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Propanil metabolism in rice: A comparison of propanil amidase activities in rice plants and callus cultures
Propanil
Metabolism in Rice: A Comparison Amidase Activities in Rice Plants Callus Cultures
Received
.Trdy
17, 1074;
accepted
Oc*toher
of Propanil and
18, 1974
Propanil amidase is an enzvn~e fotmd in rinse, which hydrolyses the herbicide 3,4dichloropropionanilide, propan~l. The activity of t,he enzyme as measured in rice plants was found to be two to fonr times greater in plants with four leaves than in plants with fewer than four leaves. The higher amidase acti\ ity of the four-leaf planls was localized in the unexpanded leaves. Hive root calIlls suspension in cult rues also demonstrated propanil amidase activity. In vzbo experiments indicated that the herbicide was metabolized by the t,issrle cult\ue. Propanil amidase activity as determined in oil~o, however, was detected only after t,he culture had de\-eloped to late stnt,ionary phase.
The acylanilide hrrhicid(l 8,5-dichloropropionanilidc? (propanil) is widely used to control wwds such as barnyard grass (Echinochloa crmqalli I,.) in fields trf rice ((fryza satiua, I,.). Propanil is srlect.ively toxic, wit,h rice showing resistance (1). This rcsistancc has hwn attrihutcd to the abilit,y of rice to rapidly degrade thn herbicide (Z-.5). The enzyme which hydrolyzes propanil in riw plant8s has bwn partialI> characterized (4-G). It, has bwn found t,o bc particulate (5) and is sPnsit8iw to inhihition by carbamate insecticides (6). Based on enzyme activity in ~jifw, St,ill and Kuzirian (5) cst,imated t#hat three-leaf rice plant,s dcgradr t,hc hcrbicidc 4 to 10 tinm faster than ricr weds. This suggested than t,hcl ratw of mctabolisrn drpc>nd upon the age of the plant. To investigate this ph~~lt~lM!l~J~ll further we havct measured the ICV& of amidasr activity in homogenates of rice plants of various gro\sth st,agc>s.
In st,udying 1n~tabtJh pathways it, is critical that a defined sgntrm be employed. Although grwnhouse-grown plants do not8 rrprescnt such a system, thry have been usrd in all rtportcad studies on propanil mrtabolism in rice (‘L-6, 11). In order to create a more closely CoIltrokd syst~fn for such studies in riw, we have dcvcloped rice callus susp(%sion cultures (7). Use of any such system for study necessit#at,esestahlishing it,s validity as a model for the whole plant. In order to do this WPhave compared propanil metabolism in rice suspension cult,urcs to that of the plant. 1\1.4TERI.4I,S
ilNI)
XIETHOI)S
:lJatenYals.Seeds of rice (Oryza sativa 1,. var Starbonnet) were sown in wvoodcn flats containing vermiculit8e in a greenhouse. The grcenhousc leas maintained such that maximum dag temperatures ranged bctwcen 7.3OFwinter and 95OF summw and night tcnqwraturcs were not lomrr than
172
ItAY
AXD
58°F. Incattdrsccnt~ lighting \vas applied when necessary to mairtt,ain long da! conditions (16 hr). Plant)s ww watcrrd on alternate days with tap n-atcr or a routinr: ttutricnt solution. I’izpamhitr of hfmofgetia2e.s J’rwrrr rice p/a)rts. Thr arrial portions of riw plants \v(w harvcstcd, washed in d&jtrizc>d natcr, blotted dry, and five \v?w cut iIlt0 small picccs less than 0.5 cm in lwgth. All of the following steps wwc ptkrfortttod on at1 iw bat,h. l’h~ cut riw was ground in a chilled mortar ivith 1.5 ml of a bufftbr containing 50 mill sodiunt phwphattl buffclr, 1 md/ Ka2EDTA, 0.1% v!v mercaptoc,thartoJ, and 300/, v/v glycerol. The pH of the buff(Jr was S.O. Aftckr grinding t,o hrcak up large picws of plant material, the homogenate was subjrctr>d to sonic oscillatirjn (150 W for .i min). The> homagenote was filtcrcd through cdight. layers of cheesecloth and cwbrifugc>d for 15 mitt at 75Oq at 4°C. The rcwlting supwtiatantS IV:M called the crude homogcnatc~. In preparing honiogcnatcXs of caxpartded Icavcs, lt>af bladrs ww twisrd at the Jigule attd homogenizrd as descritwd abovc. Homogcnatw of urtc>xpandrd lravw wcrc prrpawd by carcfulIy stripping away th(J (Lxpandcd l(~avc~s and t,hGr sheaths. The urtcxpattdcd l(~avw wcrc thrln honlogc~nizrd as d(wribcd abow. Prepamtifttr
of
Iromo~euatm
,/‘)wJ))
cal1u.s
szsspe~lsio,~ cz~ltuws. CMls ww harvested Oil filtw papw in a Uiichnrr funttc~J and \\-ash(>d with swrral portions of d&jnizcld \vatw. Execss wat,clr was wmowd \vit.h suction and the fresh wight dctwmitwd. The tissw nas transfrrwd to a chilled stainlrss-stwl J,ogeman hand homogc~rtizw and homogcttiz~d in a volume> of huffw qua1 to twice the ueight of th(L tissue. The buffer contained 0.3 iI/ tnattnitol, 10 mX tHorpholitloI)rOI)atlt-sUIfotliC acid ( L\JOJ’S), 1 tnil1 I\‘arEDTA, 0.1% \v/'v hovittca serum albumin (MA), and 0.1 y. (v/v) 2-mercapto&hanol. Thr pH was 7.5. Th(~ tissw was passed through the homogenizer several
STILL
timw and the rwulting hornogc~ttat,c~ wtttrifugrd at 3OOq for 5 min. The supcrnatant n-as called the crudr, h~JlllOgf?Il~tf?. LI rtridase assay. Th(A assay mixtuw for the d~t’c~rniittatiott of amidase act,ivity cObsists of I ml of wtz~mc~ and 4 ml of 50 m61 phosphatfl huffw (pH T..i). Thtx buff(tr contaittr>d propartil such that, the: final coticc~titratioti in 3 ml of tlit> wactioti mix was 22.5 pcld/. Samplw of twhnical grade propanil Ivct-c obt)aitwd from thtl Kohm and Haas (‘o., l~hiladnlphia, J’A (HTA:\J I<‘-3-2) and from .\Jonsant.c) (‘O., Ht. J,ouis, ,\iO (Jtogw). Thrsc samples ivwc gwatot than BSY, purity and used \vithout furth(>r purification. The? rract’ion was started b> tht> addition of (~ttz~m~~, and the rcactiott mix was incubated with shaking at 30°C’. Th(+ waction n-as t,crmittatrad by twnoving a l-ml aliyuot of t tw wactiott tnix and adding it to 6 ml of an acid solutiort COBtaining 0.96 N H(I and 1.7 N aec+ic acid. III caws in which a hwvy prwipitatc~ formed, thck acid solution \vas wntrifug(!d and the pellrt dixcardrd. Thr amount of 3,4-dichloroanilinr (3,4-D(:A) in the acid solution was dt~trrmincd coloritnctricall;v by tht? Bratton-AJarshall waction as dcset&d bp P~asc~ (8). Otw unit of c~tz~rntt activit,>- \\-a~ defined as the amount of c9iz~mc~ twluiwd to produce 1 nmolr (Jf 3,4-DCYA p(‘r hour. Protein \vas dct,c~rmitwd by th(a m&hod of I,ow~~ cl al. (9). /ti
uiw
76ptake
of
propatti
bJ
s?6spetrsiotr.
cullwes. T\\-0 milligrams of propattil were added t.o the culture mt,dium prior to The 1~11s ww grown as strrilizat,ion. dwcribod by I,icab et al. (7j and at’ various timw harwsttd t,v suction on a Riichtwr funncal. The mcldium \vas tlxtractcd several timw kvith qua1 volumes of c>thyl awtatc?. Thts fresh \wight of thr tissue was detcrmined aftw which it \vas stirwd for at1 hour wit,h a small volumr of ct,h>ll awtatC. This clxt>ract was t.hen combined with that of thca mcldium and thr volume reduced itr ~aruo to 1 ml. The amounts of propanil and 3,4-DCA in the: extracts were detcr-
PI{OPANIL
METABOLISM
mlnc~d by gas chromat,ography (Tracer .\Iicrc+!I’c,k Gas C’hromatography ; 1.8-m stainlrss-stwl column packed \vit,h 5,% I~XOl on Gas Clhrom Q, N-100 mesh; rnrriw : 7.5 ml,/min S, ; flame ionization d(%clctor ; temperature : loo”-20°C at lO”(!.!min). Untlw thrw conditions propanil had a. retcwtion t,irne of 11 min and 3,4-DC’A .5 min. Tht~ limits of d(+c,ction for both propanil and :3,1-DC’A \V:LS 20 ppm. l’c,ak awas \vCrf’ m(>atsurrd with a planimc~trr and collc~cIlt,rat,iorl of’ substrate or product IV&S d(ltcwninc~d fro111 standard curvw. tlEC;ULTS ,
Table 1 sho~vs the propanil amidase activity in various growth st,agrs of rice plants. The grO\\th stage \vas defiwd ha the numhcr of clxpandcd lravw On the plant. I2lrasuwments \v(w mad? owr a pctriod of 30 days using t\vo flats of riw. l’hc valuw in the t,ablt> wprcscwt tjlw mcan of srveral drtrrminat~ioiis from clach stage. l‘h~ awragfh amidastr activitiw of plants with frb\vtar than four lravw \vw(’ found to b(x 11.2 unitJti,/mg protein. That, of plants \vith four lcavw \vas x3.s uIiits/nig prOttiI1. ‘1’1-1~ diffrrcww bc>twwn th(> m(‘ans \vas significant at the 1% lewl. ICrom these wsults it car1 tw sw11 fhat thtt Ill(‘aIi TARLK
Amidase
(:rowth stage Units #‘ml homogenate I 2 3 4 --~-__-.-
1
46.0 4o..i 20.0 I Ili.T,
act,ivit)
Units ,‘g fresh wt 138 115 s2 347.5 _____~
ITni t,s img protein 13.4 !I.7 1 I .s x3.7
I&’
173
Rl(‘E
TABLI‘:
2
I’mpanil A miduse Acliviiy in Hornoyenutes qf haws from Four-ldaf Rice Plunts. One Enz!jme Unit Eq~ta1.s the Amonnt of Eruyrn~ Rayuiretl for the Fombation oJ’ 1 nmole of cY,.$-fl(‘A per HI
spwific activity of plants with four l(~avc:s \vas thrw times greater than that in plants with fwver than four lraws. Sinw the amidase activit,y incrcascd S-fold with the appearance of the fourth lwf, it, \vas of int,wet to daterminr if t,he enzymr act,ivitJ of the fourth leaf was higher than that Of the other t’hrw lravw. Table 2 shows the wsult,s of assays of thr various leaves for amidase activity. By the time that fourth leaf was fully ctxpanded the first lc,af had bewmr scw~swnt and, thcwfow, \vas not, assayed. The amidasr lwrls of the third and fourth lcavrs wwc’ found to bc similar to that of thr whole plant \\-ith frwr than four Iravcs on a frrsh wright) basis and in tclrms of total activity in the homogc>natr>. 7’hr activity 011 a protrin basis, although highr~r than that of plants with fewr than four lravcs, \vas not as high as that of whole, four-leaf plants. k’rom thcsct dat,a \V(’ concludrd t’hat the higher ruzynct activity of four-leaf plant)s was not, dw to higher activity in thr fourth Iraf. A characteristic of many grassrs is that th(l lraf sheaths in t,he aerial portion of plant form a tube \\-hich r~nclosrs the> unc~xpandrd I~avw. Since it, apprarrd that the high(lr amidase act,ivity of four-leaf plants was not dw t80 highrr amidaw act,ivity in thp fourt)h lraf, the activit,y of thr uwxpandrd lraws \vas detprminc>d. l’ablr 3 summarizw thcx results of th(w expwiments. As can ho sren, the uctivit3
171
lt~x-
TABLE
AND
3
I’ropunil Amidasr Activity of Eqantled Leuf Hlatks and linerpanrletl Leaves. One Enzyme 7!nit Eqds the Amount of Enzyme Keqrl,irrtlfo~ thr Formation qf 1 nmolv of .i’,&llt’A per HI
STILL
28 hr for wJntro1 cult,urcts containing no propanil. The cultures COntaiIling propanil also showrld so111e reduction in final frwh \I-Aght (2 g) as compawd to that of th(l ccJIl~rOlS
(l..i
I'ropar,il
Homogenate
amidase Units/ml homogenate
activitv
Units. fresh
__Expanded leaf blades Unexpanded leaves Leaves 5 and 6
72 2.50
21 ti
Cl.4 ci2.5
of the uncxpandrd Ieaws is about four times greater t’han the cxpandcd leaves and about twice as high as that, Of four-leaf plants. D&rmination of amidaw activit) in leaves five and six aftrr they expanded revealed achivity comparablt~ to that, of the unexpanded leaves on a pr(Actin basis. We concludrl then that tho higher act,ivity of t)he four-leaf plant is localiwd in the lcavrs which appear after the fourth hlaf. That is, at SOrJW time during devclopmcnt of t,hr fourth leaf tjhc ,voungrar developing l~~avrs pruduce cithcr mow or-a more active propanil amidaw. It, uiuo uptake callus suspel,sim
of
r)roparlil
by
rice
actiuify
of
vice
twi
The fwzymr, was assaycAdin crude homogcwatrs of the tissue cultuw. Thc~ awrage amidasc activity A\-as found to h(, 14.4 units/mg protein. This is comparable to th(b activit,y of plants with fwwr t,han four Icavw and is consistent \\-ith th(> finding of I’rclar and Still (6) that riw rOcltS haw lrss amidaw activity than riw l(bavc>sin mont,h-old plants. ItI \vas of intrrwt then to dctt>rmine if th(> amidaw activity of rice tissw in cultuw, likt> t)hat of t)hc rice plant, would change \vith the agcl of the culture. Tho amidasc activit,y of the suspt>nsion culture \vas mr~asurcd at, various timw during the callus
g IJnit,s/rny wt proteirl
g). awidasr
susI~e~~.sio~r
cultures.
‘I
?od
cultwe. The kinct,ics of propanil uptake by rice root callus suspension cult,ures are shown in P’ig. 1. C:ulturc flasks contairwd 9.2 pmolw of propanil (2 mg) in 50 ml of culture medium and \I-cre inoculated at zero time with approximatrly :?.iOmg of tissur. Tht> amount of extract’ahlc propanil drcwasrd 1incJarly up to 16X hr and by 192 hr no propanil could be detected. 3,4-D(IA first appeared in the extract,s at 96 hr, reaching a peak of 2 pmolcs at approximately 1-H hr and decreased afterwards. Thr cfftct of propanil on t,issw growth is shown in I:ig. 2. The doubling time for the cultures grown in the presence of propanil increased to 48 hr as compared to
Time
b’ra.
(hr,)
1. c’ptake of propand from cl6ltz6re medil6tn by root calll6.5 suspension clrllures. Solid line: Propanil; dashed line: 3,4-DCA. Each point represents the a~rage of two $asks. Each jtask initially contained 9.2 ptncles of prOpand in 50 Nd of CdtlLre medium and was inoculated with abol6t 350 mg of suspension cc6lture at zero time. rice
PROPANIL
48
96 Time
144
192
METABOLISM
2,
(hours)
I~Iu. 2. The growth kinetics of rice callus suspension o~lturrs in thr presence of propmil. Initially 9 my of propanil were atdrlerl to 50 ml Gf cullwe medium at 2~1’0 /imp. The ordinate is a log plot of the ,jresh wright of the tissw. The abscissa represents hours a,fter inocrrlntiorl. The controls receivrrl no propnnil. Solid linr: with propnnil; dashed lint: control.
IN
175
RICE
Leaves five and six wrc found to contain higher amidase activity than leaves one t,hrough four. The same was true of t,hc immature unexpanded leaves of the fourleaf plant,. We concluded that the higher amidase activity of four-lraf plants \vas the result of higher amidaw activity in th(l unexpanded lravw. Incwaws in c’nzymcl activity may 1-wthe WSUh Of PllZylllC actiVatiOI1 Or de 1101’0 synthesis of the protein. Proof of cithrr of the altcrnativcs in regard to t,hP changw in propanil amidasc activity obscrwd in thcb rice plant requires purificatjion of thn mzynr. Our attempts to do so have not, met as yet with compl(+ succws. I’wliminary studies, ho\vwcr, using inhibitors of protrin synthesis suggwt that> the rise in amidase activity obwrvrd in riw tissw culture is the rrsultf of de UOZUI synthesis of t’hc c~nzymc~(12). The change in amidasc
growth cycle and the rosult,s arc’ sho\vn in I;ig. 3. Thr cnzymc n-as not detwt~ahlc until 132 hr. At 168 hr the activity row dramatically to about 13 units/mg protein and by 192 hr it dropped to 4 unit,s/mg protcki. C3bniparison of curve for activit) and growth show that the increase in amidasc activity of the t,issur in culture occurs \vh(bn the, wlls arc’ in stationaq pllwsc~.
The data pwsentcd show that) the propanil amidase activity in rice plants with four or more leaves is three tin-w greater than in plants with fcw-rr than four kavrs. The greater amidasc activity of four-leaf plants should he correlated with dcvclopmcnt of rwistancc toward propanil hy these plants. This would contradict the fichld studies \vhich show\-cdthat, plants in the third and fourth lraf stagw aho\wd grclatclr scnsitivit,y toward propanil tjoxicity than plants with only one or t\vo l~aws (1).
0
13~.
72
96 Time
120 (hrr)
144
168
3. The kinetics of appearance OJ” proptmil actiuity in outlr hornogpnatus o.f rice root callus suspension cdluws. Cirltrrrrs were inocdaletl at zero ti,me. Each point rrpresents the avrrag~ amidase acfivity from few jlasks qf ocltures. The abscissa represents lime in hours njler inoculation and ihe ordinafe, aniitlase uctivity in tanils per my protein. One unit of enzyme activit!y is the amount oJ rnzqmc’ requirefl! for lhr formafion of 1 nmolr oj 3, ~-ZX’iI prr hr. amidasP
3 76
RAY
AIYD
activity in the plant, occurs during the expansion of the fourth leaf and data prescnt,cd in this report show that high lcvrls of amidaw act,ivity aw prcwnt) in the fifth 1Paf hcforr it expands. Thr~ fact that this leaf is already prcwnt hPforc thcl expansion of the fourth indicat,cLs that the Iwcl of amidaw act,ivity incwaws in the fifth leaf during t,hc expansion of the fourth leaf. This suggests that, prcJpalii1 amidaw is under snmp typp of regulatory control in t#he rice plant. Use of a tissue cult>urr as a model for st,udying metabolism in plants requires that the two systems be comparablt~. Several findings support the USC of riw root callus suspcxnsion culturw as a mod(ll system for the study of propanil mc+abolism in the rice plant. First the dat,a from uptake cxxprrimcbnts (Fig. 1) indicate that thr tissue in cultuw drws take up propanil and mc~taholizc~ it, releasing 3,4-DCA. The amount of 3,4-DCA cxtrackd ~vas not ryuivalwt to the amount of propanil addcld on a moldybasis, suggwting further mctaholisrn of 3,4-DC’A. D(‘A mc~tabolism has hwn sho\vn to owur in th(> rice plant (2, 11). This is furth(,r support,cld by the fact that the amount of caxtractabl(k 3,4-DCA begins to dccrcase after 144 hr. Thr finding that, 3,4-DCYA first appclawd hctwecn 48 and 96 hr indicatw t,hat th(a tissue in culture ran mc~taholizc~ propanil i,l viva at this growth stage‘. III vitro prOpand amidase activit,y, howrwr, was undctectahle until aftcar 120 hr (l;ig. 2). Xo irl Z&XI propanil amidasr activity could be detected in cultures gro\vn in the prrsencc of propanil for 72 hr. ‘l’hrsc rwults suggest that \oung cultuws possc~samidasr activity below the d&ction limit of th(l i/c z!i~w assay. Albrrnativcly, the young tissue culture may possessinhibitors which art’ released in the (lnzymc preparation or the product (DCA) may hrcomt~ und(~tc~ct~ah!f: in the colorimctric assay. Secondly, the ability of both rice plants and tissw culture to mc>taholizc pmpanil
STILL
dclpcnds upon thr agcb of thck plant and on the age of cultuw (Table 1, Fig. 2). Thirdly, the hydrolysis of propunil in bckh the plant, and thcx rnodcl is cwzymic, with t,hc wtivitiw Ioc3lizcd in siniiktr particulatr fraction. 111 addition, the, (‘IizJ.tiic front both sourws is scwsit,iw to inhibition t,y cnrbamatc~ inwcticidw. WC, fwl thca similaritiw bclt\vccw propanil nit’tabolinm in the> riw plant and rice root callus SUS~~PllSi~Jll cultuws arc’ sufficicwt~ to justify tht> USC’ of the callus cultuw as a model for t,hcl study of propanil mc%abolism in t,h(> riw plant. WC \vill report in grclatw d(+ail lxtcir on other studies \vith the callus culturw. Th(w includr the mrtahfrlic fatct of 3,4DC‘A and the intrxcrllular ~IlcaliZatilJIl of propanil amidaw. ACKNO\VLEI)G\fEN’I
This work was supported by Ilegional I:esearc*h l<‘tmd NE-.?:<, United States IIepartment of Agricllltllre ; paper of the jollrnal series, Sew .Jrrsq Agricldt.lxral Il:xperiment StaGon. REFERENCE
i
I. I<. .J. Smith, :i,4-dichlol,opropiorlarlilide for (YDIItrol of barnyard grass in rice fields, Il-r&s 9, :318 (19B1). 2. (:. (:. WI, Metabolism of :3,4-dic!hloropropiollanilide in plants: metabolic fate of the 3,4dichloroaniline moiety, S(,i~nce 159, !I!)2 (1 YW). 3. (:. (;. St ill, Met,abolism of :3,4-dic,hloropt,opir)IIanilide in plants : metabolic* fate of 1he plopionic acid moiety, Plunk Physiol. 43, .i4:3 (lOfir(). 4. It. Y. Yih, I>. H. McItae, and H. E’. \Vilson, The mechanism of selective action of 3,4dichloropropionanilide, Plan{ Physiol. 43, 1291 (l%iX). .j. C. C. Still and 0. Iiuxirian, I:nzyme detoxification of 4,4-dichloropropinnanilide in rice and in herbicide selecbarnyard grass, a factor tivity, ~Yrcttrrr (/,ondm) 2 16, 799 (I !)ti7). ti. I). S. I:rear and (:. (+. Still, The metabolism of :j,4-dicl-lloloprcIpionsrlilide in plants. Partial pllrificntion and properties of an aryl avylan~idase from rive, Phg/och~n/islr!/ 7, !)I:1 (1968). 7. H. B. I&b, T. B. Ray and C. C. Still, (irowth of rice root callus suspension cultures, Plani Ph~ysiol. 51, 1140 (1973).
PROPANIL
S. IT. J. Pease, Separation monuron and diuron (‘hrrn. 10, 279 (1962). 9. 0. H. Lowry, 1<. H. I;olin (I!,.il).
phenol
and determination residues, J. iicrr.
N. J. Rosebrough,
Randall,
Prot,ein reagent,
METABOLISM
A. I,. Farr, measurements
.r. Hid.
C’h~nr.
of Food and with
193,
26.5
IN
RIC’E
Iii
10. Ii.
T&an, “Pinnt~ Anatomy,” p. KS, Wiley, Sew York, 1967. 11. Il. H. Tih, 1). H. McRae, and H. F. &%orr, Metabolism of 3,4-dichloropropionanilide : X,4dich!oroaniline lignin complex in rive plants, Science
72.
T.
161,
B. Ray preparati,,n.
376
and
(196X).
C.
C.
Still,
nranltscrir>t
in
Metabolism in Rice: A Comparison Amidase Activities in Rice Plants Callus Cultures
Received
.Trdy
17, 1074;
accepted
Oc*toher
of Propanil and
18, 1974
Propanil amidase is an enzvn~e fotmd in rinse, which hydrolyses the herbicide 3,4dichloropropionanilide, propan~l. The activity of t,he enzyme as measured in rice plants was found to be two to fonr times greater in plants with four leaves than in plants with fewer than four leaves. The higher amidase acti\ ity of the four-leaf planls was localized in the unexpanded leaves. Hive root calIlls suspension in cult rues also demonstrated propanil amidase activity. In vzbo experiments indicated that the herbicide was metabolized by the t,issrle cult\ue. Propanil amidase activity as determined in oil~o, however, was detected only after t,he culture had de\-eloped to late stnt,ionary phase.
The acylanilide hrrhicid(l 8,5-dichloropropionanilidc? (propanil) is widely used to control wwds such as barnyard grass (Echinochloa crmqalli I,.) in fields trf rice ((fryza satiua, I,.). Propanil is srlect.ively toxic, wit,h rice showing resistance (1). This rcsistancc has hwn attrihutcd to the abilit,y of rice to rapidly degrade thn herbicide (Z-.5). The enzyme which hydrolyzes propanil in riw plant8s has bwn partialI> characterized (4-G). It, has bwn found t,o bc particulate (5) and is sPnsit8iw to inhihition by carbamate insecticides (6). Based on enzyme activity in ~jifw, St,ill and Kuzirian (5) cst,imated t#hat three-leaf rice plant,s dcgradr t,hc hcrbicidc 4 to 10 tinm faster than ricr weds. This suggested than t,hcl ratw of mctabolisrn drpc>nd upon the age of the plant. To investigate this ph~~lt~lM!l~J~ll further we havct measured the ICV& of amidasr activity in homogenates of rice plants of various gro\sth st,agc>s.
In st,udying 1n~tabtJh pathways it, is critical that a defined sgntrm be employed. Although grwnhouse-grown plants do not8 rrprescnt such a system, thry have been usrd in all rtportcad studies on propanil mrtabolism in rice (‘L-6, 11). In order to create a more closely CoIltrokd syst~fn for such studies in riw, we have dcvcloped rice callus susp(%sion cultures (7). Use of any such system for study necessit#at,esestahlishing it,s validity as a model for the whole plant. In order to do this WPhave compared propanil metabolism in rice suspension cult,urcs to that of the plant. 1\1.4TERI.4I,S
ilNI)
XIETHOI)S
:lJatenYals.Seeds of rice (Oryza sativa 1,. var Starbonnet) were sown in wvoodcn flats containing vermiculit8e in a greenhouse. The grcenhousc leas maintained such that maximum dag temperatures ranged bctwcen 7.3OFwinter and 95OF summw and night tcnqwraturcs were not lomrr than
172
ItAY
AXD
58°F. Incattdrsccnt~ lighting \vas applied when necessary to mairtt,ain long da! conditions (16 hr). Plant)s ww watcrrd on alternate days with tap n-atcr or a routinr: ttutricnt solution. I’izpamhitr of hfmofgetia2e.s J’rwrrr rice p/a)rts. Thr arrial portions of riw plants \v(w harvcstcd, washed in d&jtrizc>d natcr, blotted dry, and five \v?w cut iIlt0 small picccs less than 0.5 cm in lwgth. All of the following steps wwc ptkrfortttod on at1 iw bat,h. l’h~ cut riw was ground in a chilled mortar ivith 1.5 ml of a bufftbr containing 50 mill sodiunt phwphattl buffclr, 1 md/ Ka2EDTA, 0.1% v!v mercaptoc,thartoJ, and 300/, v/v glycerol. The pH of the buff(Jr was S.O. Aftckr grinding t,o hrcak up large picws of plant material, the homogenate was subjrctr>d to sonic oscillatirjn (150 W for .i min). The> homagenote was filtcrcd through cdight. layers of cheesecloth and cwbrifugc>d for 15 mitt at 75Oq at 4°C. The rcwlting supwtiatantS IV:M called the crude homogcnatc~. In preparing honiogcnatcXs of caxpartded Icavcs, lt>af bladrs ww twisrd at the Jigule attd homogenizrd as descritwd abovc. Homogcnatw of urtc>xpandrd lravw wcrc prrpawd by carcfulIy stripping away th(J (Lxpandcd l(~avc~s and t,hGr sheaths. The urtcxpattdcd l(~avw wcrc thrln honlogc~nizrd as d(wribcd abow. Prepamtifttr
of
Iromo~euatm
,/‘)wJ))
cal1u.s
szsspe~lsio,~ cz~ltuws. CMls ww harvested Oil filtw papw in a Uiichnrr funttc~J and \\-ash(>d with swrral portions of d&jnizcld \vatw. Execss wat,clr was wmowd \vit.h suction and the fresh wight dctwmitwd. The tissw nas transfrrwd to a chilled stainlrss-stwl J,ogeman hand homogc~rtizw and homogcttiz~d in a volume> of huffw qua1 to twice the ueight of th(L tissue. The buffer contained 0.3 iI/ tnattnitol, 10 mX tHorpholitloI)rOI)atlt-sUIfotliC acid ( L\JOJ’S), 1 tnil1 I\‘arEDTA, 0.1% \v/'v hovittca serum albumin (MA), and 0.1 y. (v/v) 2-mercapto&hanol. Thr pH was 7.5. Th(~ tissw was passed through the homogenizer several
STILL
timw and the rwulting hornogc~ttat,c~ wtttrifugrd at 3OOq for 5 min. The supcrnatant n-as called the crudr, h~JlllOgf?Il~tf?. LI rtridase assay. Th(A assay mixtuw for the d~t’c~rniittatiott of amidase act,ivity cObsists of I ml of wtz~mc~ and 4 ml of 50 m61 phosphatfl huffw (pH T..i). Thtx buff(tr contaittr>d propartil such that, the: final coticc~titratioti in 3 ml of tlit> wactioti mix was 22.5 pcld/. Samplw of twhnical grade propanil Ivct-c obt)aitwd from thtl Kohm and Haas (‘o., l~hiladnlphia, J’A (HTA:\J I<‘-3-2) and from .\Jonsant.c) (‘O., Ht. J,ouis, ,\iO (Jtogw). Thrsc samples ivwc gwatot than BSY, purity and used \vithout furth(>r purification. The? rract’ion was started b> tht> addition of (~ttz~m~~, and the rcactiott mix was incubated with shaking at 30°C’. Th(+ waction n-as t,crmittatrad by twnoving a l-ml aliyuot of t tw wactiott tnix and adding it to 6 ml of an acid solutiort COBtaining 0.96 N H(I and 1.7 N aec+ic acid. III caws in which a hwvy prwipitatc~ formed, thck acid solution \vas wntrifug(!d and the pellrt dixcardrd. Thr amount of 3,4-dichloroanilinr (3,4-D(:A) in the acid solution was dt~trrmincd coloritnctricall;v by tht? Bratton-AJarshall waction as dcset&d bp P~asc~ (8). Otw unit of c~tz~rntt activit,>- \\-a~ defined as the amount of c9iz~mc~ twluiwd to produce 1 nmolr (Jf 3,4-DCYA p(‘r hour. Protein \vas dct,c~rmitwd by th(a m&hod of I,ow~~ cl al. (9). /ti
uiw
76ptake
of
propatti
bJ
s?6spetrsiotr.
cullwes. T\\-0 milligrams of propattil were added t.o the culture mt,dium prior to The 1~11s ww grown as strrilizat,ion. dwcribod by I,icab et al. (7j and at’ various timw harwsttd t,v suction on a Riichtwr funncal. The mcldium \vas tlxtractcd several timw kvith qua1 volumes of c>thyl awtatc?. Thts fresh \wight of thr tissue was detcrmined aftw which it \vas stirwd for at1 hour wit,h a small volumr of ct,h>ll awtatC. This clxt>ract was t.hen combined with that of thca mcldium and thr volume reduced itr ~aruo to 1 ml. The amounts of propanil and 3,4-DCA in the: extracts were detcr-
PI{OPANIL
METABOLISM
mlnc~d by gas chromat,ography (Tracer .\Iicrc+!I’c,k Gas C’hromatography ; 1.8-m stainlrss-stwl column packed \vit,h 5,% I~XOl on Gas Clhrom Q, N-100 mesh; rnrriw : 7.5 ml,/min S, ; flame ionization d(%clctor ; temperature : loo”-20°C at lO”(!.!min). Untlw thrw conditions propanil had a. retcwtion t,irne of 11 min and 3,4-DC’A .5 min. Tht~ limits of d(+c,ction for both propanil and :3,1-DC’A \V:LS 20 ppm. l’c,ak awas \vCrf’ m(>atsurrd with a planimc~trr and collc~cIlt,rat,iorl of’ substrate or product IV&S d(ltcwninc~d fro111 standard curvw. tlEC;ULTS ,
Table 1 sho~vs the propanil amidase activity in various growth st,agrs of rice plants. The grO\\th stage \vas defiwd ha the numhcr of clxpandcd lravw On the plant. I2lrasuwments \v(w mad? owr a pctriod of 30 days using t\vo flats of riw. l’hc valuw in the t,ablt> wprcscwt tjlw mcan of srveral drtrrminat~ioiis from clach stage. l‘h~ awragfh amidastr activitiw of plants with frb\vtar than four lravw \vw(’ found to b(x 11.2 unitJti,/mg protein. That, of plants \vith four lcavw \vas x3.s uIiits/nig prOttiI1. ‘1’1-1~ diffrrcww bc>twwn th(> m(‘ans \vas significant at the 1% lewl. ICrom these wsults it car1 tw sw11 fhat thtt Ill(‘aIi TARLK
Amidase
(:rowth stage Units #‘ml homogenate I 2 3 4 --~-__-.-
1
46.0 4o..i 20.0 I Ili.T,
act,ivit)
Units ,‘g fresh wt 138 115 s2 347.5 _____~
ITni t,s img protein 13.4 !I.7 1 I .s x3.7
I&’
173
Rl(‘E
TABLI‘:
2
I’mpanil A miduse Acliviiy in Hornoyenutes qf haws from Four-ldaf Rice Plunts. One Enz!jme Unit Eq~ta1.s the Amonnt of Eruyrn~ Rayuiretl for the Fombation oJ’ 1 nmole of cY,.$-fl(‘A per HI
spwific activity of plants with four l(~avc:s \vas thrw times greater than that in plants with fwver than four lraws. Sinw the amidase activit,y incrcascd S-fold with the appearance of the fourth lwf, it, \vas of int,wet to daterminr if t,he enzymr act,ivitJ of the fourth leaf was higher than that Of the other t’hrw lravw. Table 2 shows the wsult,s of assays of thr various leaves for amidase activity. By the time that fourth leaf was fully ctxpanded the first lc,af had bewmr scw~swnt and, thcwfow, \vas not, assayed. The amidasr lwrls of the third and fourth lcavrs wwc’ found to bc similar to that of thr whole plant \\-ith frwr than four Iravcs on a frrsh wright) basis and in tclrms of total activity in the homogc>natr>. 7’hr activity 011 a protrin basis, although highr~r than that of plants with fewr than four lravcs, \vas not as high as that of whole, four-leaf plants. k’rom thcsct dat,a \V(’ concludrd t’hat the higher ruzynct activity of four-leaf plant)s was not, dw to higher activity in thr fourth Iraf. A characteristic of many grassrs is that th(l lraf sheaths in t,he aerial portion of plant form a tube \\-hich r~nclosrs the> unc~xpandrd I~avw. Since it, apprarrd that the high(lr amidase act,ivity of four-leaf plants was not dw t80 highrr amidaw act,ivity in thp fourt)h lraf, the activit,y of thr uwxpandrd lraws \vas detprminc>d. l’ablr 3 summarizw thcx results of th(w expwiments. As can ho sren, the uctivit3
171
lt~x-
TABLE
AND
3
I’ropunil Amidasr Activity of Eqantled Leuf Hlatks and linerpanrletl Leaves. One Enzyme 7!nit Eqds the Amount of Enzyme Keqrl,irrtlfo~ thr Formation qf 1 nmolv of .i’,&llt’A per HI
STILL
28 hr for wJntro1 cult,urcts containing no propanil. The cultures COntaiIling propanil also showrld so111e reduction in final frwh \I-Aght (2 g) as compawd to that of th(l ccJIl~rOlS
(l..i
I'ropar,il
Homogenate
amidase Units/ml homogenate
activitv
Units. fresh
__Expanded leaf blades Unexpanded leaves Leaves 5 and 6
72 2.50
21 ti
Cl.4 ci2.5
of the uncxpandrd Ieaws is about four times greater t’han the cxpandcd leaves and about twice as high as that, Of four-leaf plants. D&rmination of amidaw activit) in leaves five and six aftrr they expanded revealed achivity comparablt~ to that, of the unexpanded leaves on a pr(Actin basis. We concludrl then that tho higher act,ivity of t)he four-leaf plant is localiwd in the lcavrs which appear after the fourth hlaf. That is, at SOrJW time during devclopmcnt of t,hr fourth leaf tjhc ,voungrar developing l~~avrs pruduce cithcr mow or-a more active propanil amidaw. It, uiuo uptake callus suspel,sim
of
r)roparlil
by
rice
actiuify
of
vice
twi
The fwzymr, was assaycAdin crude homogcwatrs of the tissue cultuw. Thc~ awrage amidasc activity A\-as found to h(, 14.4 units/mg protein. This is comparable to th(b activit,y of plants with fwwr t,han four Icavw and is consistent \\-ith th(> finding of I’rclar and Still (6) that riw rOcltS haw lrss amidaw activity than riw l(bavc>sin mont,h-old plants. ItI \vas of intrrwt then to dctt>rmine if th(> amidaw activity of rice tissw in cultuw, likt> t)hat of t)hc rice plant, would change \vith the agcl of the culture. Tho amidasc activit,y of the suspt>nsion culture \vas mr~asurcd at, various timw during the callus
g IJnit,s/rny wt proteirl
g). awidasr
susI~e~~.sio~r
cultures.
‘I
?od
cultwe. The kinct,ics of propanil uptake by rice root callus suspension cult,ures are shown in P’ig. 1. C:ulturc flasks contairwd 9.2 pmolw of propanil (2 mg) in 50 ml of culture medium and \I-cre inoculated at zero time with approximatrly :?.iOmg of tissur. Tht> amount of extract’ahlc propanil drcwasrd 1incJarly up to 16X hr and by 192 hr no propanil could be detected. 3,4-D(IA first appeared in the extract,s at 96 hr, reaching a peak of 2 pmolcs at approximately 1-H hr and decreased afterwards. Thr cfftct of propanil on t,issw growth is shown in I:ig. 2. The doubling time for the cultures grown in the presence of propanil increased to 48 hr as compared to
Time
b’ra.
(hr,)
1. c’ptake of propand from cl6ltz6re medil6tn by root calll6.5 suspension clrllures. Solid line: Propanil; dashed line: 3,4-DCA. Each point represents the a~rage of two $asks. Each jtask initially contained 9.2 ptncles of prOpand in 50 Nd of CdtlLre medium and was inoculated with abol6t 350 mg of suspension cc6lture at zero time. rice
PROPANIL
48
96 Time
144
192
METABOLISM
2,
(hours)
I~Iu. 2. The growth kinetics of rice callus suspension o~lturrs in thr presence of propmil. Initially 9 my of propanil were atdrlerl to 50 ml Gf cullwe medium at 2~1’0 /imp. The ordinate is a log plot of the ,jresh wright of the tissw. The abscissa represents hours a,fter inocrrlntiorl. The controls receivrrl no propnnil. Solid linr: with propnnil; dashed lint: control.
IN
175
RICE
Leaves five and six wrc found to contain higher amidase activity than leaves one t,hrough four. The same was true of t,hc immature unexpanded leaves of the fourleaf plant,. We concluded that the higher amidase activity of four-lraf plants \vas the result of higher amidaw activity in th(l unexpanded lravw. Incwaws in c’nzymcl activity may 1-wthe WSUh Of PllZylllC actiVatiOI1 Or de 1101’0 synthesis of the protein. Proof of cithrr of the altcrnativcs in regard to t,hP changw in propanil amidasc activity obscrwd in thcb rice plant requires purificatjion of thn mzynr. Our attempts to do so have not, met as yet with compl(+ succws. I’wliminary studies, ho\vwcr, using inhibitors of protrin synthesis suggwt that> the rise in amidase activity obwrvrd in riw tissw culture is the rrsultf of de UOZUI synthesis of t’hc c~nzymc~(12). The change in amidasc
growth cycle and the rosult,s arc’ sho\vn in I;ig. 3. Thr cnzymc n-as not detwt~ahlc until 132 hr. At 168 hr the activity row dramatically to about 13 units/mg protein and by 192 hr it dropped to 4 unit,s/mg protcki. C3bniparison of curve for activit) and growth show that the increase in amidasc activity of the t,issur in culture occurs \vh(bn the, wlls arc’ in stationaq pllwsc~.
The data pwsentcd show that) the propanil amidase activity in rice plants with four or more leaves is three tin-w greater than in plants with fcw-rr than four kavrs. The greater amidasc activity of four-leaf plants should he correlated with dcvclopmcnt of rwistancc toward propanil hy these plants. This would contradict the fichld studies \vhich show\-cdthat, plants in the third and fourth lraf stagw aho\wd grclatclr scnsitivit,y toward propanil tjoxicity than plants with only one or t\vo l~aws (1).
0
13~.
72
96 Time
120 (hrr)
144
168
3. The kinetics of appearance OJ” proptmil actiuity in outlr hornogpnatus o.f rice root callus suspension cdluws. Cirltrrrrs were inocdaletl at zero ti,me. Each point rrpresents the avrrag~ amidase acfivity from few jlasks qf ocltures. The abscissa represents lime in hours njler inoculation and ihe ordinafe, aniitlase uctivity in tanils per my protein. One unit of enzyme activit!y is the amount oJ rnzqmc’ requirefl! for lhr formafion of 1 nmolr oj 3, ~-ZX’iI prr hr. amidasP
3 76
RAY
AIYD
activity in the plant, occurs during the expansion of the fourth leaf and data prescnt,cd in this report show that high lcvrls of amidaw act,ivity aw prcwnt) in the fifth 1Paf hcforr it expands. Thr~ fact that this leaf is already prcwnt hPforc thcl expansion of the fourth indicat,cLs that the Iwcl of amidaw act,ivity incwaws in the fifth leaf during t,hc expansion of the fourth leaf. This suggests that, prcJpalii1 amidaw is under snmp typp of regulatory control in t#he rice plant. Use of a tissue cult>urr as a model for st,udying metabolism in plants requires that the two systems be comparablt~. Several findings support the USC of riw root callus suspcxnsion culturw as a mod(ll system for the study of propanil mc+abolism in the rice plant. First the dat,a from uptake cxxprrimcbnts (Fig. 1) indicate that thr tissue in cultuw drws take up propanil and mc~taholizc~ it, releasing 3,4-DCA. The amount of 3,4-DCA cxtrackd ~vas not ryuivalwt to the amount of propanil addcld on a moldybasis, suggwting further mctaholisrn of 3,4-DC’A. D(‘A mc~tabolism has hwn sho\vn to owur in th(> rice plant (2, 11). This is furth(,r support,cld by the fact that the amount of caxtractabl(k 3,4-DCA begins to dccrcase after 144 hr. Thr finding that, 3,4-DCYA first appclawd hctwecn 48 and 96 hr indicatw t,hat th(a tissue in culture ran mc~taholizc~ propanil i,l viva at this growth stage‘. III vitro prOpand amidase activit,y, howrwr, was undctectahle until aftcar 120 hr (l;ig. 2). Xo irl Z&XI propanil amidasr activity could be detected in cultures gro\vn in the prrsencc of propanil for 72 hr. ‘l’hrsc rwults suggest that \oung cultuws possc~samidasr activity below the d&ction limit of th(l i/c z!i~w assay. Albrrnativcly, the young tissue culture may possessinhibitors which art’ released in the (lnzymc preparation or the product (DCA) may hrcomt~ und(~tc~ct~ah!f: in the colorimctric assay. Secondly, the ability of both rice plants and tissw culture to mc>taholizc pmpanil
STILL
dclpcnds upon thr agcb of thck plant and on the age of cultuw (Table 1, Fig. 2). Thirdly, the hydrolysis of propunil in bckh the plant, and thcx rnodcl is cwzymic, with t,hc wtivitiw Ioc3lizcd in siniiktr particulatr fraction. 111 addition, the, (‘IizJ.tiic front both sourws is scwsit,iw to inhibition t,y cnrbamatc~ inwcticidw. WC, fwl thca similaritiw bclt\vccw propanil nit’tabolinm in the> riw plant and rice root callus SUS~~PllSi~Jll cultuws arc’ sufficicwt~ to justify tht> USC’ of the callus cultuw as a model for t,hcl study of propanil mc%abolism in t,h(> riw plant. WC \vill report in grclatw d(+ail lxtcir on other studies \vith the callus culturw. Th(w includr the mrtahfrlic fatct of 3,4DC‘A and the intrxcrllular ~IlcaliZatilJIl of propanil amidaw. ACKNO\VLEI)G\fEN’I
This work was supported by Ilegional I:esearc*h l<‘tmd NE-.?:<, United States IIepartment of Agricllltllre ; paper of the jollrnal series, Sew .Jrrsq Agricldt.lxral Il:xperiment StaGon. REFERENCE
i
I. I<. .J. Smith, :i,4-dichlol,opropiorlarlilide for (YDIItrol of barnyard grass in rice fields, Il-r&s 9, :318 (19B1). 2. (:. (:. WI, Metabolism of :3,4-dic!hloropropiollanilide in plants: metabolic fate of the 3,4dichloroaniline moiety, S(,i~nce 159, !I!)2 (1 YW). 3. (:. (;. St ill, Met,abolism of :3,4-dic,hloropt,opir)IIanilide in plants : metabolic* fate of 1he plopionic acid moiety, Plunk Physiol. 43, .i4:3 (lOfir(). 4. It. Y. Yih, I>. H. McItae, and H. E’. \Vilson, The mechanism of selective action of 3,4dichloropropionanilide, Plan{ Physiol. 43, 1291 (l%iX). .j. C. C. Still and 0. Iiuxirian, I:nzyme detoxification of 4,4-dichloropropinnanilide in rice and in herbicide selecbarnyard grass, a factor tivity, ~Yrcttrrr (/,ondm) 2 16, 799 (I !)ti7). ti. I). S. I:rear and (:. (+. Still, The metabolism of :j,4-dicl-lloloprcIpionsrlilide in plants. Partial pllrificntion and properties of an aryl avylan~idase from rive, Phg/och~n/islr!/ 7, !)I:1 (1968). 7. H. B. I&b, T. B. Ray and C. C. Still, (irowth of rice root callus suspension cultures, Plani Ph~ysiol. 51, 1140 (1973).
PROPANIL
S. IT. J. Pease, Separation monuron and diuron (‘hrrn. 10, 279 (1962). 9. 0. H. Lowry, 1<. H. I;olin (I!,.il).
phenol
and determination residues, J. iicrr.
N. J. Rosebrough,
Randall,
Prot,ein reagent,
METABOLISM
A. I,. Farr, measurements
.r. Hid.
C’h~nr.
of Food and with
193,
26.5
IN
RIC’E
Iii
10. Ii.
T&an, “Pinnt~ Anatomy,” p. KS, Wiley, Sew York, 1967. 11. Il. H. Tih, 1). H. McRae, and H. F. &%orr, Metabolism of 3,4-dichloropropionanilide : X,4dich!oroaniline lignin complex in rive plants, Science
72.
T.
161,
B. Ray preparati,,n.
376
and
(196X).
C.
C.
Still,
nranltscrir>t
in