The Effects of Victorin on Cells and Cell Fractions.

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Louisiana State University LSU Digital Commons LSU Historical Dissertations and eses Graduate School 1964 e Effects of Victorin on Cells and Cell Fractions. Homer Selton Black Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: hps://digitalcommons.lsu.edu/gradschool_disstheses is Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and eses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. Recommended Citation Black, Homer Selton, "e Effects of Victorin on Cells and Cell Fractions." (1964). LSU Historical Dissertations and eses. 934. hps://digitalcommons.lsu.edu/gradschool_disstheses/934

Transcript of The Effects of Victorin on Cells and Cell Fractions.

Page 1: The Effects of Victorin on Cells and Cell Fractions.

Louisiana State UniversityLSU Digital Commons

LSU Historical Dissertations and Theses Graduate School

1964

The Effects of Victorin on Cells and Cell Fractions.Homer Selton BlackLouisiana State University and Agricultural & Mechanical College

Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses

This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion inLSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please [email protected].

Recommended CitationBlack, Homer Selton, "The Effects of Victorin on Cells and Cell Fractions." (1964). LSU Historical Dissertations and Theses. 934.https://digitalcommons.lsu.edu/gradschool_disstheses/934

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This d issertation has been 64—13,247 m icrofilm ed exactly as received

BLACK, Homer Selton, 1935- THE EFFECTS OF VICTORIN ON CELLS AND CELL FRACTIONS.

Louisiana State U niversity, Ph.D ., 1964 Botany

University Microfilms, Inc., Ann Arbor, Michigan

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THE EFFECTS OF VICTORIN ON CELLS AND CELL FRACTIONS

A D isserta tion

Submitted to the G raduate Faculty of the Louisiana S tate U niversity and

A gricultural and M echanical C ollege in p artia l fulfillm ent of the

requirem ents for the degree of Doctor of Philosophy

in

The Department of Botany and P lant Pathology

byHomer Selton Black

B .S . , Texas A & M C o lleg e , 1956 M .E d ., Sam Houston S tate Teachers C o llege , 1960

M ay, 1964

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ACKNOWLEDGEMENTS

Sincere apprec ia tion is extended to D r. Harry E. W heeler for a d v ic e ,

helpful su g g e s tio n s , extrem e p a tie n c e , and critic ism w hich provided

the guidance and stim ulus for th is re se a rc h . A ppreciation is ex tended

to Dr. S . J . P . C h ilto n , Chairm an of th e Departm ent o f Botany and

P lant Pathology, who made fa c ilitie s a v a ila b le , to D r. Thomas P .

Pirone and D r. A. D. Larson for helpful su g g estio n s during the course

of th is re se a rc h , and to D r. John Baker and D r. I . L. Forbes for help

in th e preparation of th is m anuscrip t.

The suggestions and help of fellow graduate s tu d en ts are reco g ­

n ized and ap p rec ia ted by the w rite r .

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TABLE OF CONTENTS

P ag e

ACKNOWLEDGEMENTS.................................................................................... ii

LIST OF TABLES................................................................................................ iv

LIST OF FIGURES............................................................................................. vi

ABSTRACT............................................................................................................. v ii

INTRODUCTION................................................................................................ 1

REVIEW OF UTERATURE................................................................................. 2

MATERIALS AND METHODS........................................................................... 6

T is s u e ............................................................................................................. 6Victor in and V ic to rin - tre a tm e n t........................................................... 6Perm eability M e a s u re m e n ts ................................................................. 6R espiration M e a su re m e n ts .................................................................... 9P reparation of M itochondrial F r a c t io n s ............................................ 9Q uan tita tive A n a ly s i s .............................................................................. 10Enzyme A s s a y s .......................................................................................... 13

RESULTS................................................................................................................ 16

Perm eability M e a s u re m e n ts ................................................................. 16C hem ical A n a ly s i s .................................................................................... 36Effect of V ictorin on O xidation and P h o sp h o ry la tio n ................... 40

DISCUSSION AND CONCLUSIONS.............................................................. 46

SUMMARY............................................................................................................. 64

UTERATURE C IT E D .......................................................................................... 66

V IT A ...................................................................................................................... 69

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LIST OF TABLES

TABLE P age

1. E lectro ly te lo ss from naturally in fec ted t i s s u e ................. 17

2. P lasm olytic te s ts of a r tif ic ia lly inocu lated p la n ts . . . . 18

3 . Leaf Recrement: After 4 Hr uptake of 1:40 v ic to rinand w a te r .......................................................................................... 18

4 . Effect of high and low oxygen ten s io n s on e lec tro ly tere le a se of v ic to rin -trea ted t i s s u e s ......................................... 20

5. E lectro ly te re le a se and reaccum ulation by t is s u e .............. 21

6 . Effect of 2 , 4 -d in itropheno l on reaccum ulation byt i s s u e ................................................................................................ 24

7 . Effect of shaking on the resp ira to ry ra te of v ic to rin -trea ted t i s s u e s ................................................................................. 26

8 . R elease and reaccum ulation of e lec tro ly te by v ic to rin -trea ted m ito ch o n d ria .................................................................... 27

9 . R elease and reaccum ulation o f e lec tro ly te s bym itochondria from v ic to rin -trea te d t i s s u e s ......................... 30

10. Effect of v ic to rin on boiled m itochondria andm itochondria from re s is ta n t p l a n t s ......................................... 31

11. Effect of d ifferen t g a se s on m itochondrial re le a se andreaccum ulation of e le c tro ly te s .................................................. 33

12. Inh ib ition of m itochondrial e lec tro ly te reaccum ulationby 2 , 4 -d in itro p h en o l.................................................................... 34

13. Effect of sugar on re le a se and reaccum ulation ofe lec tro ly te s by m itochondria..................................................... 37

14. C o n d u c tiv ities of so lu tions in w hich v ic to rin -tre a te dtis s u e s w ere s h a k e n .................................................................... 38

15. P hosphory la tive-ox idative ra tio s of m itochondria ex trac ted in various concen tra tions of su cro se from v ic to rin -tre a te d t is s u e .

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TABLE P age

16. The effect of various concentrations of malonate on phosphory lative-oxidative ra tios of mitochondria from v ic to rin -trea ted t i s s u e ................................................................... 44

17. Effect of various concentrations of NaF on the phosphory lative-oxidative ra tio s of mitochondriafrom v ic to rin -trea ted t i s s u e .......................................................... 45

v

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LIST OF FIGURES

FIGURE P a g e

1. Effect of v ic to rin on m itochondrial su cc in ic dehydro­g en ase a c t i v i t y .................................................................................... 41

2 . E lectro ly te lo s s from natu ra lly in fec ted t i s s u e ................ 47

3 . Effect of high and lo*« oxygen ten s io n s on e lec tro ly telo ss from v ic to rin -trea ted t i s s u e ............................................ 48

4 . E lectro ly te re le a se and reaccum ulation by healthyt i s s u e ................................................................................................ 49

5 . Effect of 2 , 4 -d in itropheno l on t is s u e reaccum ulation . . 50

6 . R elease and reaccum ulation of e lec tro ly te s byv ic to rin -tre a te d m ito c h o n d ria .................................................. 52

7 . R elease and reaccum ulation of e lec tro ly te s bym itochondria from v ic to rin -trea te d t is s u e ............................ 54

8 . E ffects of v ic to rin on e lec tro ly te re le a se andreaccum ulation by m itochondria from re s is ta n t t i s s u e ................................................................................................ 55

9 . Inh ib ition of m itochondrial e lec tro ly te by 2 ,4 -d in itro p h en o l.................................................................................... 56

10. E lectro ly te re le a se by bo iled m itochondria .......................... 57

1 1 . Effect of high and low oxygen te n s io n s on e le c tro ­ly te lo s s and reaccum ulation by v ic to rin -trea ted m ito ch o n d ria .................................................................................... 58

12. Inh ib ition of m itochondrial e lec tro ly te reaccum ula­tio n by s u g a r .................................................................................... 60

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ABSTRACT

The effect of H elm inthosporlum v lc to rlae and v ic to rin on the

perm eability o f su sc ep tib le oat t is s u e s and th e e ffec ts of v ic to rin on

m itochondrial perm eab ility , ox ida tion , and phosphory lation w ere s tu d ie d .

Helm inthosporlum v lc to r la e . as w e ll a s v ic to rin , c a u sed changes

in perm eability of su scep tib le oa t t i s s u e s . V lctorln-induced perm eability

changes occurred under both high and low oxygen te n s io n s . Furtherm ore,

v ic to rin not only cau sed a more rap id lo s s of e lec tro ly te s th an th a t

w hich occurred in contro l t is s u e s but a lso preven ted th e reaccum ulation

of e lec tro ly te s w hich normally occurred .

Solu tions in w hich v ic to rin -trea ted t is s u e s had been shaken w ere

ana lyzed for to ta l n itrogen , ca rb o h y d ra tes , amino a c id s , p ro te in , in ­

organic and organic phosphorous, sodium , ca lc ium , and p o ta ss iu m .

After 15 m inutes of leach ing in d is t ille d w ate r trea ted t is s u e s lo s t

20 per cen t more nitrogen than co n tro ls . Treated t is s u e s lo s t two

tim es more inorganic phosphorous, n itrogen , ca rb o h y d ra tes , p ro te in ,

and po tassium a fte r four hours th an did c o n tro ls . The lo s s e s in c re ased

in m agnitude w ith tim e . No organic phosphorous w as d e tec ted in so lu ­

tio n s in w hich tre a ted or contro l t is s u e s w ere shaken and only sm all

d iffe rences in sodium and calcium w ere o b se rv ed .

V icto rin -trea ted m itochondria and m itochondria from v ic to rin -

tre a ted t i s s u e s , w hen com pared to co n tro ls , show ed no change in

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perm eability . S uccin ic dehydrogenase ac tiv ity of v ic to rin -trea te d m ito­

chondria w as sim ilar to th a t of co n tro ls . H ow ever, phosphory la tive-

ox idative ra tio s of m itochondria from v ic to rin -tre a te d t is s u e s w ere

sig n ifican tly low er than c o n tro ls . Rates of oxygen uptake betw een

m itochondria from trea ted and contro l t is s u e s w ere not s ig n ifican tly

d iffe ren t. These data in d ica te th a t in v ic to rin -trea te d t is s u e s e ith er

an in c reased breakdow n ra te of h igh-energy phosphate compounds

occurs or th a t th e effic iency of phosphorylative s te p s is red u ced .

Either c a se could account for the in c reased resp ira to ry ra te s of

v ic to rin -trea ted t i s s u e s .

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INTRODUCTION

During the p a s t d e c a d e , p lan t p a th o lo g is ts have g radually p la ced

more em phasis on in v e s tig a tio n s in to th e physio logy of d is e a s e d p lan t

t i s s u e s . V icto rin , th e sp e c if ic tox in produced by H elm inthosporlum

v lc to r la e . h as prom ised to be a v a lu ab le to o l for in v e s tig a tio n s of th is

n a tu re . Not only i s v ic to rin highly a c tiv e , s p e c if ic , and re sp o n s ib le

for a l l th e symptoms of th e d is e a s e (Victoria b lig h t of o a ts ) , but i t s

u se e lim in a tes any com plicating e ffec ts ca u se d by th e p re se n ce of th e

c a u s a l o rg an ism . In v e s tig a tio n s in to th e e ffec ts of v ic to rin have had

an im portant bearing in v a lid a tio n of th e to x in theory of p lan t d is e a s e .

In su sc e p tib le o a t t i s s u e s v ic to rin c a u se s b iochem ical changes

w h ich ap p ear to b e common to many p lan t d i s e a s e s . Forem ost among

th e s e are resp ira to ry and perm eab ility c h a n g e s . V icto rin -induced

resp ira to ry in c re a se s have b een s tu d ied but co n c lu s iv e ev id en ce of

th e natu re of such ch an g es is la c k in g . Perm eab ility in c re a se s have

a lso b een o b serv ed in th is syndrom e. The experim ents d esc rib ed h ere in

w ere d es ig n ed w ith th e purpose of gain ing an in s ig h t in to th e natu re o f

c e r ta in of th e s e b iochem ical ch an g es and to de term ine , if p o s s ib le ,

th e re la tio n sh ip of th e s e changes to one an o th e r.

1

%

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REVIEW OF UTERATURE

In 1927, the United S tates Department of Agriculture introduced a

variety of Avena bvzantlna from South America into the United S tates for

the purpose of breeding for res is tan ce to oat crown rust caused by

Puccinia coronata. This new varie ty , named V ictoria, exhibited a

hypersensitive type of res is tan ce to crown rust and consequently

derivatives from cro sses with other agronomic varie ties became widely

planted throughout the oat growing reg ions. However, in 1944, Meehan

and Murphy (21, 22) iso la ted a Helminthosoorium s p . from germ inator-

grown oat seed lings and the same year iso la tions were made from oats

in the field as w ell as from Timothy. In two years the spread of th is *

new d isease had atta ined epiphytotic proportion and had caused

much destruction and reduction in y ie ld s . Reactions of oat varie ties

to th is fungus were te s ted and i t w as found tha t varie ties possessing

"v ic toria-type" resis tan ce to crown rust were highly suscep tib le

w hereas varie ties lacking th is re s is tan ce were not (14, 23). Because

the fungus exhibited such specificity it w as given the name

Helminthosporlum vlctorlae (22),

When progeny from F2 populations of hybrids, each involving

one Victoria crow n-rust re s is tan t paren t, were te s ted against

Helminthosporlum v lc to rla e . the ra tio of suscep tib ility to res is tan ce

w as 3:1 . When te s ted against Puccinia coronata the ratio of resis tan ce

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to su scep tib ility w as 3 :1 . Murphy and M eehan (25) suggested tha t the

genes controlling V ictoria-type re s is tan ce to crown rust and su scep tib ility

to Helminthosporium vlc to rlae were linked . Because he could not

separa te h y p ersen sitiv en ess to Puccinia coronata and su scep tib ility to

Helminthosporium v ic to riae in c ro s s e s , Litzenberger (15) suggested the

p resence of a sing le gene w ith p le iotropic effects as the determ ining

factor for re s is tan c e and su scep tib ility . W heeler and Luke (38) d e ­

v ised a method of screening large populations of seed lings for J j.

v ic toriae re s is ta n c e . Using th is method Luke et a l . (17) were ab le to

obtain mutants w hich exhibited th e typ ica l V ictoria-type of re s is tan c e

to crown ru st as w ell a s being re s is tan t to H . v ic to ria e . These workers

concluded th a t th e gene tic re la tionsh ip betw een P . coronata re s is tan c e

and JLi v ic to riae su scep tib ility w as not contro lled by a sing le gene but

conditioned by a number of g e n e s .

Soon afte r iso la tio n and iden tifica tion of the new fungus it w as

postu la ted tha t a tox ic m aterial sec re ted by the fungus might be re ­

sponsib le for the c h a rac te ris tic yellow fo liar s trip es (23). M eehan and

Murphy w ere ab le to show th a t a toxin w as indeed responsib le and

its extrem e sp ec ific ity and high ac tiv ity w as dem onstrated by th e se

workers a s w ell as by L itzenberger (14). The la tte r postu lated th a t

su scep tib ility to H. v ic to riae a nd re s is ta n c e to P . coronata w as d e ­

pendent upon com parable types of reac tions induced by tox ins (15).

Luke and W heeler (16) devised a medium favorable for tox in

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production and w ere a b le to re la te to x in p roduction to p a th o g e n ic ity .

This along w ith i ts ex trem e sp e c if ic i ty and high a c tiv i ty in d ic a te d th a t

th e to x in w as p rim arily , if not s o le ly , re sp o n s ib le for th e sym ptom s of

th e d i s e a s e . P tin g le and Braun (29) c o n c en tra ted th e to x in and su g ­

g e s te d i t w a s com posed of tw o com pounds - one w h ich th e y nam ed

v lc to x ln ln e and a seco n d thought to be p o ly p ep tid e in n a tu re . They

su g g e s te d th a t v ic to x in ln e w as th e to x ic p r in c ip le . L a te r, h o w ev e r,

S cheffer and P ring le (31) co n c lu d ed th a t v ic to x in ln e p lay ed no part in

th e d is e a s e syndrom e. The s p e c if ic to x in , g iv en th e nam e v ic to rin

by Luke and W h ee le r (16), is ex trem ely u n s ta b le and a l l e ffo rts th u s

fa r to purify i t have f a i le d .

B ecau se v ic to rin ex h ib ited ex trem e s p e c if ic i ty , h igh a c t iv i ty ,

and p roduced a l l th e v is ib le sym ptom s of V icto ria b l ig h t , i t not on ly

le n t support to th e to x in theo ry of p lan t d is e a s e bu t p rom ised to be a

v a lu a b le to o l in th e stu d y of d is e a s e p h y sio lo g y and th e n a tu re of

r e s is ta n c e . In an effort to determ ine th e b a s is for r e s is ta n c e ,

Romanko (30) found no q u a n tita tiv e d iffe re n ce s in v ic to rin up take

or d iffe re n ce s in v ic to rin a c tiv i ty of re s id u a l so lu tio n s ex p o sed to

s u s c e p tib le and r e s is ta n t t i s s u e s . From th e s e d a ta h e co n c lu d ed th a t

re s is ta n t t i s s u e s in a c tiv a te d or bound th e to x in . O n th e b a s is of

h is to lo g ic a l s tu d ie s Paddock (26) su g g e s te d th a t r e s is ta n t t i s s u e s

form s u b s ta n c e s a n ta g o n is tic to th e fu n g u s . H ow ever, he o ffe rs no

ex p la n a tio n a s to th e natu re o f r e s is ta n c e to th e to x in .

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A sid e from p ro d u c in g a l l th e v is ib le sym ptom s of V ic to ria b l ig h t ,

v ic to r in p ro d u c e s a num ber of b io c h e m ic a l sym ptom s com m on to th e

d i s e a s e . Rom anko (30) found th a t v ic to r in c a u s e d in c r e a s e d r e s p i r a ­

t io n in s u s c e p t ib le t i s s u e s . Krupka (11) co n firm ed R om anko 's f in d in g s

an d in v e s t ig a te d th e n a tu re o f th e e le v a te d r e s p ir a t io n . H is d a ta s u g ­

g e s te d th a t v ic to r in - in d u c e d re s p ir a t io n w a s m e d ia te d th ro u g h a NaF -

s e n s i t iv e p a th w ay a n d th a t th e h e x o s e -m o n o p h o sp h a te p a th w a y w a s

not in v o lv e d . W hen h o m o g en a te s w e re ex am in ed fo r p o s s ib le c h a n g e s ,

in c re a s e d r a te s o f a s c o rb a te o x id a tio n w e re d e te c te d (1 0 ). No d if fe r ­

e n c e s w e re found in K reb 's c y c le o x id a t io n s . The n a tu re of v ic to r in -

in d u c e d r e s p ir a t io n i s s t i l l u n c e r ta in .

S in c e T h a tc h e r (32) f ir s t in v e s t ig a te d th e o sm o tic a n d p e rm e a b ili ty

r e la t io n s h ip s o f c e r ta in h o s t - p a r a s l te a s s o c ia t io n s in 1939 , v e ry l i t t l e

in te r e s t h a s b e e n sh o w n in th e ro le o f p e rm e a b ili ty in p a th o g e n e s is .

H o w ev er, v a r io u s o th e r to x in s , p a r t ic u la r ly ly c o m ara sm in and fu s a r ic

a c id , h a v e b e e n im p lic a te d in p e rm e a b ili ty c h a n g e s o f th e h o s t ( 6 , 9 ,

2 7 , 3 9 ) . In 1961 W h e e le r (35) re p o rte d th a t v ic to r in in d u c e d p e r ­

m e a b ility c h a n g e s in s u s c e p t ib le o a t t i s s u e s . As w a s th e c a s e w ith

o th e r e f f e c ts p ro d u ced by v ic to r in , th e p e rm e a b ili ty c h a n g e s w e re

s p e c if ic for s u s c e p t ib le t i s s u e s . B lack an d W h e e le r (3 , 4) s u g g e s te d

a p o s s ib le r e la t io n s h ip b e tw e e n v ic to r in - in d u c e d r e s p ir a t io n a n d p e r ­

m e a b ility c h a n g e s . W h ile no d ir e c t e v id e n c e h a s b e e n fo rthcom ing

w h ich d e f in e s th e m ech an ism (s) o f p e rm e a b ili ty c h a n g e s , c e r ta in ly th e

im p o rta n c e o f th e s e c h a n g e s in d i s e a s e d t i s s u e sh o u ld n o t b e o v e r lo o k e d .

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MATERIALS AND METHODS

The princip le experim ental methods involved perm eability t e s t s ,

m anometric te ch n iq u e s , and co lo rim etric or spectrophotom etric p ro­

cedures .

T issue

O ats w ere the source of a l l p lant t i s s u e . V ictorgrain 48-93 w as

the source of su scep tib le t is s u e w ith Camellia and a re s is ta n t Victor­

grain m utant (CI-7418) serv ing as sources of re s is ta n t t i s s u e . In the

labora to ry , oat seed w ere soaked 30 m inutes in tap \ w a te r, p laced on

moist paper tow els in 6 -inch petri p la te s , and germ inated in an in cu ­

bator at 27°C . In the g reen h o u se , th e seed w ere p lan ted under 1/4

inch of so il in 8-inch c lay p o ts .

V ictorin and v ic to rln -trea tm en t

C ulture f iltra te s contain ing v ic to rin w ere ob tained by the methods

prev iously d escrib ed by Luke and W heeler (16). W hen t is s u e w as p re ­

tre a te d , the stem s w ere p laced in appropriate d ilu tions o f v ic to rin or

contro l so lu tions and allow ed an illum inated 2 -hour uptake period 1- f t .

from a fro sted 60-w att b u lb . O therw ise the appropriate d ilu tio n s of

v ic to rin w ere app lied d irec tly to the t is s u e or m itochondria.

Perm eability m easurem ents

For e lec tro ly tic conductiv ity m easurem ents, t is s u e w as w rapped

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in cheeseclo th bags bound with rubber b an d s . The bags containing

trea ted and control t is s u e w ere rinsed together in d is tille d w ater to

bring them to the same conductiv ity . They w ere then p laced in 500 ml

flasks containing 100 ml of d is tille d w ater or the appropriate so lu tion .

The flask s were shaken at the rate of 120 strokes per minute w ith a

ho rizon ta l-stroke m echanical sh ak er. At designated time in tervals

a liquo ts w ere removed from the flask s and the conductiv ity m easured

with a W heatstone c ircu it conductiv ity b ridge. The aliquo ts w ere then

returned to th e ir respective flask s on the sh ak er. In the experim ents

involving m itochondria, 5 ml of the final m itochondrial suspension

w as added to the flask s containing the appropriate so lu tio n s . W hen

the effect of g a se s on e lec tro ly te lo ss and reaccum ulation w as stud ied ,

the flask s were c lo sed with a stopper p o ssess in g an in le t-o u tle t v a lv e ,

evacuated w ith a vacuum pump, and filled w ith the gas to be s tu d ied .

The controls were prepared in the same manner except after being

evacuated the in le t valve w as opened to the atm osphere. All g la s s ­

ware used in th e se experim ents w as se ria lly rinsed s ix tim es w ith

d is tilled w a te r. Sodium chloride w as used to ad ju st co n d u c tiv itie s .

Exceptions to th e se procedures are noted in the tab les or d iscu ssed

in r e s u l t s .

Plasm olytic te s ts were made using the methods of Thatcher (32)

and M achlis and Torrey (20). P lan ts , grown in the g reenhouse, were

inoculated afte r 25 days w ith a suspension of Helminthosporium

Page 18: The Effects of Victorin on Cells and Cell Fractions.

8

vic to riae sp o res . The spore suspension w as prepared by w ashing the

surface of petri d ish cu ltu res with s te rilized d is tille d w ate r, filtering

through a th in cotton filte r, and w ashing five tim es by centrifuging

and resuspend ing . The w ashing removed any v ic torin w hich might

have been present on the sp o re s . The final spore suspension con ­

ta ined about 70 spores per low power f ie ld . Inoculations were made

w ith a hypodermic sy ringe. W ater w as in jec ted in to control p la n ts .

Leaves were harvested after symptoms had appeared w hich w as usually

after four d ay s . Shallow cu ts were made across the leaf surface with

a sharp razor b lade and th e epiderm al layer stripped o ff . When a

number of su ffic ien tly th in sec tio n s had been obtained they were

imm ersed in graded se rie s of sucrose so lu tio n s. After 30 minutes

the s trip s were examined m icroscopically and the so lu tion in which

50% of the c e lls were plasm olyzed w as considered iso to n ic . The ce ll

osm otic p ressure w as determ ined by the following formula: Osm otic

p ressure (Atms.) - ^ where M = concentration o f the ex ­

te rna l so lu tion at inc ip ien t p lasm olysls and T = abso lu te tem perature.

Once the osm otic p ressure had been determ ined, fresh sec tio n s were

plasm olyzed in a sucrose so lu tion tw ice the iso to n ic concen tra tion .

After 30 minutes the t is s u e s trip s w ere transferred to a so lu tion 1/2

th a t of the iso to n ic concentration .and the tim e (t) required for 50%

deplasm olysis determ ined. The abso lu te perm eability to the so lu te

w as determ ined by the following formula: Ps » d /4 t where d * average

c e ll diam eter and t - tim e required for d ep lasm olysis .

Page 19: The Effects of Victorin on Cells and Cell Fractions.

Leaf recrem ent w as m easured by a m odification of G aum ann's

method (9). Leaf t is s u e w as cu t and w ashed thoroughly in d is tille d

w ate r. The stem s were p laced in d is tilled w ater or a 1:40 dilu tion of

v ictorin and allow ed a 4-hour uptake period . After the treatm ent

period, 200 mg of the t is s u e th a t had not come in con tact w ith victorin

or w ater w as cut and p laced in 100 ml of d is tille d w ater of known con­

ductiv ity in 500 ml f la sk s . The flask s were shaken for 10 minutes

afte r which the conductiv ity w as m easured.

R espiration measurem ents

Standard manometric procedures w ere employed to m easure ra tes

of oxygen uptake (33). R espiration ra tes of tis su e were expressed on

a fresh weight b a s is . M itochondrial resp iratory ra tes w ere either ex ­

p ressed as to ta l oxygen uptake per flask or on a to ta l nitrogen b a s is .

All resp iratory stud ies were conducted a t 25°C .

Preparation of m itochondrial fractions

E tiolated leaf t is s u e w as rinsed in d is tilled w ate r, cut in to 1/2

inch segm ents, and ch illed for 20 m inutes. The tis s u e w as then p laced

in a Servall O m ni-m ixer homogenizing chamber containing a volume of

extracting media equal to tw ice the w eight of the t i s s u e . The ex trac ­

tion media com position is noted in the ta b le s or re s u lts . During

hom ogenization the homogenizing chamber w as p laced in an ice b a th .

If the mitochondria w ere being prepared for conductiv ity te s ts the tis su e

Page 20: The Effects of Victorin on Cells and Cell Fractions.

10

w as homogenized three minutes at a pow erstat se tting of 60 . O therw ise

tis su e w as homogenized for one m inute. The homogenate w as strained

through two layers of cheeseclo th and centrifuged for 10 minutes a t

500 x G in a Servall refrigerated centriiuge. The resu lting supernatant

w as centrifuged for 15 minutes at 10,000 x G . The p e lle t , containing

the m itochondria, w as resuspended either in buffer or w ater with a g la ss

p es tle and recentrifuged at 10,000 x G for 15 m inutes. The resulting

pe lle t w as resuspended in a Potter-Elvehjem tis su e hom ogenizer and

made up to the desired final volum e. A11 m anipulations w ere conducted

a t 0- 2° C . using ch illed ap p a ra tu s .

Q uantita tive an a ly sis

Total nitrogen w as determ ined either by N esserliza tio n or the

M icro-Kjedahl method. M itochondrial sam ples th a t w ere to be analyzed

by M icro-Kjedahl were dilu ted 10-fold and d igested over a g as flame

under a hood. The d igestion mixture for M icro-Kjedahl co n s is ted of

500 ml of w a te r, 500 ml of concentrated H2SO4 , and 75 grams of

Na^SO^. Eight ml of th is mixture w as added to Kjedahl flasks co n ­

tain ing the dilu ted sam ple. One Hengar granule w as added and the

sam ple d igested until c le a r . It w as then cooled and 25 ml of d is tille d

w ater added. Upon d is tilla tio n the sample w as co llec ted in 10 ml

of 2% boric a c id , using methyl red ind ica to r, and back titra ted w ith

H2SO4 of known norm ality. For N esserliza tio n 1 ml of the tis s u e

bathing solutions w as d igested w ith an equal volume of 2N H2SO4

Page 21: The Effects of Victorin on Cells and Cell Fractions.

11

w hich contained 0 .2 grams of CuSe03 per l i te r . After d igestion 2 ml

of w ater and 5 ml of N ess le r 's reagent w as added to each sam ple.

The mixture w as allow ed to stand 15 minutes for color developm ent

and then transm ittancy w as determ ined at 490 mu with a Beckman

Spectronic 20 colorim eter. A standard curve w as prepared by adding

N e ss le r 's reagent to various dilu tions of ammonium chloride (0.3820

grams o f ammonium chloride d ilu ted to one lite r in w ater contains

0 .1 mg of nitrogen per m l). N ess le r 's reagent w as prepared in the

following manner: Solution A w as prepared by adding 100 grams of

mercuric iodide and 70 grams of potassium iodide to 400 ml of w a te r.

Solution B w as prepared by adding 100 grams of NaOH to 500 ml of

w ate r. Solution A and Solution B w ere mixed and made to a final

volume of one l i te r . After a precip itan t had formed the supernatant

w as poured off for u s e .

Total carbohydrates w ere determ ined by th e anthrone t e s t .

Anthrone so lu tion w as prepared by d isso lv ing 2 grams of reagent grade

anthrone in one lite r of 95% H2S 0 4 . Three ml of the te s t sam ple were

mixed w ith 6 ml of reagent in matched te s t tu b e s . The tubes w ere

p laced in a boiling w ater bath for three m inu tes, co o led , and the

color m easured a t 620 mu. A standard curve w as prepared from

standards containing 0 , 12, 60, and 120 micrograms of g lu c o se .

Inorganic phosphorous w as determ ined by the method of F iske

and Subbarow (8) a s modified in Bausch and Lomb's c lin ica l m anual.

Page 22: The Effects of Victorin on Cells and Cell Fractions.

12

One ml of th e t i s s u e bath ing so lu tio n w as d ilu ted 10-fold w ith d is t i l le d

w a te r . Two ml of 30% tr ic h lo ro a c e tic a c id w as ad d ed , m ixed, and

a llow ed to s tan d for 5 m inutes a f te r w hich it w as f ilte re d and 8 ml of

th e f iltra te rem oved to ano ther tu b e . One ml of m olybdate re a g e n t,

0 .4 ml of 1 ,2 ,4 -am in o n ap th o l-su lfo n ic a c id , and 0 .6 ml of d is t i l le d

w a te r w ere added to th e f iltra te and a llow ed to s tand for 6 m inutes

befo re m easuring co lo r in te n s ity a t 700 mu.

In order to m easure to ta l phosphorous* bath ing so lu tio n sam ples

w ere d ig e s te d w ith 0 .4 ml of 10 NH2SO4 in an oven a t 160°C . O ne drop

of 30% hydrogen peroxide w as added to com plete th e d ig e s tio n . The

sam ples w ere coo led and determ inations made for ino rgan ic p h o spho rous.

O rganic phosphorous w as ta k e n a s th e d iffe rence betw een to ta l and in ­

organic ph o sp h o ro u s.

Amino a c id s and p ep tid es w ere determ ined by th e u ltrav io le t

copper s a lt m icrom ethod d e sc rib ed by C olow ick and Kaplan (7). Sodium

bo ra te b u ffe r, pH 9 .1 to 9 .2 , w as p repared by d isso lv in g 4 0 .3 grams

of anhydrous N a2B4 0 7 in 4 l i te r s of w a te r and f ilte r in g . Six gram s o f

N aC l w as d is so lv e d in 100 ml of th e f i l t r a te . F ive ml of th e te s t s o lu ­

tio n w as added to 5 ml of buffer in a cen trifu g e tu b e . O n e -ten th ml of

a 0 .05M C u C l2 so lu tio n w as ad d ed , th e tu b e s to p p e re d , sh a k e n , and

allow ed to s tan d for 10 m in u te s . The su sp e n s io n w as th e n cen trifu g ed

for 8 m inutes in an In te rn a tio n a l c l in ic a l c e n tr ifu g e . The su p e rn a tan t

w as poured off in to a cu v e tte and tran sm itta n cy m easured a t 230 mu

Page 23: The Effects of Victorin on Cells and Cell Fractions.

13

in a Beckman DB spectrophotom eter. A ca lib ra tion curve w as prepared

using 3 1 .2 5 , 6 2 .5 0 , 125, 250, and 500 uM alan ine so lu tio n s . The

blank co n s is ted of 5 ml of the te s t so lu tion and 5 ml of buffer.

Protein w as estim ated by an u ltrav io le t absorption method (7).

Bathing so lu tion sam ples w ere p laced in cuvettes and the op tica l density

m easured a t 260 and 280 mu. The following formula w as used to estim ate

protein: Protein (mg/ml) * 1.55D2Q0- ®*^®^260.

Sodium, po tassium , and calcium w ere determ ined by flame pho­

tom etric m ethods. Some leach a te sam ples w ere passed through a 2 cm

column of Dowex 2-X8 anion exchanger before being an a ly zed . Samples

w ere p laced in 5 -ml beakers and atom ized with a Beckman DB flame

photom etric apparatus using an oxygen-acety lene flame of about 1 1/2

inch heigh t. Sodium w as determ ined a t 589 mu w ith a s l i t w idth of

0 .05 mm,' potassium a t 767 mu and s lit w idth of 0 .2 0 mm; and calcium

at 423 mu and a 0 .05 mm s li t w id th . Fuel se ttin g s of 15 psi of oxygen

and 3 psi of acety lene were used for a ll t e s t s . Q uan tities of ca tio n

w ere computed from standard c u rv e s .

Enzvme a s sa y s

Succin ic dehydrogenase w as determ ined by the method of Price

and Thlmann (28). One and on e-h a lf grams of e tio la ted 3 day o ld ep ico ty ls

w as Immersed in 1:20 d ilu tions of v ic torin for 2 hou rs. They w ere then

m acerated in a ch illed mortar in 3 ml of buffer co n sistin g o f 0^2M sucrose

and 0.02M phosphate at pH 7 .0 . The m acerate w as made up to 5 ml

Page 24: The Effects of Victorin on Cells and Cell Fractions.

with buffer and strained through four lay ers of ch e esec lo th . After frac ­

tional centrifugation the m itochondrial p e lle t w as resuspended in buffer

to a volume of 5 .0 ml. O ne-half ml of th e m itochondrial su spension w as

added to th e side arms of Thunberg tu b e s . The main chamber contained

final concentrations of the following: 0.25M su cc in a te , 0.5M phosphate

a t pH 6 . 8 , 0 .2 mg of g e la tin per ml, 1 x 10"4 M 2 , 6 -dichlorophenolendo-

phenol, and w ater to a final volume of 4 .0 ml. The tubes w ere evacuated

in an ice w ater b a th . They were then equilib rated 5 minutes at 30°G after

w hich th e mitochondrial suspension w as tipped in to the main cham ber.

Transm ittancy w as determ ined every 5 m inutes in a Spectronic 20

colorim eter a t 640 mu.

Q ualitative te s ts for succin ic dehydrogenase were made by a h is to -

chem ical method sim ilar to that of Avers (2). Seedlings which had d e­

veloped primary roots of about 5 mm length w ere p laced in 1:100 d ilu tions

of v ictorin or in w ater in petri d ish es and allow ed to rem ain for four or

eight h o u rs . The seed lings were then p laced in a reac tion mixture con­

taining equal parts of 0 .1M phosphate buffer pH 7 .3 , 0 .2M sodium

su c c in a te , 0.1% blue te trazo lium , and d is tille d w ate r. They were

allow ed to incubate for 1 1/2 hours a t 37°C . C ontrols w ere run w ith no

substra te to determ ine endogenous r a te s . After incubation squash mounts

of the root tip were made and exam ined m icroscop ica lly .

P hosphorylative-oxidative ra tio s w ere determ ined by the method

of Bonner and M illerd (5). M itochondria w ere ex tracted from tis su e

Page 25: The Effects of Victorin on Cells and Cell Fractions.

15

w hich had been p re trea ted w ith w ater or 1:50 d ilu tions of v ic to rin . After

d iffe ren tia l cen trifugation 0 .5 ml of the m itochondrial p reparation w as

added to 2 .5 ml of th e reac tion mixture in th e main cham ber of ch illed

W arburg f la s k s . The fla sk s w ere equ ilib ra ted 5 m inutes a t 25°C and

oxygen uptake determ ined m anom etrically . C ontrol f la sk s w ere e ith e r

rem oved at zero tim e and the reac tio n h a lted by th e add ition of co ld 1M

trich lo ro ace tic ac id or the su b stra te w as e lim inated from the reac tio n

m ixture. In th e la tte r c a se both contro l and experim ental f la sk s w ere

removed after 30 m inutes and th e reac tio n s h a lted by removing 1 ml

a liquo ts from th e fla sk s and mixing them w ith an equal volume of cold

1M trich lo ro ace tic a c id . T he mixture w as then cen trifuged at 9 ,5 0 0 x

G for 15 m inu tes. The supernatan t w as d ilu ted 3 -fo ld and determ ina­

tio n s made for inorganic p h o sp h a te . R atios w ere com puted by dividing

m icrom oles of phosphate e s te r if ied by m icroatoms of oxygen tak en up .

Extracting media for the m itochondria usua lly c o n s is te d of 0 .6M sucrose

and 0 .01M phosphate a t pH 7 .2 . All m anipulations w ere conducted at

0 -2 °C . The reac tio n mixture w as com posed of th e following:

(Final concen tra tions b ased on a 3 ml volume)

0 .2M su cro se 0 .0 1M p h o sp h a te , pH 7 .1 0 .02M a lp h a -k e to g lu ta ra te

0 .1M g lucose 0 .01M NaF 1 mg Coenzym e 1

10 ad en o sin e triphospha te 1 mg H exokinase

10~3M MgSC4 0 .01M M alonate

E xceptions to the ex trac tion and reac tio n media are noted in th e t a b le s .

Page 26: The Effects of Victorin on Cells and Cell Fractions.

RESULTS

I . Perm eability M easurem ents

As v ic to rin w as known to cau se in c reased perm eability of s u s ­

cep tib le t is s u e s it becam e n ecessa ry to determ ine w hether the fungus

w ould a lso produce such ch a n g es . P lan ts w ere grown in the laboratory

from re s is ta n t seed and in fes ted and c lean su scep tib le seed un til about

12 days w hen symptoms ap p eared . One gram lo ts of le a f t is s u e were

h a rv e s ted , p laced in d is tille d w a te r, and conductiv ity te s ts made of

th e bathing so lu tio n s . As shown in Tables 1-1A the co n d u c tiv itie s of

th e so lu tions from d ise a se d tis su e , w ere about 70% g rea te r a fte r 8 hours

th an th o se from hea lthy and re s is ta n t t i s s u e s . This in c re a se in d ic a te s

th a t the fungus a lso c a u se s perm eability changes w hich are sp ec ific

for su sc ep tib le t i s s u e .

P lasm oly tic te s ts were made to further a sc e r ta in w hether the fungus

w as ab le to c a u se perm eability c h a n g e s . A su sp en sio n ^ Helm inthosporium

v lc to rlae spo res w as ino cu la ted in to th e stem s of p lan ts grown in the

g reen h o u se . After symptoms appeared the le av es w ere h arv ested and the

te s ts m ade. Table 2 show s th a t th e osm otic p ressu re of d ise a se d p lan ts

d e c re a se d w hereas ab so lu te perm eability to th e te s te d so lu te In creased

by 215%. Thus th e fungus cau sed a consid erab le in c re a se in p e r­

m eability of h o st t is s u e w hich w as far removed from th e s ite of in o cu la ­

tio n .

16

Page 27: The Effects of Victorin on Cells and Cell Fractions.

17

Table 1. Electrolyte loss from naturally Infected tissue .^ (MHOS x 10®)

_ _ 15 Min 1 Hr_ 2 Hr 4 Hr 6 Hr 6 HrRes, Res Res Res Res Res

Ex p - p k Why PIS g k ffthy p is • gk Hf t v Pis g k Hthy p is g k ffitaL P is g k Hthy Pis

. 9 4 6 1 . 0 0 6 1 . 0 2 6 1 . 0 8 9 1 . 1 1 1 1 . 1 7 9 1 , 15 6 1 . 15 6 1 . 3 0 4 1 , 2 2 8 1 . 2 0 4 1 . 4 4 3 1 . 2 5 3 1 . 2 5 3 1 , 70 2

11 . 1 1 1 1 . 1 1 1 1 . 0 6 7 1 , 2 5 3 1 . 2 0 4 1 . 3 3 1 1 . 30 4 1, 253 1 . 5 0 3 1 , 3 8 6 1 . 3 0 4 1 . 7 0 2 1 . 4 73 1 . 3 6 3 2 , 0 1 9

I1 . 0 4 1 1 . 0 4 8 1 . 0 3 9 1 . 1 7 4 1 , 1 4 9 1 . 2 5 4 1 . 23 8 1 . 1 9 6 1 . 4 1 7 1 , 3 1 5 1 . 2 4 5 1 . 5 9 3 1 , 3 6 3 1 . 2 9 8 1 . 8 8 5

. 559 . 572 . . 7 2 6 . 667 . 661 ,946 . 756 .756 1 , 0 8 9 , 821 . 8 2 1 1 . 2 2 8 , 873 . 873 1 . 3 0 4 . 909 . 909 1 , 41 4

. 741 . 667 1 . 0 6 7 . 891 . 756 1 . 7 3 8 . 965 . 821 2 , 2 5 6 1 . 0 4 6 . 8 7 3 2 . 9 2 9 1 . 0 6 9 . 927 3 . 5 1 9 1 . 13 3 , 965 4 . 1 8 0I

. 6 56 . 6 2 1 . 845 . 788 . 731 1 . 2 2 3 . 871 , 799 1 , 5 0 0 . 9 4 4 . 8 5 6 1 . 8 4 0 . 9 7 6 . 9 0 3 2 . 1 0 9 1 , 0 2 3 . 9 4 0 2 . 4 0 9

. 488 . 535 . 891 , 572 . 639 1 . 1 5 6 . 598 .667 1 . 3 3 1 . 639 . 71 0 1 . 5 3 4 . 667 . 7 5 6 1 . 6 6 7 . 681 , 772 1 , 7 3 8

. 547 . 625 1 . 1 1 1 . 639 , 726 1 . 5 9 9 , 667 . 772 2 . 0 1 9 . 7 1 0 . 805 2 . 5 9 3 . 741 . 8 3 8 3 . 0 1 0 , 756 . 873 3 . 3 3 4tJ

. 515 . 565 1 . 0 0 3 . 603 . 668 1 , 35 3 . 635 . 702 1 . 6 1 8 . 667 . 742 1 , 9 4 3 . 701 . 783 2 . 1 6 9 . 716 , 606 2 , 3 6 4

^Upper numbers represent the range of the conductivity values whereas the lower number represents the mean.

Table LA. Electrolyte loss from naturally infected tissu e .

Release Curve Slope x 10® Reaccumulation Curve Slopes Res Res

Exp. Ck Hthy P is_ _ _ _ _ _ _ _ Qk_ _ _ Hthy Pis

1 18.7 14.5 84.9

2 47.4 41.2 201.8

3 25.9 31.1 175.6

Page 28: The Effects of Victorin on Cells and Cell Fractions.

18

Table 2 . P lasm oly tlc t e s t s o f a r tif ic ia lly in o cu la ted p la n ts .

O sm otic Solu te_____________________ P ressu re___________ Iso to n lc itv ________ P erm eability

atm s M

H ealthy 5 .2 2 0 .2 1 .475

D ise a se d 4 .5 1 0 .19 1 .028

Further experim en ts w ere conducted to determ ine w hether v ic to rin

w ould c a u se perm eab ility changes to th e ex ten t th a t In c reased am ounts

of m a te ria ls w ould be lo s t from th e c e l ls and ca rried w ith th e tra n sp ira ­

tio n stream to th e ou te r le a f su rface s w here they cou ld be d e te c te d .

T issu e w as p re trea ted and se c tio n s w ere h a rv es ted from a re a s w hich had

not come in co n tac t w ith trea tm en t so lu tio n s . T hese se c tio n s w ere

p laced in d is t i l le d w a te r and a f te r being sh ak en for a short p e rio d , th e

co n d u c tiv ity of th e bath ing so lu tio n s w as m easu red . As show n in

Table 3 the co n d u c tiv ity of so lu tio n s w hich co n ta in ed v ic to rin - tre a te d

t i s s u e w as 38% above th a t of th e c o n tro ls . The re le a s e curve slope

of th e tre a te d t is s u e w as nearly tw ice th a t of th e c o n tro ls . Thus v ic to rin

is ab le to affec t th e lo s s of e le c tro ly te s from t i s s u e through the t r a n s ­

p ira tio n s tream .

Table 3 . Leaf Recrem ent: After 4 Hr up take of 1:40 v ic to rin and w a te r .

MHOS x 105 R elease curve sloDe x 103

T est w ater .244

C ontro l .361 11.7

T reated .454 2 1 .0

Page 29: The Effects of Victorin on Cells and Cell Fractions.

To learn more of the nature of the perm eability increase caused by

v ictorin , the effect of varied oxygen tensions w as Investiga ted . Con­

ductivity te s ts were made in the presence of a ir , oxygen, and nitrogen.

T issue w as shaken in 1000-fold dilutions of v ic to rin . Experiments 3 and

4 of Table. 4 were separate control experim ents using deactivated victorin

and d is tilled w ate r. As shown in experiments 1 and 2 of Table 4 , victorin

produced its characte ristic effect in an atm osphere of a ir . However,

high or low oxygen tensions seemed to produce no effect on the pattern

of e lectro ly te lo s s . This ind icates that the lo ss of electro ly tes from

treated and d iseased tis su e is a passive process and not dependent upon

oxidative p ro c e s se s .

The pattern of natural electrolyte reaccum ulation by healthy oat

tis su e was stud ied . Conductivity te s ts were made of the bathing so lu ­

tions of healthy t is s u e . In every case except experiment 7 of

Tables 5-5A there occurred a re lease of electro ly te followed b y Jre -

accam ulation. Reaccumulation usually began after about 6 hours . In

some ca ses it occurred as early as 4 hours and as la te as 10 hours.

Thus it would appear tha t victorin not only cau ses an increased magni­

tude of e lectro ly te lo ss but a lso prevents the reaccum ulation of e lec tro ­

ly te .

As the inh ib itor, 2 ,4 -d in itrophenol, is known to in terfere with the

sa lt accum ulation of storage tis su e s it w as decided to study i ts effect

on the reaccum ulation of e lectro ly tes by oat t is s u e s . Conductivity te s ts

Page 30: The Effects of Victorin on Cells and Cell Fractions.

20

Table 4 . Effect of high .and low oxygen ten sio n s on e lec tro ly te re le a se of v ic to rin -trea ted t is s u e s . * (MHOS x 10$)

Ex d . O ther Variables Starting Conductivitv 8 Hr

Air ° 2 n 2

1 T issue shaken in 1000-fold dilu tion of toxin under a ir, Og or N2 for 8 Hr

1.253

3.2463.617

3.399

3.8274.444

4.109

3.5194.057

3.839

2

1.633

5.2396.058

5.512

4 .7365.239

5.077

3 .9404.289

4.056

DT H20 DT h 2o

3 T issue shaken in 1000-fold dilu tion of deactiva ted v ic torin and in d is tille d H20

1.738 .891 2.6552.721

2.677

1.1791.386

1.273

h 2o Tox h 2o Tox

4 T issue shaken in 1000-fold d ilu tion of v ic to rin and in d is tille d H2O

1.026 .9093.5193 .940

3.762

6.0586.296

6.137

l Upper numbers rep resen t the range of the conductiv ity values w hereas th e lower number rep resen ts the m ean.

Page 31: The Effects of Victorin on Cells and Cell Fractions.

21

Table 5 . E lectrolyte re le a se and reaccum ulation by t is s u e .* (MHOS x 10^)

Other 15 HoursExd . Variables Min 1 2 4 6 8 10 12

5 Gm lo ts 1.633 2.157 2.593 3.617 3.940 3.827 3.827d t t is su e 1.813 2.473 2.929 4.308 4.586 4.895 4.736

1 1:1000deactiva ted 1.697 2.264 2.735 3.929 4.323 4.376 4.285victorin

10 Gm lo ts 1.331 1.933 2.473 3.334 3.827 3.081 2.929 2.768of tis su e 1.599 2.419 3.246 4.444 4.444 4 .180 4.057 3.940

2 h 2o1.507 2.205 2.819 3.754 4.115 3.665 3.529 3.403

11 day old 1.026 1.414 1.975 2.109 1.633 1.304 1.006tis su e 1.228 1.702 2.419 2.473 1.975 1.534 1.179

3 h 2o1.145 1.597 2.213 2.303 1.796 1.406 1.096

12 day old .873 1.156 1.386 1.738 1.778 1.566 1.386 1.228tis su e 1.204 1.566 1.892 2.419 2.532 2.205 2.019 1.933

4 h 2°m§ 1.024 1.382 1.679 2.171 2.228 1.945 1.744 1.633

11 day old .726 1.111 1.534 1.738 1.363 1.228 1.133 1.089tis su e 1.278 2.362 3.617 5.062 4.895 3.719 3.081 2.721

5 h 2o.881 1.509 2.124 2.745 2.507 2.082 1.796 1.606

12 day old .772 1.204 1.386 1.599 1.813 1.331 1.133 .946tis su e .927 1.473 1.738 2.109 2.157 1.702 1.443 1.253

6 h 2o.809 1.354 1.590 1.865 1.965 1.494 1.279 1.089

17 day old 2.419 3.827 5.425 6 .826 8.143 10.530 13.700tis su e 2.788 4 .586 6 .826 8.993 10.530 15.870 19.080

7 h 2o2.579 4 .080 5.892 7.751 9.558 13.717 15.843

*Upper numbers represen t the range of the conductivity values w hereas the lower number rep resen ts the m ean.

Page 32: The Effects of Victorin on Cells and Cell Fractions.

22

Table 5A. E lectrolyte re le a se and reaccum ulation by t is s u e .

Ex p . R elease Curve Slope x IQ** Reaccum ulation Curve Slope

1 274.8 - 45 .5

2 453.6 -1 1 8 .7

3 201.4 - 201.2

4 209.4 - 99 .2

5 497 .1 -1 4 2 .4

6 201.0 -1 4 6 .0

7 1.1

Page 33: The Effects of Victorin on Cells and Cell Fractions.

23

w ere made w ith 10 gram sam ples of leaf t is su e w hich were p laced d irectly

in d is tille d w ater. The resu lts are shown in the firs t two experim ents in

Tables 6- 6A. Reaccum ulation w as blocked when 10"3M 2 ,4 -d in itrophenol

w as added to the bathing so lu tio n s. The inhibitor w as added after 6 hours

when reaccum ulation usually b eg an . W hen 2 gram sam ples of tis su e were

a llow ed a 2-hour uptake of 1:40 d ilu tions of v ic to rin , deactivated_3

victorin , 10 M 2 ,4 -d in itropheno l, and 1:40 dilu tions of v ictorin plus .3

10 M 2 ,4 -d in itropheno l, only v ic torin blocked reaccum ulation . The

inhib itor did not block reaccum ulation a t the applied le v e l. R elease of

e lec tro ly tes from the combined treatm ent of v ictorin and 2 ,4-d in itrophenol

w as not much different from tha t of v ic to rin a lo n e .

The effect of leaching on the resp ira tion ra te of v ic torin -treated

t is s u e s w as stud ied by preparing tis su e in the same manner as tha t

for conductiv ity t e s t s . The tis su e w as p laced d irec tly in v ictorin

so lu tions and shaken . As seen in Table 7 , after 2 hours the ra te s of

oxygen uptake by trea ted tis s u e w as considerab ly lower than the con ­

tro ls . Thus shaking su scep tib le t is su e in d ilu tions of v ictorin a lte rs

th e resp iratory p a tte rn .

To determ ine w hether v ictorin caused perm eability changes in

mitochondria as it did in in tac t c e l l s , conductivity te s ts w ere made

of mitochondria suspended in d is tille d w ater d ilu tions of v ic to rin .

M itochondria were ex trac ted , w ashed , and resuspended in d is tille d

w ate r. C onductivity of the so lu tions w as m easured at various time

in te rv a ls . Tables 8 - 8A show th a t both v ic torin and control treatm ents

Page 34: The Effects of Victorin on Cells and Cell Fractions.

Table 6 , Effect of 2 ,4 -dlnitrophenol on reaccum ulation by t i s s u e d (MHOS x 10'*)

15 Min Life______ Life---------- iTox Tox Tox

Exp. Other Variables Ck Tox DNP DNP Ck Tox DNP DNP Ck Tox DNP DNP Ck To?

12 day old tis su e .612 .612 ..838 .927 1.026 1.204 1.204H2010"3m 2 ,4 -d in ltro -

.652 .838 1.006 1.304 1.253 1.633 1.443

phenol added at .629 .706 .924 1.107 1.137 1.408 1.3086 Hr_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

15 day old tis su e .625 .652 1.278 1.15610_3m 2 ,4 -d ln ltro - .741 .756 1.414 1.386

2 phenol added a t .6 Hr .683 .706 1.346 1.265

12 day old tis su e .625 .855 .625 .710 1.278 3.424 1.443 2.788 1.778 5 .62 gm lo t s , 2 Hr .821 .873 .652 .741 1.813 3.334 1.566 2.857 2.363 6 .0

3 up take, 1:40 Tox,2 .070 5 .8DT, 10"3M 2 ,4 - .723 .864 .639 .726 1,546 3.379 1.505 2.823

dlnitrophenol, Tox p lus 1Q"3M 2 ,4 - dlnltrophenol

17 day old tis su e .559 .772 .523 ,772 .756 1.414 .696 1.414 1.089 2.362 .927 2.655 1.813 4 .3

4 H ».710 1.026 .756 .855 1.156 1.738 1.179 1.534 1.778 3.010 1.667 2.788 2.857 6.5

.640 .890 .668 .827 .993 1.606 .988 1.484 1.478 2.743 1.376 2.699 2.326 5 .4

15 day old tis su e ,511 .652 .523 .535 .891 1.156 ,772 .927 1.278 2.064 1.026 1.566 1.852 4 .40 5 x 10’ 3 M 2 ,4 -

5 dlnitrophenol added.667 .681 .625 .598 1.414 1.331 1.566 1.133 3.010 2,593 3.081 1.892 4 .180 6 .2

to 2 ,4 -d in itro - phenol treatm ent at 8 Hr

.597 .662 .578 .556 1.090 1.238 1.028 .996 1.864 2.396 2.057 1.686 3.065 5.2

lUpper numbers represent the range of the conductivity values whereas the lower number rep resen ts the mean,

1.7021.892

1.797

1.5031.892

1.724

2.4192.788

2 .604

Page 35: The Effects of Victorin on Cells and Cell Fractions.

24

IH r_ _ _ _ _ _ _ _ _ y j r __ _ _ _ _ _ 8 Hr_ _ _ _ _ _ _ _ _ _ _ _ _ 10-H r_ _ _ _ _ 12 Hr_ _ _ _ _ _ _ _Tox Tox Tox Tox Tox

ix DNP DNP Ck Tox DNP DNP Ck Tox DNP DNP_ Ck Tox DNP DNP Ck Tox DNP DNP.

1.331 1.228 1.304 .855 2 ,362 .726 2.788 .667 2.9291.852 1.363 1.852 .985 2 .721 .855 3.081 .756 3 .519

1.617 1.298 1.585 .942 2 ,559 .801 2,909 .721 3 .176

2.064 2 .473 2.1092.721 2 .857 3 .010

2.364 2.665 2 .531

2.419 2 ,109 2 .3622.788 3 .01 0 2 .788

2.604 2 .550 2 .575

2.109 2.362 2 .2053.163 2.788 3 .01 0

2.642 2.575 2 .691

523 1.852 5 .062 1.975 7 .437 2 .064 6 .825058 2.064 5 ,23 9 2 .721 8 ,146 2 .308 7 . 118

341 1.958 5 .151 2 .348 7 .792 2 .18 6 6 .972

2 . 109 8 .989 2 .362 8 .54 7 1 .892 10.5823 .163 9 .472 2 .593 9 .472 2 .7 8 8 12.786

2 .63 6 9 .231 2 .47 8 9 .00 9 2 .3 0 4 11.684

2.157 1.778 1 .892 13,7022,532 2 .593 2 .25 6 14.762

2.345 10,582 2 .186 14.762 2 .07 4 14,232

308 1.253 5 .425 1.933 5 .425 1.363 6 .825556 2.362 6 .556 3 .163 8 .989 2 .593 8 .146

496 1.849 5 .938 2 .523 7 .079 2 .007 7 .608

2 .019 10.582 1.363 14.762 2 .019 11.960 1 .331 15.9873 .334 17.416 3 .719 19.091 3 .424 19.091 4 .30 8 19.091

2 .6 6 9 14.66,2 2 .298 16.613 2 .767 16.156 2 .4 8 4 17.022

444 1.443 3 .010 2 .308 10.001 1.633 4 .444295 4 .308 6 .058 4 .596 10.582 4 .73 6 8 .146

236 3.193 4 .02 6 3 .734 10.195 3 .559 5 .884

2 .256 10.582 1.566 8 .1 4 6 2 ,419 12.7864 .73 6 15.987 4 .58 6 10.582 4 .05 7 17.416

3 .644 12.601 3 .487 9 .08 9 3 .2 7 0 14.635

1.813 10.001 3 .208 14.762 2 .06 4 11,2355 .062 12.786 3 .827 19i091 6 .295 14.762

3 .979 10.929 3 .456 16.613 4 .8 0 6 12.652

Page 36: The Effects of Victorin on Cells and Cell Fractions.

25

Table 6A. Effect of 2 ,4 -d in itrophenol on reaccum ulation by t is s u e .

Ex d .

R elease Curve Slope X 10^ Reaccum ulation Curve Slope

Ck Tox DNPToxDNP

ToxCk Tox DNP DNP

1 181.1 210.2 - 73.4

2 344.7 168.9 - 22.5

3 246.8 1182.8 237.3 1149.4 -112 .5 - -1 0 1 .0 -

4 181.0 1299.2 154.6 1378.3 _

5 545.6 1357.5 518.4 1029.4 -1 1 6 .0 - - 36.0 -

Page 37: The Effects of Victorin on Cells and Cell Fractions.

26

Table 7 . Effect of shaking on the respiratory rate of v ic to rin -trea ted t i s s u e s .

1 Hr Dry W t. 2 Hr 5 Hr

Shaking(1:500/2 Hrs) (Controls)

Treated as % of Control

470.4485.1

96 .9

453.7622 .0

72 .9

(1:500/2 1/2 Hrs) (Control)

Treated as % of Control

429.7430.9

99.7

392.5389.4100.8

(1:500/2 1/2 Hrs) (Control)

Treated as % of Control

352.4330.5106.6

355 .2 311.5 114.0

(1:500/3 Hrs)(Control)

Treated as % of Control

339.9544.6

62.4

161.9337.3

47 .9

(1:40)(Control)

Treated as % of Control

107.6537.6

20.0

12.920.3

8 .9394.2

2 .3

- 8 .9 385.3

0 .0

Uptake(1:40/4 Hr)(Control)

Treated a s % of Control

623.0296.9209.8

14.520 .4

562.5288.2195.2

415.4253.3164.0

Uptake (washed)(1:40)(Control)

Treated as % of Control

623.8351.8 177.3

12.320 .4

594.5273.7217.2

506.8234.6216.0

Uptake(1:40)(Control)

Treated as % of Control

598.2303.7196.9

550.7282.5194.9

Page 38: The Effects of Victorin on Cells and Cell Fractions.

Table 8. Release and reaccumulation of electrolyte by victorin-treated mitochondria, 1 (MHOS x 10$)

__ 15 Min 1 Hr 2 Hr _Exd. Other Variables H2O DT Tox H2O DT Tox W2U. DT TOX. _ H?0

12 day old tissue

1

.805

.838.796.805

.855

.891.838.855

.891

.946.891.909

.909

.985

.821 .802 .867 .849 .915 .903 .947

9

1.089 1*133 *

1.1111.133

1.1561.204

1.1791.204

1.110 1.122 1.186 1.198

3

1.0061.046

1.0061.026

1.0891.133

1.0671.111

1.0671.133

1.0891.133

1.019 1.019 1.104 1.089 1.096 1.111

4

.927

.985.985

1.0061.0261.111

1.0671.133

1.1111.204

1.1331.228

.959 .992 1.068 1.096 1.149 1.180

Fries salt solution was used to bring the H2O to

5 the same conductivity as the toxin solution.

1.0061.046

1.026

.9851.006

.999

1.0891.133

1.111

1.0671.089

1.082

1.1561.253

1.204

1.1561.228

1.196

1*13;1.20'

1. 17:

» If

6

.891

.909.891.909

.9461.006

.946

.985.965

1.026.985

1*026.87;.90!

.903 .897 .972 .965 .999 1.006 .881

Deactivated victorin and victorin solutions diluted

7 1: 10,000 - both adjusted with equal volumes of KOH and HC1

1.5341.566

1.556 1.534

1.5341.566

1.556

1.5991.702

1.656 1.633

1.5991.633

1.622

1*6671.813

1.764

1.7021.738

1.714

1.7021.738

1.726

1.701.89

1.79

lupper numbers represent the range of the conductivity values whereas the lower number represents the mean.

Page 39: The Effects of Victorin on Cells and Cell Fractions.

27

4 Hr 8 Hr 10 Hr 12 Hr (or last reading)PT Tox 1*2̂ PT Tox H26 PT Tox H2O dt Tox H2° DT Tox

95

.946

.985.909.965

.927

.965.838.909

.821

.838

l?Hr.838.927

.547

.559

7 .949 .940 .952 .873 .829 .869 .551

1.304 1.386 1.386 1.414

1.359 1.407

1.111 1.133 1.228 1.204

1.167 1.179

.997- .838 1.026 .909

1.005 .878

.946 .7561.006 .788

.981 .772

.927 .7561.006 .788

.969 .776

.927 .855,965 .873

.946 .861

.909 .726

.965 .756

.940 .746

.891 .710

.946 .788

.921 .751

.909 .7881.006 .927

.960 .857

1.006 .9271.156 1.067

1.091 1.000

1.111 .965 1.133 .756 1.067 .6961.253 1.067 1.278 .788 ' 1.133 .855

1.173 1.013 1.205 .772 1.096 .759

13 1.156 .788 .909 .681 .756 .667 .726 .598 .726)4 1.179 .909 .946 .772 .805 .696 .788 .625 .788

'2 1.164 .869 .927 .731 .778 .686 .746 .616 .752

'3)9

.476

.559

)5 .909 .523

.667 .4<11

.696 .443.598.625

.681 .432 .612

)2 1.778 1.73d 1.386 1.228 1.386 .946 .805 .873 .821 .772 .805 .772n 1.813 1.778 1.473 1.443 1.414 .985 .909 .909 .927 .788 .909 .788

n 1.789 1.765 1.415 1.371 1.405 .959 .868 .891 .874 .788 .783 .856 .783 .772

Page 40: The Effects of Victorin on Cells and Cell Fractions.

28

Table 8A. R elease and reaccum ulation of e lec tro ly tes by v ic to rin - trea ted m itochondria.

R elease C urve Slope x 103 Reaccum ulation Curve Slope!xp. HsO DT Tox m o DT Tox

1 33.6 26 .1 - 8 .7 - 57 .3

2 66.4 76 .0 -4 8 .8 -1 0 5 .8

3 113.3 52 .6 -2 6 .1 - 91.3

4 31.7 107.4 - 27 .3 - 42 .1

5 101.7 112.6 - 58.5 - 56 .3

6 54 .9 62 .3 - 94 .5 - 65 .7

7 62.7 68.0 55 .7 -116 .9 -2 5 .8 - 24 .1

Page 41: The Effects of Victorin on Cells and Cell Fractions.

29

lo s t e lec tro ly tes for a short period of tim e after which reaccum ulation

occurred . No sign ifican t d ifferences in e lectro ly te lo ss occurred b e ­

tw een trea tm en ts . Experiments 5 , 6 , and 7 were conducted to d e te r­

mine w hether sa lt level or specific sa lts might influence the ra te of

e lec tro ly te exchange. In experim ents 5 and 6 d ilu tions of Fries sa lts

w ere te s te d . In experim ent 7 , d ilu tions of v ictorin and deactiva ted

v ictorin which contained the same quan tities of s a lts w ere te s te d .

There were no sign ifican t d ifferences in ra te of e lec tro ly te lo ss or

reaccum ulation . From th e se data i t appears that v ic torin has no"

d irec t effect on the perm eability of m itochondria.

Because of the p o ssib ility th a t perm eability changes could

occur Ind irec tly , mitochondria were ex tracted from pretrea ted tis s u e

and conductiv ity te s ts m ade. Although in experim ent 2 of Tables 9 -

9A the control fa iled to reaccum ulate at the same ra te a s the v ic to rin

trea tm ent, n ev e rth e le ss , reaccum ulation did occur betw een the eighth

and ten th hour. Victorin fa iled to halt reaccum ulation in either ex ­

perim ent. Thus it would seem th a t v ictorin does not ind irec tly cause

perm eability changes in mitochondria th a t can be de tec ted with the

experim ental methods em ployed.

Further efforts to determ ine w hether v ic to rin , per s e , had an

effect on m itochondrial perm eability w ere made by te s tin g mitochondria

from re s is ta n t p lan ts and boiled mitochondria from su scep tib le t i s s u e s .

Tables 10-10A show tha t a sim ilar pattern of e lec tro ly te re le a se and

reaccum ulation occurred w ith mitochondria from re s is ta n t sources a s

Page 42: The Effects of Victorin on Cells and Cell Fractions.

3 0

Table 9 . Release and reaccumulation of electrolytes by mitochondria from victorin-treated tissues ,1 (MHOS x io ')

Exo. Other Variables15 Min 1 Hr 2 Hr 4 Hr j f l r 8 Hr... 10 Hr 12 Hr (or last read!no

H20 Tox tyO Tox h 2o Tox h 2o Tox h 2£) Tox H20 Tox H20 Tox H20 Tox

11 day old tissue ,559 .572 ,612 .639 ,401 ,401 ,390 ,4012 Hr uptake

1,572 .612 .625 ,696 .411 ,422 ,401 ,422

1.566 .594 ,619 ,662 ,585 ,612 ,432 ,465 .406 ,408 .396 .408

(17 Hr)14 day old tissue 1,156 1,278 1,331 1.331 1,443 1,473 1,443 1.566 1.353 1,473 1,473 ,6523 Hr uptake 1.179 1,304 1,363 1.363 1,473 1,534 1,473 1,667 1,386 1.599 1,566 ,681

L

1,168 1,179 1.278 1,291 1,347 1,347 1,458 1,504 1.458 1,617 1,375 1.536 1.519 ,667

^Upper numbers represent the range of the conductivity values whereas the lower number represents the mean,

Table 9A, Release and reaccumulation of electrolytes by mitochondria from victorin-treated tissue ,

Exp,_ _ _ _ _ _ Ck Tox

1 30,3 38,9

2 20,9 56.5

*27.9 - 42,3

- *105,6

Page 43: The Effects of Victorin on Cells and Cell Fractions.

3 1

Table 10, Effect of victorin on boiled mitochondria and mitochondria from resistant p la n ts ,1 (MHOS x 10s )

Ex p . Other Variables15 Min 1 Hr 2 Hr 4 Hr 6 Hr 8 Hr 10 Hr 12 Hr

Res, Sus, Res, Sus. Res, Sus, Res. Sus, Res, Sus. Res, Sus, Res. Sus. Res. Sus.21 gms of Camellia and ,838 ,838 .891 ,891 ,927 ,909 .805 ,821 ,572 ,585 .559 ,535 ,547 ,535Vg 48*93 were tested to ,855 .855 ,927 ,909 ,965 ,946 ,821 ,830 ,585 ,639 ,598 ,572 ,639 ,598

1 determine whether thetoxin ge t se had an .849 .844 ,909 .903 ,946 ,927 ,816 ,932 ,581 ,607 ,576 ,551 ,590 ,560effect.

BolL Cont. Boll. Cont, Bpll. Cont. Boll, Cont, Boll, Cont, Boll, Boll. Cont, Boll, Cont.43,5 gms tissue made 1,304 1,473 1,363 1,503 1,443 1,503 1,534 1,503 1,566 1,566 1,473 1,633 1,386 1,738 1,204to 32 ml, Mitochondria 1,414 1.534 1,503 1,566 1.599 1,599 1,738 1,599 1,778 1,702 1,667 1,778 1,534 1.992 1,304

2 were boiled 3 mln.1,503 1,349 1.504 1,427 1,535 1.525 1,551 1.646 1,551 1,682 1,634 1,591 1,706 1.485 1,815 1,262

typper numbers represent the range of the conductivity values whereas the lower number represents the mean.

Table 10A. Effect of victorin on boiled mitochondria and mitochondria from resistant plants,

Release Curve Slope x T ? Reaccumulation Curve Slope Exp._ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

Res, Sus. Res, Sus,

25,9 22,1 •61,7 ■62.7

m l Cont. m l Cont,

26,6 57,9 - •70,0

Page 44: The Effects of Victorin on Cells and Cell Fractions.

32

tha t w hich w as found to occur in mitochondria from su scep tib le so u rces.

When m itochondria were boiled th ree minutes the ab ility to reaccum ulate

e lec tro ly tes w as d estroyed . Experiment 2 of Tables 10-1QA show that

boiled mitochondria continually lo st e lec tro ly tes during the experi­

m ent. These experim ents further ind icate tha t v ic torin produces no

d irect effect on m itochondrial perm eability . Also it appears that

mitochondria must be In tac t and capable of m etabolic p ro cesses in

order to reaccum ulate e lec tro ly te s .

As the data in Tables 10-10A ind icated tha t reaccum ulation by

m itochondria w as dependent upon m etabolic p ro c e s se s , th e effect of

oxygen ten sio n on reaccum ulation w as stu d ied . M itochondria were

p laced in flask s under atm ospheres of a ir , oxygen, and nitrogen.

The conductiv ities of the te s t so lu tions (1:1000 victorin) w ere , as

shown in experim ents 1 and 2 of Table 11, not s ign ifican tly different

afte r 12 or 13 hours. It appears th a t high or low oxygen ten sio n s have

no effect on mitochondrial e lec tro ly te re le a se or reaccum ulation .

Because the m etabolic inhibitor 2 ,4-d in itropheno l w as known

to prevent sa lt accum ulation by storage t i s s u e s , it w as te s te d to d e ­

term ine w hether i t would a lso prevent reaccum ulation by oat mito­

chondria . Experiments 4 and 5 of Tables 12-12A show th a t concen­

tra tions of 1 x 10~^M stopped reaccum ulation . C oncentrations below

th is level failed to do so . This in d ica tes th a t th e p rocess of e lectro ly te

reaccum ulation is dependent upon uninterrupted phosphorylative and

oxidative m etabolism .

Page 45: The Effects of Victorin on Cells and Cell Fractions.

Table 11. Effect of different g ases on mitochondrial re lease and reaccum ulation of e le c tro ly te s .1 (MHOS x 105)

2 Hr 4 Hr 6 Hr 8 Hr 10 Hr 12 Hr (or la s t reading)Exp. Other Variable T ■ Aair T . air T ■ Aair T ■ Aair T . air DT . Aair T0Xair t o x N2 T03fc>2

1

13 day old t is s u e , each flask evacu­ated and filled with the desired gas

.9651.067

1.016

.772 .821

.891 .891

.832 .862

.873

.927

.897

o

14 day old tis su e 1.3311.386

1.4731.534

1.3861.473

1.2531.386

1.1111.253

(13 Hr) .855 .927 .985 .965

1.0261.111

c1.368 1.514 1.444 1.323 1.181 .922 .952 1.068

^ p p e r numbers represent the range of the conductivity values w hereas the lower number represen ts the mean.

GJGO

Page 46: The Effects of Victorin on Cells and Cell Fractions.

Table 12. Inhibition of mitochondrial electrolyte reaccumulation by 2,4- dinitrophenol. 1 (MHOS x 10&)

15 Min1

1 Hr 2 HrExd. Other Variables h2o DT Tox m2° DT Tox h2o DT Tox H;

47 Gms tissue made to a final volume of 40 ml

1 10"® M 2,4-dlnitrophenol added at 8 Hrs ,909 .909

.891

.909

.897 .946 .946

.946

.985

.972 1.026

1.0261.046

1.033

1.0061.046

1.026 1.

35 Gms tissue made to final volume of 32 ml

2 10"5 M 2,4-dlnitrophenol added to 2 flasks* of each treatment

.9851.006

.999

.927

.965

.946

1.0261.089

1.054

.9851.006

.992

1.0891.111

1.104 1.046

All tox treatments,1.5 x 10‘^M 2,4-dinitro-

3 phenol added at 4 Hr. Final conc. 2 x 10"5 M added at 6 Hr

Tox.965

1.006

.979

Tox. DNP .985

1.006

,999

Tox1.0461.111

1.068

Tox, DNP 1.046 1.067

1.060

Tox1.1111.179

1.134

t<?* m 1.111 1.133

1.118

1 x 10~3 m 2,4-dinitro- phenol added at 4 Hrs

4

.9461.067

1.0461.067

1.1791.204

1.1791.204

1.013 1.053 1.187 1.187

1 x 10 ̂ M 2,4-dinitro­phenol added at 4 Hrs

5

1.0671,089

1.1331.179

1.2281.278

1.2531.278

1.082 1.111 1.156 1.179 1.253 1.261

^Upper numbers represent the range of the conductivity values whereas the lower number represents the mean.

Page 47: The Effects of Victorin on Cells and Cell Fractions.

34

4 Hr 6 Hr 8 Hr 10 Hr 12 Hr2° DT Tox ^2° DT Tox H2° DT Tox h2° DT Tox h2u DT Tox

1.0461.067

1.0261.067

1.0461.089

1.0461.089

1.0261.046

1.0061.046

.873

.891.772.821

.046 1.053 1.046 1.067 1.067 1.067 1.046 1.039 1.019 .927 .879 .788

1.1331.156

1.0461.067

1.1331.156

1.0671.111

1.0891.133

1.0671.089

1.0891,133

1.0061.067

.965

.985

1.148 1.060 1.141 1.089 1.111 1.082 1.089* 1.089* 1.111 1.089

1.046* 1.046* 1.037 .975

Tox1.1561.253

T<?* m1.1561.179

I22L1.1561.253

TffltBNP1.1331.156

Tox Tox. DNP 1.026 1.089 1.089 1.111

Tox Tox. DNP .741 .873 .772 .946

1.188 1.164 1.196 1.141 1.061 1.096 .756 .923

1.4431.473

1.0061.156

2.3082.473

.813

.8382.5322.721

1.453 1.473 1.089 2.400 .824 2.595

1.3311.386

1.3861.414

.667

.7412.3082.419

1.386 1.395 .711 2.363

Page 48: The Effects of Victorin on Cells and Cell Fractions.

35

Table 12A. Inhibition of mitochondrial e lectro ly te reaccum ulation by 2 ,4 -d in itro p h en o l.

3Exp._______R elease Curve Slopes x 10 Reaccum ulation Curve Slope

H?Q PT Tox H?.0 DT Tox

1 27.5 27.5 29.6 -2 3 .3 -3 1 .3 -46 .5

2 39.7 24.9 w DNP w /o DNP

- 12.8-1 3 .9

- 7 .2 -1 9 .0

ToxToxPNP Tox

ToxDNP

3 37.7 44 .0 - 110.0 - 40 .2

4 117.3 112.0 -15 7 .3 +280.5

5 81 .1 75.7 - 84 .4 + 121.0

Page 49: The Effects of Victorin on Cells and Cell Fractions.

36

B ecause the osm otic condition of the m itochondria is im portant in

regard to enzym atic a c tiv itie s ( experim ents involving various sch ed u les

of su cro se and w ater m edia were conducted . Experim ents 2 , 3 , and 4

of Tables 13-13A show th a t when m itochondria were e x tra c te d , w ash ed ,

and resuspended in sucrose no reaccum ulation occurred reg a rd le ss of

com position of the te s t so lu tio n . In any other schedu le reaccum ulation

o ccu rred . Rates of reaccum ulation of contro l and trea ted m itochondria

v a ried .

II . C hem ical A nalysis

A nalysis of d is tille d w ater in w hich trea ted t is s u e w as shaken

revealed in c re a se s of numerous m a te ria ls . Two-gram sam ples of 13-day

old green lea f t is s u e w ere rin sed sev era l tim es in d is tille d w ater and

p retrea ted w ith 1:50 d ilu tions of v ic to rin or d eac tiv a ted v ic to rin . The

sam ples w ere bound in ch eesec lo th b a g s , p laced in f la sk s contain ing

100 ml of d is tille d w a te r, and sh ak en . Enough fla sk s of each treatm ent

w ere prepared to allow removal of one flask at each tim e in te rv a l.

C onductiv ity w as m easured and the bath ing so lu tio n s stored in the

refrigerato r un til an a ly zed . A nalysis of lab ile m aterials w ere performed

soon a fte r a l l sam ples had been c o lle c te d . R esults are shown in

Tables 14-14A. After 15 m inutes of leach ing in d is t i l le d w ater tre a te d t is s u e s

lo s t 20% more nitrogen th an co n tro ls . . Treated t is s u e s lo s t two tim es

more inorganic phosphorous, n itrogen , ca rb o h y d ra tes , p ro te in , and

po tassium after 4 hours than did c o n tro ls . After 24 hours 10-fold

Page 50: The Effects of Victorin on Cells and Cell Fractions.

Table 13. Effect of sugar on release and reaccumulation of electrolytes by mitochondria A {MHOS x 10$)

15 Min 1 Hr 2 HrExp. Other Variables h2o DT Tox Sd to o DT Tox h2o DT Tox

Mitochondria were ex­ * .946 .891 1.006 .946 1.026 .985tracted in sucrose,

1 washed and suspended in ^ O . 1* and 10 ml of susnension were used

.965 .927 1.067 1.026 1.111 1.067

Mitochondria were ex­tracted, washed and

1.1111.133

1.0461.067

1.21.2

2 suspended in sucrose1.089 1.126 1.053 1.179 1.204 1.111 1.228 1.253 1.156 1.2

Mitochondria were ex­ .351 .341 .390 .380 .441 .4tracted, washed and sus- .380 .351 .443 .390 .454 .5

3 pended in sucrose. Tissuegiven 2 Hr up-take of 1:40 .364 .344 .418 .387 .439 .411 .4tox before extraction

Same as above except 1.111 1.253 1.253 1.358 1.358 1.5mitochondria were placed 1.156 1.304 1.278 1.414 1.414 1.6

4 in toxin diluted in sucrose.114 Gm tissue made to 18 1.134 1,133 1.279 1.266 1.386 1.386 1.6ml, 4 ml prep added to

_ rt75 ml. held at 25°C.

Mitochondria extracted .891 .946 .927in sucrose, washed and .909 .965 .985

5 suspended in water.13 day old tissue. 1.067 .897 .891 1.089 .927 .927 1.133 .952 .953

Mitochondria extracted, 1.738 1.778 1.852 1.6washed and suspended in

6 H2O. Tox diluted in soln.1.778 1.852 1.933 l.S

which( contained 1/2 conc. 1.633 of Fries sucrose and salts diluted 1000-fold.

1.765 1.814 1.892 1.6

^pper numbers represent the range of the conductivity values whereas the lower number represents the mean.

Page 51: The Effects of Victorin on Cells and Cell Fractions.

37

4 Hr Q Hr 8 fir IQ Hr 12 Hr (or last readino)0 DT Tox DT Tox H2b DT Tox H20 DT Tox H20 DT Tox

1.067 1.026 1.067 1.026 1.067 1.026 1.046 1.0061SLHr

1.026 .965

1.179 1.133 1.204 1.179 1.006 1.026 .791 .873 .873 .710

253278

1.2781.304

1.1791.204

1.2531.331

1.3041.331

1.2531.278

1.3631.386

1.3631.386

1.3041.333

266 1.295 1.166 1.304 1.331 1.279 1.318 1.363 1.269 1.375 1.371 1.333

432511

.432

.465.535.681

.523

.612.665.855

.625

.741

477 .447 .609 .569 .759 .682

599668

1.5661.599

2.3622.473

2.1092.144

3.0043.424

2.7212.655

4.1804.736

4.0523.827

634 1,583 2.418 2.127 3.214 2.688 4.458 3.939

.946

.985.946.985

.946

.965.927.965

.909

.946.891.946

.891

.909.855.891

.891

.909.788.821

133 .972 .959 1.133 .952 .939 1.133 .921 .915 1.111 .897 .867 1.089 .897 .805

852 1.892 1.599 1.852 1.253 1.702 .681 1.414 .553 1.363933 2.019 1.667 1.975 1.386 1.778 .710 1.534 .598 1.503

892 1.948 1.622 1.906 1.342 1.739 .,700 1.484 .570 1.436

Page 52: The Effects of Victorin on Cells and Cell Fractions.

38

Table 13A. Effect of sugar on re le a se and reaccum ulation of e lec tro ly te s by m itochondria.

OExp.______ R elease Curve Slope x 10 Reaccum ulation Curve Slope

DT H ?0 Tox DT H 20 Tox

1 32 .3 3 6 .0 - 4 .5 - 6 .8

41 .6 43 .8 -1 0 3 .3 -67 .0

2 25 .1 29 .3 28 .7 - - -

3 50 .9 50 .1 - -

4 282.9 238 .8 - -

5 20.0 37.7 18.1 - 12.5 - 4 .4 -1 9 .2

6 69 .1 48 .9 -1 6 5 .3 -6 4 .0

Table 14. C onductiv ities of so lu tions in w hich v ic to rin -tre a te d t is s u e s w ere shaken (MHOS x 10®).

D eactiva ted Victorin Victorin

15 M in. .805 1.006

.927 1.067

4 Hrs .985 2.655

1.204 2 ,593

8 Hrs 1.179 3 .246

1.089 4 .057

24 H rs .772

1.204

17.400

2 1 . 1 1 0

Page 53: The Effects of Victorin on Cells and Cell Fractions.

39

Table 14A. C hem ical an a ly s is of so lu tions in w hich v ic to rin and d e ­ac tiv a ted v ic to rin -trea te d t is s u e s w ere sh ak en .

” 15

HQ

d t 8 D124 T O ^ s t o x 4 t o x m

True Inorganic Phosphorous fma/100 ml)

_1 - - - - 0.11

0.12

0.21

0 .16

0 .70

0 .7 0

O rganicPhosphorous

- - - - - - - -

Total N itrogen (g m s/ml)

1.63

1.80

1 .60

1.95

1.70

2 .5 0

2 .25

3 .7 0

2 .03

2 .10

4 .2 0

4 .7 0

6 .95

9 .4 0

4 0 .8

34 .5

TotalCarbohydrate (uam s/3 ml)

6 .0

ia^ o

7 .0

16.0

10.0

15 .0

16 .0

19.0

13 .0

12.0

2 7 .0

2 7 .0

2 8 .0

3 8 .0

3 3 .0

3 9 .0

Amino Acids (uM Eq.) 62 .5 100.0 108.0 274 .0 31 .3 127.0 258 .0 665 .0

Sodium (PPM) 2 .5 2 .0 1.0 < 1.0 2 .5 2 .0 2 .5 3 .5

3 .5 3 .0 2 .5 5 .0 3 .0 3 .0 3 .5 4 .0

Potassium(PPM)

6 .0

.2 .5

6 .0

2 .5

8 .5

2 .5

6 .0

3 .0

6 .0

2 .5

11.0

6 .0

11.0

12.5

2 1 .0

23 .5

C alcium (PPM) 4 .0 4 .0 4 .0 4 .0 4 .0 4 .0 4 .0 4 .0

2 .5 2 .5 2 .5 4 .5 2 .5 2 .5 2 .5 2 15

Protein (mg/ml) 0 .0429 0.1115 0.1355 0 .2160 0 .0382 0.2620 0 .2896 1.155

1Quantitifes w ere below th e lim its of the t e s t .

Page 54: The Effects of Victorin on Cells and Cell Fractions.

40

in c re a se s in inorganic phosphorous, 30-fo ld In c rea se s in to ta l n itrogen ,

2 -fo ld in c re a se s in to ta l carbohydrate , 2 1/ 2 -fo ld in c re a se s in amino

ac id s and p e p tid e s , 5 -fo ld in c re a se s in p ro te in , and 7-fo ld in c re a se s

in po tassium occurred in bathing so lu tions o f trea ted t i s s u e s . No

changes in organic phosphorous were d e te c te d . No g reat d ifferences

in sodium or calcium were noted excep t in one experim ent w hen 2-fo ld

in c re a se s of calcium occurred betw een the eighth and tw enty-fourth hour

in control bath ing so lu tio n s .

III. Effect of V ictorin on O xidation and Phosphorylation

Succin ic dehydrogenase w as m easured q u a lita tiv e ly and q u an ti­

ta tiv e ly . M itochondria w ere ex trac ted from young ep ico ty ls (co leop-

t i le s and primary le a f) . S uccin ic dehydrogenase w as m easured

q u an tita tiv e ly by th e Thunberg m ethod. Although th e a c tiv itie s of

trea ted m itochondria w ere s ligh tly low er than c o n tro ls , the d if­

ferences w ere not s ig n ific an t. R esults are rep resen ted in Figure 1.

For q u a lita tiv e a s sa y s su cc in ic dehydrogenase ac tiv ity w as

estim ated by h istochem ica l te s ts of ro o ts . V ictorin trea ted roots

showed v irtua lly no a c tiv ity . W hen roots from re s is ta n t sou rces w ere

te s te d v ic to rin exh ib ited no n o ticeab le e f fe c t . Although the treatm ent

w as rather sev ere and the tim e of treatm ent w as long , v ic to rin produced

an inhibitory effec t th a t w as sp ec if ic for su sc ep tib le ro o ts . Further

in v estig a tio n s should be conducted a t shorter treatm ent periods and

at higher d ilu tions of v ic to rin .

Page 55: The Effects of Victorin on Cells and Cell Fractions.

% T

rans

mis

sion

55

50

45

40

35

30

25

Time (M in.)

Figure 1. Effect of victorin on mitochondrial succin ic dehydrogenase a c tiv ity .

ControlVictorin

Page 56: The Effects of Victorin on Cells and Cell Fractions.

Phosphorylative-oxidative ra tio s were determ ined for m itochondria

ex tracted from trea ted t is s u e . The effect of various concentrations of

su c ro se , m alonate, and sodium flouride on P/O ra tios w as te s te d .

The re su lts of over 40 experim ents, represen ted in Table 15, are

duplicated in Tables 16 and 17 but have been rearranged according to

other experim ental v a riab le s . In alm ost every ca se the P/O ratios of

mitochondria from trea ted tis su e were lower than co n tro ls . Because of

variab ility betw een experim ents and the fewer number of trea ted te s t s ,

com parisons were not made betw een treatm ents for each se t of experi­

m ental co n d itio n s. However there were sign ifican t d ifferences a t the

5% level betw een the means of the combined trea ted and control ra tio s .

These d ifferences were not s ign ifican t a t the 1% le v e l.

Page 57: The Effects of Victorin on Cells and Cell Fractions.

43

Table 15. Phosphorylative-oxidative ra tio s of mitochondria ex tracted in various concentrations of sucrose from v ic to rin -trea ted t i s s u e .

Control V ictorin-treatedSucrose AP AO P/O AP AO P /P0.2M 28.80 21.20 1.36*

9.32 5.98 1.56*9.32 8.57 1.09*

12.71 5 .98 2.13*0 5 .8 0 0*4.42 5.22 0.85* 4 .67 5 .90 0.79*

Avg. 8 .79 1.170.5M 0 13.9 0* 5 .30 16.90 P .31*0.6M 20.08 5 .44 3.68 6 .98 7.94 0.88

18.60 5.52 3.37 6 .98 8 .5 0 0.8229.90 12.50 2.39* 14.40 12.45 1.15*

9 .30 7 .60 1.229 .30 7.48 1.242.32 5 .08 0.469 .30 3.12 2.98

10.501 7.60 1.376.97 9 .10 0.770 6 .52 00 8 .3 0 0*0 7.68 06.97 6 .16 1.13 2.32 5 .8 0 0.402.32 7.22 0.31

17.10 8.25 2.08* 0 4 .89 0*0 6.78 0 0 4 .1 0 0

Avg. 7.15 1.31 7 .28 0.540 .8 M 1.70 8 .6 0 0 .20 12.20 11.40 1.07

8.25 6 .14 1.34* 3 .09 8 .56 0.36*11.30 10.90 1.04 7.85 9 .3 0 0.859 .30 7 .40 1.26 1.16 9 .60 0.128 .14 8 .3 0 0.98 6 .96 9 .8 0 0.719.05 10.10 0.822 .50 8 .0 0 0.318 .1 0 7 .4 0 1.09

10.04 6 .7 0 1.564.65 4 .56 1.02

Avg. 7 .81 0.96 9 .73 0.62

* P / 0 ra tio b a s e d o n m icrom oles p h o sp h a te e s te r i f ie d an d m icroatom so x y g en ta k e n up p e r hour pe r mg N .

Page 58: The Effects of Victorin on Cells and Cell Fractions.

44

Table 16. The effec t of various concen tra tions of m alonate on phosphory- la tlv e -o x id a tiv e ra tio s of m itochondria from vifctorin-treated t i s s u e .

Control_________ V ic to rin -trea ted* P AO P/O * P AO P /O

No 28 .80 21 .20 1.36*M alonate 9 .32 5 .9 8 1.56*

9 .32 8 .57 1.09*12.71 5 .9 8 2.13*

0 5 .8 0 0*4 .42 5 .22 0.85* 4 .67 5 .9 0 0.790 13.90 0* 5 .30 16.90 0 .31

Ava. 9 .52 1.00 11.40 0,550.005 M 29.90 12.50 2 .39* 14.40 12.45 1.15*

1.70 8 .6 0 0 .20 12.20 11.40 1.078 .25 6 .1 4 1.34* 3 .09 8 .5 6 0.36*

11.30 10.90 1.04 7 .85 9 .3 0 0 .85Ava. 9 .54 1.24 10.43 0 .86

0 .0 1 M 20.08 5 .4 4 3 .68 6 .98 7 .94 0 .8818.60 5 .52 3.37 6 .98 8 .5 0 0 .829 .3 0 7 .4 0 1.26 1.16 9 .6 0 0 .128 .14 8 .3 0 0 .98 6 .9 6 9 .8 0 0 .719 .05 10.10 0.822 .5 0 8 .0 0 0.318 .1 0 7 .4 0 1.09

10.04 6 .7 0 1.564 .65 4 .5 6 1.029 .3 0 7 .6 0 1.229 .3 0 7 .48 1.242 .32 5 .08 0.469 .3 0 3 .12 2 .98

10.50 7 .6 0 1.376 .97 9 .1 0 0.770 6 .52 00 8 .3 0 00 7 .68 06 .97 6 .1 6 1.13 2 .32 5 .8 0 0 .4 02.32 7 .22 0 .31

17.10 8 .25 2.08* 0 4 .8 9 0*0 6 .78 0 0 4 .1 0 0

Avg. 7 .31 1.12 8 .4 4 0 .41

♦ P /O ra t io b a s e d o n m ic ro m o les p h o sp h a te e s te r i f ie d an d m ic ro a to m so x y g e n ta k e n up p e r h o u r p e r mg N .

Page 59: The Effects of Victorin on Cells and Cell Fractions.

45

Table 17. Effect of various concen tra tions of NaF on the phosphorylative ox idative ra tio s of m itochondria from v ic to rin -trea te d t i s s u e .

Control_________ V ic to rin -trea tedAP AO P /O A P A Q P /Q

0.001M 2 8 .80 21 .20 1.36*9 .32 5 .9 8 1.56*9 .3 2 8 .57 1.09*

12.71 5 .98 2.13*0 5 .8 0 0*4 .42 5 .22 0.85* 4 .67 5 .9 0 0.79*0 13.90 0* 5 .3 0 16.90 0.31*

Avg. * - 9 .5 2 1.00 11.40 0.55_0.004M 20.08 5 .4 4 3 .68 6 .98 7 .94 0.88

18.60 5 .52 3 .37 6 .98 8 .5 0 0.8229 .90 12.50 2 .39 14.40 12.45 1.1510.50 7 .6 0 1.376 .9 7 9 .1 0 0 .770 7 .68 0

Ava. 7 .97 1.93 9 .63 0 .950.007M 0 8 .3 0 0*0.01M 1.70 8 .6 0 0 .20 12.20 11.40 1.07

8 .2 5 6 .14 1.34* 3 .0 9 8 .5 6 0.36*11.30 10.90 1.04 9 .3 0 7 .85 0.859 .3 0 7 .4 0 1.26 1.16 9 .6 0 0 .128 .1 4 8 .3 0 0 .98 6 .96 9 .8 0 0 .719 .05 10.10 0 .822 .5 0 8 .0 0 0.318 .1 0 7 .4 0 1.09

10.04 6 .7 0 1.564 .65 4 .5 6 1.029 .3 0 7 .6 0 1.229 .3 0 7 .48 1.249 .3 0 3 .12 2 .980 6 .5 2 06 .97 6 .1 6 1.13 2 .32 5 .8 0 0 .4 02 .32 7 .22 0 .31

17.10 8 .25 2.08* 0 4 .8 9 00 6 .7 8 0 0 4 .1 0 0

Ava. 7 .2 9 1.03 7 .75 0 .440.02M 2.32 5 .0 8 0.46

* P / 0 ra t io b a s e d o n m icro m o les p h o sp h a te e s te r i f le d a n d m icroa tom so x y g e n ta k e n up p e r hour p e r mg N .

Page 60: The Effects of Victorin on Cells and Cell Fractions.

D ISC U SSIO N AND C O N C LU SIO N S

The p erm eab ility ex p erim en ts w ith n a tu ra lly in fe c te d and in o c u la te d0

s u s c e p tib le t i s s u e s w ere an e x te n s io n of p rev ious work w ith v ic to r in -

t r e a te d t i s s u e s (35). To f it K rupka 's c r ite r ia of th e ro le of to x in s in

p la n t d is e a s e (11), both fungus and to x in m ust p roduce s im ila r sym ptom s.

F igure 2 show s th a t H elm lnthosporium v ic to r la e . lik e v ic to r in , i s c a p ab le

o f c a u s in g e le c tro ly te r e le a s e from s u s c e p tib le t i s s u e s . F igure 3 show s

th a t h igh o r low oxygen te n s io n s produced no e ffe c t on e le c tro ly te lo s s .

W ith v ic to r in - t re a te d t i s s u e s , e le c tro ly te lo s s occu rred ev en under

an a e ro b ic c o n d itio n s . W h ee le r and Black (37) had p rev io u s ly rep o rted

th a t th e tem p era tu re c o e ff ic ie n t of th e e le c tro ly te lo s s w as s im ila r to

th a t ex p e c te d o f a p h y s ic a l p r o c e s s . T herefore i t a p p e a rs th a t th e

e le c tro ly te lo s s from v ic to r in - t re a te d an d n a tu ra lly in fe c te d s u s c e p tib le

t i s s u e s i s a p a s s iv e p ro c e s s and not d ep en d en t upon o x id a tiv e

m e tab o lism .

C o n d u c tiv itie s of d is t i l le d w a te r s o lu t io n s , in w hich h ea lth y

t i s s u e s w ere s h a k e n , in c re a se d for a sh o rt p e rio d a f te r w h ich a d e ­

c re a s e in co n d u c tiv ity o c c u rre d . F igure 4 show s th e ty p ic a l p a tte rn

of e le c tro ly te flux by h e a lth y t i s s u e s . As s e e n in F igure 5 , th e d e ­

c re a s e in co n d u c tiv ity of th ese , so lu tio n s i s p rev en ted by 2 ,4 -

d ln itro p h en o l - a n in h ib ito r know n to b lock s a l t a c cu m u la tio n .

L ik e w ise , v ic to rin not on ly c a u s e s a more rap id r e le a s e of e le c tro ly te s

46

Page 61: The Effects of Victorin on Cells and Cell Fractions.

MHO

S x

10A Infected

o Healthy

o Resistant

2.500

2.000

1.500

1.000

■O—-

0.500

Time (Hrs)Min

Figure 2 . E lec tro ly te lo s s from n a tu ra lly in fe c te d t i s s u e .

Page 62: The Effects of Victorin on Cells and Cell Fractions.

MHO

S x

10s

a - O2 (Victorin)• - N2 (Victorin}# - Air {Victorin}4.000

3 . 00C.

2.00C.

Time (Hrs)

F igure 3 . E ffect o f high and low oxygen te n s io n s on e le c tro ly te lo s s from v ic to r intre a te d t i s s u e .

I*00

Page 63: The Effects of Victorin on Cells and Cell Fractions.

MHO

S x

105

2 .50

2 .0 00

1.50C .

1.00C .

M in. Time (Hrs)

Figure 4 . E le c tro ly te r e le a s e an d rea cc u m u la tio n by h e a lth y t i s s u e .

CO

Page 64: The Effects of Victorin on Cells and Cell Fractions.

MHO

S x

10

o - 2 ,4-d in itrophenol

e - Control

3.000

m2.000

1.000

11 12IS 104 92 3 5 7 81 6M in. Time (Hrs)

Figure 5 . E ffect o f 2 ,4 -d in itro p h e n o l on t i s s u e re a c c u m u la tio n .

Page 65: The Effects of Victorin on Cells and Cell Fractions.

51

but p reven ts th e c h a ra c te ris tic d ec re a se In conductiv ity w hich occurs

w ith healthy t i s s u e . Furtherm ore, in some in s tan ce s w ith hea lthy t is s u e

the conductiv ity d e c re a se s to a value le s s th an th a t of th e sta rting co n ­

d u c tiv ity . This su g g e s ts th a t the d ec re a se in conductiv ity of th e so lu ­

tio n s in w hich healthy t is s u e s are bathed rep resen ts a reaccum ulation

of e lec tro ly te by th e t i s s u e . Thus th e effec t on perm eability by both

v ic to rin and th e fungus appears to be of a dual n a tu re . Not only do both

ca u se a more rap id lo s s of e lec tro ly te but they a lso p rev en t e lec tro ly te

reaccum ulation w hich occu rs in hea lthy t i s s u e .

Although v ic to rin , e ith e r d irec tly or In d irec tly , s tim u la tes the

resp ira to ry cen te rs to a h igher lev e l of a c tiv ity , the point of ac tio n

of th e tox in is unknow n. In an effort to d e lim it, w ith in th e c e l l , the

s i te of ac tio n of v ic to rin w hich re su lts in In c reased ac tiv ity of th e

resp ira to ry c e n te r s , te s ts w ere made to determ ine w hether v ic to rin

produced an e ffec t on the perm eability of m itochondria sim ilar to th a t

produced on w hole t i s s u e . Figure 6 show s th a t w hen m itochondria w ere

tre a ted w ith v ic to rin they lo s t e lec tro ly te s for a short period of time

a fte r w hich reaccum ulation occurred . The pa tte rn of e lec tro ly te flux

w as not s ig n ifican tly d ifferen t from th a t of th e c o n tro ls . S tud ies on

th e enzym atic a c tiv itie s of v ic to rin -trea te d m itochondria showed th a t

the tox in had no app rec iab le e f f e c t . Thus no apparen t d irec t e ffec t on

th e resp ira to ry ce n te rs w as e v id e n t.

B ecause th e e ffec t of v ic to rin on th e resp ira to ry ce n te rs could be

Page 66: The Effects of Victorin on Cells and Cell Fractions.

MHO

S x

105

/

- D eactivated victorin1.400

a - Victorin

1.300

1.200

1 .100

1 . 000.

0.90C.

10 11 12987653 4Min. Time (Hrs)

Figure 6 . Release and reaccum ulation of electro ly tes by victorin-treated mitochondria.

cnto

Page 67: The Effects of Victorin on Cells and Cell Fractions.

53

an in d irec t o n e , mitochondria from v ic to rin - tre a te d t i s s u e s w ere exam ined .

Figure 7 show s th a t no n o ticeab le perm eability d iffe rence occurred b e ­

tw een tre a te d and con tro l m itochondria . Furtherm ore, a s se e n in

Figure 8 , no d iffe ren ces in th e p a tte rn of e lec tro ly te flux occurred

betw een v ic to rin - tre a te d m itochondrial su sp en s io n s from re s is ta n t or

su sc e p tib le s o u rc e s .

S ince 2 ,4 -d in itro p h en o l b locked reaccum ulation of e le c tro ly te s by

t i s s u e s in much th e sam e m anner a s v ic to rin , i t s e ffec t on m itochondria

w as s tu d ie d . Figure 9 show s th a t th is in h ib ito r p rev en ts reaccum ulation

of e le c tro ly te s by m itochondria . T hese data su g g e s t th a t v ic to rin ,

w hich does not p reven t reaccu m u la tio n , does not a c t in a p a ra lle l

m anner. Further s tu d ie s of th e nature of th e reaccum ulation p ro cess

w ere conducted w ith b o iled m itochondria and m itochondria under varied

oxygen te n s io n s . Figure 10 show s th a t w hen th e in teg rity of th e m ito­

chondria i s d es tro y ed by b o ilin g , reaccum ulation does not o c c u r . This

further supports th e id ea th a t v ic to rin does not d isrup t th e se m i-

p erm eab illty of m itochondrial m em branes nor d irec tly in te rfe re w ith

normal m etabo lic p r o c e s s e s . W hen m itochondria w ere tre a te d under

high and low oxygen te n s io n s , how ever, no apparen t e ffec t w as s e e n .

As show n in Figure 11, conduc tiv ity v a lu es for m itochondrial su sp e n ­

s io n s under high and low oxygen te n s io n s w ere nearly th e sam e for

th o se in a i r . Although th e s e d a ta appear to co n trad ic t th o se w hich

support th e id ea of m etabo lica lly co n tro lled reaccu m u la tio n , th is may

Page 68: The Effects of Victorin on Cells and Cell Fractions.

MHO

S x

10

o - H y O Control

a - Victorin

0.600

in

0.500

0.400

Time (Hrs)Min

Figure 7 . Release and reaccum ulation of e lectro ly tes by mitochondria from v ic torin-treated t is s u e .

Page 69: The Effects of Victorin on Cells and Cell Fractions.

MHO

S x

10

• - R esistant (Victorin)

a - Susceptible (victorin)0.900

0.800

0.700

0.600

Time (Hrs)M in.

Figure 8 . Effects of victorin on electro ly te re lease and reaccum ulation by mitochondria from re s is tan t t is s u e .

cncn

Page 70: The Effects of Victorin on Cells and Cell Fractions.

MHO

S x

10s

2 500 ■ ° ~ Control (Victorin)d ■ 2 ,4-dinitrophenol (Victorin)

2 . 0 00 .

1.50C.

1.00C-

Time (Hrs)Min

Figure 9. Inhibition of mitochondrial electrolyte reaccum ulation by 2 ,4 -d in itrophenol.

cncn

Page 71: The Effects of Victorin on Cells and Cell Fractions.

o - Boiled (Victorin)

a - Unboiled (Victorin)1.900

1.800

1.700

1.600

caQ 1.500

1.400

1.300

1 .200

3 4 7 8 1015 1 52 6 9 11 12M in. Time (Hrs)

Figure 10. Electrolyte re lease by boiled m itochondria.

Page 72: The Effects of Victorin on Cells and Cell Fractions.

- Air (Victorin)

- N2 (Victorin)

- 02 (Victorin)

1.50C.

1 .40C.

1.300-

o1 . 20C■

CQ

£ 1. 100.

l.OOC.

0 .9 0 ( .

Time (Hrs)

Figure 11. Effect of high and low oxygen tensions on electro ly te lo ss and reaccum ulation by v ic to rin -trea ted m itochondria.

Vioo

Page 73: The Effects of Victorin on Cells and Cell Fractions.

59

not be n ecessa rily the c a s e . Under the lim its of the experim ental

m ethods, a ll oxygen from the nitrogen filled flask s may not have been

rem oved. Also no conductivity m easurem ents of m itochondrial su sp en ­

sions under oxygen and nitrogen were made until term ination of the

experim ent. It is p o ssib le th a t Interm ediate m easurem ents would have

shown tha t th ese treatm ents did not behave sim ilarly to th o se exposed

to the a tm osphere . Furthermore there are suggestions in the lite ra tu re

tha t ion carrier po ten tia ls may be generated under optimum conditions

and u tilized when conditions become le s s favorable for resp ira tion

(12, 19).

Because changes in m itochondrial perm eability induced by ex trac ­

tion in hypertonic media are known to effect the ac tiv ity of several

enzym atic re a c tio n s , trea ted mitochondria and mitochondria from

trea ted t is s u e w ere ex trac ted , w ashed , su sp en d ed , and shaken in

various schedu les of sucrose and w ate r. Figure 12 shows th a t m ito­

chondria ex trac ted , w ashed , and suspended In sucrose did not r e -

accum ulate e lec tro ly tes regard less of the com position of the te s t

so lu tio n . The reason for th is behavior is unknown. Several exp lana­

tio n s are p o ss ib le . F irs t, ex traction in hypertonic media may cause

an irreversib le exosm osis of e le c tro ly te s . It is a lso p o ssib le tha t

w hen mitochondria w ere m anipulated in sucrose and then p laced in

w ater te s t so lu tio n s , equilibrium w as not a tta in ed during th e te s t

period . Another po ssib le explanation is tha t sucrose in te rferes w ith

Page 74: The Effects of Victorin on Cells and Cell Fractions.

1.300

in2 1.200 X w O X 2

Sucrose + :

o - Victorin o _ H2O^ - D eactivated Victorin

1.100

Time (Hrs)Min.

Figure 12. Inhibition of mitochondrial e lectro ly te reaccum ulation by sugar.

01o

Page 75: The Effects of Victorin on Cells and Cell Fractions.

61

phosphorylative s tep s necessary for ion accum ulation. M iddleton (24)

reported tha t resp irato ry stim ulations sim ilar to those induced by

2 .4 -d in itropheno l occurred in storage tis su e s in the p resence of

g lu co se . Lehninger (13) found th a t sucrose inh ib ited an in term ediate

reaction of oxidative-phosphory lation which w as shared by the adenosine

triphosphate-d riven con trac tile mechanism of m itochondrial m embranes.

He suggested tha t sucrose might ac t a s an a rtif ic ia l accep tor in an e s ­

sen tia l enzym atic group transfer reaction or th a t it b locks a c c e ss of

adenosine triphosphate to membrane s ite s concerned in con trac tion .

Thus it is p o ssib le tha t sugars are involved in inhibiting a phosphoryla­

tiv e step w hich is n ecessary for m itochondrial e lec tro ly te reaccum ula­

tio n .

Although Uritani and Akazawa (34) have d iscu ssed the p o ssib le

m echanism s of e levated resp ira tion in d isease d p lan ts th e b a s is for

th e se in c rease s is s t i l l unknown. One important mechanism may be

uncoupled phosphory la tive-ox idation . As d iscu ssed previously there

appeared to be no d irect or ind irect effect by victorin on the per­

m eability of m itochondria. Although victorin prevented reaccum ulation

by tis s u e s much the same as the uncoupling ag en t, 2 ,4 -d in itro p h en o l,

no such effect by v ic to rin occurred w ith m itochondria. Hence it could

be concluded th a t v ic torin did not produce a d irec t or ind irec t un­

coupling effect on the m itochondria. However, Krupka showed tha t

2 .4 -d in itropheno l did not stim ulate resp ira tion in v ic to rin -trea ted

Page 76: The Effects of Victorin on Cells and Cell Fractions.

62

t is s u e s indicating tha t oxidation in those t is s u e s w as not lim ited by

th e level of inorganic phosphate accep to rs . Also when P/O ra tios of

mitochondria from v ic to rin -trea ted tis su e s w ere determ ined they were

considerably lower than c o n tro ls . Superficially th ese data appear

con trad icto ry . R esults of th is natu re , how ever, do not d istingu ish

w hether v ictorin is acting in the sam e manner as 2 ,4 -d in itropheno l or

w hether other m echanism s are involved . It is po ssib le th a t after p re­

treatm ent w ith v ic to rin , ce llu la r organization is so d isrupted tha t lo ss

of phosphorylative reac tan ts re su lts in lower lev e ls of adenosine t r i ­

phosphate and in effect "u n co u p les .'' The fact tha t v ic torin cau ses

lo s s e s of inorganic phosphorous from tis s u e s lends some support to

th is id e a . It is a lso po ssib le th a t treatm ent w ith victorin un leashes

or ac tiv a tes adenosine triphosphatases and thus brings about depletion

of high energy com pounds. Before uncoupling can be elim inated a s the

mechanism of v ic torin -induced resp ira tio n , the direct effect of victorin

on P/O ra t io s , adenosine triphosphatase a c t iv i t ie s , and the re la tive

q u an tities of adenosine triphosphate and adenosine d iphosphate in

trea ted and control t is s u e s need to be determ ined.

It is w ell known th a t s a lts are capable of causing in c reased

resp ira tio n (18). W heeler and Black (36) suggested th a t changes in

perm eability might re su lt in changes in ra tes of sa lt uptake and co n ­

cen tra tions in various parts of the c e l l . These workers suggested a

p o ssib le re la tionsh ip betw een perm eability ohanges and respiratory

Page 77: The Effects of Victorin on Cells and Cell Fractions.

63

in c re a s e s . It w as p o stu la ted th a t v ic to rin might in c re a se the per­

m eability of the to n o p la s t, a s w ell a s the e c to p la s t, th u s allow ing s a lts

and other m aterials to leak from the vacuole through the cytoplasm w here

they come in con tac t w ith the resp ira to ry c e n te r s . The re su lt might be

com parable to s a l t resp ira tio n or sim ply enhance resp ira tio n by fu rn ish ­

ing a read ily u tiliz a b le s u b s tra te . The fac t tha t la rge q u an titie s of

p o tassiu m , ca rb o h y d ra tes , amino a c id s , and o ther m aterials are lo s t

from v ic to rin -trea ted t is s u e s tend to support th is th eo ry . Furtherm ore,

Amador and W heeler (1) have shown th a t th e resp iratory ra te s of p re trea ted

t i s s u e s shaken in w ater w ere considerab ly lower than th o se tre a te d in the

sam e way but le ft standing in a ir . This in d ica tes th a t m ateria ls w hich

enhance re sp ira tio n are quickly lo s t from th e c e lls and thus re su lts in

low ered resp ira to ry r a te s . It may be extrem ely d ifficu lt to determ ine

the ca u se of e lev a ted resp ira tio n in v ic to rin -trea ted t i s s u e s , e sp e c ia lly

a s the p o ss ib ility s t i l l e x is ts of compounded e ffec ts ra th er than a sing le

c a u s e .

Page 78: The Effects of Victorin on Cells and Cell Fractions.

SUMMARY

Helmlnthosporium victorlae w as shown to increase the perm eability

of suscep tib le oat t is su e s in a manner sim ilar to that of v ictorin .

V ictorin-induced perm eability changes in tis su e occurred under

both high and low oxygen te n s io n s .

The conductivity of the solutions in w hich v ic to rin -trea ted tis su e s

were shaken not only increased more rapidly but failed to show a

following period of decrease as exhibited in co n tro ls .

V ictorin-treated tis su e s shaken in d is tilled w ater lo st large quanti­

tie s of n itrogen, carbohydrates, protein , amino a c id s , inorganic

phosphorous, and potassium .

No changes in the perm eability of v ic to rin -trea ted mitochondria

were observed. After a short period of e lectro ly te re lease both

trea ted and control mitochondria reaccum ulated e lec tro ly te s .

No changes in the perm eability of mitochondria extracted from

v ic to rin -trea ted tis su e s were observed.

After 12-13 hours the conductiv ities of v ic to rin -trea ted mitochondrial

suspensions under various atm ospheres were not significantly differ­

en t. These data ind icate tha t high or low oxygen tensions have no

effect on re lease and reaccum ulation of e lectro ly tes by v ic to rin -

treated mitochondria.

S ucrose, 2 ,4 -d in itropheno l, and boiling prevented the electro ly te

reaccum ulation which normally occurred in m itochondria.

Page 79: The Effects of Victorin on Cells and Cell Fractions.

No s ig n ific an t changes occurred in m itochondrial su c c in ic d e ­

hydrogenase ac tiv ity a fte r trea tm en t w ith v ic to rin .

P h o sp h o ry la tiv e -o x id a tiv e ra tio s of m itochondria from v ic to rin - tre a te d

t i s s u e w ere s ig n ific an tly low er th an c o n tro ls .

Page 80: The Effects of Victorin on Cells and Cell Fractions.

LITERATURE CITED

1. Amador, E. J . , and H . W heeler. 1963. Effect of leach ing on re sp ira ­tion of v ic to rin -trea ted oat t i s s u e s . Phytopath . 53: 621.

2 . A vers, C . J . 1956. H istochem ical lo ca liza tio n o f enzyme ac tiv ity inth e root epiderm is o f Phleum p ra te n s e . Am. J . Bot. 45: 609-613 .

3 . B lack, H . S . , and H . W heele r. 1962a. Perm eability changes a s s o ­c ia ted w ith V ictoria b ligh t of o a ts . P hytopath . 52: 4 .

4 . B lack, H . S . , and H . W heele r. 1962b. R elease and reaccum ulationof e lec tro ly te s by c e lls and c e ll frac tio n s afte r treatm ent w ith v ic to rin . Phytopath . 52: 725.

5 . Bonner, J . , and A. M illerd . 1953. O xidative phosphory lation byp lan t m itochondria , Arch. Biochem . an d B io p h y s . 42: 135-148.

6 . C o llin s , R. P . , and R. P . S cheffer. 1958. R espiratory re sp o n se sand system ic e ffec ts in F usarium -in fected tom ato p la n ts .Phy topath . 48: 349-355 .

7 . C olow ick , S . P . , and N . O . K aplan. 1957. M ethods in Enzymology.III . Academic P ress In c . New York.

8 . F isk e , C . H . , and Y. Subbarow . 1925. The co lorim etric determ ina­tio n of phosphorus. J . B iol. C hem . 66: 375-400 .

9 . G&umann, E. 1958. The m echanism of fu saric ac id in ju ry .P hytopath . 48: 670-686 .

10. Grimm, R. B ., and H . W heeler. 1963. R espiratory and enzym aticchanges in V ictoria b ligh t of o a ts . Phy topath . 53: 436-440 .

11. Krupka, L. R. 1959. M etabolism of o a ts su sc ep tib le toHelm inthosporium v lc to riae and v ic to rin . Phy topath . 49: 587-594 .

12. L a tie s , G . C . 1959* The genera tion of la te n t- io n - tra n sp o rtc a p a c ity . P roc. N . A. S . 45: 163-172.

13. Lehninger, A. L. 1961. Inh ib ition of ATP-induced con trac tionof m itochondria by polyhydroxy l ie com pounds. J . B io . C hem . (Tokyo). 49: 553-560 .

66

Page 81: The Effects of Victorin on Cells and Cell Fractions.

67

14. L itz en b e rg e r , S . C . , and H . C . M urphy. 1947. M ethods for d e ­te rm in ing r e s is ta n c e of o a ts to H elm lnthosporlum v lc to r la e . P h y to p a th . 37: 7 9 0 -8 0 0 .

15. L itzen b erg e r, S . C . 1949. N ature o f su s c e p tib il i ty toH elm lnthosporlum v lc to rla e and re s is ta n c e to P u cc in ia co ro n a ta in v ic to ria o a t s . P h y to p a th . 39: 3 0 0 -3 1 8 .

16. L uke, H . H . , and H . E . W h e e le r . 1955. Toxin p roduction byH elm lnthosporlum v lc to r la e . P h y to p a th . 45: 4 5 3 -4 5 8 .

17. L uke, H . H . , H . E. W h e e le r , an d A. T . W a lla c e . 1960. V ic to ria -ty p e r e s is ta n c e to crow n ru s t s e p a ra te d from s u s c e p tib il i ty to H elm lnthosporlum b lig h t o f o a t s . P h y to p a th . 50: 2 0 5 -2 0 9 .

18. L undegardh , H . 1955. M echan ism of a b so rp tio n , tra n sp o r t,a c cu m u la tio n , and se c re tio n of io n s . Ann. R ev. P lan t P h y s io l.6: 1 -2 4 .

19. M acD onald , L. R . , and G . C . L a tie s . 1962. O xygen e lec tro d em easu rem en ts of p o ta to s l ic e re sp ira tio n a t 0 ° C . J . E x p tl. B ot. 13: 4 3 5 -4 4 2 .

2 0 . M a c h lis , L . , and J . G . T orrey . 1959. P lan ts in A ction . W . H .Freem an and C o . S an F ra n c isc o .

2 1 . M eeh an , F . , and H . C . M urphy. 1946. A n ew H elm lnthosporlumd is e a s e of o a t s . P h y to p a th . 36: 4 0 6 .

2 2 . M eeh an , F . , and H . C . M urphy. 1946. A new H elm lnthosporlumb lig h t of o a t s . S c ie n ce 104: 4 1 3 -4 1 4 .

2 3 . M eeh an , F . , and H . C . M urphy. 1947. D iffe ren tia l p h y to to x ic ityo f m e tab o lic b y -p ro d u c ts o f H elm ln thosporlum v lc to r la e . S c ien ce 106: 2 7 0 -2 7 1 .

2 4 . M id d le to n , L . J . 1956. S a lt re sp ira tio n in sto rage t i s s u e . J .New P h y to l. 55: 117 -1 1 9 .

2 5 . M urphy, H . C . , and F . M eeh an . 1946. R eac tio n of o a t v a r ie t ie sto a new s p e c ie s of H elm ln thospo rlum . P h y to p a th . 36: 4 0 7 .

2 6 . P ad d o ck , W . C . 1953. H is to lo g ic a l study of s u s c e p t-p a th o g e nre la tio n s h ip s b e tw een H elm ln thosporlum v lc to rla e M . and M . an d s e e d lin g o a t le a v e s . N . Y. (C ornell) A gr. E xp t. S ta .M em oir 315 .

Page 82: The Effects of Victorin on Cells and Cell Fractions.

68

27. Paquin, R . , and E. R. W aygood. 1957. The effect of Fusariumtoxins on the enzym atic ac tiv ity of tomato hypocotyl m ito­chondria . C an . J . Bot. 35: 207-218.

28. P rice , C . A ., and K. V. Thlmann. 1954. The estim ation of d e ­hydrogenases in p lant t i s s u e . Plant Physio l. 29: 113-124.

29 . P ringle, R. B ., and A. C . Braun. 1957. The iso la tio n of thetoxin of Helmlnthosporium v lc to rla e . Phytopath. 47: 369-371.

30. Romanko, R. R. 1959. Physio logical b a s is for re s is tan c e of oatsto Victoria b lig h t. Phytopath. 49: 32 -36 .

31. Scheffer, R. P . , and R. B. P ringle. 1963. Toxicity of V ictoxinine,Phytopath. 53: 558-561.

32. T hatcher, F . S . 1939. Osm otic and perm eability re la tions in thenutrition of fungus p a ra s ite s . Am. J . Bot. 26: 449-458.

33 . U ^ b re it, W . W . , R. H . Burris, and J. F . S tauffer. 1957. M ano-m etric T echniques. Burgess Publishing C o . , M inneapolis, M inn.

34 . U ritan i, I . , and T . A kazaw a. 1959. A lteration of the respiratorypattern in in fec ted p la n ts , p . 349-390. In_J. G . H orsfall and A. E. Dimond (Ed.), Plant Pathology. I . Academic P re ss , New York.

35. W heeler, H. E. 1961. Perm eability changes caused by vfctorin .Phytopath. 51: 646.

36. W heeler, H . , and H. S . B lack. 1962. C hanges in perm eabilityinduced by v ic to rin . Science 137:.983-984.

37. W heeler, H ., and H . S . B lack. 1963. Effects of Helmlnthosporlumvic to riae and v ictorin upon perm eability . Am. J . Bot. 50: 686-693.

38. W heeler, H . E . , and H . H . Luke. 1955. M ass screening ford is e a s e -re s is ta n t mutants in o a ts . Science 122: 1229.

39. W heeler, H . , and H . H . Luke. 1963. M icrobial toxins in plantd is e a s e . Ann. Rev. M icrobiol. 17: 223-242.

Page 83: The Effects of Victorin on Cells and Cell Fractions.

VITA

Homer Selton Black w as born September 9 , 1935, in Port Arthur,

T exas. He w as reared near N ederland, T exas, where he a ttended public

schools and received a high school diploma in M ay, 1952. He entered

Texas A gricultural and M echanical C ollege in Septem ber, 1952, and

received a B .S . degree in Animal Husbandry In 1956. After m ilitary

serv ice in the U . S . Air Force, 1956-1959, he entered Sam Houston

S tate Teachers C ollege and received a M .Ed. in A gricultural Education

in August, 1960. In Septem ber, 1960, he entered Louisiana S tate

U niversity and is now a cand idate for the Doctor of Philosophy degree .

69

Page 84: The Effects of Victorin on Cells and Cell Fractions.

Candidate:

Major Field:

Title of Thesis:

EXAMINATION AND THESIS REPORT

Homer S e l t o n B l a c k

Botany

T he E f f e c t s o f V i c t o r i n on C e l l s a n d C e l l F r a c t i o n s

Approved:

Major Professor and Chairman

Dean of the Graduate School

EXAMINING COMMITTEE:

Date of Examination:

May 5, 1964