only M a d v m t for t k hrrbicidcr. whmec a small percentage d ethanol in water was used u a mlvenl lor !her fwr herbicida in the Ahton d ol. (19681 study. Alcohol increased the inhibi- tory dfed d d flour herbicides on proteinu. activity. wen tbcugh u l r d d at t h mnantrations d hnd noelfed on the activity in the a b c e d herb& rid& 'Ihc influcna d aloohol is a n i d e n d to be mlated to i n r d mcm. branc pnnmbi l i i . which resulted in higtar macmtratiau d th*r bntri- c*L , ' t .I '

Thr prrdeirtaw i u~ lu t~d II~ Pt.nrrr a d Arhtlm (19671 and t l dipptidasr rhrrilc.trr~rfil I)! AS~~IIII mnd I)ahmen 11967) are pnhahly tlw principal en. ~ y n r , rnln~~lsi l~lr lor pretrinaw and tl~pptidrvr activity measured In thrse studuz. Thr wt i \ i l \ (rl lnah c r l t h * x rnzynur increase with time during gcr. tninuti~n Th.\ nlqxar to Ix r y t ~ t h t ~ i ~ d de nocio and their synthesis rrm trollnl II\ a d~tnulu>, p n h l ~ l t a ~.yt<binirl. from axis (Rnwr and Ashtcnl. IWNi. IHh7: U'llr! and Arl~lon. 1967; Szr ard Ashton, lWl1. Tbrrt~lure, in ~I~II~IIIII OII t l ~ v r l ~ ~ ~ m n t of d~lxplidaw aetlvitg by thc wvrn l herblrrtlm could i r~\oI\r (lln- 11r IIIO~P ~~wrhilnim~s. Possibly, llw hed~icidn i@rrferr with dwt.1. oq)nwn~t or trarulxrrt d thr hc~rnlo~r that c~~ntrols syllthsi$d the. dilwpticlar In t l r nfiylnkse. Altrmalivrl!. the) rnu, interfere with sy$hmls 01 thc.clipp t~(law 111 t l r cot>kulor~r. Intrrlrrrn<c with d~lwytitlax bnthsis could be I)rltught ulralt 11) IIIII~III~ICIII 01 any 01 the wveral rwnt ial @nmponmts 111 pro trills! t)llr*sir-r.y . l)NA. HNA. ATP. or rilxsorw.

I'rofdtl rynthrais Inhll>itl~s~ of pmtrin s!nthrsis in h m ~ w b e n i a (Srsbonia nelfalu tHal.) (i~r!.l hylx~mtyls alwlior barley ~ ~ ~ ~ r o ~ r t i I e s b ~ chlorprupham. (:l)AA. and PII~IIIIII~I~I wrn. wpurttvl by Mann r t al. (196 . Moreldnd r t al. t l U i Y i ~ i ~ w n ~ ~ l that prvlrin synthesw in m h n hylxwot~ was inhil l itd by ~l~lorlrr~npl~anr. fr-l~ilc.. (:I)IC(:. dirunlba. and iiii~hlcdx.nil. %he mulls of t h m twtr \tutltc&s ,Irv nclt III ayrc~.~rn~t with mp+ar<l tcr the inhil>iUcm of prr~trin syn Ihc-sir 11) (:l)AA. (:I)I<(:, d ~ r l d1rhll4unil. Ill tko studies Iq Mann el 81. 11965). (:I)E(: alrl ~ l ic . l~ l~r lx~n~l d ~ l Ilot l l l l~ ib~t pr(~trin synthr~is, whrwas thq did in1

the nwurrh rqxirlcvl I)! hloreland rl al. (lYfi91 Cn)nvewlv. CDAA ditl n11l in- 11il11l prolrill antI11%i3 ill lhr dudies by Mann r t al. 119651. We r l a~ l i nd varia- tint1 1vlwtv~11 Iwotrlnnu. alxl dipptidaw activity dwelopment in quash crltyklcn~r w~th (1)AA: it i~~h~l r i ts pn*einar a~+i\~it! but not dipeptidaw ac- ti\ it\ M~~relusd rt HI. (1969) reported that w~me d thr diffewncw lxtwwn lhrir wwurrll and tlmt cJ Mi~nn r t ul. (1965) ran pmibly be attributd to dif- I rmt~ im in r~~ntrotrut i t~r~s d thr ht*rbicitkr tested. Hwevrr, differcnws in the rsl\mt uw l to d ~ s J \ r thr herbicides. as wll 8s syrries differenw, also may huvr Iml invol\.nl. M~IIII rt nl. (19651 usrd ethanol and Mowland r t al. I IY(iYI ustd dilmthvl s~~lftmick (1)MSOI. Pmiouslg wr discussed the inlluencp crl rthanol (MI thr c.lfcct of bromoxynil. bcnsul~de. CDEC. and ehlorpropham cm pn*einuw erc-li\it! d quash c~~tykhms. Tlw report by Mann et al. 119651 supports the ~ n n c r p ) that inhibition ddevelopment ol divptida~e activity in quash c w t y k l r ~ n s by Ienac, dilrilmba, dichlobenil, a d endothall mny be due lo inliibitiw~ d protein s3nlhesis. These investigators did not investigate t ke f - fed d 2.4-D. nnptalam bmmoxynil, or bmmacil on protein synthesis. Pmribly. inhibition d dipeptidoa adivity by thae herbicides is aim via p m tein synthesis. Homver, one w both of stidin included picloram. rmihuh. or strazinr. which did not inhibit protein synthesis. thenlore. t k herbicides mud inhibit dipphd.r via nmr mechanism dber t b n protein synthuii

RNA r y n t h h RNA synhda pmbably ic q u i d for dcvelopmcnt d dipcpllbr activity in qwh My-. Mdmnd d al. (1969) Investiyted the iduena d rvcral d the herbicicba used in our study on RNA lynthesln in wrn mesDedyl x c t h (using "Cottic acid Incorpormtion) and In mybean h-l rdh (using "CATP inrorporrtion). Only two d tho horbicida that inhibited dipcptidase activity, and which urm tested far RNA synthis. inhibited RNA synthesis. Thrrdore. amitmk, dichlobmil, fnuc, dicamh. and piclornm inhibition d diprptidan u probably not via th. inhibition d RNA synthesis. Atrazinc inhibited RNA synthesis in the "MTP rury, k t was Hmulatory in the "C-orotatr anuy and inhibited d i p c p l h activ~ty. Ikrdorc, thc inhibition of dipptidar activity by atnrinr may Ix via RNA synthrsis. Chlorpropham inhibited RNA synthnb wry significantly in both assay^, but b d only 0 slight inhibitory efforl cm dipcptidur activity. Other herbicides that inhibited dipptidan activity, but that wcrr not tnted for thrir influence on RNA ynthrris and thedorr could be ading via RNA, are 2.4.D. naptalam. bromacil, enddhall, and bromonynil.

ATPsynthcrlr. Compounds that interfere with ATY prdudion by a t l i n ~ an unmuplers d oxidative phorphorylation or as electron tranqwrt inhibitrrn muld inhibit development of dipeptidax activity HerbieidCS that inhibit dipptidar activity, and which have k n m p l l d to uncouple ~~xidutivc phoaphorylation, are 2.4-D (Switur. 1957: l~ t l i ka r el 01.. IBRH), nuptulnm (b l ikar e( el.. 1968), diehlobcnil iFoy and Pmnrr. IWSI. and dicsmtrn (Poy and Penner. 1985).

In a study on the influmrr d dichlobenil endothall, and bromoxynil on cytokinin contml of prdwlytic activity in quash mtylcdcnu. it war s u ~ g ~ e d that dichlobmil inhibited cytokinin synthesis or trsnrp~rt, rduthall inhibited RNA metabolism, and bmmoxynil interfend with the rytokinin system and protein synthcsis(Penner and Ashton. 19681.

Dipcptidan activity d dylednu of quash increased during germinatirm. Sevcrsl herbicide inhibited this inerewe to various dqqrsn. Hcrblc&s inhib iting dcvelopmont d dipeptidare activity by 70 prcent or mom were 2,4-D. dicamba. fenac, and napalam Thoz inhibiting dipeptidor activity by 63 to 34 p a n t m n picbram, dichlobmil, bromcaynil, amitrole, atmzim. mdrt thall. and bmoncil. Thor causing only sligbt inhibition, 24 to 16 percent. wnr dipbummid, chlorpmphmm. and hinunl in Monwon war amtbl ly the same as the omtrol. However. &lapon, benarlide, OM, and a)EC wnc m m h o t dimulatory. When 0anp.d witb p m i w l y npMtal herbicide activity on pmtrmsre adivity. herbicidn thrr inhibited dfpplbr activity did not aarsrrily affect prateinmadivity to the =me &gm.

HORMONAL RIXUUTION OF DIPEPIIDASE

Hormonrl wgulntion d dipptidaa in squash cotyledm was observed by Su ad Ashton (1971). They showed that quash ondr r equ id the presence d the rmbryoaxir lor maximum drvelc~pment d dipptida* activity. and that t h rytokinin BA muld replaw the embryo axis. Protein* isolated from the vvmc t ime was fwnd to be rprilically nmtrolld by cydtinin (Penner and Arhtcbn. 1887). Thac hmmzymcy arc clmely related in pwein digestion dur- ing savl gernlination. H&wer, the properties and t iqcuurse studies of diptp~dase are differcnl from thme of proteinsoe. Dipc@idax may or may twa show thr same specificity, The question of whether &ipcptidasv is con- trolled hy BA alone or is also controlkd by other growt&egu~ators was the sul)jwt id this invrstigntion.

Rrub Morpholodral reapom to h o r n . All four honnap-BA. CA, IAA.

and ABA (abxisic acid)-nffccted thc growth d dli&. No quantitative measurement of there mponsr was taken, but the msp@m are described below. Thm rrsponas a p p d to tw more pronounced at the higber c o n m tration(5 x 10-'M) thanat the lower mnntration(5 x IT M).

Intart mda. BA.treated weds had shortened hypxwls, and t k grnwth of the pr in~un rtuts was ~reatly inhibited. IAA also reduced the growth of pri- maw rmts, us wll as the development of root hain. ABA almurt stopped 4 grmninmtion. Hwwer. (;A promoted &ling growth; the sdlings had l a r ~ r cu(vlrdonr, longer hypotyls. and longer primary rmts compared to thr mtmls.

D 6 f ~ l . k l f colylnlonr. BA. IAA, and CA induced the enlargement of distal- half cmlylnknu. Among thcre t h w hormones. BA s h o d the great& effect. I)islnl-hall rotylcdon? treated with BA were b ided and curled. IM and CA i ~ ~ c n a a d the size without changing the form. Distal-half cotyledons incubated is ABA rxhihitnl almmt no expansion.

E f f d oJ hownow on dipcpttdav rwctim. The enzyme was prepared in the usunl manner by extraction from cotyledons d m t r o l plantr grown in water. A quantity d h o r m w solution was added to the enzyme. Enzyme ac- tivity was then meoruml and eonparad to the curtrol. The l s u l t s showed that d i w i b u r activity was ~t affectsd by the p -ad hormoaer

E j b t of h o r m ~ ~ ~ m d d o p m d ojdlpsp~dopc. Dipeptidnrc activity of sguuh mtykdonr either fm intact anb: or cxciad eotylcdom, i n d during acd gemination (Sze and ~htaq 1971). Titis incnase can be altered by PQiuy( the hormones. The mlb a n pmental as a mlrtivc pmmtsge d the mntrol and mn statistically a ~ ~ l y d by cw~pkte madomized design ClhMe 1-21. Thc control obtained fa t * ' ^ d * i wit , , ' ' ' ,-

I W 0.' 88 flab M.3r

Vshn .rpmmd as rrlatln prmlaw . .. -.- - - ." -- .. . - . . . . .. . . . - , . . .

(;A IAA ARA

.. . mlnd by Duncan's Mull~pCRa~Tnt A l l rtnnpriwnrn lmdr wllhm Ik w m nblumn

'Enzym sctaity was nr( mmtld fvr n n l u m m r m a l , If th~r had hrm dm. v r l u l wluW hare bcm d t h~ghvr Ihanlhcrr rrprrlnl

mones was arhitrariiy el as 100 prrvnt; throthrr v n l w wrrecrlrulatnl rrr percentage d this value. Thc rwults d this rrprrl lwnl an- prpvntnl ill llw follcnving live sctiom.

Partbl control ofdlpepHdasrarNvity by mbryoarla. Tluddta froln th r rx perimnts at zero mmnntrati~m of horrnrmea rhvwecl that en'yrw nrtnlty <I( distal-half cwty lnk ,~ was about 75 percrnt d that of thr contr<~l ITukrlr 1-21. Therefore, withuut t k proximal half, which k l ud r r Ihceml~ryoaxi*, mxvmr activity was mlucetl ahjut 25 prccnt. Sin- ctdylrtlun Ionrun In hrth rl~stsl. half and proximai half are similar. the rdudion of rnzvmr orlivity in distill. half suggests thut part ol thcenzynu. adivity in thr crr(vkdol1 is crmtrollrd ly thrembrvu axis.

~ f f c c t i f o f ~ ~ on dipeptiduu, promohon a d InhlbiNon The rrducl~un uf m zyme act~vity in distal-half cotyledmu can be rep lad by the eddihun d 0.4 (Tab& l-~).'The -Its dmondnted that by &p ly ing BA 5 x lo* M or S x 10- M the enzyme activity in distaChalf cotyledom developed to the lame kvel as in the control. However, at the higher cunmtntion. 5 x lo* M. 0.4 inhibited mzym development in intarl 4 s . Similar dwrvationc were made by Su and Ashtun ( I 97 1).

EHuct of G( on diueotiaku, mumotion. CA rbowcd a stm p r d i v e d - fectk d i k i dase &!qmn; in disisl-hiill wtyledonr flabk j -2). By I& ing CA. mtymactivity in did-hi i lva increarsd drastically. As -red tu the zero momtration, CA at bdh amantrstbnrapprmrimkly doublal the enzyme activity. In intad 4. CA a h d i d y anhand enzyme rdlvity at

the higher concantration. Thus CA, m well u BA, u a stimulus that can pm mutedippidare drvclopment and can replace theeffrci of the embryo axis.

Efferr o/lM and A M on dipcptkfum inhibition. In intact d, the l m r mcmtrr t ion d IAA had no effect m the enzyme activity: at the higher m w cmtrrtinn it was slightly inhibitory. For the distal-hall cotyledons, both cow c rm l ra th &na rd dipptidaw development (Table 1-2). Results similar to IAA w r e alm obrrvd in auxiwtype hrrbicidn, dicamba, and 2.4-D; t k a n rqwrtd in Tvblr 1-4. ABA rlightly rcduml the enzyme activity in intact mdr at thr ltnvrr eonrcntration, 5 x IU* M. At the l&her mnmtration, 5 x 1W' M. ABA decnBsrd the enzymr activity about M)@rc?nt. Enzyme ac- tivity in both intact & and distal-half colylrdonr wC abnut at the same level, rxcvpt at zero conmtratton: there was no differen4 in activity betwen 5 x 10- M mnmtration and zero concentration in dhal-half cotyledons. Thrr rml ts indicatr that in the intact system the lowr&ncenlrution of ABA stoppd the influrnw d thr embryo axis. The action of h b r y o axis war alsn s t o p d at thr higher cvncentralion of ABA and there wa&n additional inhib itinn nf thir enzymr'r devck)pmnent with cotyledons per st;

Effect of RA + GA o t ~ diprptidalp. This experiment + d e s i d to deter. mine thr interaction d CA and RA on the development $1 dipept~daw. Two cm~wntratioru d BA were combined with two cunrcntratlons of GA. The data frcm each cumbination were rompared lo the additive vdue of the individual n1mrx)nents. If there was any additive or synergistic effect, the activity of a ~wmbiratum should lw equal to or grenter than t h total activity of their mm pbnmtr. Table 1-3 su~ests that none d thr rombinatbns was additive or synrr~islir. Thrreforr, hflh BA and CA appar to a d at the same biochemical rite in this svstcm.

Portia1 ronhol o/diprpHdav by thr embryo axin Thmr~ mlrltr shw that 25 prcmt d thr dipeptidux artivitv t f i thr intac? ucnlr was ernrtrolltd by thr emllwo axis for a 4-day Krrn~inuttoc~ priod. Ftn a Siiay y~rm~rimti~rn. it tun- trtrl ld 40 prrpnt of t k activity (Sn u r ~ l Arht~m. 19711 ( in r t i r d i fk r rn in may partly acvlunt lor this apparrnt laryr diffrrtl~cv tn addttion tc~ the differ. cnw rn agr. Sn and Ashton suggmtd thut u ntirnulus for the clnbry~r axis war rmdR1 to initiatr maxinlal vnrynr uctivity. oncl tlw. dinlulur wns lcn~rnltc~ Iw r!.trrkinin. Howvrr, from th* rcwltr d our nprrtl~u-nt, il 1.i n~nc.ludirl thut (;A as wrll us BA p n l ~ n ~ ~ t n l the dcvrlopmrnl d thr artivitv ed this rn7ynw In addition. IAA. ARA. und c~ther laiiors may Ir implrcatnl III thr rryr~luli~r~r. Apparrnll!. the lunclioa of thi* c~rnhryo dxir in rr)ntndling this cnrptw 1s claitr <u~mplicalnl. In ordrr ttr lllrthpr ur~brttand th13 pknon~*nim, it wtnrlrl Ir nc.cursan tcr kmrw (In. istrrartlr~n alllong the. horna~nw. t k i r cl\ruotitat~v~. changes during urulli~~pl eCvt.lrrpnn-nt. find tbrir clittril~uticrn. Sarh ctaclirs wrre hrycmd the s c t p t r l our ~nvr*tig.it IIIII

Ejjvcr of hnrmonrs on drtrlrrprnrnf of dlprpNdanr. 111 tlistal.hal1 rt~lylt.- rlr~m, drveloprnrnt cd cl~yrptlclaa war vtin~ulrtr~l by (;A arrd RA and itrhil~itrcl h) IAA and ABA. Thr rxprrimr~rt, which r t ~ r n l ~ l ~ n ~ l the twolrornr~rwu RA or111 (:A, was ccmdurtnl tcr up if thrir rrrrnb~~rnl ac+icm wu\ uddltivr or rynrrg~rtic. Slnc? their site of artion (asoutlirxul Irlow) is cl~ll~rt~rrt, nrw wcnllil asrulrus that the artlon would Ir otlditivr. Tlw artiol~i of rytokinir~ tab prtuvn rynthrria 1%

thought to b asuriatml with tHNA (Fox. 1906. (klttv and Hu%~(~ll. iH(i9I. (;A is blieved to be involvtd in the prcdurti~~n d rsH%A molcr.nlrr on I)NA ttbnl- plate (Chrisprls and \'amer. 19670). 7711s idcrrmat~n~i s~rggrnts that thr.ws tw~r hormones dn not art nn t k sdmc rite Therdorr, additlvr or rynt-ryirlir dlrrts appear ps i b l e when the two hormoru-s a n c~a~ l~ i r~c r l . Hc~w~.vcr. III the wr. rent investigation, additive or synergistic rc*pcmsn: wrrr not fr~trncl. Thrc mav b because there was ctmtid~.ral~le var~at~im.'rhc. varratirwr war n d a~uu.d 11). chan~ing the environsmt, hrcauw tnvironmmtal conditiom wrw krpt con. dant, ro it may be due to genetic diflcrmtw. The phenotypic. diffrwrlir in In. ture and ndor d thr wed cuat was previuusly oh~rvcd and drwri tn~l (S7r. 1970). Although d s used were wIe~t(I1 k ~ r uniformity, it wa% nc~I ~mmib l~ ~II

obtain identical cxprirnenhl material lor uu i nn givtnrxprimnlt. Scluartl. a monoeciour plant, is often crwpullinated. and 1u.n~r crrtain variatic~lrs in p o l y p s are produced. Therefore. endcamous factor.i invcllvd in thc ryntlwt- sir d this enzyme can be alkred and can c a w diffrrential drvelynnent 114 (11- pcptidase activity. Other factors (e.g.. herbicides ard inrrdicibs) also rnav have been involved before the secds were brought into the IaIwatory.

Bdh GA and BA appear to induv development d d i m t i d m activity in distal-half cotyledons, but they rhow different elfed$ on the intact mfs. In irc tact reeds. CA promotes the enzyme activity a d radling pycnvd, slightly; RA inhibits thew phenomena. T I differences f o r k substunen my bcdw to

their dUfefmnt phyr iol~~icaI rnlrr and linnlly are related to the endogenous hormtlnsl balnnw. I t has t w n w~~.esied that cyttkinin may cwnteract a ger- minatiun inhihitnr in barley ucda (Paleg. 1965). It has been shown that in the b ~ r l v alcurt~ne layer, the inhibititm IJI alpha-amylase *thesis by ABA can he prtiallv overtume by additiwn of a large amount of CA (Chrispels and Varwr. 1967b). In intart barley weds, pnniuetion of alpha-amylase is iw hibl td hy ABA. and this illhibition is only slightly r e v e d bv the addition d mcpn (;A, Hcwrvrr, when a cylrkinin such as kinetin or BA is added lo the iw hihi td systeln, th~mzylm, prtduct~on, as wll as gern~imtion, is almost fully m u w r r l (Khan. 1969). Therelore, nctinp. as an antap.t)qst of a germination inhihitor, ttw cytnkintns w m rrucntial for thr funcligling of endtgenws C;A(s). the induirrlr) of alpha-amylase synthnis IPaleg.iilY65). Khan (1969) wportnl thnt in rml)nc+lrss half.& kinetin failed to dieve ABA inhibition td alpha-i~mvlar. hut his datn ~ h w d tbat by adding t k alpha-amylasr ur.tivlt\ m~)vrr tr l to t k saws level as in thc c~mtn~l. T s, h ~ t h RA and GA ma! nrunt~~ract ABA inllibiti~)n. Thr interaction among three hormtmn. (;A. BA, awl ARA. was no1 ~nvc-sl~gitnl III this stud). but hr antagonism may a w d r in this rqolntt~ry s)steni.

f IAA is inrapablr d causing an incrraw III thr protcoYytic activity (t'enwr

iind Adllon, I%7l, hut it inhih~ts d*vrlt~p~nmt d diprp(ldase in intact A s n~n l in distnl.hull cot!ldorw. Thr action td auxin has Lan r rv i rwd by Key (I%%; duxln cattwr an incr~ase in DNA. HNA, and prolein synthesis at hor- nalnal Irvrla. An inliil~itrd tlt~vrlopmmt $ diprptidas~ aetivity hy the higher n)~arnlrution of 1A.A ma! not Ir due to auxin elf<-cts d DNA. BNA, or probin r!ntlnsis. I~ut rat1n.r to thr arru~nulation td inhibitory mtabolites. Data pre- mt - I i i n rwr nep)rt 61 11ut spcifically su~cs t thir. howevrr.

R~t rhrn~~ca l aspcts of the arti(m of ABA have been r w i w ~ l rrmntly (Ad- din~tt, 19721. Many %ludies indicate that ABA ncnrcumpetilively interacts with otlnsr grt~uth hor~ncs~rs. Ttw r~sults d this experiment art- inudequatr to ac- t ~ u n l lor a qwcific~ ABA-hor~rn>nr interaction. However. ABA nullilied the el- fw l of thr en~lwyo axrr .it lowrr cwncmtrations and arvpped the lurther t b ~ r l r ~ m m t d tlw 1-nzynr activity in the cotyleclon at the highcr conccntra- Iton. ABA ma) munlernc? tlw elfrct d BA or CA (as discussed above).

Dipeptidss activity of cutykdons of quash increasd during s&d germina- tion. The embryo axis contmlled about 25 percent o l this development for 4 day grrmination. Four plant-growth regulakn (CA, BA. 1.4.4, and ABA), each with two mcentrat im. 5 lo* M and 5 x 10- M, mrr examined for their dled on the dipptidu. activity. Nmd t b regulators affected the activity pa u d the isolated dipptidsr. The p m r e d tbe gmwth reguhtors d u ~ g the genni~iation proms c a t d varkurc degreas d dfeds on d i p e p t h dedapmmt In intad radr. BA and Uh inhibited dipptidase activiiy at the

higher mnnntralion. 5 x 10-' M: ARA &cnl theactivity at both cuntrtitra- tionr. S x 10- M and 5 x 10-' M: CA. hwrvrr. mhancnl its drvrltqxtwnt at the hipfVr concentration, 5 x IV' M. In didal.hall ndylrdcwlr, RA atnl (;A h#h stimulatrd tht. enzyme dcvrlopnunt: I lcw*rr, in n~mltinatlirn t h q shrwnl no additive or symrg~stir dfwt, ARA n~ppn-sd the cnryme urtiviv at the higher concentrution, and IAA ~nh ih i ld I tu I-nr!nu activity at IuAh Iwels.

INFI.UWCF. OF HERBICIUES ON HOHMONAI. (:OHTROI, OF DIPEPl'IDASE

Prnmr ant1 A~htoa ilY671 rtsportcd that protrinaw artiv~ty III squanh ci~tylade~a incrrdsd sevrralk~ld durlnp. ~c*rl~rin~lion. dr~tl that it wuc; rpwifically rcrntrollccl 11) ytokinin Sc*vrrul hc.rl~~r~d~*r 1n11il)it~l thca clrvrlup nwnt of this prr~teinust* actlvily IAshttn~rl ul.. IRRWI RA n)~rltl rrrlutv the. ilr hibltion produced by dirhldwn~l andt~ndcrtlull(Rnnrr nnrl Aahttra. 1968)

Iliprptidvse. om of thr protrcrlvt~c mlymes 111 wlua.rh c~~tylnlosr, n l u ~ in. crrsscos wvrrulfold during thr ~I~~~I~IIJ~IOII isre and A,l~l~,n. 1971). HnuIt~ 01 prc.\ illus ~xprrimmts rhowed that d~ lwp t~c la~ ~n ig l~ t lus n,r~trollcd II? (;A un~l RA, and that th- presenw of diffrrcnt I rrb~cit ln In t l r rulturr ntllutics duritig Rcrn~innt~(n~ causd varit~ls r l fc~ts on rlr\rlnpnu~nt (11 this~nxymr

Five herbiridcs klund to inhibit thr development d clipptidiru in thr cotyledons d intact seeds were: dicamba. 2,4.D, naptalam, dichldmil, und endothall. The purposed thirnrprriment wsrto investi~irte whrthrr t h m five herbicides might alter the hormt~nal control d dipeptidirv 6vrlgrnent.

h l t c Morphologtcal mponae to herbicides, Bath RA and GA stimulated the nr.

pansion d distal-half cotyledons. As compad to thr control treatrnml. nap talam lo-' M. 2.4-D 104 M. and dicamba lWa M had no effect on growth d this storage tissue, nor were tmic symptom obrrved. When BA or GA W ~ I

mmbined with one of the three herbicides. BA or CA-stimulstkK1 war urn channed. However. dichlobcnil mmuletelv & o d the emwth d the distaC haif &I&. and thedfca ~ B A and C'A k i i m u ~ e t G n W ~ S P ~ X J nullified. Additionally, d ich loh i l c o d exudation d o gummy matarial from the timu.

Enddhall i n j u d the distal-half mtyledau--the margins and atrfaar of this time d to be burned. Developam( was inhibited by &hall alonc, and expansion induced by CA and M wnr ah reduced by endothall.

E f l d of kcrMddr~ ar CPUF w~ctlrm. EPch hcrblcide wsr added to an mr)rmeducion p q r e d from m t r d plants grown in water. Raulh dpwcd that activity d the d i i wnr not affected by the hnblddcr

T . H I - 4 . I J * n n d ~ n ~ l ~ d . I d i p p t i d u ~ t . Y J u r m a . pmmd *I &live v a p ' . . . . . . . . . ...... - -.. . .- ......

Valw l 'wr lnnnl ( % I . . . . -. ..-

Indl~dwl rrmtmmr l I $ l 1u10 (:AS x l l l dM 168 1 HASx l l P M 18.3 I)nua,bu IlY'M 2.4-0 10"M

la 5

hr)**l*rn Ill* M l ~ ~ v h l ~ ~ m l l IIlV'ht

g ;: pa:ruhrl~mll IU+M 2l n

('omh,lotion hrotmmf I l r r ~ t t l u l 0 " M t (:A S s l0"M ~>I<~I~IM IlY'kl t HAS w lfl.'M 2.l.V 10-M +(:AS# IW'M

1 2.4.1) I0 "M + B A S X I I V M l @ 3 Nupulr~s IW'M +(;.A 5 x 10"hf 1 *.I) Nap~Iant IO"M t HA l s Ill-'M I) t rhkl l r t~~l IO*M + (:A S 8 Ill-'M I)trhh4n~1l lO..M t R S I IOPM l l D ll t:nkx!rll l(Y1M t (:A S x IW'M 27% 5 Elxk*hull lO.'hl t HA 6 r lO" hf 3*15

. . . . . . . . . . . . . -.-~-.- .- -.

' \ a h a IJ trrutnmtl~ crl H,O. (;A S r 10'' M. BA 5 x IIV' M a n the rvenprof 20 rrplnrrlnrnr \ 'a~~ttWllvl l l l l lgIwatl lmtl~ arr Ilr u\rruCr$ flrlr ~plaulmns

Ejfrrt oj hrrbiridrr on hormonal ronfrol ojdipcptidarr dcmlopmmt. Nap trlam at 10-M and 2.4.D at 10-'M duceddipeptida~&velopment activity significantly (Tablr 1-4). The addition d C A and BA mmpletely nullified the inhibition produd by these two herbicides.

Dicanibu at 10- M also significantly redud enzyme development; hw. ever, only BA prrvetded this inhibitiun (Table 1-4). Effect of GA on inhibition wasnot signiiicnnt.

Dichlolwnil lo4 M showed little inhibition d dipeplidase development (Tablr 1-4). The mmbination d BA and dicblohi l i n c r e d enzyme adivi. ty mow than did the BA treatment; addition of C A to dichldKnil s u p p d the enzyme deurlupmpnt. S i m thr value of control treatmcnb was much higher than usunl in this nperimenl the rrlativepmntagc d BA treatment wasmmcquently lower. The lhedlert d BA on the promotion d enzyme activity was m&al by arprimntal ermr.

W h a l l markedly dfeded di- devclopnent 1-4). but a m~klwable variation in the data mods it impay'bk to determine itr interne

ticm with CA or BA. This alsn dmuml thr d f ~ ? of RA cm thr pmmotbn d dipepiclaw. More replicatiarc arr d l to d a m the nprimental error.

It is difficult lo find any n)rrelation Iwtwccn the rnlnrp-nwnt (II distsl.half n~tylednrandmzymeactivity. This u pr:hably bctvuw chrmlral trcatmrntr dfectrd not only the synthesis d dipptidar hut also the many othrr m d m . bolic reactions invdved in the enlargrnunt d this aorage tinw, lr~trrlerrnw ol dipptidas synthesis by thrs herbiridcl is prnbubly due to thrir rllcrt m one or more d st least three areas ol metahollsm: protrin synthnir, HNA vn. thesis, or ATP synthesis. Time limitalitm for this m n r r h prevents invcstiga. tion to determine the pos&k inhibition siMrl for each hrrtriridr Ilnw~vrr. different interactions altrred the rnzyme synthrsis whrn plant.grcmth regulator BA or CA war added to ttw hrrbicih.

Dicamba and 2,4-D reduced the rnzyme activity in distsl.haII cotyldnnn and intact ndr. The influence ol thesr two auxiwtyp herhicidn cm dipep tidar development appears to br similar to IAA, a naturally wcurring uuxin. Addition of CA or BA to these two herbicik counkracts their inhihition lo u artain degree (Table 1-4); the promotive effect of CA and BA on elrryme ac. tivity was alsn diminished by the herbicidts. The intcractioru thus alpnr to he complicated and probably involve more than one of thr ~ ~ m p r n n l s in. volved in the synthesis of this enzyme. Both 2,4-D and dicamha were found to inhibit the ATP synthesis (totlikar a nl.. 1968): hmvwrr, in r m t r e v l m (Key. 1969; H a m and Slifc. 1971; Ashton and Crafts. 1973). auxin has alw been reported to caw an oveproduction dRNA. to promotr ahyltw forms tion, and to change the pattern d dcvelcpment d endofrnour cytdrinin. In treated plants. 2.4-D was nlsn found to changr the amount of metallic im. vitamins, and amino acids available lor cnaymr synthesis. While the majnr factofis) in the interaction is not known, p r b p all ol t k fsrton srr direct. ly or indirectly involved in the action.

Naptalam aim inhibited enzyme development in distal-half dyledunr and intact 4. Addition d CA and BA to naptalam yielded mulb similar to thoae p rev idy d i d for 2.4-D. Naptabm rcpnrtedly has link dlect on protein lynthc*l in barley and &anis raL(Mam et PI., 1965), but ha btm shown to inhibit oxidative phosphorylation in cabbage mitochondria (Lotlllar et al.. 1988). Therefore, it is suggerted that naptalam r u p p m ~ the dipep t i dm mainly through the inhibition d AT? synthesis, and that it interacts with CA or BA at a different site.

Dichlobmil and mdothsll bdh inhibited thc dippHdPw in intact d. Hmvsr, dkhhbmil M little effect m dipcptidue dcvelopmcn( in the dirtPCbaU fotrkdons, and &hall i n m d mym activity in thc tbuc. Thir fbat tha. h an intcncth bstmcn the aobryo 4 s and h two ~~ altbougb dnta &id from th fombintbn trmtmnt~wsrs

u variabk that no canclrulon as to the interaction d two herbicida with the two homwmr was plrrible. A p p ~ m t l y then was an unstable factorls) inter- fering in thnc exprimrnts. Further kinetics studla would br nmarary to &a this factor.

Fivr herbicides wm e x a m i d for their influma a dipptida~c activity a d growth d the didal-half c o t y b d quash dur iq incubation. None of t k herbicides a f fdn l the enzymatic reaction pn rcol the irolated d i p p tidam. Herbicides in the &lture alution cawed diffelerm(cllectr on the growth and thedrvclopment ddipeptidar activity, although i t b difficult tolind any correlaliun 1*hvm, growth d didal.halva and mzy activity. Dicamba. '2.4.D. and naptalarn showed no effect on d u d enzym drveiopment significantly. The addition d BA or CA qliwed part of the in. hibition produced by t k three herbicides. Dichl*l hardly inhibited dipeptidar activity but stoppd growth d this ti-. &lothall damaged the distal-halves but incrcaxd enzyme activity. How these 60 herbicih interact with CA and BA is not known.

Diprplidua uctivily ol squash cotyledons increased during d germina- tion and was strongly inhibited by cycloheximide, a p r d i n synthesis in- hibitor; dr nova ynthrsir d this enzyme was suggcrkd (Szr and Ashton, 1971). Howver, such an inhibitor study is in itself not pnal d dr novo syn. t k ~ s d an induml enzyme. Activation d a latent m z y m may k indirectly depmdcnt upon protein synthrsis (Filner d el., IW). The effect d pmtein synthesis inhibitors on activation d enzyme activity was shown by Shain and Mayn (1968) in an in uiuo system. They fwd that pmtein synthesis in. hibiton partially inhibited d c n l q m m t d trypsidike enzyme activity in Id- tuamdsandrmylopdin-I,6glucorklpr in pears.

Thc question d whrther dipptidar is synthcrized dc novo or activated during the ral germination was the prrpa~c of the lollowing expriment. Ttx radioactive amino acid incorporation into pmtein method wpr wed for thia irr Wigation.

Sods wne genainahd in water tk first 96 batp and thcn placed in 'C lcucim for 24 hours. Table 1-5 and Figun 1- I show the d h Table 1-5 mDva that the &ic activity of the anzymc i n c d with cad, prrification

and Fgurr 1-1 show that ths paL d ndiolcfivity was in a a l h agrsemn( witb tk peak d auymc adivity. The iarrrore in the spcilic d b

T . L L 1 - S . ~ t i o s d ~ u * 1 & ~ r i a d d u . u k i n ( o t h r ~ ~ - . C N b - ~ & l i r d h ~ ~ i n ~ k14bm*Incd.~Cnn&tim

11) 121 131 ' (41 I S I . 16) (71 181 18) Spcdlc FoUd Eiutmr Sprdlc Fddd Pwro* Prr- A -11lc unttr mr\- rnzymc d d &

P m h n .rt~\lt) nd- p r anmtn prirc m Fnu- w m l Cpn'ml 121111 vrcarmtr ml t5~11, rat- -- rrram - In 4 8 . W 3652 9 4 I .m 3.813 0078 1 .w l a 0 0 1000 Anhactll l l:.WX) 4.031 2265 2 65 I 1 0 4 0 0620 7 9 0 382 1279 s r p h n ~ r r ~ t ~ r 1.831 ( IC~ 36.3 ; 4s; 3120 I;M 2 1 7 0 504 5 % DEAEIl\'l 0.075 45 a00 7 5 5 3764 WIS6 - 5 0 1121

- - - . -. . -- 123

* I &- I mldO.1 MKMimOnhrrr

activity during purification. as mll as the pak d radioactivity in active ~n

zym wion. provides dI'OfIg?~idmm d & naro synlbis I$ this dippliclar.

In orckr to use the radioactive amino acid methad to prove the dr nocio syn. thesis d an enzyme, it is nmrrsry to show that la) radioactive amino arid is incorporated into the enzymr prdein. and (bl the amount d radiwrtivr amino acid that is incorporated into the enzymr prdein i s greater than thal found in other proteins. The data show that. in thr DEAR frac.(itm. "(>Irucinr is incorporated into the purified d i m i d a r . and that them ir a gmatrr incor- poration d "Gleucinc into dipptidas than int11 other pr~drinr. Sincr "G lcucine can be metabolized dur iq the incubation. the greater amcwnt d "C incorporation into the enzyme brromcl (hr mod impurtant criterion fnr demonstration d the dr nouo synthesisof an enymr In this rtudy. t h greater amount of "Gincorporation is evi&nl from the puttern d rad~wetivr distribution in the DEAE fraction and from the compriu~n of qrcil ic radie activity in each step of mzymr purilicatia. This rvidcnrr i a disnranl in thr following two paragraphr

Rodiwrtfw dismbunon in DEAE frarfion. Vamr iIW/ inculrutnl embwc~less barlq hall-& in "Cphnylmlnnirw xrl1111cm for 18-20 hcurr. and alpha-arnylasc was then extrartd and frai4icaald IIV DEAI< c h r ~ ~ n l a t ~ ~ raphy. The result showed that, in thc pmnc,e III (;A, alpha.arnylnx ~IIII-

stitutd a major fraction of the radioactivity, and I l~st, without (;A, t k r r war no radioactivity in alpha-amylax. I t was mnc luk l thst, in mpmw t r~ (;A. alpha-amylase was p r o d u d in thr aleurm layer by dr wvo uynthrsir. I41r.v. ever, in an intact system an inductive agent is not r r qu id , as. for rnamplr, in de n w o synthesic of imrltritax in germinating panul rotylrtkrm as reported by Cimtka-Rychter and Cherry 11968). Thsy germinated intad p n u t A s in "Crmonstitutd amino acids for 4 daysmnd followed this by frncticmation d a 20 to 35 percent ammonium sulfate prqwatiun on a Sephadn C.200 arl. umn. The result showed thst the active enzyme fracticm coincided wilt1 a large peak d radioactivity. They mb ined thae nrults with the result from the density-labcling methcd and concluded t b l irucitritaw war rynthcr id & m o during red germination Data from this study &maatrated that t k ac- tiveenzym -ion coincided with a peak d radiictivity in E A E Iroction. Therefon, tbe dippt idar appears to be lyntherii & m e . SpcdpC radiwcti&y ojmch s u p ojmyme purfficatla. Specific radioac.

tivity (SR) war wed by Rotman and Spkgelman (1954) for ds(crmination d the& m o synthai d bda-galactorkluc in E. coll. ' b y mmpod SR value ( c p d ~ N) and specific mrym activity (enzyme activitylminlpg NJ d eacb step d enzym purification a d f a d thst. u the purification dmryme pro gmat. the SR v a l w &read. Thcy q g u t r d that thc nrldual radhctivC tyw. ; - = v t ' - , r t - 1 , I ' " 1 ' ~ m h t i w matcrhL

14 HAWAII ACRILIJLILJRALL*C ClnUWIIl hIA1 ION

H m , m n z y m t i c mtarnination with radioactivity may amunt for thr transformation of "Camino acids during the incubation. In this study SR valuca i n c m d with ea& step of purification. This Increase in SR value. which crrmprrcd the amount d "G impra t ion into dUfercnt proteiru. also ruppurtg thrdr m o syntheru concept. Ashton and Dnhmcn(1967) s h e d by wing vcrticsl acrylami& PI rlecfmphonris and ulttsmntrifugation thal fraction IV (DUE fracti~m) m t a i d only m p d i n . The gel electro phomir pattern for thr other fract io~ s h e d that these were at least three proteinr in fraction Ill. four proteins in fraction 11, an#iw proteins in frac- lion I.

Thn expriments provide strong evidence for dm quo synthsis of this dip#idase in squash cotyledons during germination, askgg&ed by the p m tein synthesis inhibitor studies of Siz and Ashton ( 1971). eurther confirmatory evidenw I w de nouo synthesis d this enzyme could bcp*ided by demonstra- ting "Grmino acid incorporation thmughwt the p o l h t i d e chain and uti- lizing the dcnsity.Iabcling technique for protein syntbis: however, these p d r urn nut mential.

CENERAI, DISCUSSION

In quash &, reserve protein is one d the storage fwdc in cotyledons. and the p m n m d the embryo axis is q u i d lor t k briakdown of this m N e material during germination (Wiley and Ashton. 1967). For complete hydwlvsis the intact m r v e protein is first attacked by prateinas. and this yields smaller pptides then a series d pptidasn berome active and further d m r m p the peptides into amino acids. The entire quenw d metabdic step d this hydrolysis has not bnn fully elucidated, but this study and ptwiws findings provide important information lor understanding thc p m rro: As stqppted by Pcnner and Ashton (1967). both diprptidase and proteim a x am synthesized dr now. Gmparing the timcoune development d t h e two enzymn, it i s a p p m t that their synthesis coincides with thc pmcea of hvdrolyris. That is, pmki~sedevelopmnt is initiated at an early staged ger- mination lthr l int day d genninatim). and dipepths is initiated somewhat later (the third day of germination). This sequential d w c 1 r . p ~ d two m zyma may be mntmllsd by their honnaurl specificity and inhibitor intaao tiom. Only BA can stimulate the prcbum activity, according to P- and Ahton (1967); however, our data indimte that bDtb BA and CA arc abk to @nhan~thrhcdrpepthrynthan DuringdwearIy *ged gemination, rcds have inhibihm that inhiiit artaia lashbdic maionr and m2ym synthcsir. Since cyhdtinim a* amsided the mDat *in agent for rmmtemcting tbc gcrmirvltion inhibitors (Khan. 19711, t&y m y x m dual &that is, tby

my promote protein synthesis as wrll u rmntrrrd the d lwt d inhibitors. Hamer , mRNA is required for protein synthesis. It has b m strtnl that the entire apparatus nmpt mRNA nmgpry Iw protein synthnir ir lunrtional in the reb (Marcus and Feley. 1964). Mn*nger RNA is ronqitk.ml to Ix autC vatd during the first 24.hour germinution: prior to it5 activation. cmymra cannot be synthesized.

The catabolism d mrrve protein is prticularly important in nrull that have protein as their principal storage product, rincv dorap protein nclt unly mpplies amino acids lor protein synthnia but aim pnlvidm rvwrgy for clthrr metabolic reactions. Thus, herbic~dn that inhibit any cd the e n q m in tho system could prevent red germination. The data p r r m t d prrvhnaly in the -Zion rntitled "Elfect of.Hsrbicidt-son Diprptidar Activily." which indiral. ed rveral herbicidesdecreaxd thrdipptidar nynthnb, when c o m b i d with information r e p r t d by Prnner and Ashton (1968) sbiw that inhihition of both dipeptidase and proteinae or were inhibition of ow cd thew rnrylna was correlated with the inhibition crl quash red grrminat~on. The inhibition of dipeptidase (see previous action entitled "lnflumcv d Hcrhiriilrr on Hor. monal CMItrol d Dipptidaw") or p ru te iw (Penncr and Anhlon. 1968) In. d u d by some herbicides was cuunteractd to a nrtain d q r w by Iln. utlditiun of nrtain horrnoner. However. the nversbility in enrymr ac?lvlty did ncd result in thr recovery of raed germination. This indiratrb that 1~1th her~n(mr and herbicides affect more than onrmetabolic sitr, and thrrdt~rr thrir srltvpr nism may be different in each system

SUMMARY

Effst of Herbit$& m Diprptk Activity

The dipeptidaae activity of thp cotyledumof thesquash (Cuturbfta mrlm Duch. 'Hubbard') increased durina aerminrtion. Several herbicidrs inhilritd this increar in various degrees. Thov herbicidcr inhibiting the drvlrlopmmt d d i p e p t i h activity 70 percmt or more were 2.4-D, dicamba, f m c , and naptalam. Those inhibiting dipeptidase activity by 83 to 34 prcmt w m picloram, dichlobmil, hromoxynil, am%mle, atrazin, nwblhall. and bromacil. Thor sawing only slight inhibition. 24 to 16 prcmt, wen diphenamid. chlorpmphpm, and trifluraltn. Monuron was amtlal ly the same as the wntml, H m r , dalapon. h l i d e . CDAA, and CDK were ximewhat stiiulatory. When compared with previwdy rqhnted hc@ici& activity on pmteinue activity, thar krbieidn t b t inhibited d l p e p t i h ac- tivity did mt neansrily sf fat proleins+ activity to thewme dcyle.

H d WtimdDipplidu Dipcptik activity d odyledom d r p d inmascd during rsd germin,

tion The embryo axis rmtdM abut 25 prrcmt d tbb devdqnrmt for 4-

I6 HAWAII ACflICIILIIJHALrA,?411Mf1k~ SI A I ION

day ~ermhtion. Four planbgmwth reguhtors (CA. BA. IM. and ABA), each with two mmtrationr, 5 x 10- M a d 5 x lo-' M, wenexamined for their dfed on the dipeptidnr activity. N m d t k ngulaton offeted the activity pn am d the imlated dipeptidar. The p n m n d growth regulators during the gcrmi~tion p- c a d various d e p c a of dl& on the diprptidpn devdopmmt. In intact sedr, BA and IAA inhibitd dipptidar activity at the higher cunrmtration, 5 x 10- M; ABA m l u d the actidty at both connntra. lions, 5 x 10' M and 5 x 10.' M; howrver, CA mhancqd its developmnt at the higher concentration. 5 x 10.' M. In distiil.half mCylcdonr. BA and CA bath dimulald the mzym development; howwrr. theitmmbination showed no additivr or synergistic effect. ABA s u p p d the &cptidar activity at Lhr higher mncentration, and IAA showed an inhibitionbthe activity at both kvelr.

Five herbicide mn examined for their influenn on &e dipeptidase activi- ty and the growth of the distal-half cutyledons of squab during the incuba- tion. N m of thex herbicides affected the enzymatic #action per w d the idatrd diprptidasc. The p m n r r d the hrhicides ib the rulture solution caused diffrrent dfertt on tbe growth and the drvelopn~~t of dipeptidase ac. tivity. Hmvevrr, it isdifficult to find any correlation b e e n growth d distal. halve and the enzyme act~vity. Dicsmba, 2.4-D, and naptalam showed no ef- fort on growth but did reducrp enzyme development significantly. The addition d BA or CA nlirvrd a wrtain degree of inhibition produd by t k three herbicides. Dichlobrnil inhibited the dipptid.4-w activity very link; however, it stopped the growth of this t h e . Endothall damaged the distal-halves, but it pmmoted enzyme activity. Data on interaction of these two herbicides with GA and BA a n inconclusive.

Ds now S y n t h m k d D i i 1 ~

By using the radioactive amino acid incorporation method. this study pm v i h s rhonn cvidmu for the theorv of de now svnthais d this dioeotidsre in squash 4kdont during germinah as sugg&d by Sm and ~ k t k (197 I 1. This thsory is hrd on the fact that the amount of "Clwine incorporated in to dipeptidnr was greater than that found in the other pmte i i The greater -nt d "Gincorporation war dnaautratd by (a) the action enzyme r+ ccdncided with a peak of radioactivity in the DEAE fraction a d (b) the specific ndiolctivity d the erurymc incna.4 with ach step d purifia- tion The mult also indicah that t h e is a pmitivc agrcuad bamca the rate d "Gkucine incorporation into the dipetdPr and thc rate d d i p p tkbxdmkpmnt.

AddiM. F. T. 1972. &cdmnicrl rger(r dlhs rdlan d a h i d c acid. b D. j. Can. Ed. Phnt Cmvth Sulnlmcsr 1970. MIn, HcMslberg. New Yorl: Sprbpr Vnh& pp. 272-280.

A h . F. M.. a d A. S. ash. 1973. Moda d Actbn d Hrrbicida. New Yo&: Wlby-IntuncUna. pp 89.178.249.277.401. - and W. J. hhmm. 1967. Pur l f lc r th and rh.nmriulbn d dipsptldra ImmCucurMta MAW mtykdau.Phytochembty8:1215-1225. . ud j. L DcWllt. 1971. H . u d d d W r r r alwnl far herbkidr mnh. Abdr. 1971 Meet. W q d Scl. Sac. Amur.. pp 79-80. Abdr. no. IS1. - D. P m r , and S. Hdfnun. 18(18. Elfed d awnl hmbkklsl on prdrdytk sctlvilydqwrhaedlingr. W d S c i . I6:IW-171.

b~ler , E.. and C. D. Wills. 196.3. Weds d light rnd 2.4.D m glulrthbn rsductu. and other NN)P.utili~@ enzyma d s r c i d M o n colylrdmr. Pnr . Olrlrhumr Awd.Sci. 47:l-7. iWdAbdr. 181137, A b r . m. $75).

Burkblter. A.. ud M. C. Carter. 1869. Ribomckaw kvell a d m l l l r nw d wody piants lo 2.4D. Abdr. I96Q Meet. W d k l . h. A m . . Abdr. no. 56 l W d Abdr. 20: 193. Abdr, no. 1305).

Chrirpsels. M. 1.. and j. E. Vanrr. 19670. Clbtcdlic acld.enhanccd synthml~ and mkrw d rlphm-amylase and rlbonuclow by isolated b r k y alruronc Iryen. Plant Phyrlol. 42:388-406 . a n d . 1967b. Hormonal mntrd d mzym spthar: on t h modsd re. tbn d g ibbd l i c acid and ah is in in a h o w laym d h r k y . Plant Phyrld. 42: 1008-1016.

Devlin. R. M.. and R. P. Cunnln&m. 1970. lb inhibitlm d #lMl ic mid iduc. tion d alphpamylar activity In barley a d a p r m by mrtaln herbkida. W e d Ra. 10:316-320.

Fan, D. F,. and C. A. MsclrNrn. 1966, Conbol d w l l u h r adhity by intklkamtir acid. G~ud ianJ . Bd. M1025-1034.

Filnm. P.. J. L Wny, d J. E. V a m . 1969, &mu indudion In hi+ plant#. S c h 165:358-367.

Pox. J. E 1966. Inmrporath d a kinin. N.Bbazyhdcnln intodubk RNA. Plant Phynd. 41:75-82.

Foy. C. L. ud D. P a r . 1965. Elfed d iablMtorr and hehicider on t r b r h n y l k add vde d d l a b 0Xldptbn by irdntsd ~ ~ m b s r lllil~~hndt'k. W d l 13:226-23 1.

F d V. K 1953. Hsrbieide mcbim mob d netinn alher thsa afyl olyrlkyt nci& J. Agr. F m d h 1:47-51.

Cdtar. M. L. ud R L R u d l . 1889. Rated modtlwlora In cymrhu t n lde r RNA: r dbdha&e&ngrlbomul&d& J. W. Bkl. 39:145-147.

C M W l y e h t a , L . u d J . H. Qrry. 1ObB.DslowsynthmkdLDcYrU.cInp.nY (Am&& hypqaa L) oDlyhdmL PLurt Pbyrid. 43:653-65%.

CI.nbu.K.T..J.CWrLdma.dT.A.&JLIQBB.Wdbrvn(ar@bh ~ r a o c M d a 1 ~ l d ~ d l a b . W P h y s b l . 4 1 : 2 8 % 2 M .

, K. R. Caykr. and J. C. Waldm. 1988. Uf& d auxin and gibbrrrllr acid m the rrgulatim of m z y m lynthmh in sugarcane d m tim. In F. Wightman and C Smdkld. FA.. Biorhrmidry ud Phyriobgy d Plmt GmMh Sub ~ ( I M - . Oltawa: Rungr P w , pp 433-442.

Hmm. J. 0.. and F. W. Slifc. 1971. Rok I$ RNA metablien in the adim d auxin. hcrhiridm Ruiduc Rrvirws 2559-67.

Hllttm. J. 1. .. L L. Jahm, awl H. M. Hull. 1963. Mwhan im of herMcidr action Ann. Rw PIanlPhvrlol. 14:353-384.

Kry. ) I. I%9. Hnrm~mul and nuclric ucid mlsbalism hnn. Rev Plant Physiol. 20:449-474.

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chapter 2. Effects of Hehicides

on Bud Sprouting of Purple Nutsedge R. K. Nishimoto

The objcctivcs of this study were(a) todetermin t b hsir d bud dormancy in purple nutsedge-U dormancy can be overmmr and sprouting initiated ar d e s i d , the effect of repeated herbicide awlicationi on the sprouting ability of thest dormant buds on tubers can be cktormincd: and (b) to campnre the el. fectr d repatmi foliar applications ul glyphasvte IN-(phosphonumsthyl) glycinc] and wera l other herbicidn on ths growth nnd reproduction of pur- ple n u d g e .

Purple n u d k (Cypmw rotudw) is tk most troublesome wml of the tropical world, mainly hccaur of its biolqicsl adapability. Thia weed is a perennial, porrrring an extenrive vegetativc structure d rhiwma and tubcrr that am alro propneptive; t h w tubers may remain dormant in the mil, and this further favors its survival. ~ l l t h w facton have ma& ermdicatbn d n u d g e difficult. and to dpte normnomid control &hod i~ available.

Purple nutsedge appean tolerant to m y herbicida primarily becaw mom rhooh will r p w t if olie is killed by M i c i d a . Varying love11 d control hnve been achieved by the ur of 2.4-0 [(2,4dichlomphcnuoly)acetic acidl, dicamh (3,6dichloro+anicic ncid). amitrole ~3-ami1y).ttrlrwle), and MSMA ( m o n d i u m methammnste) (Hewer. 1983; Mamlhaes et el., 1968: Hamilton, 1971). h t l y , glyphmte w u reported to-dufe pvpk nu- rtanda in Tanunia (Mllgnmbo and Tmy. 19731. Ewn d tuber ides

hnr km athated that hd& p&nted at b m qpcing wlU prodwe 2.1 million plPntrand4.4 million tubem per 0.4 ha in I year (Hairumr. 1962).

TbcdcvcloprratddormpntRtknrmblaDutrsdgctoodpPtonappfng mdtmbd variourcultura, tbur mnkLg itrerdicetiondifficult. Black t u h exhibit the p t a t dsgree d dormancy, whik yarql wbite tubsn wad crrdly. Dampnt tubm h v e from I to I3 bdr (Udri. 1 W ) . Only one or two bulrlpadun&rruitrbkmodiths,t&tbcothcndaaynt.~f

budr inhibit sprouting d other buds on the Ism tubrr (Muzik and Cruzado. 1953). Similarly.Jsngaardet al. (19711 nded that u p thedeath of the grow ing plant's foliage, inhibition of tuber sprcnrting was relieved, and sprouting of otbrr buda occurred. Diarding the tubers also stimulated sprouting d dor. msnt buds near the cut surfan (Muzik and C~rado. 1055).

An cndogrnotu pmmotrr.inhibitor complen is com~nanly believed to cnn- tml dormancy in rmfr and resting buds (Wareing and Saundrrs. 19711. The endc~~laus pn~motrrr may include gibberellins (CAI a d cytokinins. Many inhibitors are pntulatd to tr present, but thox mast oRn cited are abscisic arid M A ) and phet~lic compounds. The role of cytok In In wleasing dor. rnancy. in mmc c a n i s to antagonize action d an e n b a r inh ih ih In thr alwniv of endogvnws inhibitors, cytokinin i s diop@ble. Fishrr (1971) nlggrsted that a high lrvcl d the endogencnrs inhibigr gibberellic arid, trw!hrr with a deficirnt? d +&inin. c a d bud ihibition in Cyperw altrmlfbllw L.

Bud dormsncy in nutspdp i s suspectd by many to be c(ue to the presence of inhibitors (Ueki. 119). Washing tubers More spro@ting incrrad the numlrr rrf sprouts pnduid p r tuber. suggesting that th) inhibitor was water soluble. Brr~er and Day (117) rrportd that salicylicPcid war the major ccrmporrnt ammg thr many inhibitors found in the lr$ilge d purple nuts- rdge; h v r r . it was not found in the tubers. Jangaard rt a!. (19711 thought that ABA n~ight be the inhibitor umiated with nutsrdg~hrtrr dormancy, but they were unable todrttWt i t in the dnrmant tutrrs(perswal n~mmunication). Jackson ct PI. tl9711 rrportrd that a h ~ l r ~ and ethyhnstimulated bud sprouting in nutadgc.

BUD SPROUTING OF PURPLE NUlSEDGE TUBERS

BA (N, Bbnuyladenine) at 50 and 300 ppm stimulated bud sprouting, pm ducing two to t h e timn as many pmuh p r treatment as thr cwtmlr Vabk 2-11, In the mntrols, only one long apical spmut was usually formed. Iw c d BA ccmuntrations inhibited mot formation and d e e d mean length d spT0ut.s None d the other growth regulators trsted stimulated spmutiq. Apparently, sprout st imuhti i occurred only w l m tubers were iw cubahd continuously in a BA rolutim (Table 2-2). Higher tunperaturn pm d u d mom bud sprouts (Tabk 2-3); at the I m r temperature rqime. BA- stimulated sprouting occurred only at 50 ppm.

As argemd by data in Tabk 2-2. BA is appyently nguind as an agent for tbe sproutin# procm rather than being mereiy a trigger mrsb.nirm to rcker dormancy. Further. thm is an implication that dormancy in IILI~&~

ia due lo a deficiency d thi spmutirg .eat, cawing an unfavorable b a h in tk pmmh-inhibitor ampbx inthe tuber.

Tab?-2 . EMndrukiiprpknut+~ whmm IWppntl l UA l u r d i n m ~ l@nall lhw . ...... ~ -

'ipnml, p"Iwrllln11' T lmr fh~ ln l .......................

lnrr tpl4lbll - OIw~lrr ~(mlnAl I 4 U r 3 13 H r fi 140" I2 l b 5 r 24 ISlu 240 IBA rmln~l) ' 29 0 I, ..................... -. -

'Mranr wtlhin a column hnmp u dlffmt~l pdw.r~p( krlrr arc ~(yl~lnanl lv dillrmvl IP<O (IS! IDunrm'r Mull~plr Hanw lntl.

'Tubmrrmr~d mBAuhtrndurm~thr I ( 1L~~d lhr l lu ly

SOURCE: TwCI 11.1973

On the other hand, M A inhibited Iprrnting at higher r~ l rm l ra t i tw (Table 2-4). When tuben were pretreated with BA and Ister incubated in ABA, spmuting was also inhibited, but to a lnrr drlgsc. Further c x p r c to BA stimulated sprouting. T h e data sum that dormancy in nutdgc tuben m y be due to a hormonal imbalana, favorlng such arbatsnm as M A . BA b the muggested pmmoter in thb y-.

Growth d plants from tuben prdnsted with 100 ppm BA did no( differ significantly fmm untreated hlkn when mensuring p~~ such Y

flowering time. number d infiorrrmca, and number and tdal Imqth d rhimmer after 5% wedu qnbk 2-5). Hovurver. amtinuan BA applierHom induced a tuft-type growth, witb hoe dark g m bra. wnr , . I . . . . . . I, " , ,

34 HAWAII ACRILIICIL~R~L~~, .*IN n t 41 A IION

c m d BA lnduced production d numnour short, diagsdropic rhimma. In untreated plants, rhiumrr wen branched, w h n a r no bnnchiag w u ob d with BA tmatnmt.

BA. as well u PBA (Bbmzybmim9 (Mrahydmrsn2-yl)-9H-plrinej a h induced rprouting d tuben in wit when applied u a drench. Fmm 70 to 80 percmt d the burh a, the tuben sprouted when grown in mil with either

24.C dry: G n n n t r a t b 17.C nigh1

Tmtmml (ppml ..-.- - -. . ., . - - - - -- .. - - - --- Wdrr m t d 2 3 . 6 ~ : 6.0 a Uhnd Nnlrul 2 2 . 8 ~ ; 3.8 a B A I 9 2 b l S 8 a B A 10 20.6 r : 50. BA 50 314di. 13.0 b B A 300 46.4 c 5.0 a -- 'Mwly withm a d m n lrunns r dlrmnt poltrrlp htrr am s@icantl! dBI-t (P<0.05) Ihncan's Multupk Rang?tnl.

X)UH(E Tat* r1 , 1973.

Spmucrpr t ru tmnt (nalplant)'

Cunmtra lm B A + BA+ Tmalmmr (ppm) --- ABA ABA' AM-BA' l r m

BAuwHml' IM 22.3 r 22.3 a W1nrrmtrnl 10.3 a A M I 13.54 18.5 a 22.3 a 3.8 hBA 10 8.61 21.8. 14.8. 3.0 ABA I W O.Sb 8.5b 15.3. 7.2

T . b * 2 - 1 . F A n X d m H M M i u + b d I I A o l , & ~ ~ ....... - ................ -..-

C n w h d asid +a' .. .. ........... ---..-...... ...., -.... ,

MNII

B A hqnhd p,rrmn

rmmnlntm Sbxlls lnflurmnrn Rhatmr Lnq,tl~ r h ~ n u m Ippm) inrj rplmtl IIUB /fludl imrplndl lnlrnl tmnrl .. " ......................................................

0 11.Z.b 4.1. 19 l ah 1346, 476 10 9 0 " 2Rr 14 Uw l ( i l r Ma

100 1 7 7 ~ 0 2 h 3RBc I*. 21 Ir 100 104, OSh 349, 1115r 24b

............... ...-..... .-. . .- 'Mranr wuhm r ~*rlumn Irannr u ddlrnt p ~ @ i I ~ k r uw ilp~l~vuntlr rllllrmlt IP<O 051 (Duncan's Mull~p!+ Hanp w)

SOURCE Trucfrl . 1973

Figure 2-1. B i y ol actiiity d acidic &er exlraci I m IS g C. rotundw lufrn (A. 9. C) and tk various canpment~ d ~nhibilorflfrum lO$C. roundlu tubcn(l). 8. u r i q the w h t mkaplik assq and. with ~ h c addition d I pprn BA, tuifig Ihr C. rohndw excised bud m a y (F). EI(rhCL. mrs run m 7LC and hvrbpd with Ihr fdhwing Iolvml- A-butand:.a(ir a&wstn 15:4:1); B-Chbm/~:&hyf satateamtic acid (WWS); C i q m p u a l : a m r r m l u m hydmjdrrmrn (lW.l4:B); D-mkm(orm:aatk acid (85:51: and + F-dhclhyl udate~atlc rcid l*S:2L TlaCd~mFindiahrdlia~~rd.Tbs@bndmrl;nMAblbwr b y a d i d ~ m t & X u b F m m T e o e t ~ l . , 1974.

BA or PBA, whik only 25 percent d the bucb sprouted in the control. Further. mom. BA activity in the soil lasted for hm than I 4.

Whm Inhibitom m m extracted from tubers. only the acidic ether extract rhomd activitv. a d thu fraction was further investigated. TLC (thin laver chronutagra&yl rpsratlon d theatrsct indicated t&t more than one inhib Itory mbstaner was p m n t in nu t rdp hbem(Figure2-I), and, based on the slmil~rlty d Ihc Rf "alum in the solvent Iystem uwd (&opropanol:ammonium hydmxi&water [100:14:6] hereafter rderred to a5 PAW), the inhibitors were colkdivc.ly rslerred to as the inhibitor $ complex d mrlier workers (knet . Clarkad Kdford. 1953; Hembcrg. 1958: SmIai. 195k:Holst. 1971: Waning a d Snunden. 19711. Hechromatography and bioasro@thir$ inhibitor corn plen tcdrd by the excised nut&@ bud assay (~ igurd 2-1. F) gave broadly similar results to the wheat mleoptik assay (Figure 2-b El. Five zones (Rf 0.6 to 1.01 inhibitrd bud sprouting, and the mar&er ABA torresponded to Rf 0.9 (Figure2- I, 0.

Figun 2-2 shows a comparison of the activity of A and the inhibitors from nut* tubcn. Fifty percent inhibition of whe!f**plile elongation war obtained with the inhibitor $ compkx fmm 3 g ofhben (Figure 2-2. A. with ABA at 0.5 ppm IFigure2-2. D), and with t h ~ RIOB comporwnt from 6 g

Figm 2-2. hdivity d inhibitors lm C. mhrndur tubers a d ABA. A-inhibilor fl mapkr; B-Rf 0.0 cimponmt d inhlbilurp mqka further c h m n ~ ~ r a p k d with ~ h r ~ r t h y l l e d s k a m i c acid (M:5:2k udC D-A-

~ k l ~ . a n d B a o d D w l c h ~ ~ c b s d b u d , d C ~ m h u d u r l n d w i ~ t k

d tuben (Figure 2-2. A). Fifty p r m t inhibition d bud spmutiq was ob. trincd with Rf 0.9mmpavnt from 3 g d t u b m ( F i y m 2-2. B) rnd w i t h ~ B ~ at0.5 ppm(Fiyn2-2. D).

Inhibitor P fmm PAW significantly inhibitale*clad bud spmutln&plrtlal. ly or completely, depending on tho wigM d tuberr axtractmi. 7hr rddition d 100 ppm BA to the extract nlicvsd partial rpmut Inhlbltion but wer indfcc. tivcon mmpletely inhibited budr.

Bdsprouting inhibition indumd by t b variow nrmpxrnts d inhihitor P was a h relieved by treatment with BA; thw none d the mmpnrants c a d complete inhibition. Inhibitors utractsd from 10 g d tuben inhibited bud sprouting even with BA p m n t . On hundred ppm BA did not rwnrc b d sprouting inhibition. for the inhibitors pmrnt at Rf 0.9, which shwld have included any cndogcnow ABA (Table 2-6). Bud sprwling war werely inhibited by exogenous ABA, and this inhibition war mvwlsd with varying amounts of BA application. lnhibitiun d budr by 250 pprn ABA war not m r r d by 100 ppm BA. The gas liquid chmmatography (GLC) analysis d the BSA IN. OBis

(trimethylsilyl) acetamide] treated inhibitor p compkx indicated the p m r e of a mmponent having an identical retention time 120 min) to the standard trimethylsilyl derivative d ABA (TMSABA). U p further TIC purificatim of the inhibitor ,9 mmpkx in dher:ethyl snlate:anlic acid (50:5:2), the materials eluted from Rf 0,9 d the chronmtcgram wen nnvlyvd alone and with added ABA after silylation. The ABA added to the eluate war coincident with a peak in the unfortifiedelunte(Fi(yum2-3). CLC s ~ l y s b d the uthn i s hibitory z m (Rf 0.6. 0.7, and 0.8) s u e d that they did not m t a i n any ABA.like mbstana. This i i c k n a . supported by the b i o ~ p ~ y s d thc elustn.

T a ' . b * 2 d . W k a d o a p a n n f n m s ~ ~ ~ ~ ~ l u r p l r ~ ( u b n n d u m u b g * b l i ~ . d l r r ; l a h r L i d ( 5 0 : 1 ~ 2 1 , a n d d I A a , p r ( 4 d n r i d W d d w h d l * -- . .-

Per- d I d s qulltd'

# d y Inlhtea Ib.1 ABA none d the minn annparnh d inhibitor $ cocn phr .

Spraying r drlp d r T K plate d tho inhibitor p compkn with diizotiad pnitmmiline (DPNA) produd I ckep $low wIonHan bebum Rf 0.5 and 0.8. w i r y that m w d thae rubotsnar wen phenolics. A similar chmrutqlnm *rip prayed with n nrrraoHClnh8nol reagenl showed a pink.vW color r t W 0.6 and violet at WO.8, thus indicating that them might be dC or trihydmy phenola. A li&t gray spot obtained at Rf 0.7 when a drip was mated with 2 percent aqucau, ferric chloride WIS again indicative of a phenolic compound.

Although thir work mtsblWIcd the p m n m d &c inhibitor p camplex (including ABA a d p W r J in purpk nutrdgt ha*, this is not sufficient evidence to mlatmtirte its poaible phyriological ref) in causing tuber dor- mancy, The m l b suggut a hypotbais implicating a w i b l e relationship be hvmn inhibitor $ or ABA and bud dormancy d tulf~rtrs in this species. BA rsveml d the inhibitory SfIwts c a d by the inhibil)r p complex and ABA

i

F iyn 2-3. Cu cbmrm(ognrmd a dlybtedeluate fmm RI 0.9 comp.mt d inhi& tw$mmpbad C. tvtundwlubu~ m d u o n m t u hctbnvthyle&kaat ic acid 1W5:2), w i t h [A) a with (B) tb ukUtim d adurl M A . F m Tm ct nL, 1974.

mpporb the hypothesis that a balance bC(Ymn inhibiton md w e n m tmb dormancy in nutsedge tubn. T m m m t d bulr with inhibitor p or ABA hifh the belam toward ~pmuting inhibition: addltiun d BA to tho apmutiq media permits qmuting. In this hypathnis, the mk d 8A in releasing dor. mancy n to antagmizr thendoganow inhibitor rctim.

This hypothesis explaining dormancy in purpk nutrdgt ruggrda that dur. man1 tuben are deficient in a cy Jinin. and this la& to an imbaluncr in thw promoter-inhibitor ccnnp'm favoring tho inhibition. ABA und thr t&r wn. ponents d the inhibitor 1 mmplex are likely natural inhit~itors. Acvumulutian d a cyiokinin, or dimppearanm d thc inhibitors. wc~llcl tn nrcrasry to restore P favoruble balancv d growth primden, vllwing rpmutirg to occur, The effect d nogentnu BA is to counteract tho inhihitor neliun in u mpnmr similar to that postulatid for tkact im da rytokinin.

RESPONSE OF PVRPLI; NVISEDCE TO REPEATED APPLICATIONS OF HERBICIDES

Glyphmate d u d purple nutdgestPnd, in the fkld(Tablr 2-7). Thrm- ond and third glyphmate treatments n d d the stun& substantinlly but did not completely eliminate the population, 'he importun* of cumpldr elimina tion in an infested field was clrarly ckmarvtruted in this nprlment (Tobk 2-71, When the field war Mt undisturbai for 10 d s nfkr t k third treat- ment, t k nutsedge population increased to about 35 and i Q prcmt d the control, which demonstrates the mrtabbhmmt pntential d uncnntrolkd nutsedge.

NurnbndplPntslnu.ffl I m'l ---- - - - . - ... . .- . 4& 4 d 1 4& I O d

Ralr .herfirst after& altrr~hid dlnthld HnMrkje &&a) Imtmnd lmtnrnt IWmmI IwMmmI

GI- i 32.8 1.1) 2 3 13.8 Gb- i 14.5 1.11 1.2 1.3 MSIU 35.0 13.1 22.8 22.8 hw I 31.5 40.3 37.3 80.5 Dlcrmb 1 40.3 23.5 54.3 32 J Codrd 44.3 47.3 w.3 w.0 L9). 0.05 in 240 4S.S 21.0

-lmt.lrorH.M-, ~ Z s l d m a a l . 1 8 7 4 .

MSMA dgrllicantly dud the mt#lge &and 4 wddrr after the rcand t m h m t : at 0 t h tima, bowever, i t did not differ fmm tbc control. Parqurt (I.ldhmthyl-4,4' bipyridinium lonl and dicnmba had no d f d on the n* star&.

Ahcr the field war mtovated (working the mil thoroughly to a depth d IS cm) on cmnpkion of the l int stap d the a p r i m t , the initial nubedge stand dkl nd differ behu&o treatments (Table 2-61. This similarity may be d r to revera1 facton. Rdovating the dl may have r a i d dormant tuben lrom the lomr ail Icvelr, which had not bsrm under C influmn d the k r - bicide treatments. Sine the field was left untreahd lq 10 weeks More rote vating, the nutnedge my have &ablihal italf in)he treated plots. This allowed lor the pmduction of ncw reproductive anrqum, and t k st~c tunrrpmuted alter thr licld was mtovntcd. 1

Thr duction of the n u k d p stand due toglyphom# treatncnts in them- owl duse of the experiment (Table 2-8) was similar tqthat described earlier (Table 2-7). Likwix, paraquat and dicambn had 4 dl& on the stand. Repeatrd application$ of MSMA d u d it to a level&imilar to that of the glyphusPR trratmmts. This m l t supports earlier h a t i o n s d Hamilton (197 11 that qmtd applications d MSMA a n n-y to mntml nutadge.

In addition todestmying the leaves and reducing t w a n t population, the effect d npatrd applications d g l y p h t e and MSMA was to d u ~ r tuber production (Table 2-91, Treatments with g l y p b t e and MSMA reduced tuber production by 92 and 88 prmnt of the mntrol, mpectivrly. Paraquat and dicnmba did not mluw tuber pmduction. Thr number d tubers that sprouted after treatment with glyphosate and

MSMA was d u d when cumpad with the m t m l (Table 2-9). Thar data

Clyphovh Clyphput MSMA P.lqul D i n 6 Gntml LSD' 0.0s

T ~ ¶ - ~ ~ ~ J ~ ~ & ~ I & I ~ ~ ~ ~ ~ H * Y I ~ - .--*.-.- "-

T v h n blt prrrm'

TJrnpmhd Spnw~prp. withBA tratuwd' v(.hbhhlr

W& lk*) (mm I 1%) (mrplml - .- . 2 :Ee 14.0 0 4 5

14 5 33 3 1 MSMA z u1 .a 17 3 6 P.nqual I 11.5 (Is 3 2 Dlramb. I 82 0 I 7 3 5 Crmd 172.0 82 44 LU)' 0.05 1110 20 lu -- - - - . . - . - -. . . - .. . .. -- . . .. -. 'Tubers <hainad lm lhrlrppr 13 cm d nil. *Ten tuhm p r petri dirh rpnh~d with 100 ppni BA.

s@Ukarddlknnrr

SOURCE Zandslrarl a1 . 1974

seem to indicate that foliar applicntiwu of glyphowtr and MSMA alto of. fected tuber viability, as measured by BA~indud sprouting. Tsbrrn whnm viability was not sffe&xl produrrd as many sprouts as thr mntrul (Tabk 2-9). indicating that buds on thex sprnutd tubers had nnt accumulvtd tmic levels of the herbicider.

In the gmhowe, glyphmate reduced the fmh weight d green leaves u( purple nutsedge and the number of sprout# pmdund from thc original tuhcn, new tubers, and basal bulbs (Table 2- 10). V k l examination ol nunsprouting tubers indicated that same of their ti- were necrotic, suggesting t b t g l y p b t e was translocmted and sffecied the viability d original and new- developing tubers.

Differentin1 translocation of glyphom@ c d explain why sum tubcn were killed and others were not, An establbhed purpk nut* a n d cmirCc

d s m p h inlerronncasd system d plants, rhiuma, bus1 bulbs, and tuben (Ram& and B u m . 1925). Plant, may a r k directly from the tuber or from rhimm* at varying distanw fmn the tuber. On the basis d distance. diffemtt fkgm nl trowlocation d fdiorly applied herbicides to under- ground ltructum muld be expected. A h , amc tubm wen p r h b l y alnsdy dormant at the timr of application, and Ica tramlocation to then tubers wwld br r*pcM.

SUMMARY

Varicms mnmtrations of BA were wed to purple nutwdp tubrrs. BA al 50 to 300 pprn sti tinuws p r m m of BA during the sprouting p nificant sprout stimulation. ABA wunteracted when tubrrs were treated with ABA after B mnrkdly pnstor with days at 33% and nigh 24.C und nightsat 17'C. Growth d plantsorigi~tid#fmm tubers pretreated with 100 ppm BA did not differ significantly from 1% controls. Sustained BA upplications at I00 and 200 ppm produd numer& plants with tuft.* ~rowth habit, delayed flowering, and redud the number ol inllomrnces. Numerous short, diagootmpic rhizomes wen producal.

BA and PEA stimulated bud qmuting of purple nlltscd$ in soil. BA adivi. 1). in roil was less t b n I week.

The acidir &her fraction of methanol extracts from C. rofundua tubns m n l a i d inhibitory suhenms descriW as inhibitor $. Inhibitor j inhibited sprouting of a c i d nutpdge buds and elongation d wheat mlmptilcs. B d sprouting inhibition by inhibitor fl was merpd by the application of BA. A p plications d ABA also inhibited sprouting d excipd buds, and this was similarly w e d by BA applicatimr. Chmmatographic evidence suggested that inhibitor f l misted mainly d phenolic materials and pmtibly ABA as a minor mponent.

Purple nubedge plants wen treated in botb the grunhour and the field with gl+te. In the field. glypbrnste trutmmts at 2 and 4 k&a w m r0mp.d with avetol herbi& for nu* mntrol in nputed applica- tions wcr an 8-month pemd After kve~al application% glypbosate reduced the number d plants per unit ua. After rotovation and reapplintkh glyphmrte and MSMA plots bad femr plants s;ld tubers per amit area than contmls. Thsr tubers wcrr also lar vtblc than tubcn fma rmtml pbtr. la the gmhuse, f d wyht d k.va sprouts per ori@al t h . and sprouts per new tuber were reduced by 4 kgh glyphmate.

b ~ t C l r & . T. A.. a d N. P. Kdlard. 1953. Chmnut~gaphy d t h p w t h 6 st- in plant arm&. Natun 17I:BIS-047.

b. C.. and 8. E. Day. 1987. DMnurry, Faw(h inhibition, and tuhrriaatlm d nu* u dfcrted by phalqarbdr. Pmr. AdmParillc Wanl L i Sor Gd. 1:123.

Fisher, j. 8. 1971. Control d bud inhibition In C v . Plant. 97:257-268. Ilmmiltm. K. C. 1971. bpahd ldiar applintimr d MSMA on purplr nut*.

Wad Sci. 18:675. Hauser, E. W. 1982. Edabllrhmnt d nut* f m rpwplmtrd tulm. W d

10.315-321. . 1863. Rerpnr d purpk nut* to amitmk. 2.4.D. and Fmt.. W a b

11:251-252. Hemberg. T. 1958. The signilicanw d thr inhibitor # iwnpln In thr mt p r i d d thr

potatotuber. Phyriol. Plnnt. 11:815-828. Hdd, U. 8. 1971. Some pmptties d inhibitor! from Solanurn fubrraum rompred

to abrcirir acid. Phytbl. Plant. 24392-396 Jackm. E. K.. N. 0. langaard and A. L. J a m 1971 Thr airnulath d nut+

tuber sprouting with clhylm. Plant Phyrbl. 1Suppl.I 4787 jangaard. N. 0.. M. khcrl, and R. H. Schidentrin. 1971. The rnk d phmolla and

akisic acid in nubdgr tuber dormancy. WsnlLl. IP:17-20 Magnlhan. A. C.. F. M. b h t n , and C. L. FQ. 1988. Trawloc~tion and late d

dicamb. in plrpk nutdgc. W d Sci. 16:240-245. Magambo. M. J. S., and P. j. Tnfy. 1973. Canbd d purpk nulsdg (Cypnu rohuc

dud with glyphmate. Pmc. Ada~P~cil ic W d k i . Sor. Cad. 4:191-194. Muzik, T. 1.. and H.J. Cmudo. 1953. The dee( d Z,+D ~m sprout lomutien In

Cypnu rohdw. Amn. J. Rd. 40507-512. RPnade, S. 0.. and W. BUM. 1925.Thr eradiation d Cgpmu rohndw. Indm Dsp.

Aa. Memoin M. Ser. 13:99- 192. Sulai. I. 1959. Quantitative changad yowthpromutiq and lnhibtti~rulntancm in

thepotntotuhcntrntsd with rhditt. Phylbl. Plant. 12:237-244. Tm. C. K. H.. L E. Bedken and R. K. Nishimto. 1973. Bud rpmuting a d I(mwth

dpurplerwhsdgealtnad byhzylsdmir*. Wadki . 2I:19-23. , R. K. Nihimdo. and C. S. Tpng. 1974. Bwl inhibition d C y p w rofundw L

tukn by inhibltor fl or sbKWc acld md tbe reversal d h e Llf& by N,C bauyldni~~~. W a d k . 14173-179.

Ueki. K. 1980. Shdia on rmtrd d nutrdge m gnminrtian d a tuba. Pme. A ~ l r n P d k WadSei. Soc. Cad. 2355-369.

Wuein#, P. F.. d R. F. Surndar. 1971. Ha- and dommncy. Am RN. Plslll Phytid. 22:261-288.

Zdrtra. 8. H., C. K. H. Tm, a d R. K. Nbhlmdo 1974. R q m m d purple nul mdgetorrps.lal r p p l i e r t b n r d g l ~ WssdSci. 22:130-232.

HAWAII ACRlCllLNRALEXPWMEKTSTAllON

Chapter 3. Action of Sandoz 6706

on Carotenoid Synthesis and Chlanplasts P. G. Bartels

Sandoz 6706 [&chlor*S-(dimet pyridazinone] is an nprirnental pramerpnn contml d many b rdp (Cypmu p.), in cotton fields. Cotton plants 6706 in their roots but tramlocate I- d the herbic cam and mybean plants. Tbis differential translocnt plnin the diffemcm in the tolerana d &ton (residantf, cam (very sumpti- ble). and sybsan (~snptible) tohndoz 6706 (Strang a d Rogers. 1974). The chemical dnrctun d S a n k 6706 is similar to that d the krbicide

pyrazon [5-amind.chlor02-phmyl-3(2H)~ri&zi~~ The initial visible symptom dSandoz 6706 toxicity to plants, hmvrver. is uhlike that d p y n m but is similar to symptoms c a d by the herbicider amitrole and dichlormate. The ultr.atructural study by A n d e m and Schaclling (1970) revealed that the earliesl symptom d pynm injury is swelling d the chlomplast in mature bean learn no immediate bleaching d the leaf titare w u obrwed after pyraun treatment. A n d e m and Schaelling (19701 a l a rcporhd a marked incrense in the number and aim d osmiophilic plortoglobuli in thr chlor* plad. Accumulation d plutoglobuli m y nsult from an inhibition d thyla- koid formation In m t n d . Sandor 6706 hw a pronounad affect on the chloroplad pigments. New foliage f d in the light after treatment with sublethal application d Sandos 6706 is either a blachnd whlte or an& cyanh md. as detemined by the plant via' caplcitia for anthocyanin p m ducHoh Slndoz 6706 dom not inhibit d germination or mnrml leaf emegsnce.

Hilton et 11. (1969) studied the allact d Sudor,6706 on established gcsl lttvsraddevdopin(ltirwred4diffmntrpeciaTbyfaudhtthc Hill &ion a d C Q fixation wm inhibited in olda establihrd lam w h liptd up& and utUkatioa wen affected in yamg tbar. They% ahdthSudor6708m*ht~ctuadireftinhrmtordrneady

stage d bioaynthair d *opmdd I p l b fmm acetate. This M i c M c m y rla inhlbit utilization d lipids twesary for iwrrmtbn d h m l h r r m n b r w ot other structural components d the chlomplu(. Hilhn d al. (1971) raportal that SPndoz 6706 reduced the amount d fatty Kki found in pdrr lip& (p Iaddipidd d chlompl.sts and i n c d tho amount in ntmpolar lipids. whlk having link dfect on the tdsl content d b d fatty acids. AIM, S . h 6706 redud the amount d linolmic acM nlrtivo to linolsic acid In k.va and chloroplast*. h tomphcml awlate. phytd, fanrrol, q w l m . unukrated fatty aci&. and fatty acidmethyl den m l d circumvent some d thca Inhibi. tory effects of S a h 6708, especially on h a i d pigment accumulation and fatty acid buildup. It war Mtrd that tdfluoromrthyl ~bstitution on the phmyl ring d Sanda 6706 was ncnrrpry for its herbicidal activity (Hilton d al., 1969).

Carcdmids protect t ~ l l s of photrsynthtic badrria Imm lethal ph~t t t indutvd reacticms. In exprimcnts with bacterial cells larking nor~nsl cnna- moids, hartcriorhlnrophyll was blrached and vrlls werr dtdn~ycd when I& were illuminated under aeroh~c rnnditemsi w~rmal rnrotrnt~idq prwrntd bl~aching and pho~ukstruvtim of cvllr ~CohmRarirr and Stonier. 1058; Fuller and An&rr)n. 1958). Cart$rnoids alaproteiq c r l l s n l n~nphot~wvnthr- tic bacteria from damage due to phtawxidation cutalvlrd try adlrr I~ght- absorbing pigments(Mathewr, lW1.

Kohl (1902) was firrt to mggnt that carntsnoidr might protect chlorophyll. The protective adion is now believed dur lo mrotmoidc with ninc nmjugntd double bondr or more quenching excital st* of chlorophyll (Fujlmori and Livingston. 1957). Krinsky (1967) reported tbst carotmoida could mnwr m- ygen from excited chlorophyll-oxygen mmphnr vla a cardrnoidcpnrldr cy- ck. This would pmmt photoexidation or bkaching d the chlorophyll. and mldexplain the protediveactwn d carotemidr.

There is much evidcncc extending the protective theary d nrotmoidr to higher plants. Korki and Smith (1951) fwnd that a bleachcd mutant of corn was actually a c a r d d i m mutant. This mutant syntbizcd pre to&lomphyll(i&) in the dark and converted it tochknophylMl&) upon IIlumi. nation. With cattinuow illumination undsr r d c cundition howww, whatever chlorophyll the mutant lynlllaid vu lod. Anhron and Robert- son (1860) lrtwmd this mutmnt to haw r block bchrrm the u m d procur. sor phytoene and mrml cardemids. Walks (1867) f a d that a d b w n mutant that dkl not haw normal c a d pi- m n t a i d dirnrpbsd plsrtidr Thir mutant I d normnl e t i i h when p ~ n (D th. dark. Whm tbe m h n t wu e x p d to light. t h y W a d va ick m m I I y

t h m q h d the pluti, but mnthusd illumination rrruhsd in plaid disiup t h a d blsrching d the nudlower d i n g s . FaludCbniel d rl. (1965) found two other photo*nritive mutanh d oom with b lo th in the carolemid. synthalriq ystem. All t k camtsnoid.free mutantr armmuhted chbrr, phyll unkt dim light but were bkactud whm a p d to bright light.

Variau mechanisms d adion for hcrbicidw have hm pro+ by dif. ferent mearchen (Ashton and Crafts. 1973). However. only recently has the lnhlbition d cr- been cnnsidmd a piblemcchanirm d action d m r r l chumicrlly unmlrted herbicidm (Burns et nl, 1971). Burns ct al. (1971) have shown that tnahnant d wheat sca#ings with amitrole (hmincwtriawla). dichlonnate (3.4dichbrokenzyl ~hylcarbamate), and pyricbr (2.3.S.triclord-pyridinol) multed in inhibit* d the normal bi* synthetic pathway of cardemids and. as a conralwn4 in photafnt~ct ion d chlorophyll and disruption d chloroplast. Theit work shomd that

plants.

Light. and dark.grown wheat sd l i n l p synthesize and accumulate camt- emid in their plastids. M w i n (1971) prop& that t b pigments were bio synthesized from mwalonic acid and iqentenyl pyrophosphate via granyl gcranyl pyrophosphatc, two molecules d which condeare to form phytoene. In a perics d dehydrogenation reactions, phyian is cbnnged to cardenoid rcmtainlng fully mnjugateddoubb W. Cyclizationd t k carotrnoids oc- curs nxt. Specifically, carotmopm*s is believed to promd as follows: phy. tanr ( t hm cmjugatcd doubk bonds) + phytdluen (five) - t-carotene ( s m ) - neurosprrrne (nine) - carotenes and mnthophylb (ekven) ( G d win. 1971).

Our meprch shows that w r o t e extradm from untmted wbmt seedlings haw absorption d m . d 477,450. and 427 nm (Figure 3-11, which arc characteristic of mmtnr. The @amtane u m a n h s t h rn& from 13 pg/g fmh weight (FW) in darkgrow. pknh to 26 (rglg FW in 1500 f t - c g m conhd planh (Tabk3-I). I n theuI tnvbk(W) part d the spechum lFigun 3-11. rbooption muim d 264 and 260 nm were &wed. The aimpound r r r p a u l M c f w ~ a o b o l b P ~ * s d ~ t k L d e n t i f i i b u t t h e d ~ obmrved a n not thwe d phytmw (Davis, 1965). The concmtmtion d xa* thophylh R& fmn 53 to W &w FW (Tabla 3- I). Wbm the crmhaoib d

Smh6708 1 o-'

'Tach value Is rn avtrag d ib drhrmwlim 'Nudnrrmuu~irm unr Mdr

control dark- and light-grown plmnb were subjrchd to thirrlnyer chmmatw raphy W) on silica gel G, four csrotenoH pilpnmts were rporutul and identifld ps &carotene. IuteIn. violoxnnthin. and mounthin.

c.mtapidcdS~da67o(L.~mtsdSQdlhy The rpsdnl rnrlyrir (flyn 3- 1) d thc arotau fraction fmm elthcf dark.

1 ft-c, M IS00 h-cS.ndm87OEtmtd asd)lng(lO+a l ( r M ) h o w d that the rpftrs wem identical and that ths w l d p6gmt& (100-500 nm) were abant. Sandor 8706 c a d thc arrumul.bbn d hvo colorlnr ubx&nar. Thwe were judged to h phytom and phytdlww on the hdc d abraptbn maxima at 288. 285, and 275 om and 367, 341. and 331 nm Isliff(ivtly Rkvb. lQ6S). The mceritntion d phytaaa WM 43 fldg FW for dark-grown aadlinp and 61 (glg FW for I ft-c-gmwn srdtlngr mrbb 3-1); them was a h a dight aeauaulrtkm d pbytonume(S.Op#gFW) In t h m d i n g . The rsduead conrmtrstbn (lO&g FW) d phytosns in phnts gmwn under 1500 h-cwaspmbnbly ~urdbythsphobbmdhmdpbytaoa.TLCdthlrsw hct lbausdrnodorsdca~plgarnut*butafkwarontb.ndw.~ ob.avsd undra UV I* m fluomlw bDd motafnsd PMMm d ghy- tdhaas(srjudpdbytbc.karption~J.Indditkmlothgsrrbrtnaa* h r * . dW(1974)fand~poryph.lltorasintdwhatsalllng.

I WAVELENGTH, nm I Figurr 3- 1. Abmrpbn rpelra d the nrotm fraction (hexate) Imm mntml and Saw daz 67W!rmtd w b t &lings gmwn eilher in the dnrk a at 1 It-c or 1500 ft* d light. Sendm 6708 cuncmtratim w u 10- M.

Serdlings treated with LO4 M Sandoz 6706 had pcrrotaw! as well a6 phy t a m and phyiduene. The connntntiun d pcamtms in t h e heedlings was about 7 pg/g flebb 3- I), which was kn than that found in motrol d i n g s . Sdlingr tmatd with lo-' and 104 M Ssndoz 6706 did not contain detw tabk guntitier d phytome and phytdluene, but had normal amounts of carotmids Thac mlts indicate that 10" and lo* M 6706 mre most dfm tlve in inhlbitlngpesmtnw biosyntkb whik not affecting ths growth of the asdlingr

Tabk 3-2 shoun that both treated a d control anlliagsgnmr at I f t - c m t a i d ctoooplrrol a d cblomphyll. The wwmtrationr d each in the 6706 heated asdlinlpwwc about 82 rad 80 percent. respectively, d b found in tbe cwnhol dings. Tho drlomphyll d thwe heated plaots contrincd the phytd arttr. In &A-lpown redlw 67!l6-tmted rsdlings W abmt 79 p s r c s a l u m u c h ~ t o o o p h o r o l r r t h c c m h d . I n ~ ~ d b r l p l p a r m a t Imtt.c,bbbchlomphyePad.Jooqr&rdwsrrdrast~l~~.Sina umtanoid pilgnaab am a k t (a thae leaves, pbobodatnretioa d Q

chlorophyll and a-towpherol probably ormrrcd. A n d e m and Robsrtrcn (1960) suggested that carotmoids act as "chemical bufferr" to protect chlorophyll from phot*oxi&tion. They repnrted thnt a camtmoid.bm albino mutant of corn (whit-3) pmluccd chlorophyll when grown in dim light (0.5 ft-c); however, exposure d this mutant to bright light in the p m r r of air resulted in destruction d chlorophyll.

GENERAL DISCUSSION OF MODEOF ACIION OF SAND02 8708

Our m a r c h shows thnt Sandoz 6708 cauad lcm d carotma and x a n t b phylls as well as accumulation d phytoene and phytdluenc. To explain theae effects. it is suggested that 67bs acts both as m inhibitor d dehydrogmtion mctiom leading from phytoene to unrsturakd acyclic carotmokb and as a stimulator d phytoene formation. The actiond 6706 in stimulating phytoene formation m y not be direct. P m a a in the rantrol radlingsd tbecnd p d uct b.carotene might deprra the synthesis d phytoon. In treated d l i n p where the &carotene conantration is nil, r r e k d fedback Inhibition might occur. If the cycliution reaction were inhibited. an acnunulation d mlod acyclic enrotenoids such as lycoprr and (-cnrotm might be ex- pected. Figure 3- 1 shows that thir did mt oaur, <-carotene hu h lhnvn to accumulate in whcst d i n @ treated with diehlormate, amittml, d pyriclor(&unrd PI., 1971). Tba abknadcycliccnmtsm&bIn67~tre~Ud rcdllag ia probably due to m inhibition d dcbydrotpotbn nrdiaa m r - ring bdom cyclizotbn. h A z i z and Koren (1974) Mpcntcd that &.a- e d t s r as d l u phytdhmw and pbytoav in 6 7 W m t e d w h a t d lings, they arm &at the herbicide acb ma a cycliution lnblbfbr in urn t y g a m b r o t b e r t h a n o n j w t t b e d e h y ~ i o n h p d ~ b c ~ ~ . * ( q- . I - w r r p 'h 4t edB0~rA&andKofa1(lB74)mrybc

50 HAWAII ACNL~L#UMLU[IZIIIMLH~ SIATION

srplrlnmd by the fad that Ihc wbsrt d i n g s ~n tm*d and grown in a d& l m t mum. Our d i n g # wsn gsnnfnted and gm*n in pari d l h a cow h i n h d u t b m d Sandos 8708 or distilla1 water, wbik BmAriz and Komn (107+)lacorporated 8708 into the d l . Tba mil may alter the herbicide. andlor the mncentntkm d hnbiclde reaching the mbdlinga may vary. Tky found t h t ehlomphyll and mmtenoklr arrumulrted for the first 7 &yr and then Mined. In our work, no m l 4 crmtmoid pi- were accumulated at any time.

A n o h hypothda, prop*sd by Hilton d al. 0971). nrggah that the prlmary adlon d 6708 m y not be mtriehd to carotmokl pathways pn r, but rather the abmnced mmtemkb may be only m d m r a l earrqucnca d an inhibition affecting other chlomplut lipid ts. T k suthon

biayntheda d lroprsnoii lipidr Our results howevei do not W r t this hypothsrlr If Iropmid blcaynthmb w u inhibitd at 3 asrly stage. then the synthadr d colorlm umtemids, the phytd chain d ch(bmphyll. and the ride b i n d etompheml rhwld have been inhibited d n a originate fmm a cmmon precunx-i... bopcntmyl p y q h q h a t e ( L i n 191 1). We howsd (Table 3-2) that hnda 8706 had a very slighkdfecl if any on ryrr theda d etoeopheml and pbytol d chlorophyll when g+wn under dim light. Our multa indicate that Sandin 8706 exertad its dl& a camtmoid rydhe sb intarfering with dehydrcgenatlm nactlmr either b) inhibiting catalytic activity d dehydrqlaurr or by inhibiting formation d a rpcific &hydro genue.

Tba chlomphyll contart d 6 7 m h a t a l d l i n g dspsndsd upon the light Intmvittkr under which the plants w ~ a grown. At IS00 ft-c, the chlomphyll rmlsnt (Table 3-3) was Fsducsd by 97 p m n t and t h e d i n g a a p p d rsh indicating the prs+na d admyanin piemm mly. In mntmt. trsrted d i n g s gmwn at I ft-c fw 6 drya a p p e n d a bl- mbr and mntaind a b w t W ' ~ u m & ~ ~ I u d i d t h c m a t F o b ( T a ~ 3 - 3 ) . W b e n tbasplurbwernsrqpoadtolSOOh-cdI~tfwI2ha,~thy~Wpr- ant ol W r origbl chbpopbyll, whUb m t m l pltob 8aumul.tal thFsc ~ m ~ s s c b b a o p b y l l t b a n t h s y ~ l l y b d . ~ c s l o r o p h y U c o r d m t d 6 7 0 6 t m t d d d p l r k ~ ~ b w u d r t s r m h m d b y t b c l ~ i n l a n d t y l a d u whkhthsplmbwreiUumbulaLff tbeymrr~to1f t - fd l ight for12 bourSthsaallh\gbeamepka~d.mrmub(al.bau70parrad.r~ rhlpmpbyllu~hcmardr~3-3),vhil.lbaeiUumiartafwltb1500fCc dwf.lbdtolW-yI1-

~ * * - ~ , ~ * l y l ~ - c Y l l C f i d ~ n d n @ t ~ b r * l rw

aboprra CM*a(d, -- -- sub kdr

L*brmd Card 8VM t.hd 8700 *pl.a-- w ~ f a - w

~ 4 d . * d I S O O H t 190 17 IS5 I w 6 r I a p n l h s I75 10s 45 1 ~ d L y s n l h ~ . t b l l WO I9 10 1

l s u n a ~ 1SOOh.r a l t e * - - 7s 3 D u t d & p . h I l ~ a t I f i ~ 55 41 (I 1 D u t 8 & p , ( b a l ~ b a v l . ) I ~ f 6 ~ P It Y I .- ' S a s d l l n u v m m ~ * . I M P u r d w c m d a t a b a n h m d l l ~ C H k h W W l *

0.1 mid

The clmtenoid pipnmb d the 6 7 O B t W mulling wen vlmully a b mt, i rape( in d whahcr the plantcwsrrgmm mt high or law Iighl intend. tiaor in&rlmrffaMe3-3).Th influenad6708on thsribmmrl- dtion d wheat mailing gnkn unda 1500 ft-c w u Identical to that d amitmk and dichlonnatetmtal dl@ 'Ibs r i b 1 prepanlimn (Tabk 3-4) d 6706.treated dim had on& r single 80 S peak, whib mt t d plants contained two peak^ with r p p m b t e dhmtation oodficbnll d 70 S and 80 S, which reprqd ehlomplutrnd EYtq)*Dl(f rlkransl. re spcctively. In mntrut, the rlbo*mul coarpdkn (Tab* 3-4) d 6705tmtal seedlings grown for 6 &yr under I ft-c d Ugbtrhmsd ths pr&ms d bdh 70 S a d B O S ~ , h a m v n , t b c m t b d ~ B O S r i k * o m s l t o eblomplslt 70 S riboamrr for 670Btrsltal p&tr w u 4:1 u mmpd with an80Sto70SntbdS:l foramtroh,thuriadlmtiagadight~d70 Sribmma.Somedtheaedtingr1pom,for6dryr.I I f t e w a s s t h s n a p o d to 1500 fts d ligbt f a 12 bun, a d a mdimmtation anal@ rhaud that the chlomplut rIbaaasl d tbcss d i q p dinprsd whlb 80 S r l b - rrrmbudmabk 3-4).

In m t ~ to thae d t r &lad with ilkmhvtal fin& both 6706 and cmh& &rk.gnnun plants wee fcurd to haw idontin1 rlh*mul anqral. tiom mabk 3-41. and the ntio d c y t q h d c tu & h a p U ribmms VW

t b c n m ( 3 : 1 ) 1 n ~ ~ t h t 6 7 0 6 d l c l a d t f 6 a t u r u m u l r t b n d 7 0 ~ ~ & t h s d r k ~ l h t m t a r ( i o o d t h a h k . p o m ~ w r t b 1500ft- cdli&forsltber1.4,a12bauralaaltlrnpid~lodfbuUytbc & d 7 0 S e b b m p l P * r i b o r o m a p a a l p r b r t o ~ ~ I bar d f f~ th .~670~baadpLdrCT .bbJ4 ) , tbs l rU )s07osr ibo

I@t 6 dap 1 W! 11-r I+@ 6d.p at l If+ l+ttid.pal I Il.~,lhen 12 hounal 1W)OIl.c t7UtkBd.p ~rk l ;8d .yr lhm1WOft~1or lhr~ l r h r k R & p t h m l W O 1 1 ~ b r 4 k m n Dark 8d.p. thm 1500fl.t lur I 2 huun DarL8d.p IhmI2k lvns l Ik-r lmktul r ihmnw !n&tni uUh S a d 1 2 R7M for 6 b t n

I WO 1t.r - -- - - - 'Phnls -a~ICrdt*d ~n TaM 1-3

wmal ratio changed to 4:1, as compand with a 3:l lrtio for dark-grown 6706-tnatal plants. Four hours d illumination of.6706-treated plants changed the 80 S to 70s ratio to 6:l. and after 12 hoursif light only a hint of 70 S riboamn was obrrved (Tabk 3-4) in the 6706 plats. The 80 S to 70 S n(M(2: I) for control rsedlings wasabout the same for ealh light treatment.

In rontrast, when dsrk.gmwn plants were illuminated with low intensities d light (I it-c) for up to 12 houn, the 70 S ribosomes were n8t d u d , and the 80 S to 70 S ratio (2:l) was the same m that d the controls (Table 3-4). H-r, when the light intensity waa again increased (75 ft-c). the 80 S to 70 S rntiodroppd to6:l.

T h e m l b suggest that 6706 in conjunction with light c a m rapid dc- stmctian d 70 S chloroplast ribosome. To detmnine if the destruction of 70 S r ibo~~lb l w cn& by the photoactivation d the herbicide, isolated 70 S and 80 S ribmoma were incubated with 6706 in dtro unkr 1500 ft-c d lit for 6 houn. The 70 S ribrmrss were not dmtmysh and 80 S b 70 S ratios wen identical in both the mntml d tmted srtracts (Table 3-41, The ultra- stNmull effect d 6706 on tight- (1500 k c ) and darkgown rdllingf appeared to be identical with that d uninotri.zde unl didtlormrte. Pladids d light-grown pl.ntr lmcked p n a (G) and rilmmmcs, but rmbincd t h y b kakb that unn unbmched, ununvlly k g , ud anaoged p n l W to thc edge d Ih plutid smsbp. The etioplub d 6706 &&-grown phnb wm mor- phdDglcrlly idmtkal with the eHoplrres d mnhol plank a& Iuv iw~ pm Lmallu bodhr (P) ud ribomaa

Wbsa a y d d &&-gram pl&s we= apard to high light iDtadty (1500ft~forl2hun,thc4ssdl i~hruac.rsa.wBcrarthc67OB

(mYd.m m a i n d wh~tc. The P of dw rtlcglast frwn thir whttr t ima c h a e during the grwning prows Into na nggmutnl enlt d 111ybl\ dlwr. gan id , int~rconnrrting nwmhrnnr.i rather ~ B ~ I I ~IIP nornvl structllw of (:-

thvlakoids, and the strtln~a tnvrrn, ckvold of riIx>u)~an l'lx plarttd alqwi~rrtl to Ix morpholop;icaIly similar to d~rhlt~naatt.krr.(nl pl~lntr Krc)wII IIIX~I \h9 same cnnd~tions.

When d l l n g s wrrr grown under I f!.v of l~ghl Cr 6 (ln\s. tlr- pludldr IF~gurr 3-2) of mnlrul plants n~nluilml P. ri lx~urmr~. a1111 (; rtrlirturt9\, ull l lr plastlds elf 6706.trratcd plane, d l u ~ had P und r~tx~w~nw\ Itut lacked (: drur turcs(Flgure 3-3).

, , , ,i . ''2

.P

.6 days.

ody (PI.

We infer that chlorophyll pigments in the absence d carotenoidr become w p t i b l e to photooxidation. k pigments m y be converted to highly reactive molecular species, which t k n interact with and &roy Mher chloro phrt rompaunts. Our data nrgged that umtabilized photose~itized chloro phyll or p n c u m d chlomphyll m d directly or indirectly with 70 S r i b KIM and thylakuids to oxidize and destroy them Some evidence for this inference is supported by the wok d M a n d Krinsky (1967). who report that p h o t ~ n i d i a d chlorophyll could function as a photmmsiti~er that nacb with chloroplast DNA and nwlts in gavtic alteration.

SUMMARY

Ssndoz 6706 inhibited thr synthnird h d h rurotmtm and xmthc~hylls and c a d a massive ac~umulatbn d phvlcwnr in what r*dltnl(r. Ih vvnthrrir of chlorophyll and tcmqhrrol was tmt inhibited in whcut. rvrn thotlgh t l m substancrs have many hiwynthrtir renrticmv in cr~man~e with thr confimo~dr. Our results indicalr that 6706 intt-rfcml with thc dt~hylryln~uticm rrilcttmu c f the rarotenoid pathway. Chloroplad r ih lsc~nn urvl ~hylnk(~idr w r r nbmt in the light-grown treated seedlings. Utlsldbilizrd photcmt~~ i t~nd crhlortphyll may react with thesrchloroplsrt dructttrsand dmtrtr! thc.tn.

LITERATURE CITED

Andrmm. I. C.. and D. S. Rubrllm. 1060 Hdr d cunAnwkls tn pnltcrltnR chlom phyll frnmphotodfftruction. Plant Phyrtol. 35:531 -534.

Anderson. J. L. and J . P. SchvEllin~ 1970 Elfeels d pyruztm on lean chlurr~larl ultrasl~clum. W d S r i . 18,455-459.

Ashtun. F. M.. and A. S. Crrfls. 1873. M<& d Avtlt*b ail l i r r t ~ ~ c t ~ k N w Y ~ r k Wdey-lnlerscicnc~*.

&wAziz. A.. and E. Kom. 1874. I n k r f m n ~ ~ m rarul@nyycrnb as u mhuninrn id aaion d pyridpzinonr krbirtdr Sandcn 6706 Plant Phyntol. 54:l)lB-1120

Bum. E. R.. C. A Burhatun. and M C (jlrlrr. 1971. Inhib~lirw~ 181 ~mdrr r~ td qyn. thrsis as a mwhanirm d arttun of am~trok, d~rhl~vmutr, rnd pvr~vlr~r Plsnl Physiol. 47: 144-148.

Cohcc-Baz~m. C . . and R Y. 9unter 1956 Syrtlfk tnhibttnm of nrutmrrld synlhmis in r photosynthetic lrldrr!um and 11s physblc~g~rut cwnrqun~m Nature IXI 250-252.

Davis. H. H. IS65. Analysis d cnrutmotd p~lplids. In T . W. k d w t n . W . (:hmllc try and Biochemistry d Phnt Pigments. L A . Acah ic Pma, p. 4118

FaludCDnnicl. A. A,. A. Nsgy. I. Cyurjsn, and I). Faludi. IWS. Churarfrrtsttn d pigmmt-protein complmea in normal and uhbruplart mutant Irrvn. PlwXwhcm. Photobiol. 4459-367.

Fujimori. E.. and R. Livingstm. 1957. lnteradmu ul rhlr,rtxphyll In ilr triplrl dmtr with uxygen. carotene. ctc. Naturr 180: 1036-1038.

Fulkr. R. C.. and I. C. Andcrsm. 1958. Supp~m d curdmid synlheril an1 81% d- fcef on the aalvity d phd"ynthr(k badwial chromatophorn. Natum I81 252-254.

Coalwin T. W. 1971. B b y n h h d csrotmo& and plant Iritrrpnn. Biwhrm, 1. 123:293-329.

Hilton. J. L. A. S & m , J. 8. Sc. Jdm, D. M&nd and K. H. Nurrh. IWB. M&d aetimd pyridazinar M e i d a . Weed Scl. 17541-547.

J. 8. St. Jdur, U N. Chiuthnrol. Md K, H. Norri.. 1971. Interu.(ion d lip& &I nuledah and n pyriduimrr inhibitu d eh&oplul h ~ l . Plant Physiol. 48:17l-177.

Kcrhl. F. C. 1901. Untrrnrrhung Qbrr &a Cardin und win phyiolqische &dnnung In &r Pflann &din: Brgddrr BointrPgrr, p. I I .

K d i . V. M.. a d j. H. Smith. 1951. Chknophyll fomutlan In a mutant, white xed. ling.3. Arch. R & h . Bkrphyr 34: 188-195.

KrinrLy. N 1. 1Wb. Thc role d carotenoid pigmmtr as po(ertlvc o p t s agatnd pholcnmsitird oxidnt~un in rhloruplasts. In T. W. W w i n . Ed.. Biwhrmistry td ( % ~ N Q ~ u ~ ( s . Vul. I. k W Ywk: Acadmlr Pw. pp. 423-430.

Uf, j.. a d N . Krlnsky. 1887. A mutagcnicdfwtdviribkllght d i e t e d byen* nw, plgmmts in &11&10111 grddlfr. Scienw 158: 1332- 1335.

Mathrwr. M. M. 1884. Thr dfwr d low lempmtuwon the m i o n by cardmi& a~aindphcl(u*n.iliraliun i n L r r f ~ l u l m . Pho~nchrm. @otobiol. 3:75-77.

String. R H.. a t d R. L. Hogen. 1974. Brhavim and l a b d &o phrnylpyridaz~nme krbicldcr In mtlcn. mrn, and mybran. Agricultur~l +I Fmd Chrmistry 22. 1118-1125.

~k -n~ t r Pms. py 633-653.

Chapter 4. Growth-Mediated Response

of Methy leneoxindole with a Proposed Mechanism of Action for 2.4-D

V. A. McMahon and C. L. Villrrnez

The prevent study was undertaken to delrrmirr (a1 how auxlri p r~~n~) t ra tr.11 rlongation and Ib) if indoleac~tir arid (IAA) fur its ~)xi~lnt~on ~)r~durts arr directly responsible for prowtbpr~~m~ding activity. III view d thc typw responrrr that 2.4-dichlorophenoxyantir acid l2.4.DI ant1 IAA huvr III C I I ~

mun, it can b suggested that 2.4.D might dirit its rruplnr t h r ~ ~ u ~ h MJM

phapof IAA meta~lism(Zimmrrman. 1951: Lw. 19721.

D i d Wed d 2.1-D on 1n h y m Van Der Wwde ct al. (1972) demonstrated e 10- lo 3Bperwnt iw r ra r In

p1.2-glucan synthetasc activity in the p m n e d 5 pM 2.4-D In 1;i6ro. Thiam zymc is reported to have hrn isolated in plasm membrane from onirm arms. T h e authors used a differential daining technique that charrcteriltirally ataim the plant p lena membrane to identify cell fraction purily. In their paper. Van Der Wwdeet al. mention a p~arl eummunicatbn d C. L. VIC kmn, dimming stimulation of cellulose synthesis in the pmtnw d IAA In oitro. This FI ,4-glucan lynthetase activity was also l a n d in the prticulats fraction from mung bean &lings.

An midation product d I~~-5methylmcmindolr WOx)-is reportedly 10 to 1000 t ime more effective in dimuloting growth d highrr plants than 1M guli and Mq.aL 1969: Boar and Tuli, 197Za: McMahan. unpubllhud raultsl. Fucth*morr, the dsquen t redudion d A(& to nwthyloxinbk is

1

li -co, I I I W L E - ~ - L ~ ~ ~ ~ C LClD ~-WDRO~EWVLDXIHKILE

~.WDROXY*E THYL-

XIMWLE DEHYDRASE it-H20)

@ ~-MTHYLE~IEOXINDOLE

REDUCTASE t--------

i rn N

d :iADPIi + H k J-MET~YLOXINMILI 3-MET~LEIOX~~WLE

- Pig~ln 4 - 1 Oxlndolr pull~wuy d I A A mctnlnllrrn in plnntr l&m ol b and Tuli. 1972h)

ci~tul\arl I>\ ut leust tn.11 NhlIPH-linkml nwthylrnoxindole reductam (Moynl ulul Will~un~wn. IY6i) Mrthylcrxindnlr is an inrfi metabolite of MrOx. Ow of thrw dectaws, rductaw A, is nonrompctitively inhibited by 2.4-0. whilc 2.4-1) is a wrak nrmplitivr inhibitor of redwqaa 8.

1;vidcnw lor tta existc-niv of an oxindulr pathway ul IAA metabolism in higlr.r plants is found by the p m c r o l an IAA oxidation product of MeOx in a w l l - fm s!stem (Huger and Schmidt. 10681 and by the isolation of thr em r y e asmiatmi with this pathway. The propoapd scheme shown in Figure 4-1 is frum Bnsu and Tuli (1972b), who have idated a partialIy purified 3- h!~dr~ruymrthyloxindnlr dchydras that catalyzes the dehydrationof J-hydror- ymrthyloxindole (HMO) to Me&. Pmiousl). workers in the same laboratory w m abk 11) c b r s t r i t r the oxidation d IAA to HMO and McOx using ccll- Imrxtract from p a &lings (Slill et at.. 1965: Tuli and M o d . 1967).

GNnvth.Romo(hyMivity b r a 1 p m b b were maxlntnsd in udng mung bun hm rgmenh to

hd for pmmotion d dl clcquation. The pmbleou were dionbal by: (a)

carefully scketing srcdlings 3 cm tall, ainn nurimurn r l o q r t h raw d seedlings ormr brhvsen 3 and 6 cm; (h) using dim light In hnwwtin8 W n t l d mung bean; (c) prrimubating qmmts in 0.02 M potmiurn ptnnphtr tnd. frr to redua endogenous auxin kvck and (d) using rtrwk atllutions d MrO* at not water than 1 x lo4 M con~mtration, hecause MrOx n r rp~r td ~II p ~ l y merim abovr rvnmtratiocu d I x 10-' M (Hirumin and Eia~~rnun. IWI4). Thrr pmaulimry measures hnvr n ~ b w d drmondration d ymwth in 7. mm stem segments a c i d from t k growin(l tip d rtiolald mung hrvn m L lings(figun4-2).

F i m 4-2. Uled d M a and IM m dmplim a( rl!mlatcd mum bran r(cm

mr*I (7 nun). Segmmts me cut in dim light, p m m h l n l 4 lun in 0.02 M phoc phac tddfer (pH 8.51. .,d npoad to auxin whh tbe m a cmmtmtlm d buHn fw IS lum mm- d C I , . ~ in ~knsl~bn m~ msh d h a ~~I~a~csd (D.@Y. inghnr Wponl -anaveralpvalufa 10-r

H A W A I I A<+. IlLULl I J I W ~ X R R I M E K I S T A ~ ~ N

Apical ,qmcnb d mung bean stem If mm) were incubated in either IAA or Max at various wncentratiom. Figure 4-2 shows that the optimum mn- nntrnlion d IAA I11r growth was 10 to 100 pM, while M& showed optimum crmnnntratlon at 0.1 lo 0.01 @-a thoussndfold diffennc~ in concentration. Thw ~ " t h studin werr bcst mediated by monK ban &lings 3 to 5 cm tall. Hcsultr s u ~ a t thnt M& may be the subdance that promots growth in mung ban stems.

McOx was prepared after the prondun d Hinma8 and Bauman (1964). IAA war nac-rd with N-hmmosuccinimidc to vitld a ratrvstallilrd ~roduct d 3.bromooxindolc-3-anlir acid, This prnduct'is mnvcft;d to M&X (Figure 4-31 in the pnacnw of water (this is readily acmmpli$4 by paper chroma togruphy in 955, vlv water/isopropanol). The reaction +curs simultanwusly, and unwanted products d 3.methylwtindole (Figure 4$, Rf 0.861 and mi- bly a polymeric form of MeOx (Rf -0.08 in 955, y(v waterliropmpanol, paper chromatography) are separated from MEOY M a is readily visualized under ultraviolet (UV) light and subsequently d i w l v d in dilute solution in distilled wafer,

A POSSIBLE MECHANISM OF ACTION FOR 2,4.D

I t is unwrlain whethr 2,4.D in low connntratiom erqrts i$ effwts on wll elongation dinrtly, or whdher 2,)-D might change the level d endogenous auxin. Sin@ M&, rather than IAA, i s likely to be the natural growtb mediator horniolr. the endngenous level of M& possibly is the auxin of rrlermcc. To test this hypothesis, chlomgenic acid (a growth inhibitor that als) blocks uxidation ol IAA to MrOx) could be employed to inhibit the cow version d IAA to M& in stem segments uf mung bean, and sufficiently aged, treated qmcnts should lack M&.datrd growth. Combinations d 2,4.D with MeOx and appropriab mntrols should provide widenm d direet or in dinct dfects of Z4.D on growth; if the 2,4-D effect is indirect, MeOx should have a lorypr growthpromoting effect than its cwtrol (assuming that MeOx rcdurtasn a n inhibited bv 2.4-0).

M(hRd1it AC.1 ION OF .%t.IM. 11-J) IIEHI)I(:ll)53

Basu. P. S.. and V. TuIi. IY72a AIIXIII a r t ~ % ~ l \ d 3.mctt1\ knn~~.~~xh& ~ ~ t r l r u t I'[rl~t Phvslol. 50.4911-M2

. and . 1972h Enryrndtll drhydralka 3 . h ~ ~ l n ~ x r m t h \ I ~ ~ u 1 1 ~ l ~ 1 I r Plant Phyriol %I:W3- Wti

Hapr, A.. and H. Schm~dt IYfiHill Auxm trunqwrt uml ~ $ r ~ ~ l t n p i t ~ n t r r I DM. 111111 hnl~nflr b i ld l ln~ r m n hrmmrtotfw fur h n lrnn$pm Vcm wurtl.~hdlr#~ 118 lr,lnqb t i lw Planfa 113.347-37 1

Hinmnn. H L.. and C. P. Routnun IWi4. Hrutt in\ GII X.l,rnt~acnan~~~~n~o~L. u r ~ l I". doles. A s~mplr synrlu.s~\ d 3.brom~xm1nth,ln.) Org (:hcn~ ZB.IZ(M.. 12 15

Im. T. T 1972. ( :hut~~n tn ~ n t l o l ~ ~ u ~ ~ t i r U C I ~ ~ s ~ r l e r l u r n q n n III trbuvr.a~ t~*ru* ufter trratmrnl wit11 2.4d1rhl1rrrlph11)1vu~r(i~'~1~al Plant P t ~ r s ~ l l 49 857 H(i0

Mo+r*l. 11 S.. UII~ V. H'i l l~nmu~s. I M i M~~ l t i p l r ~-~~IIIv~~Iw~xIII~~II<~ rnlut?,tu.r I$ 0ra5. J 0101. Chrm 242.1075- 107i

St111. C. C.. C. (:. O l~ r r r . RIICI H S MIWIXI 1965. I n l~ i t~~ l c~ r ) IIXI~II~IOII pr<n111d* III 1ncl~~k.0-ntrt1c arid. mrym;rts formrtun uncl ~ l r lox~f( rut~tn~ It\ ~wu w l l l l n@~ Scwnt~ 149.I249- 1251

Tuli. V.. awl H. S. Muvtd. 1967. Inh~l>~tor) iu.idYtlun pr(~Iu(.l$ (d IIKLIIC~ u(.rtl( H( I<! 3-b~druxy11~h~lux1ndC~Ic nncl 3.mrtkknnn1n1blr r> III,III~ 11rlulx4ltn I'ln~d Physiul. 42:425-430.

. a n d . lWi9 The ralr d 3.mthvlran~1ndolr in dux~r~ urlat l J HIIII ChPm 244,491 6-4920.

Van Der Woudr, W J . C. A Lwhi, and I) J. Mar( 1872 Auxin 2.4-1) d ~ m ~ ~ l d l i < n ~ in vlvn and in vitm 01 pnlyrurchrri& .iv~~thiuts III pldsmu swmbrunr l r a w n ~ t r isolated lrom orlion Itemr. R~crhrm B~uphvs. &. Cumn~. 4h:24S 255.

Zimmerman. P. W. 1951. Furmativr rf1rr.l~ d h o r m l i k growlh q l u l o r * . I n F. Sk-. Ed.. Plant Growth Sukancn Univen~b d W~mrmsin Pmr. I~I 175-183.

HAWAII A<. I<JI.ILIRAL.L;(rr.~lMtJY 1 AIAIION

Chapter 5. Cytological and Biochemical Effectsof Trifluralin

on Mitosis P. C. Bartels, F. D. Hms, and D. & Bayer

Thc objectives d this study were: (a) to investigad the ultrastructural ef- fects d trifluralin ~~.~.~-trifluor~2,6dinitr0~,~di~~~l~tluidine) on mll division in wheat, corn, and cdtrm mot tips: (

trifluralin on the in vltro polymerization of m' k i n subunits; (c) to determine if radioactive ifluralin binds to purified pig-brain microtuhulw, and (d) to cvmpareihe mtdp of action of trifluralin with that d mlchicine in root-tipcells.

Triflural~rl is widelv uw l as a wil.activr preemerg@icv hrrl~iridc frrr con- trolling annual weeds in cotton, xiyhean, safflower, and many vrgrtabir crop. Rnt results arc normally obtained when it is incorporated in rcjil to a depth of 2 lo 4 inches. Movement d the herbicidr witbin thr,plant 1s limitcd, and it appars to be r e t a i d in the roots. No evidence was found of an active acruniulation of "C-trifluralin by mtton and soykin rmts, rather, accumu- lation wu:,attributed toadsorption incuticle, epidermis, and cell walls (Strang and Rogers. 1971). Growth of the entire plant is affected by t r i f l u r~~~n , how- ever, appamntly by interfercnc~ with mot growth and ckvelopnlenl. Triflura- lin has Ireen reported to interfew with root development in a numher ol plant species (Ashton and Crafts. 1973). Rods of treated plants appear swollen. stunted, and malformed. After 12 hwrsof trifluralin treatment. abnormal cell mlsqement (mhannd radial expansion) occurs in cells of the cortex (Mallory and Bayer. 1972). This enlargement in the region d mil elongation is thought to oroduce the mmrnonlv &d clubshad rod tim. Enhand radial ex. pansion cornpenrates lor the inhibited elongation, resulting in approximately themme volumechange for treated and untreated tiuue(Bayere4 al.. 1967). Lower application rites of trifluralin to rods appear to have nu marked d-

fect on the apical meridem d the mot but strongly inhibit formation of lateral rmts Trifluralin indurps an expanoion of the pericycle tioue. particularly in the region opparite the p m t o x y h and, at the same time, mtrids the number d layers d pericycle mlls. The net d t is the inhibited fornution d lateral mot primordia, resultingf mm wrpprard division or activity d pericyck cells

~Bayer rt al., 1967: Hacskaylo and Amuto. 191381. l i i ffhrr ratm d trlf lurrlc~~ inhibit bcih apical meridrmr a d lntrrsl M s .

0bu.rvaticms of stunting, swelling of rcwt t ip, and inhibita~r~ d Iatrrnl rrx~t formation in plants treated with lrifluralin ruw nlnrkcvl cvtoli~gtrrl rnul. function. Cylulcqical rtudirs rd t r i f lu ra l iw t rw~l rrwds $hiwed Illat mitcnco or cell division was affected or disruptcrf. In arklitlon tcr t t w lnlti~ttc a t r r ranries, differentiation cd t h vascular rvlindrr IS rcportni III urwr i~biu~rn~aI. lv near the distal p ~ r t i r n d the primay rcxr(aIMallorv awl Bayr~r. 19721 ?'tr type id mitotic figure pmai l~ng in trifl~cralin.treatd tirsw8 vvnmr to t l r l r~ ld on plant species. c~~ncvntration d hrrb~cidr rraching t k - crllr ~rmr uluvrn dl,. tributi~mn of herbicide anlong the ITII, of the tissue), and lime trl lwrlmici~lv application to the plant. In w~yban rtw~t. Talbrt ( IW5r rd~urvtd an Incrrar in mlls in proptmasr and evidencr of (111ly a few other mltotlc rtegm. l.~g~m~wnlt~ and Scott (1972) stat~d that prophar clid nutappeur to Ir ,ffrc.trd II~ lr~f lerr- lin: they fwnd prophaw appeared normal aftrr 12 hours rmf trruti~unt. lmut they alw observed an increarr in crll5 In mrtnpt~ew. Polyplu~d an11 111ult1. nuclrate cells wrrc (~bserved alter thew t i r rw had twn treated fur 24 Ir~urs. Bav~r et al. (1967) Rmund that no sinylr stag of mitcnis wub prnk~n~cnar~t III

trifluralin-treated onion rmt-tip LTIIS. Mitdicaclivity was no1 daruptnl tt1t111. same extent in all wlls, and wme appcund to undergo nc~rmul nlillni,. Jackx~n and Stetler (1973) dpervtd an iscmsr in prophaw cd trrstrd naLb sperm cells of African b l d lilies, and thdr multr c r ~ r ~ p r r d d w~ th t h findings of Ta lhr i (1965). They suwcsted that dcslmctiun crf nlic.r<~tulmulm during prcvhase may be respunsible for accumulatica rd cells in propham.

Typical mitdic aberrations found in trifhiralimtmated rnnt cells wcre c- pairs. enlarged amoeboid nuclei. micrunu&i, and plyploid plants. Ihm aberrations were similar to those observed with colchieirr, a m i l d~c pliw~rr that has been USR1 for crop imprcvemtnt thmugh dcvclopntmt d polyploid plants (Eigsti and Dustin. 1955). Colchicinc modifin micrutuh~ln in pla~lt and animal crlls, and k thwght to prevent formation of micrutubul~ by doi- cbiometrically binding to a si ted interaction through which the protein autb units combine to form mir~otubuln l~dsbnan et al.. 1968). 11 has hi rrp~rted, however. that the concentration d mlehicin required to blocl~ dl division in plant cells is 1000 times higher than the rmwnlralinn required lo block cell d i v i s i i in animal celb(Hcpkr and Pakvltz 10741.

I n this study, all division d triAuraliBmatal h t , corn, and d l o n rod-tip cells was examined at the ultrastruetural and biochemksl kveb to r'+m ' . . tt rn . 'r d -&im d t lu krbic idem micmhtbula d mat &IS.

C ~ r f # M r r e l v r l roof r r . l l \ I ' I I r . ~ \ l r ~ ~ < ~ l ~ ~ r . t l .111.tIv$i\ d r d t l p d dpt wftivul alni \IIIIN{\I, 1111, I<,~III~I\Y\ rv.i1111\ t~lwr\t,11 1)1iri11~ i i ~ i c b t (((*IL(Oti $11 II~I!O\I\, IIIII~II~~II~>II~,** v.tlrtl o l~\ t ' r \ t '~ I 111'.1r !In ~IIII<IPII\I,~I I Ilrolnatin

11) 1~11. 11rol111,tu~. .I! ~III.I.III~I.~\V. t h ~ \ \*t,rc. . ~ t t . i ~ ~ I ~ ~ ~ ~ l 10 (III~)~IIU\OIIII. L1111.!(b

I IIIIII., II'ICIIII. 5 1 i. ,III~ ~stcnded 1rwi1 p1l11 ti, I H I I I . witti11111 ~ l l r o ~ ~ ~ o \ o r ~ ~ ( , .I!-

I.II~IIIII~II~ I)IIIIIIC . I I U I ~ ~ ~ # and -%,. \IIIII~III. m : t ro t~ l l ~ t~ l (~ * u1~r1. 011.

wruJ III t l~r t11tt.rlcwr btrvrm the hwr WI\ 01 <l.i11~11tr1 cl~r t~r~ lou~l l l rm 'I'lir\

- w b t r d rougl~i~ para1lt.l III one anothr and wZrr normal III tllr planed , all llliitr T11r vrhll plate COIISIS~(+~ IJ~ manv vrrirlm Ilia1 apparrcl to lx* lualng.

In the 1ntrrph.l~ 1r.11, ni~rrotul)ulr\ w t ~ aln~ndant war lvll uallr IFIRII~T 5-21, lylm ~ a ~ m t ~ l ~ a t ~ * l \ lx*aratl~ the prunaq wall with thc~r ( i r~rntnt~r~n ur - rumfrr~nt1.11 t(r tht* vt-ll .IXI.S Thru. structur~~r, cvrtil.al m ~ r r o l ~ ~ l ) t ~ l ~ ~ \ . Ins) mn trol IIIII~II~I~I~I~ o r i r n t d t ~ ~ ~ ~ 111 tht' 1 1 ~ 1 1 wall Thrrr was no r.\~~lr.n(.r 111 any ultr.~~tr~ritur.~l d l f k r ~ ~ ~ c ~ , IU.IWIVII sp~ndlr and n ~ r t l r r l IIIIC~I~~II~III~I~\. thrrc~ Iorr,, t h ~ i n 111 IN* co11~111rri~l ~I i l f~*re~tt or11v 111 IO~~IIIIIII in t l~c <,rIl ,& ~l f~tni l rd rq~br l OII 111~ r1111.1)l ~ i~ i r ro tu t )~~ lm ha$ IMVII I>IIIIII\~IYI 11y SI>S~I>III~>I l!4(i!4\.

I 'r i/ lu~al~t~-frraf~d rrlls, rnurofubulra a b m l At any oor t in~r. wll\ '11 th? 1111% $11 rtx~tr wtsrr c~l~wrrrcl III Lx* III diflrrent stager of n11111~1,: t l ~ ~ ~ r ~ . l ~ ~ n * . thr In- itla1 I~I~~~>~N*A~~III,I* of t h ~ n~irr~~tuholrs woulrl affctrt diflrrrnt rtaun 111 vrll c l i x I~I~DII h l ~ l u ~ ~ s stc,l~p.~l whrn thr mior11hlbu11.r dlsapparrlt With tla. IIII~II~.

Ir U~III.IIIY. ~lnnhlr 111 I)~IBL.MYI. nuclear r~ l b r l op r t . l~~rmot l<~~~ ~ ~ v r ~ ~ r r r d III \III~C m l l ~ ~ n r t >1r11kl 1nt1.rphrw 1111r1rur pattrrns vharartei~rl~v 111 thr st:^^!> at whirh IIII(I~I\ W ~ I \ a~i~' \ I rd. Nl~rlrar dl\ ~1~1115 ~ ~ P ~ X Y I at (dl!, 1>rtq)ha~* 111 rar- ly nlrtapllnw* wrrt* ~ . l~ .~r ;~c t~ . r~ tn t 11). I~~~II~IIISIIIII~S rrrdng1.d III an abnurmallv

Figurr 5 -4 Nurlvtt\ a h nt~rlpar rrfarmpl~un of ,g blcrkml mrtapha.~ N d r thin L Y ~ -

Iwtlonr wrrourl Irtwwnrurh ol thr nuckar qnlrnts. indtrat~ng that thuira, ,

rleatr A I I ~ 1x14 d 1r111ur)e1tr Scak marker qua11 I.O@n x USM). 8

snldll group III tllr clear zone of cuttort rwot a l l \ ~FIXII~I. 5 - 3 ) , or by chrvmw

stern cells If nuclear envelop rc.-forn~ation wcl~rrcd. p,lyplr,id rruc 1t.i d , id Mdnr of tlic rt*-fr,rnlt.~l nurlr.i wrrc highl, 1 d ~ q 1 iFlgurr 5 41 h rnrlwmlrs In rrlls arr14nl at lair anapllar or early tr'lr,ptlvu. rcrr1a1111~r1 11, l uc~ d15tinrt gri>up Iwatrd at rarh rr~1i) l IIH. rw t wll (f;lgt~rt, 5- 51 Nt1l11 (1'1l 111.1lr vwlclc.s and sp~ndle m~crotulrulm were alwnt in ttw ~ntrrajtu. I~.tww.r~ ~J,III~~I-

trr cJlro~iiini)nles. W~Ihout VWICIPS. cvll plate flrrrndteor~ c l l c l llot U L I 111. dlid th~r rpsullcd In blnurleale crlls. Irrrnrnplrtr Ijr prrtlally lr~rrrlrd <,vII t r'r*. walls were cucas~onallv found letwcen ~ t w daughtrr rbn~mrnonlc.\ il;~gurr 5-61. rrsult~ngfr~)rn partial fusion ol urrni. lrll plalr bebrcle>.

6 hlirrc~tubule alJxnce in interphae cvlL d ~ d not qtup uceurrmcr II~ rr l l l i~ t l~ division *ttrml,ts Thr rnitotrc rrclp appeared normal until micrt~ubulr.r m e

Q u m 5-L -44,- d l ~t,lol,hau cler to dirpl*.armnmd adnahr buln. TheJf.Wllharndl& apprarj+nn n n ~ l rxtms~r~n hn$ c r a d at b t h mdrd ttw *all ~ u k r r v q ~ ~ u l $ I O p m x Ill.UO0

~ .> .,". , :

5-8. Trlflurrlrn trratmmt has nol c r u d mtnphr d l w ~ p s r n r ~ ~ r ctl IIIII ru113.

,h this nnldl>llrax dlrj\lcn~ flgtarr (:tlr~,ttu,u,rrn an. dlrr(lnl ,lrrr,y r rnrtrltl~nr but mwr<lculruh w m I<, kr radrrtmg In r l l clirrd I n c l m In,m ~ h r ~ ~ l l 1 a ~ l r 1 l ~ 5 St~<,rt P

Utfnbtubulo idrnru.1 an. a rc~ull of thnr ol,l~l~wr,r~rnlrllctr~ wllll rcqprrl trr pl.41~ r r l

r r l ton~np lor t k \ rnd! IW R ~ n ~ l j r r l ~ rb>rtmnl nr r rmall 1,f cr~~~l rac laa r l f Irrr.dk daunl Scale me&ercqualr I . O p x Ib.ilMJ

01111.r I)IK,IIII*~~~(~ 01 Ib13 r w l I?'II\ In &ton plants appeand nd k> he mor- Id~<,lr,~~c,~l l \ ~ l l l ~ . c l ~ r l 11, t r ~ l l u r d l ~ l ~ but wrr r altrrcrl In wln..~l arnl corfl r~xrts. IVIIIIIII 12 IIOIII\, .IIIII(I~III,III\ I ; ~ r p \;IVIIOII-\ ~I*\I*III~xYI III 111~ rix)l-tlp ci~lls tl:lg~lr<, 5 41. t1a.u. rr.ll\ r . r l ldr~t~ l r.i<lldll! ratlar thdn IIIII~II~CII~JII\ Also. \111111~ ,,I 1111. I.IICI~~I~\IIII, r r l l c~~ lo rn (110 ~.~\lr.r!la~. Iwirrrlc \ u < ~ l l r n ,~rlrl a p (u,.ar(~l to .I<< IIIIIII~.IIP III'X~ 1,) thc, (dl \6,1ll (FIKII~I~ l l ) i 01llt.r ~ .~ Io (~ I .~ \ l l l i c

~,r~.irn.ll~.\, \\I< 11 d\ 11l~1\01111~~, ~~l~i\t tcIs, ,III(I IIII~CII 11011drld. dltl IIII~ .Ilqw'lr to III~\IIII~~II~~III\ IIIINIIII~~IIII\ ~III.III~~~II~I(II*.

Figurq5-I0 P.UI ($1 .r < < > r r l rc*l l .(,I1 trc.,lv<l I,,, 72 l1(1111\ ).IIC~D~I.~\III~( rv114 ~ a l ~ , ~ ~ ~ ~ I . l t ) clrll~rtur b*.rlod ut.\I 111~~~I l~dl l ( ( . \ \ I u.(* wr~11171 * '114 ( H I 0

',.!$;( I 2"

11r\ Ixr.tl 11111rr(l lor &loins. To teat t h i l t ~ypdh~ . i . H;~rtr.l$ ,IIIII lllltan I 1973) dtt1~111pl?(1 to lsnlatr and l)i~rifv n~ i r r t~ t t l I~~~ le% I~IIIII (or11 rwt t111\ III trrc\tar lo uu* then. tu l~u l~n il l ~ x r l ~ t ~ r ~ r ~ ~ r t ~ r , n rxlrr8rnarrt\ u11h I~IIIII~~IIIII ur

c ~ ~ l r l r r r ~ ~ r H~.iults. h~rucber, were unmtidar~l,r\ (:<al<t~~r~iu. IIIIIIIIIIK uds uml as d ~-nsitl\e. IIII.~UI~I- td thr amnunt of h ~ l ~ i l l ~ ~ t 111 III(. 111dl1t ~ . ~ t t d ( 1%. 1t11i

l ~ l t l r c,~rlchicinr. l~intl lr~g to plnnl protein *.I\ ~,l,urvrul. tl.irt .rt~,l Sr1n11$ (1973) d l n replrtctl that t l ry n~ir ld not ~ a ~ l a t ~ . or (lrq~-ct .III\ L I I ~ I ~ I L I I H ~

lxn<ling ac.tir.rty 111 nr~nrascular ~IOIW ~d h ~ g t ~ r oli(t~t\. 7'tu ~~~JJIIIIII 111 hl~111.r plant rxtrdrh 111 \ twphru i l~ (01 is murtmon/ r~ucrotul~ul.~r pnnlrrrrtlcmr d ~ d not intcrferc. w~ th binding of raclardct~vrly laln~lr.rl ~.~~lrhlrlru. t ~ r bra111 rnlc.rcr tnbulr*. Thq roncludd that microtul~uln ~d h1ghc.r plants were prol~dtrlv prcwnt c~nl! in small quar~t~tirr and were much Imr \Yal111* tn virro. ?'Iu.rc.l~m.. Bartels and Hi l t~m I 1973) urd p~g.hraln t~,~ur as u vur r r d n~irrr l~lbulrr protein.

Figuw 5- 1 1 Ufmt of triflurvl~n and mlchicine on aggrq ion d micmtubuk wb wits. Inrubatmn medium 3 mg pmteldml: 37.C; KPO,. lcTP buffer. (0-0) nntml: (*-XI rsturntd cunmntrationd trifluralin: [A- f mlchicine.

Hart~uls and tlilton assumed that microtubules of brain tissue and rcwt tips wrrr rin~~lar, and that rrsultsobtaid with hrain microtubules were applica- ble to rcx~t tisur. h t h animnl and plant micrntubuh dlsappcar in the prwnw d n~lchirilw and at OeC (Pickett-Heaps. 1967; Borw and Olmsted. 1972). Structurillly, animal and plunt microtubules are similar-h&h are hollow cylinders abut 250 in diameter, running a straight course through tht. c)loplasn~. All tubulin appear to be similar in their molecular weight, sedi- mmtatien ca4Iicirn1, and guawsine triphosphatr binding.

Purified tubulin from pig-brain tisue (Borisy et al.. 1972) was polymerized in thr abmm or p m n d trifluralin or wlchicine The curve in Figure 5-1 1 indicates, by o p n circles, aggregation in the absnrr d herbicides or mlchicine, this mrve has a charaderistic ngmoid shape. Cdchicine (100 pM) s u p p d thcdevelopment d turbidity (Figure 5-1 I ) by inhibiting theassxi- ation d the microtubular subunits. Inmtrad. a saturated rolution d triflura- lin did not prevent aggregation d h i t s (Figure 5- 1 1). These e m were identical to the control curve.

Becawe thae linear agggrter vme nd morpbdogiwlly similar to In doe miuohlbukr. a acond rnahal c$ k dfro pdymeriratim was uacd (hrisy andolmdcd. 1972). When vkwad witb t h e ~ n m micrormp. the p& d this polymerization were found to be mirrohrbular. ~igum$-12 ka mpm mtative pictwe d minohrbukr f d In uftro in the a h m a or prrpnrd

suhmting mnc~ntrations of t r i l lu ra l~~~. 11 dsms l t ld l thrw I~rrh~clrlm rto nr~t inhibit anlnlal nncriltul~ular e>xmllly (illrh~clnc* I l W p hlj JII~ cr>\11 t~1111x.r aturr irrhlbitnl thr lormatio~i (11 ~n~rrotubulrr IHorls! und 0lnlrt1.cl. 19721

To drtrr~nlnt. whethrr h*rhir~tLs I)IIIC! 10 dnlnl~l IIIINIIIII, ~I~I~IIIC.~ IIIOLIII~ fr~)m p~&hraln tissue w.hs ~nrul>i~tnl d l O i * ( : wtlh rd~lro.rtl~\~.l\ l d l ~~ lw l "(Z

trifluralln. "(:.chlorpr~q~lia~n (lu,prt>p\l e l . r h l ~ ~ r r ~ , ~ r l ~ , ~ ~ ~ ~ I ~ ~ t t ~ l , ,111d "(:.])II)IIR. n ~ ~ c k 13.S-d1rhlorcr N- I , I~~IIIIIc~I~vI.~.~~~I~~YII~III~~II~.IIIII~L~ Aw,tr ,,I I r ~ l ~ t l l n ~ av t~v~t \ was 111r,is11rt*d IIV tl~c* gt-I f11Iri111u11 IIII*~II<UI NIII~III~ ( 8 1 r,itl~t,.t~ tl\t*I\ l a l r l t ~ l "((:-rt~lrhict~w lo I u l ~u l~n was tnkt.11 L<I rrl>rt.nt .I III,I~IIII~II~I 01 I00 pvrct-t~t 1~1ntl111g ('strig thn crllrrtibn, IIIII(~III~ 1 2 1 l l ~ c "(~~~IIIII~~I~II~III~IIII, l r ~ l l t ~ ra1111, 311d ~~,I~I.IIIIII~~, wd\ 4.4, 3 5, ~IICI 2 pr r~~r l l t , rw~n,cI~\t~l! il,tlbl~. 5 1 1 , U'rtu.nlr*rg rl dl, t 1968) ~ p o r t t d that I r ~ n ~ l ~ ~ r u l ~ ~ (11 (.I~I~IIII~IIII~ IIIIIII$ w ~ l h I

microtubular dimer. Results indicated very little interaeion betwen the her- bicides and animal tubulin, which may explain why & herbicides did not interfere with polymerization of the micmtubules. Satumting herbicidal con- cnitrntions interfered with neither polymerization 01 microtubules nor bind- ing of 'H-colchiciw (lfii) to the microtubular proteins. 'H-&lchicine bound 82,500 cpmlmg protein. Trilluralin or pronamide plus 'H-culchicine bound 82.500 cpmimg protein (the ssme amount). In this study, the amount d ml. chicinr binding war assayed by the DEAE allulon filter mcthcd.

Even thtu* wry little binding of radioactively labded herbicides to purifkd tubulin fwm pig-brain tkue was observed. '4Gtrifluralin did bind to protein in the crude 100.000 g supernatant from mrn rod tips (4300 cpm/ mgh The type or nature of the protein that bound the trifluralin is unknown. Nocvidcnce was found that the bindlng protein misted d sulfhydryl protein d mots. as ruggated by the work d Shahied and Giddms (1970). The herbC cide may have buund to a lipid component in thesupematant.

MODE OF ACnON OF W N O N M r n : r n R I E S

Obmrvatioa hdicah thrt h i n d i n tmtrnad dlan inducw a ranged d- fectsfmm wr-nomul mitosis to m d c h b i l i i e . disrupted mitosis The d t y d hinulrlids effect an mwmtukrla d mot muistem allr probably d a p a a c k m t h c h s r b l c i d e ~ ~ . ~ m & d l ~ d m t h c ~ ~ clch'r mabillty. Has .ad hyer (I9741 pmpad that tbc colrmmtbn d

trinuralin required for complete miemhrbular dirpparena-and thur eum plete blockage d mitosis-is near an squaw saturated conmtratb. W~tntc fore, the degree of aaturation achiwd would determine the thomu* d micmtubular dimppnrana and, hence, the degnc ol mitotic dimpion. In mme roots, trifluralin accumulates in oil duct gland cclla, thuc effectively blocking its movement to these dividing cellr(Mallory and Baycr. 1972).

Thc ultrastructural effects of triflumlin and of colchicine on rod nl lr ap par to be similar. Light and electron micmrap -dim wsaled that trillu- ralin produced both chrom~omal and mitotic aberrations similar to thar cauaed by colchicine (which prevents f o r n t h d microtubulcr). N d all her. bicides that produce mitotic disruptions came loa 01 microtubuln. Hepkr and Jackson (1969) nportd that propham at 10 ppmw did not destry mirr~r tubuler as did colchicine, but rather led to their disorientation in t k ccll. Ultrastructural studies showed that trifluralin at aqueour saturated cnnmntrs tions c a d disappearance of microtubula in a manner similar to colchicine. To determine if trifluralin aaed like colchidn at the biochemical level, t b herbicide was incubated with two in u(ho mkrotubular-forming cystrrns from pig-brain time. Results showed that trifluralin failed to inhibit the awrcgu. tion and polymerization d animal micratubulor The binding experimmtr. with radioactively labeled herbicidca and wlchicine, showed that t k hcrbi. cidn did not bind to animal tubulin. nor did thcy dnutura the colrhirine binding site on microtubub. Biochemical mult, showni that trifluralin did not disrupt the microtubules per rc, nor did tt exert its effect by direct interac tion with animal microtubular prdein. To date, tubulin has mt bren icolated and purified from a higher plant soura; thus. the interadion d trifluralin with higher plant tubulin has not bren studied. Current march on micro tubules (L. Wilson, pml communicatia3) weal# that plant a d animal tubulin are not identical; therefore, a chemiosl may interact with plant micro tubular protein while having little or no affiafty for animal tubulin. Fur exem ple. it taka a greater concentration of &hicine to inhibit all division in swpensiom of plant n l l r than i t don in smpsionsd animal d l r . In African blood lily liquid endmpnn alh (plant albwith no all walh). 104 M colchE cine will black all division (Hepkr and fackson, 1969). whik in human H h alls the concentration m u i m l is 1V' M iTavlor. 1985). In their R D O ~ d ektrcphoretic difference hibula bDl&d from s plant and from an animal icurcc, O l d et 01. ( 197 I ) d a t e d tubulin from m alga (CMamydp mow) and from brain the. Ekctropboretk mmpprlPn d tbc two pro(eIn mum. indicated that m pro(omr (subunit d the tubulin d i d war mm mon to htth while on fmm each waa uniqv in it# ekmphWdk mobility. Bbause purifd higber plant tubulin har mt been W for &a intaraction with tdlulurnlts, i t is pwlbk dut trinuralh hu an action mcchbtn drnilar to tbat d fdrhichr (i.c, biding to micrdubuk h d a that polynmhPtian is prventd.

18 HAWAII ACR~CVL~LIHALL~PE~I~M~~ 1 SI A I ION

tmr d microtubula war probnbly not c a d by a general deterioration d cell strueturn r r a mull d inhibition d cell divich. Pmpham blocks cell dividon but don not c a w dimppunnced m i c m t u b u l ~ l l s . The divppoaranre ol microtubules from root cells m y m l t from inhibi-

tion d the synthesis d tubulin or dysfunction d the minotubular orgal~izing cmtcr (MTOCI. Trinuralin m y act directly on the morphological unrtruc- tured MWC d mot-tip cellci.e., mdophmic reticdurn (ER) membranes thrt appear to be involved in synthesis or transport of Nbulin to and from sitr d polymerization. MTOC in higher plants appears to $a I-ly organized aggregate d smooth ER cihrnae in the polar regions@ mlls. Polar ends of the microhibukr 3tem to terminate in membra icla d the ER elc mentr (Hanzely and Schjcide, 1973). Burges an that smooth WI was involved in polymerizat bulin in when1 roots. Hanzely and Schjeide ( l was p m n t in the body ol the spindle, as well as in pola Ions. At both loca- tions, var iw types of connections or attachments wem wed between El7 ekments and microtubuln. Figun 5-10 shows that of root cells from wheat and corn was swollen and distorted in triflurali r" n a t d roots, as com- pared to controls. If the propond involvement ol ER in $licrotubul~ asrembly is correct, then an inhibition of the normal ER fundioq such as the obrwed welling, w l d explain the Ims d micmtubulcs from thetreated root mlis. A d ditional support comer; from the work d Towne (19741, who found that triflu- ralin and onyalin (3,Sdinitrc~N'.N'dipm~IsuIfanilamlde~ krbicidm caused leakage d photasynthetic products from single isolated cells, indicating that cellular membranes were altered or modifid by the herbicides. Figure 5-9 shows that treated cells have enlarged vacuolcs and that the mot cells enlarge radially, suggesting that the ~lcctive permeability of cellular inembranas may have k n affcckd. Partitioning into and binding ol trifluralin to the mem branssnwrld alter their activity.

Because triflurulin inhibits photorynthcrir and respiration (Moreland et al., 1972), the herbicides conceivably could interfere with protein synthesis through their duck on the availability or formation d energy-rich fom

IOU&, such as ATP nesW for pmtein synthesis andCTP nmmry for ply- wization d miwohtbular svbunih into micmtubuln (Borisy and Olmsted, 1972). However. Moreland et al. (1972) rrpamd that respiratory inhibitors and uncwpkm do not usually produn the multinuc~eak odls or promote the radial enlargement d ti= that m typical plant mpolues to trifluralin and to, J-hf; .

The made d action d trifluralin h dl11 unknown. Whatwer the p- may be. trifluralin achievu its effect rapidly (wlthin 3 huun) md at low con- centntions (lea than 3 x lo* MI. The a r t o m i c l l effect d trifluralin t m t - ment b aberrant all division ad cell enlargement in mot tips. Tht cyto lw ical dfect ia the disappearance d micmtubulos Imm them arras. Our m u l b suggest that the primary action mechanism d trifluralln may be related t o r n interference with o m or mom ol the following: (11 the synthnk ol tubulin; (2) the polymerization of micmtubular ~ b ~ n l t l through a d i m on Ihr MTOC; or (3) the polymerization of micrdubulsr subunits by interaction with the arb units themclves. Triflurdin'r primary mschsnim d adion, howvcr, m mains to beexplained.

Adelman. M. R.. C. G. Boriry. M. L. Shelmdri, R. C. Wrirnturg. and E. W Tuylur. 1968. Cyloplasmicfilsmentsand tubuks. FM. Pm 27:l 186-1 193.

Ashlon. F.. and A. Crafts. 1973. Dinitrwnilinu In Muln of Aclum d Ilrrb~ridrr. New York: Wileylntrrrcienn, pp. 221 -235.

Bartcls, P. G., and 1. L. H i l h . 1973. C w n p r i m d trifluralin, uryulin, prurumih, prcphsrn, and oulchicim treatmnu on miorutubuln. Pest. Bkrhcm Physcol. 3: 462-472.

Bayer. D. E.. C. L. Foy. T. E. Mallory, and E.C. Cutter. 1967. Morphulqicul and hirtological dfeetrd trffluralinw root dcvrloprrnt. Amr. J. &X 54:WS-952.

Boriry. C. G.. and J . B. Oltruced. 1972. Nuckatd rrrmbly d microfubukr in p r e h brainextraas. Srimrr 177:llM-I 197. - - and R. A. Klugmon. 1972. &I dtro nggyyllion d ryluplmic micmtubukrubunits. Pm. Nat. Acd. Sci.9:2890-21194.

Burpa, 1.. and D. H. Northe . IW8. The nlntiauhip Mmcn the mbplvrmlc mticulum and rnindubular qgregulion a d diuggqutbn. Plants I - 14.

Eigti. O.J.. and P. M i n Jr. 1955. Cdchiiiu. In Agriculture. Medicin. B%, nndCheaihy. Ams, lava: Thelava SlataCdl. Pma. t70pp.

Hacakaylo. J.. and V. A. Amto. 1888. Eifec( dMnun l in w rout# d am und d m W d k i . 16:513-515.

HIIULJY. L. d 0 . A. Sehjci&. 1973. Structunl and functimul upctl d ana8tral mitotielpindleinAU&m~~m raa(.tip&. Cyt&7:147-162.

Hart. J. W.. d D. D. hbnir 1973. C d c h i c M l ~ pmWn f m p h b a a x y b d 4 hieplant. Ptnb 1W147-152.

Hspbr. P. K.. and W. T. Indun. IW. IsopwyI N-wrbometc a k t ~ @ minohlb~caienbtbniahi*idbgadaprmrlsUldHU(IMMW~ Bnkr. J. Cdl Sd. 5727-743.

d B . h P . M h 1@74.MLemh&*..IddI*mANI.Rlv.PLnr phvrbL 25:31#-362.

M HAWAII ACRICVtTURALW(PERI~STATl0N

Hm D., ud D. &yn. 1974. Tbe dfad tdlunlin a ths ultrutruchn d divMin(( dlrdrcdamlllemdEdlm~.J.CalSd. 15:429-441.

J a b . W. T., ud D. A. Stdkr. 1973. RquMan d mW& IV. An In dho a d ultrutruet~lhdydalfsctrdtrfflun1in~CInadirn J. Bat. 51:1513-1518.

-1, E. M., d E . C. W. 1972. Ef l&~dMflunl inamHab. WdSci. 20 267-270.

Malkry, T , E., and D. E. Lyn. 1972. The dl& d trfflugin on the gmwth and dmbprmt d w o n a d d l o w e r rwb. Bd. Qz. 133

Mordd , D. E., F. S. Rrmer, and C. C. Hwmy. 1972. Inh llbn d pholaynthnh ud nrplraHan by rubrlitutd 2,Bdinitmniline he&' da. [I. Effects on re lpnas in ncird plant tirue and treated rallinp. P . Blochwn. Phynd. 2: 354-383. fo2'

Nwmmb. E. H. 1969. P b t micrdubulsl. A m O l m d , J. 8.. C. 9. Witman, K. Carism, and J.

microtubule protcinr of neumblastom ells Pmc. Nrt. Acod. ki. 68:2273-2277.

Pickett-Hap. j. D. 1867. The effect d cdchicint on the u ad~cture d dividing plmt allr, rylcm wall diffenntiath, and distribuiion cytoplsJmic micmtw buk. htl, Biol. 15:208-236.

t Sluhlsd, S. E., and J. Giddrm. 1970. Effect d cystcine on t @ a h d trifluralin on

laten1 mdr d c&on and cum, A m . ].62:306-307. Stnng, R. H., and R. 1. Rap. 1971. A micmndiasutognp(ic study d "Gt r i f lw

tin rbsorplion. WdSci. 19:363-389. Trlbert, H. E. 1885. Effectsol trifluralinonroyban mot dcvdqnnmt. Pmc. Southern

W e d Con[. 18;652. (Aktr.). Taylor. E. W. 1885. The mhanism d colchicine inhibition d mitdii. I. Kineticr d

inhibition snd the bidingd'H.colehicine. 1. Cell Biol. 25: 145-160. Town, C. 1874. Surfactant and herbicldc effects a membran prmeabilig, phdb

qntherk and u l t r s s t ~ c t u ~ d diat leaf d l $ . Muter's thesis, Univ. Ariznna. Tucsun, Arizm.

Wotmberg. R. C., C. C. Borisy. and E. W. Taylor. 1968. Tlw mlchicbbinding p m tcin d mammalian brain and its dation to mirmtubule. Biochrmidry 714466-4479.

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and Human Rcsnurces, Univrrsrty o l Ifawaii, rvr thr I!nircrJ Srdccr I)c, partmcnt of Aqict~ltttre to the exclusion ell odlerr J I ~ I rnay bcdvaiial~lc.

cOp14 of &is publicatiou ~viulstxlc without rharur lo flawaii rrrWm(l from C W t y WnU. [hlt-o[.Su~c in&ks or bulk ordcn rlrodd be xnt to lhc r\grli-rrl. mrd PuMLa60w md lafornation Oflkt, C d l c ~ c of Trrq;iJ A&ulturr and Bu. mu, Resoarce~, '2500 Dolt fibcrt. Krawe 1WI Rerun 107, tlondulu, Hawaii !t6822. P k t Ds Copy to bulk nun, ninety crnt* plus p*lllfi

NOTE: h ~ ~ d r n r u a u n l ~ a p d m d a L I h c H n u i i A p i o r l n u J P 1 ~ S u ~ d Ihc Nwii Cwpntlw Ex~muon Srma hvt u\r w q d mr)odmndiInli~ u d n ~k rnmr HAWAII INSTITUTE OF TROPICAL AORICULNRE AND HVMAN REXWRCES. C d