Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Mechanisms of diclofop methyl uptake in oat protoplasts (Avena sativa ’Cascade’) Tritter, Susan 1983

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1983_A6_7 T75.pdf [ 5.15MB ]
Metadata
JSON: 831-1.0095829.json
JSON-LD: 831-1.0095829-ld.json
RDF/XML (Pretty): 831-1.0095829-rdf.xml
RDF/JSON: 831-1.0095829-rdf.json
Turtle: 831-1.0095829-turtle.txt
N-Triples: 831-1.0095829-rdf-ntriples.txt
Original Record: 831-1.0095829-source.json
Full Text
831-1.0095829-fulltext.txt
Citation
831-1.0095829.ris

Full Text

IN  MECHANISMS OF DICLOFOP METHYL OAT PROTOPLASTS ( Avena s a t i v a  UPTAKE 'Cascade  by  SUSAN TRITTER B.SC.CAGR.), UNIVERSITY OF GUELPH. 1978  A THESIS SUBMITTED IN PARTIAL FULFILMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department o f P l a n t  We a c c e p t t h i s  Science  t h e s i s as conforming t o  the r e q u i r e d  THE U n i v e r s i t y  standard  of B r i t i s h  Columbia  A p r i l , 1983. ^(c)Susan  Tritter  jg83  In p r e s e n t i n g  this  thesis i n partial  f u l f i l m e n t of the  r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y of B r i t i s h Columbia, I agree that t h e L i b r a r y s h a l l it  freely  a v a i l a b l e f o r r e f e r e n c e and study.  agree t h a t p e r m i s s i o n f o r extensive for  financial  copying o r p u b l i c a t i o n o f t h i s  gain  Department  of  It i s thesis  s h a l l n o t be a l l o w e d w i t h o u t my  permission.  f^ftUl  The U n i v e r s i t y o f B r i t i s h 1956 Main M a l l V a n c o u v e r , Canada V6T 1Y3  (3/81)  thesis  s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my  understood that  DE-6  I further  copying of t h i s  department o r by h i s o r h e r r e p r e s e n t a t i v e s . for  make  So£kJCLjF~ Columbia  written  ii ABSTRACT  Isolated used  oat protoplasts  t o compare t h e uptake  (methyl acid  form  acid  methyl  antagonism.  up b y p r o t o p l a s t s  which  d i d not change  hour. within  herbicide  Forty 5.0  ( <  seconds  while  protoplasts  l4C-diclofop  non-mediated  difference  found  process i n which  from  uptake  Large  d i c l o f o p methyl  form  affected  concentrations. affect  membrane  permeability  taken  with  by b u r s t  or  f r a c t i o n s of the t o be a  Diclofop  i n the treatment  associated  passive,  was p a r t i t i o n e d  with  at  solution  the external  2,4-D  d i d not  concentrations  of d i c l o f o p methyl to  except a t high  methyl  was  was l i n e a r  membrane.  or d i c l o f o p uptake  Diclofop  t i m e s up t o  over  uptake  plasma membrane.  b y b y 2,4-D  level  accumulations of  10uM t o 2.0mM n o r w a s t h e c o n v e r s i o n  diclofop  rapidly  limited  appeared  phase o f the p r o t o p l a s t  was c o n v e r t e d t o t h e a c i d  f o rthe  s o l u t i o n up t o 50uM.  the herbicide  by h y d r o l a s e e n z y m e s a p p a r e n t l y  affect  was  between  uptake  was  methyl  i n t h e membrane  methyl  of the protoplast  incubation  14C-diclofop  i n the uptake  were  Diclofop  the l i p i d  surface  and t h e  to a constant  with  methyl  preparations.  methyl  protoplasts.  methyl  were  methyl  as a b a s i s methyl  d i c l o f o p uptake  Diclofop  concentration  uptake  5 seconds)  p e r c e n t o f added  T h e r e was no s i g n i f i c a n t  into  diclofop  Diclofop  significantly  same t i m e p e r i o d .  intact  of the herbicide  and t o s t u d y h e r b i c i d e  taken  the  L. 'Cascade')  diclofop (2-(4-(2,4-dichlorophenoxy)phenoxy)  2,4-D-diclofop  up  sativa  2-(4-(2,4-diehlorophenoxy)phenoxy)propanoate)  propionic  1.0  ( Avena  non-physiological  and d i c l o f o p d i d n o t  of the protoplasts  at the  adversely  iii herbicide Protoplast  c o n c e n t r a t i o n s used  i n these experiments.  p r e p a r a t i o n s p r o v e d t o be a u s e f u l and  t o s t u d y t h e u p t a k e and m e t a b o l i s m o f h e r b i c i d e s  simple system in plants.  iviv T A B L E OF  CONTENTS Page 1  INTRODUCTION LITERATURE  REVIEW  Diclofop  5  methyl  Physioloical Basis  5 effects  5  o f d i c l o f o p methyl  selectivity  7  Translocation  10  Mode o f a c t i o n  11  Antagonism  12  Herbicide  Uptake  Membrane Effects  transport  of Herbicides  M A T E R I A L S AND Plant  and T r a n s l o c a t i o n  17 on U p t a k e  and T r a n s l o c a t i o n .  METHODS  Membrane  24  Permeabilty  Atomic Diclofop  24  of Protoplasts  Neutral  absorption Methyl  Density  26 27  assay  and D i c l o f o p  Uptake  of Radiolabel  gradient  Solubility pH P r o f i l e  26  red assay  Distribution  22 24  Material  Isolation  16  fractionations  fractionation  of Hydrolase  Activity  28 30 30 32 35  V  RESULTS  AND  DISCUSSION  Protoplast  36  Viability  Membrane  Permeabilty  Diclofop  Methyl  36 38  and D i c l o f o p  Uptake  D i s t r i b u t i o n of Diclofop Methyl Oat P r o t o p l a s t s . . . . . 2,4-D E f f e c t s o n D i c l o f o p Metabolites  Methyl  49  and M e t a b o l i t e s Uptake  and  in  57 66  GENERAL D I S C U S S I O N  73  CONCLUSION  78  BIBLIOGRAPHY  79  APPENDIX  86  vi  L I S T OF  TABLES  Table  1a.  The e f f e c t o f e t h a n o l on l e a k a g e r e d dye f r o m o a t p r o t o p l a s t s  Table  1b.  The e f f e c t o f e t h a n o l on t h e K+ f r o m o a t p r o t o p l a s t s  Table  2a.  The e f f e c t o f d i c l o f o p m e t h y l on leakage o f n e u t r a l r e d dye f r o m o a t p r o t o p l a s t s  43  Table  2b.  The e f f e c t o f d i c l o f o p m e t h y l on o f K+ f r o m o a t p r o t o p l a s t s  44  Table  3a.  The e f f e c t o f d i c l o f o p on t h e l e a k a g e n e u t r a l r e d dye f r o m o a t p r o t o p l a s t s  of  Table  3b.  The  of  K+  e f f e c t o f d i c l o f o p on t h e  of  page  neutral  leakage  40  of  41  leakage  leakage  45  from oat protoplasts  46  Table  4.  T i m e p r o f i l e o f DM  uptake  50  Table  5.  Time p r o f i l e of d i c l o f o p uptake  Table  6.  U p t a k e o f DM a n d d i c l o f o p b y i n t a c t a n d burst protoplasts D i s t r i b u t i o n o f r a d i o l a b e l i n the p r o t o p l a s t suspension Percent composition of r a d i o l a b e l i n the p r o t o p l a s t s and t h e e x t e r n a l s o l u t i o n  51 54  Table  7a.  Table  7b.  Table  8.  P r o t e i n and s o l u b l e and  phosphorus content of the water i n s o l u b l e p r o t o p l a s t f r a c t i o n s ...  60  Table  9.  D i s t r i b u t i o n of r a d i o l a b e l within the p r o t o p l a s t s and the e x t e r n a l s o l u t i o n as a p e r c e n t o f t o t a l r e c o v e r a b l e r a d i o l a b e l . ...  62  Table  10.  Determination protoplast  of hydrolase  incubation  activity  58 58  in  solutions  63  Table  11.  The  e f f e c t o f 2,4-D  o n DM  uptake  67  Table  12.  The  e f f e c t o f 2,4-D  on d i c l o f o p u p t a k e  68  Table  13.  T h e e f f e c t o f 2,4-D to d i c l o f o p .  on t h e c o n v e r s i o n  of  DM 69  vii  L I S T OF  FIGURES page .  Figure  1.  Structure of diclofop methyl and d i c l o f o p  (DM)  1  F i g u r e 2.  Pathway o f d i c l o f o p methyl metabolism i n plants  F i g u r e 3-  Density  F i g u r e 4.  Flow c h a r t o f f r a c t i o n a t i o n o f p r o t o p l a s t s i n t o e x t e r n a l s o l u t i o n , w a t e r - s o l u b l e and w a t e r - i n s o l u b l e components  33  F i g u r e 5.  C o n t a i n m e n t o f n e u t r a l r e d dye i n v i a b l e protoplasts  37  F i g u r e 6.  E x c l u s i o n of Evan's protoplasts  38  F i g u r e 7.  Concentration uptake  Figure 8.  pH p r o f i l e o f h y d r o l a s e  gradient  8  f r a c t i o n a t i o n t u b e s . ...  blue dye  from v i a b l e  profile of diclofop methyl activity  31  53 71  viii ACKNOWLEDGEMENTS  I  would  Department  like  of Plant  his  encouragement  and  writing  Manitoba  the  also  support  A special  Eaton  a n d P a t Bowen  of statistical wish  t o thank  thanks  Columbia f o r  the research  t o D r . B.G. T o d d ,  f o r h i s guidance and of the thesis  proposal.  o f D r . V.C.  fortheir  time  committee,  and p a t i e n c e i n  analysis.  Hoechst  materials  the course  throughout  Professor,  m e m b e r s o f my t h e s i s  Chemical  I n c . f o rdonation of  and h e r b i c i d e s ,  and t h e Department o f P l a n t during  of British  the contributions  and Dr.P.A. J o l l i f f e ,  radiolabelled  Canada  advice  planning  g r a t e f u l l y acknowledge  discussion  University  of Agriculture,  i n the i n i t i a l  D r . G.W.  I  Science,  and v a l u a b l e  Department  Runeckles and  D r . F.B. H o l l , A s s o c i a t e  of this thesis.  suggestions I  t o thank  Science  of this thesis  and  Agriculture  f o rf i n a n c i a l research.  -1-  INTRODUCTION  Diclofop methyl (methyl 2-(4-(2,4-dichlorophenoxy) phenoxy)propanoate)  i sa s e l e c t i v e post-emergence  herbicide  which c o n t r o l s a v a r i e t y o f g r a s s y weeds i nc e r e a l a n d o i l s e e d crops.  D i c l o f o p m e t h y l (DM) i s a member o f t h e p h e n o x y - p h e n o x y  propionic  acid herbicide  series f i r s t discovered  by Hoechst  I n c . i n 1971 a n d i s t h emost e f f e c t i v e w i l d o a t h e r b i c i d e i n t h i s s e r i e s (Nestler e ta l . 1978).  S i n c e 1971 d i c l o f o p  methyl  h a s b e e n d e v e l o p e d i n t o a f o r m u l a t e d p r o d u c t now w i d e l y u s e d o n the Canadian  prairies.  The h e r b i c i d e h a st w op h y t o t o x i c f o r m s , t h e m e t h y l  ester,  diclofop methyl, andthe free acid, diclofop (Figure 1 ) .  Figure 1. Structure of d i c l o f o p methyl (DM) and diclofop. DICLOFOP METHYL (DM)  methyl-2(-4(2,4-dichlorophenoxy)phenoxy)propanoate  DICLOFOP  2-(k-(2,4-dichlorophenoxy)phenoxy)propionic  acid  -2-  The  herbicide  i sformulated  application  to plants  1979;  Todd,  1979)  theacid  form.  to  response  as the methyl  (Gorbach  o f plants  t o d i c l o f o p methyl action  2 , 4 - D , MCPA a n d d i c a m b a used  combination excellent weeds.  with  dicamba  grassy  1975;  Todd, between  The s i t e  these  cells  and r e l a t i v e l y  r e s u l t s i na  (O'Sullivan  and n a t u r e  protoplasts  of the  a n d movement a c r o s s t h e  i s impeded  homogeneous  taken  by t h e p r e s e n c e  system  up i n t o  and c e l l  wall  diffusion resistance. have  and Vanden  a r e unknown.  The u s e o f i s o l a t e d p r o t o p l a s t s  by t h e c u t i c l e  from  or with  spaces and t h e l o g i s t i c s  radiolabelled material  errors  2,4-D ( T o d d  1979)  t i s s u e s . The u s e o f i s o l a t e d p r o t o p l a s t s  adsorbed  and broadleaf  o f DM w i t h Born,  provide  o f DM a n d l o s s o f c o n t r o l o f  herbicides  o f t h e mechanism  wall, intercellular  mechanisms.  on t h e  herbicides  should  grassy  1975)  herbicides  1979).  plasmalemma o f p l a n t  simplified  hydrolysed  H o w e v e r , DM h a d n o e f f e c t o n t h e h e r b i c i d a l  Investigation  complex  action  eta l .  T h e o r e t i c a l l y ,t h e  herbicide  and Vanden Born  o f thebroadleaf  interaction  of  broadleaf  and Vanden  o f thephytotoxic  weeds.  activity  cell  areuseful  c o n t r o l o f both  MCPA ( Q u e r e s h i  (O'Sullivan  reduction  Born,  spectrum  upon  and d i c l o f o p , t h e  t o c o n t r o l weeds i n c e r e a l s .  broad  effort  unknown.  I n p r a c t i c e , t h ecombination  1979),  i ti sr a p i d l y  remains  o f DM a n d a b r o a d l e a f  However  Shimabukuro  I n s p i t e o f t h eresearch  mechanism o f p h y t o t o x i c  widely  e t a l . 1977;  (Smith,1977)  or s o i l  ester.  been used  t o study  o f handling provides  to study  constituents  uptake  a  uptake  eliminate  intercellular  In recent  of the  problems spaces or  and r e d u c e s  studies,  a n d membrane  -3transport of ions (Mettler acids (Guy  (Rubinstein et a l .  and L e o n a r d ,  and T a t t a r ,  1978, 1 9 8 0 ) . I t  1979; L i n ,  1 9 8 0 ; Guy e t  al.  1980)  1978)  has been d e m o n s t r a t e d  amino  and  sugars  that  i s o l a t i o n and p u r i f i c a t i o n p r o c e d u r e s d i d n o t i n h i b i t  the  ability  and  of c e l l s  quantitative  c o m p a r i s o n s between  indicated that altered  t o t a k e up amino a c i d s .  Qualitative  p r o t o p l a s t s and l e a f  p h y s i o l o g i c a l f u n c t i o n s are  by i s o l a t i o n p r o c e d u r e s  Published reports indicate uptake measurements  segments  apparently  (Rubinstein  not  and T a t t a r , 1978) .  problems a s s o c i a t e d w i t h  i n c l u d e maintenance of osmotic  protoplast stability,  and s e p a r a t i o n o f p r o t o p l a s t s f r o m t h e n o n - a b s o r b e d t r a c e r the  i n c u b a t i o n medium. H e r b i c i d e uptake  exposure of plant  by p l a n t s i s most o f t e n m e a s u r e d  s o l u t i o n has  t h e t i s s u e o r removed f r o m t h e  been u s e d as a m e a s u r e o f p l a n t u p t a k e .  been no d i s t i n c t i o n between h e r b i c i d e u p t a k e i n t o f r e e s p a c e s and c e l l w a l l s c e l l membrane and e n t e r s The e f f e c t s  ambient  There  the  the  N o n - p h y t o t o x i c c o n c e n t r a t i o n s o f DM (4uM) (12 h o u r s )  cuticle,  (1979). and r e l a t i v e l y  d i d not a f f e c t  growth o f  The m e t a b o l i s m o f DM i n t h e wheat c e l l  c u l t u r e s appeared  the  suspension  short  the  and r e s u l t e d i n o n l y m i n o r u l t r a s t r u c t u r a l damage  the c e l l s .  has  cells.  o f DM on t h e g r o w t h o f wheat c e l l  i n c u b a t i o n times  amount  from h e r b i c i d e which p e n e t r a t e s  c u l t u r e s was s t u d i e d by D a v i s and B r e z e a n u  culture  by  t i s s u e t o r a d i o l a b e l l e d c o m p o u n d s . The  of herbicide penetrating  al.  in  s i m i l a r to that  in intact  plants  to  suspension (Dusky  et  1980) . The o b j e c t i v e s  of the  study reported i n t h i s  thesis  include  the  development  uptake of The  o f DM  2,4-D  on  and  of a protoplast diclofop  uptake  r e s u l t s of the  their  as  system  i n o a t s and  a target  protoplasts  to i n v e s t i g a t e to determine  f o r t h e DM  effect  antagonism.  study are e v a l u a t e d i n terms  r e l e v a n c e to the mechanisms which  plants .  - 2,4-D  the  the  may  operate  of  in intact  -5-  LITERATURE REVIEW  DICLOFOP METHYL  Physiological  effects  Many a d v e r s e d i c l o f o p methyl (1979)  (DM)  application.  o b s e r v e d an i n c r e a s e  (measured after  p l a n t r e s p o n s e s have been o b s e r v e d C h r o w l e y and  in leaf  c e l l membrane  a p p l i c a t i o n o f DM t o w i l d o a t  b e f o r e v i s i b l e i n j u r y s i g n s were e v i d e n t .  after  of treated  In  tolerant  Decreased  levels  of c h l o r o p h y l l  c o i n c i d e n t appearance  and  C h l o r o s i s and  by t h e DM t r e a t m e n t ,  corresponding increase  and  a  were r e d u c e d  increase treatment.  b , and a in wild  (Chow and L a B e r g e ,  oat  1978).  photosynthates  were  by 24 and 63% r e s p e c t i v e l y .  in glucose,  (Chow,  no  96 h o u r s a f t e r  and t r a n s l o c a t i o n o f t h e  s h o o t s was o b s e r v e d  12 h o u r s  segments  o f c h l o r o s i s were o b s e r v e d  s h o o t s f o l l o w i n g DM t r e a t m e n t s Photosynthesis  leaf  wheat s p e c i e s ,  i n membrane p e r m e a b i l i t y was e v i d e n t  ATP l e v e l s  permeability  p l a n t s were n o t o b s e r v e d u n t i l 96 h o u r s  DM a p p l i c a t i o n .  reduced  Prendeville  by c o n d u c t i v i t y o f t h e e x t e r n a l s o l u t i o n )  foliar  necrosis  following  s u c r o s e and f r u c t o s e  1 9 7 6 ; Chow and L a B e r g e ,  A in  1978),  by 44% compared t o u n t r e a t e d  while  control  plants . Hoppe (1981)  s t u d i e d the e f f e c t s  o f DM on t h e  c o m p o n e n t s o f w i l d o a t and m a i z e p l a n t s . inhibited  lipid  lipid  Diclofop methyl  b i o s y n t h e s i s , reduced p h o s p h o l i p i d c o n t e n t ,  induced accumulations of carbohydrates i n t e r f e r e d w i t h membrane s t r u c t u r e .  in root tips  and  In v i e w o f t h e s e  effects  -6it  was s u g g e s t e d t h a t DM may i n t e r f e r e w i t h  fatty acid  biosynthesis. Histological wild  s t u d i e s o f a d v e n t i t i o u s r o o t t i p s o f wheat and  o a t t r e a t e d w i t h DM showed a r e d u c e d m i t o t i c  indicating  inhibition  of cell  division  methyl a r r e s t e d the c e l l s a t a stage preceeding  mitosis.  s e v e r e l y reduced a t lower exposure periods  (Owino, 1 9 7 7 ) .  i n the c e l l  The m i t o t i c i n d e x  Diclofop  cycle  i n w i l d o a t s was more  DM c o n c e n t r a t i o n s  than i n wheat.  index,  and a f t e r  shorter  Growth o f t h e e l o n g a t i o n  r e g i o n o f a d v e n t i t i o u s r o o t s o f w i l d o a t s was s i g n i f i c a n t l y inhibited  after  a 24 h o u r e x p o s u r e a s was i n i t i a t i o n  adventitious roots  (Owino, 1 9 7 7 ) .  I t was c o n c l u d e d  root meristem t i s s u e i s a s e n s i t i v e t a r g e t s i t e activity. treatment reported  A reduction  cell  inhibited and  by many a u t h o r s .  DM r e d u c e d r a d i c l e g r o w t h , i n d u c e d  arrested  a r e a s and e l o n g a t i o n division.  shoot e l o n g a t i o n  or post  concluded  necrosis i n the  z o n e s o f r o o t t i p s and  incorporated  (Wu  emergent ( F r e i s e n , 1 9 7 6 ) . proposed  the reduction  i n root  r e s u l t e d f r o m i n t e r f e r e n c e o f DM w i t h  auxin-mediated growth. induced  DM  Root d e v e l o p m e n t o f w i l d o a t s was  Shimabukuro e t a l . (1978) and  f o r DM  Hoppe ( 1 9 8 0 )  when DM was a p p l i e d e i t h e r p r e p l a n t  S a n t e l m a n n 1976)  that the  i n r o o t g r o w t h and d e v e l o p m e n t a f t e r  h a s been o b s e r v e d  meristematic  o f new  B o t h d i c l o f o p and DM i n h i b i t e d  g r o w t h i n r o o t and c o l e o p t i l e d i c l o f o p m e t h y l was an a u x i n  segments.  The  antagonist.  auxin authors  -7-  Basis  of diclofop  Diclofop control  methyl  methyl  selectivity  i s w i d e l y used  g r a s s y weed  s p e c i e s i n wheat and b a r l e y  1976). T h e b a s i s o f s e l e c t i v i t y differential (Figure Todd,  1979).  Other  differential  spray  retention  penetration  (Shimabukuro  1981;  1977),  mechanisms,  e t a l . 1979; (Todd,  (Brezeanu  e t a l . 1976; S h i m a b u k u r o  have  s t u d i e d , b u t have n o t proven  been  selectivity  between  Nevertheless, retention in  e t a l . 1979;  including  Donald  and Shimabukuro  1977) a n d t r a n s l o c a t i o n  e t a l . 1979; T o d d , 1977) t o be p r i m a r y  s u s c e p t i b l e and t o l e r a n t  i n some c a s e s ,  e g .green  methyl  selectivity  e t a l . (1977)  Gorbach  species.  foxtail,  enhanced  methyl  metabolism  included  phytotoxic diclofop  phenoxy)propanoic  acid),  (Todd,  i nwheat.  The main  positions.  phytotoxic to may  hydroxydiclofop (2-(4-  (1979)  w i t h h y d r o x y l groups later  identified  a c i d ) and t h e a t t h e 3'  as phenoxy phenol. be f o u n d  different  free  These  different  metabolic  i n theplant or present  e t a l . 1979; T o d d , r a t e s o f metabolism  referred  products  o f DM  as conjugates  1979; G o r e c k a t o these  and  an a d d i t i o n a l  metabolite, 4-(2,4-dichlorophenoxy)phenol,  (Shimabukuro The  Todd  products o f  (2-(4-(2,4-dichlorophenoxy)  non t o x i c  diclofop  role  metabolites  (2,4-dichloro-5-hydroxy-phenoxy)phenoxy)propionic o f hydroxy  spray  1977) .  studied the degradation  diclofop  6'  factors i n  a n d h e r b i c i d e p e n e t r a t i o n may h a v e a s e c o n d a r y  diclofop  isomers  compounds  1977), h e r b i c i d e  (Todd,  r o o t uptake  (Anderson  to non-phytotoxic  e t a l . 1977; S h i m a b u k u r o selective  herbicideto  i sapparently related to  r a t e s o f DM m e t a b o l i s m  2) ( G o r b a c h  Todd,  as a s e l e c t i v e  e t a l . 1981).  compounds  i n various  -8-  F i g u r e 2,  Pathway o f DM metabolism i n p l a n t s , combined from Todd ( 1 9 7 9 ) and Shimabukuro et  al.  (1979)  Cl C-COOCH.  n D i c l o f o p methyl (phytotoxic) Cl  CH.  Cl^ ^0^^\o-(J-COOH r  H Diclofop (phytotoxic) all  wheat and barley  species  CH-  °\j>°VJ T >  E s t e r conjugate  Hydroxy d i c l o f o p (non p h y t o t o x i c )  Phenoxy phenol (non-phytotoxic)  0 - C-COOH i  H 0-R  Phenoxy conjugate (non-phytotoxic)  00R  -9plants  account  In  both  converted 1977;  (1979)  to the acid  Both  between  and t o l e r a n t form  Todd,  DM i s r a p i d l y  enzymes  1979;  Todd  have h e r b i c i d a l  the h a l f - l i f e  to diclofop,  species.  species  by h y d r o l a s e  DM a n d d i c l o f o p  calculated  conversion  In  susceptible  S h i m a b u k u r o e t a l . 1979;  1980).  hours  f o rthe s e l e c t i v i t y  of diclofop  t o be 1.0  hours  (Gorbach  and Stobbe,  activity. methyl,  i n wild  et a l .  Todd  before  o a t and  1.8  i n wheat. tolerant  detoxified diclofop  1979.)  by a r y l  (Gorbach However,  hydroxylation pathway  species  the acid  diclofop  hydroxylation  t o form  e t a l . 1977;  slow  rate.  plants  occurs  via  i n susceptible  to represent  herbicidal  activity  (Shimabukuro  et a l .  species,  at a very  conjugates o f diclofop. thought  non-toxic  Shimabukuro  i n susceptible  occurs  i s irreversibly  This  a pool  of toxicant  upon h y d r o l y s i s  1979;  Goreka  e t a l . 1979;  The m a j o r  metabolic  of glycosyl  i sreversible  which  can exert  of the glycosyl  et a l .  Todd,  d e t o x i f i c a t i o n by  formation  conjugation  hydroxy  1981;  Todd,  and i s further  conjugate  1979.)  -10-  Translocation Translocation (Brezeanu and  1980;  ten  and  treated in  area  plants  directions  treatment  found  zone;  of  radiolabel  appeared  zone,  below  2.2%  et  These  a l . 1979;  herbicide areas  96  zone  radiolabel  and  susceptible  and  those  of other  of  the  Shimabukuro  et  pattern  i n the  a l . 1979;  of metabolic products  movement o f  the  1979).  ester The  was  acid  compared both  basipetal  movement, w i t h r e l a t i v e l y  large  above  below  and  and  the  treatment  zone,  absorbed  0.3%  in  the  (Shimabukuro move  accumulated  There  et a l .  The  showed  the  was  in  no  patterns of  in susceptible  diclofop  in  treatment  to  zone  (Brezeanu  negligible  Brezeanu  of  the  and  Radiolabelled  1979).  1979;).  movement  1979).  f o r DM  translocation  Todd,  the  basipetal  authors  treatment (Todd  species  DM  i n r o o t s and  direction.  rates;  from  0.7%  above  show a t e n d e n c y  out  tolerant  leaf  0 .5%  basipetal  However,  radiolabel  material  zone,  of high metabolic a c t i v i t y difference  (Todd,  recovered  treated  was  moved  the  Todd  Stobbe  translocation  had  plants  1979;  and  hours.  i n both  translocated  1979)  i n the  the  Todd  Although  been o b s e r v e d  i n the  results  i n 72  in  Todd,  radiolabel  treatment.  % of  the  1981).  recovered  translocation  translocated  detectable  (Todd,  after  has  a l . 1979;  reported higher  treatment  Todd,  preferentially  of  absorbed  hours  et  Putmann,  treatment  i s limited,  a l . (1976)  shoots.  1.6%  (1982)  the  96  i t s metabolites i s limited  Shimabukuro  the  Nalewaja  acropetal et  from  percent of  and  B o l d t and  reported only  translocated Olson  DM  et al.1976;  Stobbe,  (1980)  of  1976;  distribution species to  the  indicated acid  acropetal  amounts  in  i n r o o t s and  and  areas shoots  -11-  (Todd, 82.1% DM.  1979;).  I n w i l d o a t r o o t s 24 h o u r s a f t e r  o f r a d i o l a b e l was i n t h e a c i d In  the treatment  Similar  d i s t r i b u t i o n were o b s e r v e d  trends  i n movement  by S h i m a b u k u r o  et a l .  (1979)  d i c l o f o p and d i c l o f o p m e t h y l ,  action  in  susceptible plants.  i n h i b i t i o n was c a u s e d auxin antagonist  Todd and S t o b b e  They s u g g e s t e d  be c a u s e d  (1980)  argued  is  form.  provided  Shimabukuro  In  modes  that  by t h e a c i d d i c l o f o p .  the o p p o s i t e ,  in meristematic  A d d i t i o n a l support  of  growth stronger cellular However,  s u g g e s t i n g DM  damage and c h l o r o s i s , w h i l e  of the a c i d  of the ester  inhibitor  phytotoxic  diclofop  s h o o t and r o o t e l o n g a t i o n i n t h e m e r i s t e m a t i c  concentrations  proposal,  t h e two  had d i f f e r e n t  D i c l o f o p p r o v e d t o be more m o b i l e i n p l a n t s  areas.  t h a n DM, and regions for  exceeded  the  second  by:  et al.(1978)  r e p o r t e d d i c l o f o p was a  better  o f r o o t g r o w t h t h a n DM. susceptible  h e r b i c i d e on l e a f tissue.  Placement  resulted  in  Shimabukuro, near  al.(1979).  than d i c l o f o p whereas u l t r a s t r u c t u r a l  the u l t r a s t r u c t u r a l  inhibited  2.  and  by DM, as d i c l o f o p m e t h y l was a  damage was t h o u g h t t o  1.  et  proposed t h a t  forms,  that  as  Action  Shimabukuro  caused  f o r m compared t o 5.0%  z o n e , a b s o r b e d r a d i o l a b e l was 4 6 . 2 % DM  and 2 3 - 8 % as d i c l o f o p .  Mode o f  application,  plants  blades on t h e  selective  resulted sheath  near  the  the  shoot  leaf  meristem  i n h i b i t i o n (Hoerauf  i n d i c a t i n g the presence  t h e m e r i s t e m was n e c e s s a r y  of  i n c h l o r o s i s of the  b o t h c h l o r o s i s and g r o w t h 1979)  placement  for growth  of the  and  herbicide  inhibition.  -12Diclofop is regions of  the major m e t a b o l i t e  found i n the  f o l i a r - t r e a t e d w i l d oat  3 . D o n a l d and S h i m a b u k u r o  (1981)  w i t h d i c l o f o p and d i c l o f o p m e t h y l . was i n h i b i t e d e q u a l l y was r e s p o n s i b l e f o r expected that similar  plants  meristematic  (Todd,  1979).  i n j e c t e d w i l d oat  stems  Growth o f the t h i r d  by b o t h f o r m s o f t h e h e r b i c i d e .  i n h i b i t i o n o f new s h o o t g r o w t h ,  treatment  leaf If  it  DM  would  be  w i t h d i c l o f o p a l o n e w o u l d n o t show a  response.  Antagonism Incompatability  o f 2,4-D and DM i n t a n k m i x t u r e s  s u g g e s t e d as t h e  b a s i s o f the a n t a g o n i s t i c  these h e r b i c i d e s  (Quereshi  interaction  and Vanden B o r n ,  MCPA e s t e r  antagonistic  formulations.  In  and Vanden B o r n ,  was c o n c l u d e d t h a t f o r m u l a t i o n or  diclofop methyl.  or amine  ingredient rather  (1979)  ester  1980)  than the  it  the  antagonism.  reported a reduction  in  with  D i c l o f o p m e t h y l a l o n e r e s u l t e d i n 24% o f  MCPA e s t e r  1 5 . 0 and 7.7%  formulations  t i s s u e when MCPA was c o m b i n e d  applied herbicide penetrating  However  ester  s u r f a c t a n t s was r e s p o n s i b l e f o r  u p t a k e o f DM i n t o p l a n t  to  than the  1 9 7 9 ; Todd and S t o b b e ,  the a c t i v e  Q u e r e s h i and Vanden B o r n  presence of  in  S i n c e no c o m p l e x i n g o r d e g r a d a t i o n o f DM was  d e t e c t e d when m i x e d w i t h 2,4-D (O'Sullivan  but n o t  a d d i t i o n , MCPA amine was more  to d i c l o f o p methyl a c t i v i t y  formulation.  between  1 9 7 9 ) . A breakdown  i n t h e e m u l s i o n o f DM and MCPA amine was o b s e r v e d , the  was  tissue.  In  the  and MCPA amine p e n e t r a t i o n was  respectively  Todd and S t o b b e  the p l a n t  (1980)  24 h o u r s a f t e r and B o l d t  reduced  applications.  and Putmann  (1981)  -13reported wild  no e f f e c t o f 2,4-D o n DM u p t a k e  oat leaves.  within with  percent  of the applied  24 h o u r s o f a p p l i c a t i o n w h e t h e r  alone  into  DM w a s t a k e n  up  or i n combination  2,4-D.  Several and  Sixty  and p e n e t r a t i o n  reports  Stobbe,  tank  have  1 9 8 0 ) o r MCPA  mixtures  can reduce  diclofop.  Twenty  recovered  radiolabel  diclofop  methyl.  recovered  four  inhibition  and  Stobbe,  and  Vanden  (Quereshi the rate  hours  1980). Born  of  MCPA t o t h e c r u d e  supernatant  herbicide  demonstrated  that  t h e f o l i a g e , no a n t a g o n i s t i c  by H i l l  antagonistic  of wild  growth  were  oat  leaves  The  addition by 3 0 % .  e t a l . (1980) i n  of hydrolysis  o f DM  in  preparation.  t o f o l i a g e as a tank mix,  o r when 2,4-D w a s a p p l i e d  and r o o t  (Todd  by Q u e r e s h i  antagonistic  and Stobbe  (1980)  w h e n 2,4-D a n d DM w e r e c o m b i n e d  to  indicates  b y 2,4-D  preparations.  However, Todd  roots,  Shoot  These d a t a  i n d i c a t i n g an  to  69.6% o f  i n d i c a t i n g the presence  p u r i f i e d enzyme  oat continues,  interaction.  2,4-D,  inhibited hydrolysis  r e s u l t s were r e p o r t e d  of wild  with  extracts  to diclofop  When DM a n d 2,4-D a r e a p p l i e d growth  o f DM t o  r e s u l t s were o b t a i n e d  i n cell-free  a partially  1979) t o  z o n e was p r e s e n t a s  form.  2,4-D h a d no e f f e c t o n t h e r a t e using  Born,  (Todd  a f t e r DM a p p l i c a t i o n , 2 6 % o f  (1979) where crude  activity  o f 2,4-D  of hydrolysis  o f d i c l o f o p methyl  Similar  d i c l o f o p methyl  Conflicting  and Vanden  i n the treatment  of hydrolysis  hydrolytic  vitro  the addition  When DM w a s c o m b i n e d  of  which  that  r a d i o l a b e l was t h e e s t e r  an  converted  shown  to roots  applied  a n d DM w a s  i n t e r a c t i o n was  i n h i b i t e d by d i c l o f o p  i n t e r a c t i o n was n o t a t t h e s i t e  and  applied  observed. methyl.  of action  The  i n the  root  and shoot m e r i s t e m s ,  roots  would  concluded 2,4-D  have  that  (Todd  The  w h e n 2,4-D  addition  o f 2,4-D  quantity  of radiolabelled z o n e was  altered  b y 2,4-D.  treated  leaf  quantity  that  less  methyl  2,4-D  areas,  translocation  a  reduction  diclofop movement  i n basipetal  methyl  after  was o b s e r v e d  MCPA a p p l i c a t i o n  zone as d i c l o f o p  translocation  glucose  (Olson  suggested to diclofop  of diclofop  basipetal,  1980). O l s o n a n d and a l s o  observed  of radiolabelled However,  was u n a l t e r e d  a general  methyl  was  c h l o r o s i s and  experiments  upward  b y MCPA a n d t h e  increased.  i f MCPA w a s p l a c e d  indicating  and s h o o t  any precluded  zone  i n the  However, t h e  levels  increased  translocation  forradiolabelled  o f movement was  I t was  of diclofop  and S t o b b e ,  i n the treatment  b e l o w o r o n t h e same  plants.  MCPA t r e a t m e n t s .  movement was i n h i b i t e d  by t h e  away f r o m t h e  the roots  which  similar  of the radiolabel  amount r e m a i n i n g  basipetal  (Todd  (1982) c o n d u c t e d  the site of  Although the  o f 2,4-D.  i n higher  causing tissue  1980).  moving  reaching  resulted  symplastic  to foliage,  movement o f r a d i o l a b e l  i n hydrolysis  of the leaf  I t was  altered  the direction  i n 2,4-D-treated  destruction  Nalewaja  and S t o b b e ,  by a d d i t i o n  methyl  treatment  i n treated  o f DM w a s a l s o  herbicide  Acropetal  the reduction  after  (Todd  was e n h a n c e d  significantly  applied  to the  1980) .  unaffected,  of diclofop  response.  a n d DM w e r e  pattern  amine  application  t h e a m o u n t o f DM r e a c h i n g  and S t o b b e ,  translocation  treatment  a combined  shown an a n t a g o n i s t i c  must a c t t o r e d u c e  action  as  1.0  methyl.  cm a b o v e  1.0  A similar  and s u c r o s e non-specific  and N a l e w a j a ,  Basipetal  1982).  cm  effect  following reduction The  i n  -15inhibition  of basipetal  glucose  or sucrose  applied  topically  methyl  that  could to wild  application  treatment  sprayed  translocation be m i m i c e d  t h e downward  on t h e e n t i r e o f downward  d e p e n d e n t o n MCPA n e a r  by dark  o a t l e a v e s near  inhibited  the inhibition  of diclofop  the site  plant.  methyl,  treatments.  the site  MCPA  of diclofop  movement a s much a s a  The a u t h o r s  translocation of diclofop  concluded  b y MPCA w a s  methyl  application.  -16HERBICIDE UPTAKE AND TRANSLOCATION  The a c t i v i t y  of a systemic  ability  to reach the  factors  w h i c h l i m i t movement  efficiency  site  herbicide  of a c t i o n  symplast,  transport  The a c t i o n o f a  within  sites  (Kirkwood,  The f o l i a g e o f a p l a n t w h i c h a c t s as a b a r r i e r environment.  o f the parenchyma lamella. across  (Kirkwood,1976,  influenced polarity,  from the  underlying c e l l walls is  essential  non-toxic.  and t h e  size,  lipid  cuticle  1976).  external cellulose  1976; 1 9 7 8 ) .  directly  pH, degree o f d i s s o c i a t i o n ,  s o l u b i l t y o f the  herbicide.  s o l u b i l i t y o f the  into  herbicide  For h e r b i c i d a l t h e aqueous  activity,  phase o f  spaces of the  1976).  Binding of herbicides  l i p o p h i l i c compounds f r o m t h e  Kirkwood (1972)  f o u n d MCPB  the  apoplast  wax c a n i m m o b i l i z e t h e compound and r e n d e r  Desorption of  middle  Movement  process  considered a r a t e - l i m i t i n g step in h e r b i c i d e  (Kirkwood,  cuticle  c o n t i n u o u s w i t h the  and i n t e r c e l l u l a r  (Kirkwood,  the c u t i c u l a r  is  and i s  penetration.  lipid  metabolically  the u n d e r l y i n g  1978; P r i c e ,  which i n c r e a s e s  symplast,  with a l i p o i d a l  membrane i s a p h y s i c a l  enhance c u t i c u l a r  movement  merges w t i h  f o r m u l a t i o n and l i p i d  Any f a c t o r will  i s covered  by t h e m o l e c u l a r  the  1976).  c e l l walls  the c u t i c u l a r  penetration,  the a p o p l a s t or  between t h e p l a n t  The c u t i c l e  the  foliar-applied  t h e plasmalemma and e n t e r  efficiency  its  Thus,  of c u t i c u l a r  and r a t e o f m e t a b o l i s m and i m m o b i l i z a t i o n a t non-active  upon  t h r o u g h t h e p l a n t may l i m i t  of the h e r b i c i d e .  a b i l i t y to penetrate  dependent  i n the p l a n t .  h e r b i c i d e depends upon t h e e f f i c i e n c y the  is  within it cuticle  uptake to  be i m m o b i l e  -17and  inactive in  membranes. that  cuticle in  portions zones of  layer.  treated  ester  strongly  of  of a  plant  herbicide  m e t h y l may  remaining  indicating that  remain ester  function present  at  the  a  the  manner  the  significant cuticle  which  cuticle  into  the  the  hydrolyzed  i n the  formulations  in this  in  completely  dissolved  partition  concluded  within  1979) may  cuticular  (1981)  i s never  and  enzymes are  Hall  immobilized  e a s i l y penetrate  subsequently hydrolyze  hydrolase  l e a f are  Herbicide  lipophilic  Diclofop  (1979) a n d  i n the  methyl  (Todd,  form.  absorption  r a d i o l a b e l l e d DM  Diclofop  plants  proportion the  beans a f t e r  S i m i l a r l y Todd  large  treatment  broad  are  and  may  aqueous  i f the  in  phase.  neccessary  cuticle-cell  wall  i n t e r face . Herbicides  which  aqueous apoplast wall  to  the  apoplast leaf  can  may  d i f f u s e across  t h e n move t h r o u g h  vascular  tissue  i s strongly  the the  acropetal  following  and  margins.  Compounds w i t h i n  diffusion  and  mass f l o w  unless  cross  plasmalemma  symplast  ( P r i c e , 1976).  completely cross  the  (Peterson  apoplastic  and  the  plasmodesmata seive  to  tube  may  cells  the  the  are from  be a  the  of  and in  bound the  cell the  movement t o  apoplast to  herbicide the  action  the  move  by  into  the  plant  apoplast  phytotoxic site  space  the  can  be  herbicide i n the  must  symplast  1976). symplast I t has  connect in  to  reach  Edgington,  plasmodesmata.  they  to  Movement  water  T h e o r e t i c a l l y , no  since  plasmalemma  Compounds w i t h i n via  the  free  ( P r i c e , 1978).  tips  components or  cuticle  may  been  move f r o m suggested  that  s p e c i a l i z e d parenchyma  phloem  (Geiger,  1971).  cell  to  cell  the  cells  to  However,  the  -18research  by  suggests  sugars  Phloem has  loading  been  phloem  of  a l . (1971)  et  are  assumed  loaded  that  long  to  that  i s enhanced  conjugate  with  molecule  does not  simple  diclofop  and  conjugates  for  a  of  from  follow a  distance  and  or  (Shimabukuro  are  assimilates. that  The  in  the  Phloem  remainder  (Crisp, 1972).  lipids  It  readily  h y d r o p h i l i c balance  of  that The  d i c l o f o p , hydroxy  present  a l . 1979;  et  route.  transport  amino a c i d s .  d i c l o f o p methyl,  phenoxy phenol  apoplast.  similar  plant  lipophilic/ water  the  herbicide  normal  (1976)  Giaquinta  f u n c t i o n a l groups  favor  products  may  sugars  maintains  strongly  metabolic  by  and  i n t o phloem  herbicides  i s similar  mobility  the  Geiger  in plants 1979,  Todd,  as  sugar  Gorecka  et a l .  1981).  Membrane The  transport i n t e r a c t i o n of  determines Passage  how  easily  through  concentration  the  gradient,  a compound  Transport,  whether  carrier  indicated  specificity,  and  (Lehninger  1976).  Early  studies  permeability  or  directly  to molecular  cells  easiest  for  be  passive,  by  may  out  along  specific  the  be  lipophilic  shape.  a to  mediated  competitive  related  Uptake  molecules  cells.  by  substrate  l i p o p h i l i c i t y of  s i z e and  of  gradient.  also  kinetics,  membrane t r a n s p o r t to  plasmalemma  passive  a concentration  saturation  inhibition  on  the  a c t i v e , r e q u i r i n g energy  against  by  with  m o l e c u l e m o v e s i n t o and  a c t i v e or  inversely was  the  molecules  p l a s m a l e m m a may  accumulate  as  small  inhibitors  membrane  the  compound  into  and  Nitella  (Collander,  1954).  a  -19The r e l a t i v e  l i p o p h i l i c i t y o f t h e compound was d e t e r m i n e d  c a l c u l a t i o n o f the  l o g o f the p a r t i t i o n c o e f f i c i e n t  two phase aqueous and l i p i d  system  system i s o f t e n used to d e t e r m i n e lipophilicity 1976).  herbicides with  maximum a t values,  their  al.  1976).  m o b i l e phase  3.11,  Very  but a r e  (1969)  able Hall  higher  that because  research  lipid  c e l l membranes may compounds ( P r i c e , Most p l a n t the  cells  the  aqueous  to  at  polar  compounds  Proteins  alternate  embedded i n  pathways  f o r more  to c o n c e n t r a t i o n s  experience  a negative  potential  by e l e c t r i c a l  and  many compounds may a c c u m u l a t e  higher  the  the polar  1978).  p l a s m a membrane c r e a t e d  However,  be  can  difference chemical  g r a d i e n t s w h i c h c o n t r o l d i f f u s i o n o f m o l e c u l e s i n t o and o u t the c e l l .  log  pH 5 - 3 .  t h a n w o u l d be e x p e c t e d on  partitioning.  1976,  t o move i n t o  f o r DM, d i c l o f o p and 2 , 4 - D  has shown t h a t  provide  strongly  the  c r o s s membranes more e f f e c t i v e l y basis of  enter  calculated  and 0 . 2 9 r e s p e c t i v e l y  a  translocation  proposed  (1981)  for  for  reached  biological activity  less  1969).  partition coefficents 2.34,  across  lower  l i p o p h i l i c compounds p a r t i t i o n  (Penniston,  More r e c e n t  sole  p e r m e a b i l t y was h i g h  l o g P = 2 and d e c l i n e d a t  Penniston  and  al.  i n a b i l i t y t o p a r t i t i o n i n t o l i p i d membranes and  l i p i d membranes  of the  (Briggs et  Translocation of a herbicide  compounds have p o o r e r  symplast.  into  partition coefficent  l o g P i n t h e r a n g e o f 0-2 and was  l i p o p h i l i c i t y of  hydrophilic  the  the  s u g g e s t i n g an optimum l i p o p h i l i c i t y f o r  ( B r i g g s et  of  An o c t a n o l and w a t e r  s u g g e s t e d membrane  more p o l a r m o l e c u l e s .  in a  .  o f h e r b i c i d e s and f u n g i c i d e s  The a u t h o r s  (P)  by  than would  be e x p e c t e d  i n the if  of  cell  -20-  electrochemical uptake.  p o t e n t i a l was t h e o n l y f a c t o r  Weak a c i d s  f o r m and d i s s o c i a t e membrane  is  permeable  i n t h e c y t o p l asm ( P r i c e , t h e membrane  (Price,  1976,  i o n s and m o l e c u l e s  quantities  that  other  exceed the  compounds has  evidence  Rubery  (Poole,  p r o c e s s and a c c u m u l a t e d  a saturable, acetic  extent  transport  and 2 , 4 - D may o c c u r  in  the  Active  of herbicides for  d i f f u s i o n o f n o n - d i s s o c i a t e d 2,4-D  which of  and  However, is  available.  2,4-D e n t r y form c r o s s e d  into the  non-mediated incubation  specific  was s u g g e s t e d t h a t  by c o - t r a n s p o r t  the  medium  2,4-D was a l s o t a k e n up by  process  S m e j t e k and P a u l i s - I l l a n g e g a s k a r e  along  transport  in plants.  i n t h e c e l l when t h e  It  and  requires  ion gradient  The u n d i s s o c i a t e d  carrier-mediated  rates  studied in animals  than the c y t o p l a s m .  a c i d and 2 , 4 - D .  remain  active  at  transport  by d i f f u s i o n i n an u n s a t u r a t e d ,  was more a c i d i c  may  s i m p l e d i f f u s i o n and  d e s c r i b e d two m e c h a n i s m s  suspension-cultured c e l l s . plasmalemma  form,  through  1978).  been w i d e l y  of active  (1978)  acid  Weak b a s e s  c r o s s membranes  Active  m i c r o o r g a n i s m s and t o a l e s s e r little  in c e l l s  to c r e a t e a hydrogen  compounds d i f f u s e  organic  less  but may become c a t i o n i c  l i m i t s of  equilibrium.  energy  the  1978).  transport;  metabolic  If  f o r m and  i n the u n d i s s o c i a t e d  Compounds may a c c u m u l a t e  electrochemical  cytoplasm.  1978) .  in  undissociated  to the d i s s o c i a t e d form,  u n d i s s o c i a t e d i n the c y t o p l a s m , vacuole  i n the  to the u n d i s s o c i a t e d  or impermeable  cross  membranes  i n t h e more a l k a l i n e  permeable  accumulates also  penetrate  involved  for  uptake of  w i t h hydrogen  (1979)  indole  observed  auxin  ions. passive  t h r o u g h an a r t i f i c a l  lipid  -21bilayer  but not the d i s s o c i a t e d f o r m .  f o r mediated t r a n s p o r t o f the artifiical  bilayer  systems.  latter  The c o m p o n e n t s  required  form are not p r e s e n t  M a r t i n and E d g i n g t o n  in  (1981)  d e m o n s t r a t e d 2,4-D a c c u m u l a t i o n i n p o t a t o t u b e r d i s c s to a c o n c e n t r a t i o n 15 t i m e s g r e a t e r After  than the ambient  a freeze-thaw c y c l e to k i l l  the t i s s u e ,  solution.  the  2,4-D  c o n c e n t r a t i o n e q u i l i b r a t e d between t h e p o t a t o t i s s u e and ambient  solution.  Uptake  ( P e t e r s o n and E d g i n g t o n Edgington,  1981),  studies of other  1976)  and g l y p h o s a t e  (Martin  d i d n o t show a c c u m u l a t i o n  against  concentration gradients while Hall tuber d i s k s , against  (1981),  atrazine  and  working with  potato  r e p o r t e d d i c l o f o p m e t h y l and d i c l o f o p a c c u m u l a t i o n  a concentration gradient,  freeze-thaw c y c l e . by an a c t i v e  herbicides,  the  w h i c h was r e l e a s e d  after  a  He c o n c l u d e d t h e s e h e r b i c i d e s were t a k e n  process.  up  -22E F F E C T S OF H E R B I C I D E S ON UPTAKE AND  Uptake  and t r a n s l o c a t i o n i n p l a n t s  stimulated  by t h e a c t i o n  Paulis-Illangeskare transport  (1979)  studied  i n an a r t i f i c i a l  lipid  form  positive  and i n h i b i t e d  ions  was p r o p o s e d  2,4-D the  that  dipole  ions.  such  a decrease  that  The  the transport of  the transport  region,  membrane and d e c r e a s e d  charged  b i l a y e r system.  2,4-D m o l e c u l e s w e r e  caused  Smejek and  t h e e f f e c t s o f 2,4-D o n i o n  o f 2,4-D s t i m u l a t e d  interfacial  may be i n h i b i t e d o r  of herbicides.  undissociated  membrane  TRANSLOCATION  of negative  absorbed  within the  the orientation of the  i n the electrical  the rate  o f uptake  However, t h e s e a f f e c t s were  small  pH w h e n t h e d i s s o c i a t e d 2,4-D i o n w a s  Similarly,  Kennedy  uptake  i n maize  potential xylem only  roots  small  of excised  translocation and  describe  the bathing  a t pH 3 . 5 .  Kirkwood,  absorption nutsedge  1970,).  of ion  an i n c r e a s e  1972),  s o l u t i o n and t h e  However,  or decrease  a t pH  5.5  i n u p t a k e and  phenoxy h e r b i c i d e s .  was i n c r e a s e d  while  transport  root  observed.  picloram  t r a n s l o c a t i o n o f 2,4-D ( A g b a k o d a reduced  present.  2,4-D i n h i b i t i o n  a f t e r treatment with  and Y a h l i c h ,  pretreatment and  roots)  accumulation o f naphthalam  (Devlin the  between  d e p o l a r i z a t i o n s were  Many r e p o r t s  at a  by d e p o l a r i z a t i o n o f t h e t r a n s  (thedifference  exudate  reported  potential of  negatively  biological  (1979)  ions. I t  b y 2,4-D  treatment  and G o o d i n ,  treatment increased  1970).  o f r a d i o l a b e l l e d MCPA  Similarly,  Uptake  2,4-D s i g n i f i c a n t l y  MCPA  (Robertson reduced  a n d t r a n s l o c a t i o n o f r a d i o l a b e l l e d 2,4-D i n y e l l o w  (Bhan  e t a l . 1970) as w e l l  as reducing  translocation  -23of  TCA, c h l o r a m b e n ,  Reduction action  of translocation  as an u n c o u p l e r  reducing loading that  amitrole  decreased  available  Robertson  translocation  resulted  from  observed  destruction  parenchyma  b y 2,4-D  plugging  cells  may  of the electron  metabolic energy mechanisms.  and d i c a m b a  result  following  treatment  from  transport  and K i r k w o o d  o f phloem  1975). herbicide  chain  for translocation  with  ( 1970)  phenoxy  elements.  o f phloem v e s s e l s  after  (Chow,  and  thereby and  suggested  treatments  Eames  (1950)  proliferating  2,4-D.  -2kM A T E R I A L S AND  PLANT  METHODS  MATERIAL  Oat p l a n t s  ( Avena  sativa  L.  'Cascade') were grown  in a  o growth  chamber  16-hour  u n d e r i l l u m i n a t i o n o f 200  photoperiod  with  a 25 C d a y a n d  uE/m  /sec during  15 C n i g h t  a  temperature  regime . ISOLATION  OF  PROTOPLASTS  Oat p r o t o p l a s t s enzymatic oat  digestion  seedlings,  dark  a t 30  from  Kao  i n length.  C f o r 3.5  modification digestion  and  cell  and  The  wall  was  6 .OmM  overlaid  mM  2.0  ml  day o l d  were c u t i n t o  segments  incubated  (5000  (375units)  i n the  2  modified  units)  macerase  6.0mM C a C l  digestion,  (Calbiochem),  . 2 H 0 , 0 .7M 2  NaH PO^.H 0 2  rinsed  protoplasts  through twice  two  with  CaCl  2  was  centrifuged  2  2  (pH 7 . 0 ) . sorbitol  purified  sucrose,  The  buffer  and  ml  by  The  of cheese sorbitol  Hepes-NaOH  a t 300  discarded 0 .7M  layers  25.0  . 2 H 0 a n d 5 .OmM  was  were  o f Edwards e t a l . ( 1 9 7 9 ) .  filtered  containing  Hepes-NaOH  with  0.5%  supernatant  ml o f b u f f e r  o f 7-10  (pH 5 . 7 ) .  combined f i l t r a t e  5.0  grams  by  i n 25 m l o f e n z y m e s o l u t i o n  sorbitol,  the residue  sorbitol,  5.0  Five  segments were  of the procedures  mixture  minutes. in  0 .35M  3.0mM Mes-NaOH  Following  The  walls.  Leaf  hours  (Calbiochem),  0.35M m a n n i t o l ,  (0.7M  of cell  l e a f segments  e t a l . ( 1 9 7 4 ) c o n s i s t i n g o f 2.0%  cellulysin  cloth,  i s o l a t e d from  at the 1 to 2 l e a f stage,  0.5mm t o 1.0mm  and  were  buffer  (pH  7.0)).  X g f o r two the p e l l e t 6.0  suspension  mM  suspended  CaCl  2  .H 0 2  was c a r e f u l l y  and c e n t r i f u g e d  a t 300  X g  -25for  5.0 m i n u t e s .  interface  o f the  Purified  p r o t o p l a s t s c o l l e c t e d at  s o r b i t o l and s u c r o s e l a y e r s ,  p r o t o p l a s t s and c e l l u l a r  d e b r i s were p e l l e t e d .  were r e m o v e d , r i n s e d i n s o r b i t o l b u f f e r sorbitol In  while  the damaged  Protoplasts  and r e s u s p e n d e d  buffer. all  e x p e r i m e n t s t h e p r o t o p l a s t s u s p e n s i o n was  s t a n d a r d i z e d to a constant  c o n c e n t r a t i o n o f a p p r o x i m a t e l y one  m i l l i o n p r o t o p l a s t s per m i l l i l i t e r were made u s i n g a h e m o c y t o m e t e r . protoplast  buffer. Protein  s u s p e n s i o n was d e t e r m i n e d  method o f B r a d f o r d  (1976).  Protoplast content of  observed w i t h the  counts  the  by t h e C o o m a s s i e b l u e  dye  T h i s a s s a y was c h o s e n t o a v o i d  the  Hepes b u f f e r i n t e r f e r e n c e w i t h c o l o u r d e v e l o p m e n t t h a t Lowry p r o t e i n d e t e r m i n a t i o n .  has d e m o n s t r a t e d comparable r e s u l t s the  in  between  has  Bradford  been (1976)  the Lowry a s s a y  and  C o o m a s i e dye d e t e r m i n a t i o n . The v i a b i l i t y o f t h e p r o t o p l a s t p r e p a r a t i o n s was  by t h e e x c l u s i o n o f n o n - p e r m e a n t Okong'o-ogola, permeant  1970)  E v a n s b l u e dye ( G a f f  evaluated and  and t h e u p t a k e and c o n t a i n m e n t o f a  n e u t r a l r e d dye  (Lillie,  1969) .  -26MEMBRANE  Neutral The  PERMEABILTY  red  e f f e c t of  isolated by  assay  oat  and  protoplasts  monitoring  leakage  protoplasts. 400:1  DM  of  Isolated  (V/W)  d i c l o f o p on was  determined  neutral  oat  red  neutral  Protoplasts  were r i n s e d  i n 5 volumes  centrifuged  at  resuspended  i n 5 volumes  After  3 washes,  sorbitol Two  x g  the  hundred  Diclofop  sorbitol  final  u l of  of  dye  pellet  damaged  incubated  for  5  The  was  in  a  minutes.  sorbitol  buffer  dyed  buffer  pellet  and  and  was  recentrifuged .  suspended  protoplasts  methyl  and  diclofop  the  microlitre  amounts to  i n one  Dyed  at  0.1%  seconds.  absorbance  The at  545  ml  f o r one  supernatant nm  sorbitol and  incubated  hour  (Lillie,  and was  were  i n 95%  otherwise  were  buffer  volume  buffer.  1.0  ml  of  varying  ethanol.  prepared  ethanol,  then  The  by adding  final  diclofop treatments  was  stated. in treatment  centrifuged decanted  1969).  to  and  d i c l o f o p or  treatments  i n a l l DM  unless  protoplasts  temperature  1.0  were added  sorbitol  h e r i c i d e compounds  concentration  maintained  of  d i c l o f o p methyl,  dissolving  15  were  2 minutes.  s o l u t i o n , made up  concentrations  room  for  from  buffer.  treatment  ethanol  dye  red  of  spectrophotometricaly  protoplasts  s o l u t i o n of  300  membrane p e r m e a b i l i t y  for  at  solutions 12,000 x g  determination  at for of  -27Atomic  absorption  The v a l i d i t y confirmed K+  ions  was ul  of the neutral red permeability assay  by c o m p a r i s o n from  protoplasts after  determined  by a t o m i c  o f p r o t o p l a s t s were  DM,  to experiments  prepared  as d e s c r i b e d  protoplasts hundred with  treatment  absorption  incubated  d i c l o f o p , or ethanol.  were c e n t r i f u g e d  d i s t i l l e d water.  After  A l l samples  nm  was  determined  using  a Jarrell  atomic  absorption  spectrophotometer  comparison  Two  treatments  The  o f treatment  to a s e r i e s of standard  2.0  were  an  t o 10.0  One ml  mg/ml N a C l t o  absorption  (Fisher  with  a t 766.5  Scientific  Co.)  air-acetylene  s o l u t i o n s was K+  of  x g f o r 15 s e c o n d s .  atomic  Ash  hundred  incubation,  contained  ions.  of  or d i c l o f o p  were removed and d i l u t e d  o f K+  content  DM  concentrations  one hour  a t 12,000  ionization  T h e K+  the leakage  spectroscopy.  i n varying  reduce  flame.  with  A l l herbicide  above.  u l of supernatant  i n which  was  calculated  concentrations.  by  -28DICLOFOP METHYL AND DICLOFOP UPTAKE  Uptake of DM and d i c l o f o p  by oat p r o t o p l a s t s  was measured  using 1 4 C - d i c l o f o p methyl or 1 4 C - d i c l o f o p , u n i f o r m l y l a b e l l e d on the dioxyphenyl r i n g , 9.66  with s p e c i f i c  mCi/g r e s p e c t i v e l y  analysis  by Hoechst  (Hoechst  Inc.  h e r b i c i d e was p u r e .  Inc.).  i n d i c a t e d the  o f 19.8mCi/g and  Gas chromatographic synthesized  radiolabelled  Thin l a y e r chromatography i n a  benzene:methanol:acetic (Gorbach et a l .  activities  1977)  a c i d (85:10:5 V / V / V ) indicated  s o l v e n t system  both r a d i o l a b e l l e d and  n o n r a d i o l a b e l l e d m a t e r i a l remained pure throughout experiments. and  ethanol  The r a d i o l a b e l l e d h e r b i c i d e was d i s s o l v e d  suspension.  F i n a l concentration  permitted to exceed 1.0%  (v/v).  of ethanol was  The i n f l u x measurement  by a d d i t i o n of r a d i o l a b e l l e d h e r b i c i d e to  protoplasts After  the uptake p e r i o d , p r o t o p l a s t s  filters  placed i n s c i n t i l l a t i o n (Handifluor,  III  6881)  by s c i n t i l l a t i o n with e x t e r n a l  (Whatman GF/C) .  The f i l t e r s  and 5.0ml s c i n t i l l a t i o n was added.  spectroscopy  R a d i o a c t i v i t y was  (Tracor A n a l y t i c Mark  standardization.  without  Background counts  protoplasts,  under vacuum onto g l a s s f i b e r  were  fluid  by the a d d i t i o n of r a d i o l a b e l l e d h e r b i c i d e  s o r b i t o l buffer,  filtration  vials  by  for 10 seconds  to 15.0 ml b u f f e r ) .  Mallinckrodt Inc.)  were determined 200ul  was  the  were separated  were r i n s e d with s o r b i t o l buffer  (approximately e q u i v a l e n t  determined  not  at room temperature and under normal room l i g h t .  vacuum f i l t r a t i o n onto 2.4cm g l a s s f i b r e f i l t e r s The  i n 95%  then added i n m i c r o l i t r e amounts to 200 u l of  protoplast  initiated  the  followed  filters.  to  by  Radiolabel  -29retained the  the  on t h e s e  values  from  A time  profile  incubation  constant  filters  was d e t e r m i n e d  the treatment  and was u s e d  samples.  o f DM a n d d i c l o f o p u p t a k e  periods  from  concentration  A concentration  5 seconds  o f 2.5uM  profile  was d e t e r m i n e d f o r  t o one h o u r ,  f o r DM u p t a k e  by p r o t o p l a s t s  A m i x t u r e o f r a d i o l a b e l l e d and c o l d  was  and added  that  2.0 uM  giving  uptake  protoplasts from  of unlabelled  a total  The  herbicide  DM c o n c e n t r a t i o n  onto  was r e m o v e d  distilled  water  microscopic showed  diclofop  methyl  chloroplasts  from  and  with  0.0  t o 48 .OuM  broken into  protoplasts  Protoplasts Two h u n d r e d  resuspended  to disrupt  protoplast  and c e l l u l a r  debris.  broken  and i n t a c t  were  u l of The  i n 200ul  the protoplasts.  a n d 14C d i c l o f o p w a s d e t e r m i n e d  8 .0 uM c o n c e n t r a t i o n s preparations .  water.  and t h e p e l l e t  and a g i t a t e d  methyl  a t 1 2 , 0 0 0 X g f o r 15 s e c o n d s .  examination o f the broken  swollen  varied  exterior.  by s u s p e n s i o n i n d i s t i l l e d  supernatant  diclofop  t r e a t m e n t , and t h e  by i n t a c t  the protoplast  were c e n t r i f u g e d  was  s u s p e n s i o n so  t o d i s t i n g u i s h uptake  protoplasts  a  o f 2.0 t o 5 0 . O u M .  o f DM a n d d i c l o f o p  was c o m p a r e d  adsorption  disrupted  t o 200 u l o f p r o t o p l a s t  14C DM w a s p r e s e n t i n e a c h  concentration  using  14C DM o r 14C d i c l o f o p .  determined. prepared  to correct  Light  preparations U p t a k e o f 14C a t 2.0, 4.0, and  protoplast  -39DISTRIBUTION OF RADIOLABEL  Density  gradient  Separation carried 250ul  of protoplasts  out i n a gradient  o f 5.0%  solution layers  fractionations from  prepared  were  placed  (Figure  14C DM  Following  protoplast  suspension  component  gradient.  seconds.  The  non-absorbed  tracer  and  successive  of  pellet  sorbitol  was  a t 12,000  and d e s i g n a t e d  temperature  was  removed  tube  dispersed  onto  the  three  the  This  layers  were  was c a r e f u l l y in  o f an e q u a l  I80ul volume o f  fraction i s  180 u l a l i q u o t o f t h e  from  the treatment The  solution  pellet  was  added t o t h e decanted  the external  transferred  without  a t 1 2 , 0 0 0 X g f o r 30  X g f o r 30 s e c o n d s . was  for  of radiolabelled  o f each  A second  m i c r o l i t r e s each  s a m p l e s was  incubated  layers.. A l l three  by a d d i t i o n  180 u l o f a c e t o n e  hundred  were  layered  enzyme a c t i v i t y .  suspension  supernatant,  with  pelleted, leaving  bottom  t o as t h e p e l l e t .  discarded,  solution  followed  added t o stop  centrifuged  One  were  The  the contained  water,  I80ul  removed and  i n t h e upper  and  protoplasts  follows:  buffer  and 2 5 0 u l  suspension  incubation,  protoplasts  excised  referred  Sigma),  Tubes were c e n t r i f u g e d  by a s p i r a t i o n .  acetone  in sorbitol  s o l u t i o n a t room  were  removed  distilled  as  of silicone o i l  u l of protoplast  i n a 8 .OuM  agitation.  tubes  was  3)  hundred  one h o u r  tracer  i n the bottom o f the tubes  c o n s i s t i n g of 400ul  Four  i n 1.5ml  F i c o l K Sigma) d i s s o l v e d  (dimethyldiphenylpolysiloxane, buffer  non-absorbed  solution.(Figure  of the p e l l e t  and t h e  to s c i n t i l l a t i o n  vials  4.)  external with  -31-  Figure 3 .  Density gradient f r a c t i o n a t i o n tubes  0.7M  s o r b i t o l buffer  silicone o i l  5$ F i c o l l i n 0.7M buffer  sorbitol  -325.0  ml  An  of  scintilation  additional  100  chromatography (BDH  ul  (TLC)  Chemical).  The  fluid  s a m p l e was  separated  on  silica  plastic  plates  benzene:methanol:acetic  acid  1977)  •  Sections  reference diclofop  diclofop  Radioactivity  of  spectroscopy. calculated  with  The  in  procedure  TLC  8 .OuM  14C  into  the  ml  of  (Gorbach to  diclofop,  were c u t  out  diclofop  et  a l .  the and  and  scintillation  determined  of  hydroxy placed  in  fluid.  by  scintillation  methyl  to  diclofop  was  data.  is outlined  determined  by  previously.  TLC  and  content  determination  and  in  1.0  was  the  described  in  i n 70%  p h o s p h o r u s was  the  Bradford  procedures  temperature,  water-soluble  then  and  suspension.  of  the  This  fractions  Coomassie (1976).  perchloric by  as  of  and acid  described  determined.  blue  protein  Total Dyer at  was  the  were a l s o  Bligh  determined  three  content  fractions by  room  spectroscopy  phospholipid  by  at  were  4.  radiolabel  measured  suspension  protoplast  scintillation and  digestion  hour  solution,  Figure  insoluble  following by  for  protoplast  external  of  Protein  water-soluble  DM  components of  distribution  Inorganic  5.0  m i c r o l i t r e s of  water-insoluble  followed  methyl,  plates  in  corresponding  light,  conversion  the  hundred  extracted  UV  v/v/v)  TLC  fractionation  fractionated  Protein  plates  a l l s a m p l e s was  from  incubated  The  15cm  for  vials  Four  to  (85:10:5,  Inc.)  layer  were d e v e l o p e d  standards  scintillation  thin F 254  TLC  under  by 60  the  observed  Mallinckrodt,  gel  of  as  SolubiIty  (Handifluor,  lipid  was  (1959) 200  a modified  C.  procedure  of  -33-  Fixmre k  Flow chart o f p r o t o p l a s t f r a c t i o n a t i o n *^ f i s o l u t i o n , water-soluble and water-insoluble f r a c t i o n s .  Figure 4 .  c  o  t  h  e  e  x  t  e  n  a  p r o t o p l a s t suspension (J+OOul) I80ul centrifuge a t 12,000 X g f o r 3 0 ' through the 3 l a y e r gradient (Figure 3 )  I80ul centrifuge a t  12,000 X g for  15'  pellet discard  supernatant decant  pelljet disperse in H 0 vortex 2  add acetone (1)  c e n t r i f u g e at 12,000 X g for. 15' pellet disperse in H 0 vortex 2  add I 8 0 u l acetone (2)  (1) (2) (3)  External solution Water-insoluble f r a c t i o n water-soluble f r a c t i o n  supernatant decant add I80ul acetone (3)  -34-  Fiske  and SubbaRow ( 1 9 2 5 ) a s d e s c r i b e d  procedures  (UBC-Biochemistry  501).  14ml  o f ammonium m o l y b d a t e s o l u t i o n  20ml  concentrated  reagent  sulfuric  Samples were  placed  to cool  acid  dissolved  i n a boiling  of  laboratory  A 0.7 m l s a m p l e w a s a d d e d t o ( 2 . 2 0 g ammonium m o l y d b a t e +  and 0.6ml o f F i s k e - S u b b a R o w  ( 3 0 g NaHSO^ ; 1 g NaSO; 4 0 . 5 g  aminonaphtholsulfonic  allowed  acid)  i n a manual  1,2,4i n 200ml  water  and t h e a b s o r b a n c e r e a d  bath  distilled  water).  f o r 15 m i n u t e s ,  a t 660nm.  -35PH  PROFILE  The  h y d r o l a s e enzymes r e s p o n s i b l e f o r the  diclofop  methyl  purified  as  to d i c l o f o p  d e s c r i b e d by  lyophilized  enzyme  buffer  solutions  (Mes),  pH  (Ches), One  ml  6.0  pH of  water  enzymes  f o r use  200ul of  of  4.0,  pH  bath  ( Mops),  solution  f o r 1.0  hour  enzyme o r  denatured  i n c u b a t e d a t 30  the  of  solution  scintillation  addition  C  o f 200  s p e c t r o s c o p y as  controls .  pH  and  at each  mg.  of  pH  ( T r i c i n e ) , pH  to denature  the  hydrolase  by  DM  solution hour.  was  layer  added  The  compared  to  reaction  One  hundred  chromatograghy  described previously. methyl  in a  to a c o n c e n t r a t i o n  acetone.  thin  9.0  borate-KOH). placed  u l of  0.05M  5.0  was  f o r one  the  i n 2.0ml o f  pH  enzyme  were a n a l y s e d  a t each  8.0  Radiolabelled  conversion of diclofop  determined  Ten  phthalate),  pH  of  partially  carbonate/potassium  controls.  by  and  et a l . (1980).  (potassium  7.0  (potassium  stopped  percent  pH  as  5.OuM a n d  each  at  enzyme-buffer  boiling  Hill  extracted  p r e p a r a t i o n were d i s s o l v e d  (Mes),  10.0  were  conversion  to diclofop  to denatured  The was  enzyme  was ul and  -36RESULTS AND  DISCUSSION  PROTOPLAST VIABILITY The i n t e g r i t y of the p r o t o p l a s t membranes was  shown by the  e x c l u s i o n of Evans blue dye and containment of n e u t r a l red dye. Gaff and Okang o-ogola 1  (1970)  demonstrated that Evans blue  was  excluded from most l i v e c e l l s due to the semi-permeable p r o p e r t i e s of the plasmalemma. by Evans blue  Damaged or dead c e l l s were dyed  i n d i c a t i n g a l o s s o f c e l l membrane  integrity.  Neutral  red i s a non-toxic  stain.  In l i v e c e l l s n e u t r a l red i s taken up and contained  the  tonoplast.  preparations respectively.  permeant dye widely used as a v i t a l  Figures 5 and 6 show sample  protoplast  s t a i n e d with n e u t r a l red and Evans blue  dye  by  -37-  Figure 5.  Containment of neutral red dye i n v i a b l e oat protoplasts. ( X 300)  -38-  Figure 6.  Exclusion of Evan's blue dye from v i a b l e oat protoplasts, (x 120)  -39-  MEMBRANE  PERMEABILITY  Neutral treated of  r e d p e r m e a b i l i t y a n d K+  as a randomized  the data.  blocks  the v a r i a b l i t y  out at d i f f e r e n t  conditions leakage  used,  data  ethanol,  DM  design  experiments  for statistical  Different protoplast preparations  to partition  carried  block  leakage  both  showed  times.  inherent  Under  analysis  were  treated  the  linear  as  i n preparations experimental  t h e n e u t r a l r e d p e r m e a b i l i t y and  significant  were  the  K+  r e l a t i o n s h i p s between  and d i c l o f o p c o n c e n t r a t i o n s  and  membrane  permeabilty. Tables neutral  1a a n d  was  concentrations appendix  and  from  leakage This  of variances The  relationship  with  data  protoplasts.  reflecting  linear since showed  presented  used  t o compare  regression  o f 0.0%,  At  concentrations  The  0.1%  not a  result  Test f o r  support  linear  the linear  a not  treatment  Because o f the  means a r e  of d i c l o f o p methyl  test  small  d i f f e r e n c e between  1.0%  there  were  significant.  Duncan's m u l t i p l e range  ethanol  was  d i f f e r e n c e between t r e a t m e n t  effects  was  here  means.  and  (ANOVA i n  variances  a significant  concentrations  significant  ethanol  treatment  slopes.  of  linear  that  c a l c u l a t e d from  treatment  coefficent,  low e t h a n o l  Bartletts  leakage  significant  relationship  d e v i a t i o n s from  shallow  A  on  o f n e u t r a l r e d a n d K+  regression coefficients  small,  of ethanol  between i n c r e a s i n g  heterogeneous variances  homogeneous.  was  the effect  observed  1 a n d 2) .  homogenetity  The  show  r e d d y e a n d K+  relationship  of  1b  no  was  not  detected.  detectable  means.  on membrane p e r m e a b i l i t y a r e  -40-  Table  1a,  The dye  ethanol P e  r  c  e  n  e f f e c t o f e t h a n o l on l e a k a g e from o a t p r o t o p l a s t s .  cone.  A545  t  of neutral red  % i n c r e a s e over control  0.0  0.314a  0.00  0.1  0.313a  -0.23  1.0  0 .330a  5.19  5 .0  0.412b  31 .18  10 .0  0.502c  59.96  E a c h t r e a t m e n t v a l u e i s a m ean o f 3 e x p e r i m e n t s , r e p l i c a t i o n s per t r e a t m e n t i n each e x p e r i m e n t .  3  with  V a l u e s f o l l o w e d b y t h e same l e t t e r a r e n o t s i g n i f i c a n t l y d i f f e r e n t a t t h e 5 . 0 % l e v e l by D u n c a n ' s m u l t i p l e r a n g e test. R e g r e s s i o n e q u a t i o n o f e t h a n o l c o n c e n t r a t i o n and Y= .0191X + .3128. r= .9996  A545  '  -kl-  Tables  1b, The e f f e c t o f e t h a n o l on t h e l e a k a g e from o a t p r o t o p l a s t s .  ethanol cone. percent  ppm  K+  % increase control  0 .0  1 .02a  0.00  0.1  1.03a  0.98  1 .0  1 .00a  -2.56  10.0  2.22b  118.28  of  K+  over  T r e a t m e n t v a l u e s a r e t h e mean o f t h r e e e x p e r i m n e t s , r e p l i c a t i o n s per treatment i n each experiment.  with  3  V a l u e s f o l l o w e d b y t h e same l e t t e r s a r e n o t s i g n i f i c a n t l y d i f f e r e n t b y D u n c a n ' s m u l t i p l e r a n g e t e s t s a t t h e 5.0% level significance . Regression equation of ethanol Y = . 1 2 2 0 X + .9701 r = .9930  concentration  a n d ppm  K+  - 4 2 -  shown i n Table 2a and 2b. effect  There was no s i g n i f i c a n t  detectable  of DM on membrane permeabilty at 10 or 50uM.  DM r e s u l t e d  in a s t a t i s t i c a l l y  treatment means.  However,  significant  a significant  difference linear  As DM c o n c e n t r a t i o n was  p e r m e a b i l t y to n e u t r a l red dye and K+ increased gradual l o s s of membrane i n t e g r i t y A d d i t i o n of the a c i d ,  diclofop,  between  relationship  was observed between DM c o n c e n t r a t i o n and increased n e u t r a l red dye and K+.  Only 100uM  leakage  increased, indicating a  (ANOVA i n appendix 3 and 4). resulted  in a l e s s  pronounced e f f e c t on membrane p e r m e a b i l t i y .  The r e s u l t s  n e u t r a l red p e r m e a b i l i t y t e s t and K+ leakage  are presented  Tables 3a and 3b. observed  A significant  linear relationship  of  the in  was  between d i c l o f o p and leakage of n e u t r a l red dye and K+  (ANOVA i n appendix 5 and 6 ) . difference  However, no  between d i c l o f o p treatments  m u l t i p l e range t e s t at the 5.0% l e v e l The r e s u l t s  significant  was detected by Duncan's of  significance.  i n d i c a t e DM and d i c l o f o p have l i m i t e d e f f e c t s on  membrane p e r m e a b i l i t y under the experimental c o n d i t i o n s Significant  linear relationships  concentrations, only s l i g h t  were observed with  but the small r e g r e s s i o n  increases  c o n c e n t r a t i o n range The  of  coefficients  i n membrane p e r m e a b i l i t y over  tested.  increasing indicated the  tested.  primary purpose of the membrane p e r m e a b i l i t y t e s t s was  to e s t a b l i s h  c o n c e n t r a t i o n l i m i t s for e t h a n o l ,  which would r e s u l t permeability.  in a detectable increase  A l l herbicide solutions  as d e s c r i b e d i n the methods  section.  reduce ethanol c o n c e n t r a t i o n s  DM, and d i c l o f o p  i n membrane  were prepared i n ethanol P r e c a u t i o n s were taken  below l e v e l s which caused  to  -1+3-  Table  2a.  DM  All  The e f f e c t of n e u t r a l  cone. uM  o f d i c l o f o p m e t h y l on t h e l e a k a g e r e d dye from o a t p r o t o p l a s t s .  A545  %  i n c r e a s e over control  0 .0  0 .300a  0 .00  10 .0  0 .303a  '.1.10  50 .0  0 .318a  6.10  100 .0  0 .338b  12 .65  herbicide  treatments  c o n t a i n e d 0.1%  ethanol  T r e a t m e n t v a l u e s a r e t h e mean o f 6 e x p e r i m e n t s , r e p l i c a t e s per t r e a t m e n t i n each experiment.  with  3  V a l u e s f o l l o w e d b y t h e same l e t t e r a r e n o t s i g n i f i c a n t l y d i f f e r e n t by D u n c a n ' s m u l t i p l e r a n g e t e s t a t 5 . 0 % l e v e l o f significance . Regression  Y=3 . 8 x  10  e q u a t i o n o f DM X  +  .2993  r=  c o n c e n t r a t i o n and A545 .  .9998  Table  2b The e f f e c t s protoplasts  DM  herbicide  on  leakage  from  ppm  0.0  0.89ab  10 .0  0 .88a  50.0  1 .05bc  16.00  1 .15c  26 .00  treatments  K+  o f K+  cone. uM  100 .0  All  o f DM  c o n t a i n e d 0.1%  oat  % i n c r e a s e over control 0.00 -1 .41  ethanol  T r e a t m e n t v a l u e s a r e t h e mean o f 3 e x p e r i m e n t s r e p l i c a t e s per treatment i n each experiment.  with 3  V a l u e s f o l l o w e d by t h e same l e t t e r a r e n o t s i g n i f i c a n t l y d i f f e r e n t by D u n c a n ' s m u l t i p l e r a n g e t e s t a t t h e 5.0% l e v e l signi ficance. R e g r e s s i o n e q u a t i o n o f DM c o n c e n t r a t i o n a n d ppm Y= .0028X + .8768 r = .9795  K+  Tables  3a,  diclofop uM  All  The e f f e c t o f d i c l o f o p on t h e l e a k a g e o f n e u t r a l r e d dye from o a t p r o t p o l a s t s .  cone.  A545  % increase  0 .0  0 .220a  0 .00  10 .0  0 .229a  4 .09  50 .0  0.234a  6 .51  100 .0  0 .241a  9 .60  herbicide  treatments  over  control  c o n t a i n 0.1% e t h a n o l .  T r e a t m e n t v a l u e s a r e means o f 4 experiments, per t r e a t m e n t i n each experiment.  with 3  replicates  V a l u e s f o l l o w e d b y t h e same l e t t e r a r e n o t s i g n i f i c a n t b y Duncan's m u l t i p l e r a n g e t e s t a t t h e 5.0% l e v e l o f s i g n i f i c a n c e . R e g r e s s i o n e q u a t i o n o f d i c l o f o p c o n c e n t r a t i o n and A545 Y=1 . 8 3 7 x 10 X + .2235 r= .937  - 4 6 -  Table  3b,  The oat  diclofop uM  All  effect of diclofop protoplasts.  cone.  ppm  K+  on  %  leakage  0 .78a  0 .00  10 .0  0 .80a  2 .04  50 .0  0.79a  1.14  100 .0  0 .85a  9 .25  treatments  c o n t a i n 0.1%  from  i n c r e a s e over control  0 .0  herbicide  o f K+  ethanol  E a c h t r e a t m e n t v a l u e i s a mean o f 2 e x p e r i m e n t s , r e p l i c a t e s per t r e a t m e n t i n each experiment.  with 3  V a l u e s f o l l o w e d b y t h e same l e t t e r a r e n o t s i g n i f i c a n t a t t h e 5.0% D u n c a n ' s m u l t i p l e r a n g e t e s t . R e g r e s s i o n e q u a t i o n o f d i c l o f o p c o n c e n t r a t i o n a n d ppm Y = 6 . 3 6 x 10 X + .7797 r= .8864  K+  -47detectable Both  membrane  damage.  the neutral  absorption validity  o f K+  leakage  of these  Several prometyne,  red permeability gave  t e s t and  s i m i l a r r e s u l t s , supporting  herbicides, linuron,  including  glyphosate,  (Chrowley  reported  to increase  studies,  l o s s o f membrane  increase  i n conductivity of a solution  herbicide-treated  rates intact  in  future  DM  and e x p o s u r e  chosen  experiments.  Under  room  no d e t e c t a b l e  incubation  have r e s u l t e d Blein on  membrane  cultures.  changes  i n increased  permeabiltiy The r e l e a s e  was m e a s u r e d  i n these  of  experiments. described  conditions for  one  hour  no a g i t a t i o n , c o n c e n t r a t i o n s ( i n 0.1% e t h a n o l )  Longer may  damage.  the effects of several Acer  of  resulted  DM a n d d i c l o f o p c o n c e n t r a t i o n s  pseudoplatanus  o f f l u o r e s c e i n dye from  a f t e r one hour  direct  tests  standard  conditions,  membrane  studied  field  at the  i n membrane p e r m e a b i l i t y .  or higher  (1981,1982)  conductivity  d i d not provide  used  to evaluate  temperature,  periods  containing  of the permeability  these  by an  a p p l i c a t i o n o f DM a t  studies  5 0 u M DM a n d 1 0 0 u M d i c l o f o p o r l e s s in  was d e t e r m i n e d  membrane p e r m e a b i l i t y i n  periods  conditions  been  In those  DM e f f e c t s o n p r o t o p l a s t s  t h e s i s were  incubation,  1979) h a v e  Increased  increased  bromacil,  1980) and  permeability.  integrity  l e a f segments.  to predict  experimental this  membrane  However, t h e s e  concentrations The  cell  that  plants.  information  and P r e n d e v i l l e ,  12 h o u r s o f a f o l i a r  suggesting  dalapon,  ( C h r o w l e y and P r e n d e v i l l e ,  methyl  within  the  tests.  diclofop  occurred  atomic  incubations  herbicides  cell  cultured  i n 100uM  cells  herbicide  -48solutions.  A l t h o u g h d i c l o f o p m e t h y l was n o t t e s t e d  study f l u o r o d i f e n ,  ioxinil,  monolinuron, cycluron, increase with  fluorescein  in this  d i n o s e b , DNOC, d e s m e t r y n e ,  isoproturon leakage.  and monuron were f o u n d t o  These r e s u l t s a r e  consistent  t h e o b s e r v a t i o n s i n t h i s t h e s i s on DM and d i c l o f o p  on p r o t o p l a s t s  at similar  concentrations.  effects  -49DICLOFOP  METHYL AND  The a  time  2 . 5 u M DM  with to  DICLOFOP  profile  different protoplast  remove v a r i a b i l i t y DM  and  change  not  minutes. time  to  was  period.  significantly  i n appendix  picomoles  DM.  investigated The  at  time  Diclofop  constant  this  level  time  of  also  between  the  5.0  time  taken  up  contrasts,  periods  and  indicated  uptake  60.0  comparing a l l  at  that  any  were not  the  time  significant  are  metabolism  r a d i o l a b e l l e d DM  of  by  herbicide,  seconds  protoplasts)  blocks  protoplast  radiolabelled  between  conducted  as  Uptake values  d i c l o f o p uptake  taken  within  were  treated  from  expressed  as wa  not  time.  profile was  control  between  Possible  protoplasts  Experiments  were  d i f f e r e n t from  between  7) .  4.  freedom  (no  oat  5 seconds)  of  of  control  Differences  (ANOVA  addition  significantly  the  by  differences  I n d i v i d u a l degree  periods  control  to  r a p i d l y ( _<  following  did  i n Table  preparations  due  was  protoplasts  the  14C-DM u p t a k e  s o l u t i o n i s shown  preparations.  5.  of  UPTAKE  5.0  up  by  periods  protoplasts  seconds.  significantly were not  i s presented to  Treatment  Table  relatively  time  d i f f e r e n t , but  significant  a  in  periods  and  differences  (ANOVA  in  appendix  8) . Comparing indicate Forty into  DM  DM  and  uptake  percent  d i c l o f o p uptake  i s approximately  of  DM  in  protoplasts  in  1.0  diclofop uptake  was  values  taken for  up  the  (Tables 10  incubation  hour.  during  In a  times  4 and that  5) of  s o l u t i o n was  contrast,  s i m i l a r time  only  4.0%  the  data  diclofop.  taken  period.  d i c l o f o p make i n t e r p r e t a t i o n o f  the  up  of The data  low  -50-  Table  4  Time  profile  o f DM  uptake  Treatment  picomoles DM  control  48.4  5 seconds  198 .9  10 s e c o n d s  202.6  30  181 .5  seconds  1 minute  199 .0  10 m i n u t e s  192.2  60 m i n u t e s  167.5  T r e a t m e n t v a l u e s a r e means o f 3 e x p e r i m e n t s w i t h per t r e a t m e n t i n each e x p e r i m e n t .  3  replicates  T h e c o n t r o l r e p r e s e n t s p i c o m o l e s o f 14C-DM r e m a i n i n g o n t h e f i l t e r p a p e r , when p r o t o p l a s t s w e r e o m i t t e d f r o m t h e p r o c e d u r e DM c o n c e n t r a t i o n One p i c o m o l e  i n the incubation  i s equivalent  solution  t o 1 4 . 8 DPM  was 2.5uM  14C-DM.  -51-  Table  5 Time  profile  Treatment  control  of diclofop  uptake.  picomoles diclofop  5.8  5  seconds  23.9  10  seconds  19.9  30  seconds  2 3 .5  minute  21 .6  10  minutes  17 .6  60  minutes  25 .0  1  T r e a t m e n t v a l u e s a r e t h e means o f 3 e x p e r i m e n t s w i t h r e p l i c a t e s per treatment i n each experiment. The c o n t r o l r e p r e s e n t s p i c o m o l e s 1 4 C - d i c l o f o p f i l t e r p a p e r when p r o t o p l a s t s w e r e o m i t t e d . Diclofop  concentration  i n the incubation  One p i c o m o l e i s e q u i v a l e n t  t o 6.95 DPM  3  r e m a i n i n g on t h e  solution  was  14C-diclofop .  2,5uM  -52difficult. 3 4 9 0 DPM  For  resulted  protoplast. of  example,  This  radiolabel  Diclofop  in  DM  concentration  amount o f  Figure  DM  concentration  at  uptake  the  term  protoplasts.  cytoplasm The  of  uptake  protoplast freedom  for  to  from  i s no or  does  be  in  result  protoplasts. up  by  from  protoplasts.  2.OuM t o  relationship (appendix  solution.  .OuM  between  9). to  No  observed  50  The the  saturation  indicating  carrier-mediated  at  experiments.  the  in  this  incubation  experimental  absortion  of  DM  and  to  diclofop in  Table  were used  treatments  difference both  or  to  150DPM  the  i s proportional  was  been used  thesis  to  solution  evidence  to  membranes or  not  depend  on  diclofop  uptake  into  and  intact intact  intact  describe  by  the  distinguish  entry  or  intact  controls DM  and  uptake. DM  uptake  fragments,  viable  and  Individual  compare c o n t r o l  between  protoplast  into 6.  (burst  diclofop  burst  to  between  significant difference  protoplasts  observed  and  into  the  protoplasts.  contrasts  significant  no  has  i s presented  protoplasts)  observed  the  was  containing  be  the  uptake  incubation  these  removed  adsorption  DM  protoplasts  likely  in  There  of  concentrations  'uptake'  of  may  e f f e c t i v e l y taken  the  not  used  radiolabel  between  by  high  treatments  protoplasts surface  uptake  in  was  concentrations The  DM  solution  significant linear  DM  up  control  the  not  A  and  of  uptake DM  to  diclofop  by  profile  7.  taken  in  uptake  concentration  shown  that  low  2 ,5uM  DPM  adsorbing  is  DM  40  i s apparently  The  of  in  a  was  degree values  of (without  protoplasts). treatments However, into  A  was  there  was  intact  indicating  protoplasts.  protoplasts  burst  In  DM  uptake  contrast  significantly  - 5 3 -  F i g u r e 7.  C o n c e n t r a t i o n p r o f i l e o f d i c l o f o p methyl uptake i n oat p r o t o p l a s t s .  -54-  Table  6. U p t a k e o f DM a n d d i c l o f o p b y i n t a c t protoplasts .  concentration  2 .OuM  treatment  picomoles DM  control  and  burst  picomoles diclofop  41 .0  7.3  2 .0  burst  201 .2  14.3  2 .0  intact  210.4  24 .1  4 .0  control  6 2 .4  9 .7  4 .0  burst  3 9 6 .9  28 .1  4 .0  intact  4 3 2 .6  36 .8  8 .0  control  6 7 .0  11 .2  8 .0  bur s t  7 5 3 .3  36 .2  8 .0  intact  7 8 5 .9  42 .9  T r e a t m e n t v a l u e s a r e t h e means o f 3 e x p e r i m e n t s r e p l i c a t e s per treatment i n each e x p e r i m e n t .  with  3  Control values represent the picomoles o f herbicide o n t h e f i l t e r p a p e r when p r o t o p l a s t s w e r e o m i t t e d .  remaining  DM a n d d i c l o f o p e x p e r i m e n t s protoplast preparation .  t h e same  One p i c o m o l e i s e q u i v a l e n t 14C-dicolfop .  were  not conducted  t o 14.8 DPM  14C-DM  with  o r 6.95  DPM  -55greater This  than  uptake  difference  involving  It  counts  diclofop.  Since it  i s probably  in  this  a t very  and d i c l o f o p are the  log o f the  relatively  P=0.29 .  log  very  lipophilic  lipophilic lipid the  on i n t a c t  process.  both  Under  and 2.34  technique  herbicides  and burst  operation  repeated  protoplasts,  conditions  tested  up i n t o  that  ( l o g P ) o f DM  2,4-D, a coefficent  into  studies,  through  (1981)  and p r e f e r e n t i a l l y p a r t i t i o n  membranes.  than  Hall  were  equal  protoplast  is less  molecules.  had a p a r t i t i o n  In protoplasts  centrifugation  centrifugation  the  coefficents  a s the 5.0  This  would  uptake  tracer  into  both  intact  Vacuum  filtration  i s a useful  Methods,  and T a t t a r  gradients  into the  explain  time required  seconds.  (Rubinstein density  DM may p a r t i t i o n  fragments  t o remove unabsorbed  i nuptake  viable  DM a n d d i c l o f o p  u p t a k e o f DM a n d t h e  protoplasts  that o f  c a r r i e r mediated.  a t pH 5 . 3 .  I t was c o n c l u d e d  compounds.  protoplasts  up i n t o  than  taken  lipophilic  octanol/water  components o f c e l l  rapid  this  i s not  non-lipophilic herbicide,  with  measurements.  low rates.  d i c l o f o p t o be 3-11  and  of  a passive  depend  variable  up i s dependent upon t h e  and t h e r e f o r e  does not  conclusion,  r a p i d l y taken  thesis, diclofop i s apparently  protoplasts  found  DM i s  i s a process  i nview o f the  i s 10 t i m e s g r e a t e r  DM u p t a k e  DM u p t a k e  DM  that  concentration  Such a  wiith diclofop  T h e a m o u n t o f DM t a k e n  external  d i c l o f o p uptake  speculative  associated  c a n be c o n c l u d e d  protoplasts.  that  10) .  (ANOVA i n a p p e n d i x  protoplasts.  be c o n s i d e r e d  lowuptake  cells  may i n d i c a t e  intact viable  however, must and  by b u r s t  t o perform such as  1980) a n d  ( L i n 1980)  require  -56-  longer  time p e r i o d s .  these l a t t e r  The r a p i d u p t a k e o f DM and d i c l o f o p make  methods i n a p p r o p r i a t e  f o r studying  There a r e d i s a d v a n t a g e s t o the f i l t r a t i o n cannot  be removed f r o m t h e f i l t e r  resulting the  f r o m t h e vacuum  DM u p t a k e .  method.  Protoplasts  p a p e r s t o a s s e s s damage  filtration  procedure or to determine  c h e m i c a l form o f t h e h e r b i c i d e i n t h e p r o t o p l a s t s .  -57DISTRIBUTION  OF DM AND  The d i s t r i b u t i o n protoplast  of radiolabelled  suspension  the  applied  low  r e c o v e r y may h a v e  tubes  amount  used  been due t o l o s s  consistent The  of recovered  percent  of the recovered  pellet.  The c h e m i c a l  in  i n Table  the  external  DM.  Seventy  Only hydroxy the  supports  solution, seven  solution  taken  radiolabel  on t h e  t o reduce  the  t o be  i s shown  of the recovered  remains  percent  i n Table  was f o u n d  Eighty three  only  i n the external  diclofop  i s enzymatically converted  were  high  found  percentage  the results t h e ease  up a t a m u c h  protoplasts,  rate;  form.  as  radiolabel  In  the ester i n the  to diclofop. metabolite,  the external  a s DM  of the f i l t r a t i o n a t which  ester  solution  or  incubation period.  of radiolabel  lower  only  i n either  a 1.0 h o u r  of the radiolabel  remains  of the recovered  Sixty  radiolabel i s  i n the methyl  19.44 p e r c e n t  7a.  i n the protoplast  percent  s m a l l amounts o f t h e n o n - p h y t o t o x i c  demonstrating  the  7b.  protoplasts after The  during the  were u n s u c e s s f u l .  radiolabel  form  the protoplast pellet  external  relatively  r e c o v e r y was l o w , i t w a s f o u n d  and p r o t o p l a s t p e l l e t  presented  Efforts  of  and r e p r o d u c i b l e .  percent  solution  The  of radiolabel  i n the experiments.  although  50%.  rate  and a b s o r p t i o n o f r a d i o l a b e l  o f a d s o r p t i o n on g l a s s w a r e  Nonetheless,  The r e c o v e r y  was a p p r o x i m a t e l y  procedures  PROTOPLASTS  herbicide i n the  was d e t e r m i n e d .  radiolabel  centrifugation Pyrex  M E T A B O L I T E S I N OAT  DM  i n the protoplast  uptake  i s taken  experiments  up.  Diclofop  of the recovered  1 4 . 3 % was d i c l o f o p ,  even  was  radiolabel i n  though  analysis  -58-  Table  7a.  Distribution of radiolabel i n protoplast suspensions .  Component  percent  Protoplast External  of recovered  pellet  60.6 +  2.30  solution  39.4 +  2.30  radiolabel  V a l u e s a r e t h e mean + standard error o f four experiments 3 repeated determinations f o r each component i n each experiment.  Table  7b.  Chemical  Percent composition of r a d i o l a b e l i n the p r o t o p l a s t s and t h e e x t e r n a l s o l u t i o n a f t e r 1.0 h o u r i n c u b a t i o n .  form  Protoplast  pellet  External  solution  DM  83 .4 +  1 .06  19 .4 +  1 .44  Diclofop  14  .3  +  1 .06  77.7  +  1 .50  2  .3  +  0.33  2.8 +  0 .30  Hydroxy  with  diclofop  V a l u e s a r e t h e means + s t a n d a r d e r r o r o f 4 e x p e r i m e n t s repeated determinations i n each experiment.  with  3  -59of the e x t e r n a l radiolabel  s o l u t i o n showed t h a t a l a r g e  i n the e x t e r n a l  These data are  also  solution  consistent  with  e x p e r i m e n t s w h i c h showed r e l a t i v e l y In  a similar  recovered  radiolabel  one hour to  s t u d y Dusky et  i n the a c i d  the r e s u l t s  al.(1980)  medium.  These r e s u l t s  both the c e l l  c o n f l i c t w i t h the  protoplast former.  A major  difference  suspensions i s  the presence  after  a  found  and t h e  cell  this  the  cultures  and  o f the c e l l w a l l  in  the  constituents  r e s u l t i n g i n the a s s o c i a t i o n o f a  large  methanol-soluble  extracts. The p e l l e t  protoplasts,  was d i s p e r s e d i n d i s t i l l e d w a t e r t o r u p t u r e  and t h e n c e n t r i f u g e d  two f r a c t i o n s ;  water-insoluble two f r a c t i o n s with  extract  found i n  p r o p o r t i o n o f r a d i o l a b e l as d i c l o f o p i n t h e  into  the  extract  D i c l o f o p may be a s s o c i a t e d w i t h c e l l w a l l  i n the c e l l c u l t u r e s ,  cell  diclofop.  D i c l o f o p was  between c e l l  the  filtration  f i n d i n g s of  where DM was t h e main m e t a b o l i t e  protoplasts.  cell  of  form.  f o u n d 55% o f  i n c u b a t i o n w i t h r a d i o l a b e l l e d DM. in  of  low u p t a k e o f  in a methanol-soluble  be t h e main m e t a b o l i t e  thesis  was  percentage  water-soluble  components  fraction  bulk of  fraction  the  protoplasts  c o m p o n e n t s and  ( F i g u r e 4)  showed t h a t t h e  the w a t e r - s o l u b l e  to d i v i d e  Protein  analysis  The  o f the t o t a l  suggest  the presence  present  i n the  insoluble  e x a m i n a t i o n of the protoplast  of  phospholipid. large  fraction.  insoluble material  fragments  of  Light  the  associated  but a  This observation  quantities  of  water-insoluble  c o n t a i n e d o n l y s m a l l amounts o f p r o t e i n  percentage  the  p r o t e i n was  ( T a b l e 8)  the  lipid  large would  membranes  microscopic  showed t h e p r e s e n c e  and s w o l l e n c h l o r o p l a s t s .  It  was  of  assumed  -60-  Table  8  Protein soluble  and and  soluble  Protein  mg/ml  Phosphorus  ug/ml  phosphorus c o n t e n t of the water insoluble protoplast fractions.  fraction  insoluble  1.64  0.46  1.01  5.27  fraction  P r o t e i n c o n t e n t was d e t e r m i n e d on t h e w a t e r - s o l u b l e i n s o l u b l e f r a c t i o n s of the protoplasts. P h o s p h o r u s c o n t e n t was d e t e r m i n e d on t h e w a t e r - s o l u b l e and i n s o l u b l e f r a c t i o n s o f  and  l i p i d e x t r a c t of the p r o t o p l a s t s .  the  -61that  the  water-insoluble  and o r g a n e l l e s structures,  with  while  fraction  substantial  the  water  predominantly cytoplasmic  included protoplast  amounts  soluble  of  lipid  fraction  proteins  fragments  membrane  contained  and low m o l e c u l a r  weight  constituents . T a b l e 9 shows the protoplast pellet,  pellet  two  and t h e  thirds  of  N i n e t y one  ester  DM.  one  half  half  as  the  insoluble large  as  large  the  the  results  lipid  recovered  of  in  the  this  the  solubility  the  in  phospholipids,  contention  the  that  was  in  and  one  The r e c o v e r y fraction, is  not  water  the of  which  surprising 1981).  DM may p a r t i t i o n Most o f  the  the  and DM i n  DM ( H a l l ,  protoplasts.  protoplasts  acid  solution,  of  in  r a d i o l a b e l were  water-insoluble  high l i p i d  protoplast  approximately  pellet.  of  membranes  of  the  water-insoluble  diclofop  external  quantities  the  the  fraction  fractions  the  in  In t h e  r a d i o l a b e l was  as  protoplast  DM i n  support  from  of  present  largest  of  solution.  water-soluble  diclofop  quantities  considering These  The two  fraction  contained  percent  In the  radiolabel within  r a d i o l a b e l was  r a d i o l a b e l was  DM.  distributed  external  the  fraction. form,  d i s t r i b u t i o n of  the  into  diclofop  soluble  fraction. There the  presence  1) l e a k a g e  of  of  external  several  diclofop  diclofop  2)  hydrolysis, the  are  possible i n the  out  of  mechanism t h a t  external  solution  protoplasts  after  of  i n t r a c e l l u l a r hydrolase  solution,  or  3)  surface  of  the  plasmalemma.  rapidly  converted  to  diclofop  It in  has  plants  (Table  enzymes  enzymes on the  been  shown  explain 7b);  intracellular  leakage  hydrolase  could  that  into  external  DM i s  by a h y d r o l a s e  enzyme  -62-  Table  9  The d i s t r i b u t i o n o f r a d i o l a b e l w i t h i n p r o t o p l a s t s and t h e e x t e r n a l s o l u t i o n of total recoverable radiolabel.  External  DM  9.1  diclofop Hydroxy diclofop Total  (1)  + 0 .94  30 .5 + 2 .31  1.2 + 0 .07 40.7  +  3.87  (1) Percent composition the p r o t o p l a s t s .  Soluble  (2)  8 .6 + 0 .77 8-9+1  .41  0 .9 +  0.13  18.3+  1 .81  the as a  percent  I n s o l u b l e (3)  37.5  +  2.56  2 .7 + 0 .74 0.8  + 0 .08  41 .0 j _ 2 .80  of the e x t e r n a l s o l u t i o n  surrounding  (2) Percent c o m p o s i t i o n o f the w a t e r - s o l u b l e components t h e p r o t o p l a s t s . S e e F i g u r e 4.  of  (3) Percent c o m p o s i t i o n o f the w a t e r - i n s o l u b l e components t h e p r o t o p l a s t s . S e e F i g u r e 4. with each  of  V a l u e s a r e t h e means + standard error of four experiments 3 r e p e a t e d d e t e r m i n a t i o n s f o r each c h e m i c a l component i n experiment.  -63-  Table  Chemical  10  form  Determination of hydrolase a c t i v i t y protoplast incubation solution.  E x t e r n a l (1)  E x t e r n a l (2)  in  Sorbitol c o n t r o l (3)  DM  19.4+  1 .44  79.3  Diclofop  77 .7 +  1 .50  19 .7 + 2 .09  2.3  +  0.33  1 .0 + 0 .07  0.8  + 0 .06  Hydroxy diclofop  2.3  +  +  2.14  96 .9 +  1 .83 0.14  (1) P e r c e n t c o m p o s i t i o n o f r a d i o l a b e l i n the external s o l u t i o n a f t e r one hour i n c u b a t i o n s w i t h p r o t o p l a s t s . (2) P e r c e n t c o m p o s i t i o n o f r a d i o l a b e l i n t h e e x t e r n a l s o l u t i o n a f t e r a one hour i n c u b a t i o n w i t h t h e e x t e r n a l s o l u t i o n f r o m w h i c h p r o t o p l a s t s had been removed a f t e r one h o u r . (3)  Percent  composition  of the r a d i o l a b e l  in sorbitol  V a l u e s a r e means + s t a n d a r d e r r o r o f 4 e x p e r i m e n t s r e p e a t e d d e t e r m i n a t i o n s f o r each measurement i n each experiment.  buffer with  3  - 6 4 -  (Hill  et a l . 1980).  within  the plant  10 c o m p a r e diclofop  in sorbitol  of  after  but before  conversion  protoplast  The after were  protoplasts  addition.  i n the external removal  hydrolase  which  conversion  o f DM  protoplasts  removed  10)  had  leaked  one  20%  w h e n DM  was  or  into the  periods,  should  the percent  be c o m p a r a b l e  hour  f o r leakage  However,  the level  plasmalemma.  This  o f DM  lower  of conversion  reasonably molecules  only  enzymes  when  o f DM  would  were  of  the external  hydrolase  cultures.  activity  I t was  solution.  on t h e  that  degree  present.  removal  this  was  may  medium due  of  The  hydrolase  Dusky e t a l . (1980)  i n the c e l l - f r e e  suggested  incubated  protoplasts  be a c o n s e q u e n c e o f d i s s o c i a t i o n o f some into  and  enzymes.  located  were  1.0  to  r e s u l t i n a high  protoplast  the  solutions with  i n the absence  protoplasts  after  were  during  protoplasts  enzymes  solution  protoplasts  of intracellular  of surface  association  conversion rate  cases,  of conversion  f o r the presence  when  conversion  i n external  for  to allow  than  out of the protoplast  In both  old  of  o f an i n t r a c e l l u l a r  I f the hydrolase  protoplasts.  reported  detection  a  t h e a r g u m e n t f o r DM  i s lower  without  argues  after  to d i c l o f o p i n the external  but leaked  incubation  diclofop  were  (Table  intracellular,  one  solutions  solution  supports  hydroxy  removed  The  i n Table  solution.  present  hour  DM  in external  via extracellular activity  membrane a s s o c i a t e d external  r e s u l t s presented  to that  or with  t h e l a b e l as d i c l o f o p  added  The  enzyme  o f 14C-DM t o d i c l o f o p a n d  buffer  protoplasts,  incubation  the l o c a t i o n of t h i s  i s not known.  the conversion  containing hour  However,  from  also  4-day  to leakage  of  -65hydrolase An  enzymes  membrane  permeability  present.  enzymes  However,  these  detectable  these The  lower  i n membrane  be t h e  Increased  i n leakage  of  s o l u t i o n w h e n DM i s  the concentrations  leakage  i n t h e medium.  r e s u l t s may  b y DM.  the external  w e r e much  increases  caused  cells  o f DM a n d  than  those  ethanol  which  resulted  permeability  d o e s n o t seem  a valid  interpretation  data. r e s u l t s presented  insoluble  Comparatively,  present  d i c l o f o p were  to  propose  that  found this  of hydrolase  membranes.  small  Although taken  distribution enzymes  remains  inside  appears  t o be a s s o c i a t e d  largest  accumulations  water-insoluble  such  may  the protoplasts, with  significant  amounts  I t i s logical  be a r e s u l t o f t h e with  the protoplast  hydrolase  activity  to diclofop  solution.  activity The  radiolabel are located  f r a c t i o n a s DM a n d i n t h e e x t e r n a l  may  which  most o f t h e h y d r o l a s e  the external  of applied  the water  as t h e c e l l  solution.  associated  up b y t h e p r o t o p l a s t s  into  quantities of diclofop  some i n t r a c e l l u l a r  DM  o f DM  However,  i n the external  convert  diclofop .  only  i n the protoplasts.  of  activity  i n d i c a t e uptake  components o f t h e p r o t o p l a s t  membranes. were  since  lyzed  f o r these  an i n c r e a s e  into  experiments  herbicide-induced of  or from  permeability  may p r o d u c e  intracellular  in  cells  alternative explanation  increased  in  from  i n the  s o l u t i o n as  -662,4-D  E F F E C T S ON DM UPTAKE AND M E T A B O L I S M  The  uptake  not  affected  one  hour  taken  o f 14C-DM a n d l 4 C - d i c l o f o p by t h e  incubation  up by t h e  filtration The  procedures  incubation  experiments o f which  and  13a.  0.5N  At higher  (Tables  Individual values  without  significant (ANOVA  addition.  The  diclofop 13a).  are  To a v o i d  s u c h pH  buffer  solution  i nTables  the  Hepes  was a d j u s t e d  contrasts  11a, 12a  buffer  to  7.0  comparing  t o treatment values  between  control  with  without  into  linear  comparing  protoplasts  was n o t  o f 10uM t o  2,4-D.  e f f e c t on the  However, a t h i g h e r  means  e f f e c t o f 2,4-D  o f 2,4-D a t c o n c e n t r a t i o n s  i n t h ep r o t o p l a s t  showed a  However, c o n t r a s t s  no s i g n i f i c a n t  control  and treatment  d i f f e r e n t a t 2,4-D c o n c e n t r a t i o n s  inverse  vacuum  s o l u t i o n w a s 1 0 . 0 mM, a n d t h e  o f freedom  showed  a significant  significant  .  presented  vial  11 a n d 1 2 ) .  treatments  Radiolabel not  1 2 b , 13b).  protoplasts  addition  have  data  a  changed t h e  i n a 5.OmM H e p e s  DM a n d d i c l o f o p u p t a k e  2.OmM f r o m  not  11b,  difference  significantly  by t h e  2,4-D c o n c e n t r a t i o n s  degree  means,  11 a n d 1 2 ) .  solution  treatment  i nappendix  treatment  2,4-D d u r i n g  previously.  i n t h eincubation  pH o f e a c h  NaOH  herbicide  was removed  described  for  initial  (Tables  2,4-D w a s p r e p a r e d  concentration  o f the  p r o t o p l a s t s was  o f 2,4-D t o t h e p r o t o p l a s t s  pH o f t h e  effects,  period  protoplasts  addition  initial  addition  into  o f 0 t o 50uM  conversion  o r t h ee x t e r n a l  o f DM t o  solution  2,4-D c o n c e n t r a t i o n s  did  (Table  (Table  r e l a t i o n s h i p b e t w e e n 2,4-D  13b) a  -6?-  Table  11a  The e f f e c t  o n DM  uptake  Treatment  DM u p t a k e picomoles  control  34 .1  0  Table  o f 2,4-D  2,4-D  175 .4  10uM  210 .4  25uM  177 .6  50uM  206 .0  11b  Treatment  control 0.0  2,4-D  DM u p t a k e picomoles 21 .8 179 .1  100uM  2 0 3 .4  500uM  194 . 3  1mM  181.1  2mM  185 . 3  Values i n Tables are the results of 2 d i f f e r e n t experiments. T r e a t m e n t v a l u e s a r e t h e means o f 3 r e p l i c a t e s p e r t r e a t m e n t . C o n t r o l v a l u e s r e p r e s e n t r a d i o l a b e l r e m a i n i n g on t h e f i l t e r p a p e r when p r o t o p l a s t s w e r e o m i t t e d f r o m t h e i n c u b a t i o n solution .  -68-  Table  12a  The e f f e c t  o f 2,4-D  Treatment  control 0.0  Table  2,4-D  uptake  Diclofop uptake picomoles 9.8 16 .4  10.OuM  17.1  25 .OuM  20.0  50.OuM  17.8  12b  Treatment  control 0.0  Values Values  on d i c l o f o p  2,4-D  Diclofop uptake picomoles 4.1 13.9  100uM  12.2  500uM  9 .9  1 .OmM  10 .6  2.OmM  12.4  i n Tables are the r e s u l t s o f 2 d i f f e r e n t experiments. a r e t h e means o f 3 r e p l i c a t e s p e r t r e a t m e n t .  Controls represent radiolabel when p r o t o p l a s t s were o m i t t e d  r e m a i n i n g on t h e f i l t e r paper from t h e i n c u b a t i o n medium.  -69-  Table  13a  The e f f e c t o f 2,4-D diclofop acid.  Percent  on  of recovered  the  conversion  radiolabel  External solution .OuM  81 .5  10 .9  10  .OuM  83 .0  10 .5  25  .OuM  82 .0  11 .3  50  .OuM  81 .2  10.3  2,4-D  of  recovered r a d i o l a b e l  External solution 79 .0  12 .4  100.0  uM  78 .3  10 .4  5 0 0 .0  uM  77 .0  9 .5  1  .OmM  66 .7  8.3  2  .OmM  74 .3  8.0  The v a l u e s i n T a b l e s experiments. are  diclofop  the  13a  means o f  and  as  diclofop  Protoplast pellet  uM  Values  to  13 b  Percent  0 .0  DM  Protoplast pellet  0  Table  as  of  13b  are  the  3 replicates  per  results  of  treatment.  different  -70-  c o n c e n t r a t i o n and c o n v e r s i o n t o d i c l o f o p i n t h e p e l l e t significant  inverse  q u a d r a t i c r e l a t i o n s h i p to c o n v e r s i o n i n  external  s o l u t i o n was  that  quadratic  the  (ANOVA i n a p p e n d i x 1 3 ) .  effect  i s u n l i k e l y to r e f l e c t The d e c r e a s e  and a  It  is  the  likely  i s due t o an e x p e r i m e n t a l e r r o r  and  any i m p o r t a n t b i o l o g i c a l r e l a t i o n s h i p .  i n DM c o n v e r s i o n may be a r e s u l t o f  p h y s i o l o g i c a l a c t i o n o f 2,4-D i n t e r f e r e n c e  with  the hydrolase  activity. It  may be t h a t  the decrease  2,4-D c o n c e n t r a t i o n s i s extrusion stimulated would generate  p o s s i b l e decrease extract  (Marre et a l .  1973).  This  i n DM h y d r o l y s i s s i n c e t h e pH optimum o f  and 8 . 0  (Figure 8).  However,  was f o u n d t o measurements  pH o f t h e  e x t e r n a l s o l u t i o n showed no c h a n g e f r o m t h e  pH o f 7 . 0  d u r i n g the  1.0  hour  1979),  a higher  the ester  of  starting  .  However,  r a d i o l a b e l was  This hydrolase a c t i v i t y Hill  et a l .  It  Q u e r e s h i and Vanden B o r n  i n d i c a t i n g the presence o f a c t i v e  (1980)  2,4-D  Born, in  was  interfered with  DM was c o n v e r t e d t o d i c l o f o p i n a c r u d e  extract vitro  and Vanden  o f the recovered  the phenoxy h e r b i c i d e s  the h y d r o l a s e enzyme.  that  MCPA ( Q u e r e s h i  percentage  the  incubation.  f o r m t h a n when DM was a p p l i e d a l o n e .  suggested that of  1980) o r  an  lie  When DM a p p l i c a t i o n t o w i l d o a t s was c o m b i n e d w i t h (Todd and S t o b b e ,  effect  pH i n t h e e x t e r n a l s o l u t i o n and a  from o a t s w i t h h y d r o l a s e a c t i v i t y  between 7.0  higher  t h e r e s u l t o f i n c r e a s e d H+ i o n  by 2,4-D  a lower  i n DM c o n v e r s i o n a t  activity  (1979)  found  supernatant  hydrolase  enzymes  in  c o u l d be i n h i b i t e d by MCPA.  i s o l a t e d and p a r t i a l l y p u r i f i e d an  enzyme f r a c t i o n w i t h h y d r o l a s e  activity  from w i l d oat  plants.  -71-  Figure 8.  pH p r o f i l e of hydrolase a c t i v i t y .  Hydrolase a c t i v i t y i the percent r a d i o l a b e l as d i c l o f o p i n the enzyme extract + standard error Controlsi the percent radioabel as diclofop i n enzyme extracts boiled f o r 1.0 hour.  -72The  isolated  data of  e n z y m e was  presented  Hill  caused  et  in this  decrease  Research  into  results  response  different  reason  a wide  thesis. in  range  2,4-D  hydrolase  activity  to  field  response  studies.  physiologiclal  inhibited beans .  of  2,4-D  high  2,4-D  The  the  results of  2,4-D  diclofop.  2,4-D  often  produces  because of  varying  ranges.  concentrations  was  tested  2mM,  were o b s e r v e d ,  are  unrealistically  Ashton  responses  et  a l . (1977)  1 0 0 u M 2,4-D  in etiolated  tested  found  ( photosynthesis, lipid  where  this  decreases  in  high  growth  most  respiration,  synthesis) leaf  this  in  and  concentrations  plant  For  1mM  r a t e s and  of  (10uM).  concentrations  concentration  p r o t e i n s y n t h e s i s and at  2,4-D  consistent with  literature  concentrations  compared  synthesis,  i n the  of  by  h y d r o l y s i s to  activity  conflicting to  Only very  i n DM  the  inhibited  t h e s i s are  al.(1980).  any  not  cells  RNA  were of  red  kidney  -73GENERAL  The  use  eliminates the  of  and  upon  while  possible  1979)  only  these  examine  at  DM  rate  diclofop.  and  of  DM  highest  difference of  the  may  DM  or  is  cell  dependent  membranes.  (Chrowley plant  thesis  and  membranes, i t  i n t e r f e r e with  membrane  indicated  protoplast  untreated  d i c l o f o p on  concentrations  used  that  DM  membrane  (100uM)  concentration  from  the  known  i n membrane p e r m e a b i l i t y DM  and  level.  integrity  e f f e c t s on  d i c l o f o p were t a k e n u p t a k e was  While  concentrations process,  DM  potential  tested the  problem  symplast.  DM  ten  uptake  were inducing  protoplasts.  Any  membrane p e r m e a b i l i t y i n these  experiments  The  by  protoplasts.  greater  than  not  saturable  at  was  rate  up  times  i n these  low  membrane p e r m e a b i l i t y .  the  cellular  in  was to  uptake.  Both  uptake  of  l i m i t e d e f f e c t s on  influence  negligible  establishment  results in this  changes  penetration spaces  cellular  adverse  uptake  intracellular  the  herbicides  The  the  significant  herbicide  c h a r a c t e r i s t i c s of  have  Observed  with  direct  the  that  d i c l o f o p had  small  system,  herbicide  including d i c l o f o p methyl  processes.  integrity.  any  at  herbicides,  uptake and  of  semi-permeable  Prendeville, is  rapid  herbicide  study  with  facilitating  a protoplast  the  Since  associated  to  partitioning into  concentrations In  protoplasts  problems  cuticle  apoplast  plant  DISCUSSION  of  experiments  suggesting  for  d i c l o f o p to  and  d i c l o f o p are  observation reach both  the  of  the  d i c l o f o p uptake  latter  that  However  a  reflects may  site  passive a  indicate of  phytotoxic  action but  may  low a in  -74induce  d i f f e r e n t p h y s i o l o g i c a l responses  et  a l .  1979;  be  reflected in different sites  causing  1979).  Todd,  different plant  lipophilic  molecules  (log  3.11  P) o f  probable  that  DM  The d i f f e r e n t i a l  with  uptake  respectively occurs  lipid  phase o f p r o t o p l a s t s  the  rapid  and r e l a t i v e l y  and  intact protoplasts  factor even  determining  though  However,  physiological  than If  DM  immobilized  (1969)  effect  reported the  here  treatment  The r a t e  explain  lipophilic  molecule  o f DM i n of  noted  which would  i n plants  T h e DM  to partition  by  absorbed  i s low  be  levels  into oat  (1979)  i t may  i s converted  to diclofop  become  i n plant  spaces.  phase This  protoplasts  o f DM  ester  and Shimabukuro  by n o n s p e c i f i c  less  Penniston  (1979) . DM  less  alone.  out of the l i p i d  and i n t e r c e l l u l a r  by T o d d  be t h e o n l y  herbicide.  of lipophilicity  uptake  burst  be d i s s o c i a t e d a t  p h a s e a s was p r o p o s e d  the rapid  explain  o f d i c l o f o p uptake  o n t o membranes  apoplast  into  by b o t h  cannot  and t h e low t r a n s l o c a t i o n r a t e zone  would  proportion  permeability  compounds.  be u n a b l e  t h e aqueous  would  o f DM u p t a k e  a high  on t h e b a s i s  for lipophilic  into  This  accumulations  a charged  i n the l i p i d  membranes w o u l d and  lipid  adsorbed  very  Iti s  absorption  membranes and r e s u l t i n u p t a k e  predicted  rapidl  1981).  (Hall  g r o u p o n d i c l o f o p may  to plant  those  rate  i s a relatively  pH g i v i n g  may  thus  and d i c l o f o p a r e  membranes.  and l a r g e  uptake.  diclofop  the acid  permeable  high  However,  o f uptake  i n plants  v i a a passive  fractions containing  phospholipids.  DM  rate  (Shimabukuro  the l o g of the p a r t i t i o n c o e f f i c i e n t s  the  protoplast  of action  responses.  2.34  and  i n plants  hydrolase  out of et a l .  -75enzymes. the  Large  external  diclofop  s o l u t i o n which  produced  hydrolysis hydrolase the  or  presented  in this with  converting  DM  to  or  thesis  have  protoplasts.  the  diclofop  translocated acropetal  transport  plants,  conjugates. sugars  are  parenchyma its  cells  glycosyl  fashion  and  meristems. faster this  Geiger  rate  thesis  directly  the  data was  diclofop The  in  the  results  r e a d i l y taken that  remain  into  the  the  limited  in  of  up  most  by of  the  symplast.  phloem  and  basipetal  diclofop  the  with  may  be  translocated  to  be  and  conjugates  glycosyl  Giaquinta  from  associated  have  phloem  to  the be  into  tissue.  into site  of  a r e s u l t of  DM  and  diclofop  Diclofop  the  in  action up  that  specialized  phloem  taken  as  ester  (1976) observed  apoplast  loaded  d i c l o f o p may  of  to  expected  enter  and  of  with  .  ( 1 9 7 4 ) and  conjugates  can  by  activity  plant.  be  DM  protoplasts,  i s not  moving  i s converted  diclofop  the  of  solution.  would  However  1979)  Conjugated than  than  efflux  associated  Analysis  converted  apoplast  or  the  in  solution  hydrolase  of  i t might  diclofop  conjugates then  that  of  either  external  external  be  identified  intracellular  the  diclofop  known.  diclofop  loaded  the  diclofop  of  from  cytoplasm,  spaces  i n the  (Todd,  the  would  spaces rather  i s not  been o b s e r v e d In  in  that  apoplastic  in  surface  Therefore,  present  intercellular the  shown  by  indicates  diclofop DM  result  were  protoplasts.  intercellular  oat  Whether  the  external  intact plants,  apoplasts  from  of  diclofop  could  produced  thesis  the  of  cytoplasm  leaked  surface  associated  this  i n the  diclofop  enzymes  external  In  accumulations  a  similar  in  plant  in plants  sugar  or  at  a  moiety.  represented  only  In a  -76small is  percentage  consistent  radiolabel al.  of the recovered  with  was  other  reports  conjugated  after  where o n l y 1.0  hour  This  observation  1 .0% o f  (Todd,  recovered  1979; Dusky e t  1980) . Analysis  that  while  o f the r a d i o l a b e l i n the protoplasts  m o s t o f t h e r a d i o l a b e l was  14.27% o f t h e r e c o v e r e d diclofop. uptake  This  membranes. much  small  of diclofop  intracellular  less  f r a c t i o n may  from  hydrolysis The  than  rate  that  o f DM  absorbed  i n the external  solution.  applications  reduce  Previous reduce  sections  conversion  o f DM  than  and  h a s shown  DM  or d i c l o f o p  and Vanden  Born,  t o d i c l o f o p and  into oat protoplasts.  to the incubation  observed  pattern  protoplasts. unaffected  of uptake  The c o n v e r s i o n  b y 2,4-D  2,4-D  medium  of  apparently  a n d DM  that  i n the  result  2,4-D recovered  1979; Todd,  that  2,4-D  from  1979)  i n root may  However,  inhibit  the addition  the  t o 50 uM.  previously  also  However, a t  (1.0mM a n d 2.OmM) t h e r e  was  and  and  a f f e c t the uptake  t o d i c l o f o p was up  of  loss of control of  found  thus  found  or d i c l o f o p into oat  o f DM  concentrations  concentrations  2,4-D  d i d not a l t e r  o f DM  i s  that  of 14C-diclofop  I t has been s u g g e s t e d  o f DM  slow  s o l u t i o n , a s t h e amount  the translocation of 14C-diclofop  the  higher  t h e amount  (Quereshi  shoot meristems.  2,4-D  research  was  protoplast  hydrolysis  i s much l e s s  a loss of herbicidal activity  plant  to  a p p l i c a t i o n s of the herbicides  weeds.  form,  s o l u t i o n or because  of intracellular  external  grassy  i n the ester  be t h e r e s u l t o f t h e  the external  i n the protoplasts  Combined  indicated  r a d i o l a b e l i n the protoplast  diclofop  in  radiolabel.  a  of of  -77decrease  i n DM c o n v e r s i o n .  decreasing unchanged  pH o f t h e e x t e r n a l throughout  significant, slight, were  a n d t h e 2,4-D  physiological  was c o n c l u d e d diclofop intact  the  incubation.  h i g h compared plant  2,4-D  effects  i s unknown  capacities  of these  Born,  The r e a s o n plants  1979;  very decrease  a n d DM  Todd,  with  e t a l . 1977).  It  o f DM o r system.  In  resulted  a s DM r e c o v e r e d  i n an  from t h e  1979).  fractions  However were  f o r t h e d i s c r e p a n c y between  and  those  be t h e r e s u l t  systems.  was  this  uptake  h y d r o l a s e enzyme  b u t may  Although  i n the oat protoplast o f 2,4-D  remained  associated  (Ashton  does n o t a f f e c t  of isolated  i n intact  to those  responses  the application  by 2,4-D.  a s t h e pH  i n hydrolase activity  ( Q u e r e s h i and Vanden  unaffected  system  hour  i n the percent radiolabel  activity  2,4-D  that  solution  was n o t due t o  concentrations inducing  o r DM h y d r o l y s i s  plants  increase plants  t h e 1.0  the decrease  unrealistically  normal  This decrease  i n the protoplast  of differing  buffering  -78CONCLUSION  The  use  technique studies the  of oat  t o s t u d y DM  where  herbicide  due  difficulties surrounding  cuticle to the  separating tissues.  of the  the  between  considerable, experimental Extrapolation  diclofop  large  quantities of  the  uptake  cells  with  f o r use  isolation  these disavantages.  to  a t any  be  repeated  as  one  a  time  with  the  in plant  was  and differences.  protoplasts However  cells  to  the  protoplast  Further exploration  metabolism  arise  preparations  appropriate.  associated  of  procedures.  r e s u l t s from  be  cause  relatively  may  the e f f e c t s of such  uptake  always  that  In  used,  T h i s would  suspensions are  experiments  outweigh  warranted .  into  available  and  examine h e r b i c i d e  n a t u r e o f DM.  technical difficulties  design to reduce  simplicity  metabolism.  absorbs  different protoplasts  not  and  been  f o r the  o f t h e DM  sucessful  segments have  benefits  to  uptake  a  plant  volumes  requiring  t i s s u e may  t o be  or  Protoplast  time required  Variability  intact  small  proved  lipophilic  to h a n d l e , however  result and  and  intact plants  lipophilic  easy  protoplasts  of the  system system  is certainly  -79BIBLIOGRAPHY A g b a k o d a , C . S . O . and J . R . G o o d i n . 1 9 7 0 . A b s o r p t i o n t r a n s l o c a t i o n o f 1 4 C - l a b e l e d 2,4-D and p i c l o r a m i n Bindweed. Weed S c i . 1 8 : 1 6 8 A n d e r s o n , R . N . 1 9 7 6 . Response o f m o n o c o t y l e d o n s and HOE 2 3 4 0 8 . Weed S c i . 2 4 : 2 6 6 - 2 6 9 -  and Field  t o HOE 22870  A s h t o n , F . M . , O . T . De M i l l i e r s , R . K . G l e n n and W.B. D u e c k . 1977. The l o c a l i z a t i o n o f m e t a b o l i c s i t e s o f a c t i o n o f herbicides. P e s t i c . Biochem. P h y s i o l . 7:122 B h a n , V . M . , E . W . S t o l l e r , and F.W. S l i f e . 1 9 7 0 . T o x i c i t y , a b s o r p t i o n , t r a n s l o c a t i o n and m e t a b o l i s m o f 2,4-D i n Y e l l o w N u t s e d g e . Weed S c i . 1 8 : 7 3 3 - 7 3 7 . B l e i n , J . 1 9 8 1 . A c t i o n o f some h e r b i c i d e s o n • g r o w t h , r e s p i r a t i o n , plasmalemma i n t e g r i t y , and p r o t o n of Acer pseudoplatanus c e l l s . 1. S u b s t i t u t e d u r e a s . Biochem. P h y s i o l . 16:179-186.  extrusion Pest.  B l e i n , J . 1 9 8 2 . A c t i o n o f some h e r b i c i d e s on g r o w t h , r e s p i r a t i o n , plasmalemma i n t e g r i t y , and p r o t o n e x t r u s i o n o f Acer pseudoplatanus c e l l s . 2.Amides, diphenyl e t h e r s , n i t r i l e s , p h e n o l s , t r i a z i n e s , and u r a c i l s P e s t . Biochem. P h y s i o l . 17:156-161 . B l i g h , E . G . and W . J . D y e r . 1959- R a p i d method o f t o t a l e x t r a c t i o n and p u r i f i c a t i o n . Can. J . Biochem. P h y s i o l . 911-917.  lipid 37:  B o l d t , P . F . and A . R . P u t m a n n . 1 9 8 0 . S e l e c t i v i t y m e c h a n i s m s f o l i a r a p p l i c a t i o n s o f d i c l o f o p m e t h y l . 1. R e t e n t i o n , a b s o r p t i o n , t r a n s l o c a t i o n and v o l a t i t l i t y . Weed S c i . 28:474-477 .  for  B r a d f o r d , M.M. 1 9 7 6 . A r a p i d and s e n s i t i v e method f o r t h e q u a n t i t a t i o n of microgram q u a n t i t i e s of p r o t e i n u s i n g the p r i n c i p l e of protein-dye b i n d i n g . A n a l . Biochem. 7 2 : 2 4 8 - : 2 5 4 . B r e z e a n u , A . G . , D . G . D a v i s and R . H . S h i m a b u k u k u r o . 1 9 7 6 . U l t r a s t r u c t u r s a l e f f e c t s and t r a n s l o c a t i o n o f methyl-2-(4-(2,4-dichlorophenoxy)-phenoxy)propanoate i n wheat ( T r i t i u m a e s t i v u m ) and w i l d o a t ( Avena f a t u a ) . C a n . J . B o t . 54:2038-2047 .  -80-  B r i g g s , G .G., R.H. B r o m i l o w , R. Edmonson and M. J o h n s t o n . 1 9 7 6 . D i s t r i b u t i o n c o e f f i c i e n t s and s y s t e m i c a c t i v i t y . I n H e r b i c i d e s and f u n g i c i d e s f a c t o r s a f f e c t i n g t h e i r a c t i v i t y . E d . N.R. M c F a r l a n e . p. 1 2 9 - 1 3 4 .  Chow, P.N.P. 1975 . A b s o r p t i o n o f h e r b i c i d e s by wheat a s i n f l u e n c e d by t h e p h e n o x y compound. J . A g r i c . Food Chem. 23:730-735 . Chow, P.N.P. 1 9 7 6 . S e l e c t i v i t y and s i t e o f a c t i o n i n r e l a t i o n t o f i e l d p e r f o r m a n c e o f d i c l o f o p . Weed S c i . 2 6 : 3 5 2 - 3 5 7 . Chow, P.N.P. and D.E. L a B e r g e . 1978 . W i l d o a t h e r b i c i d e s t u d i e s 2. P h s i o l o g i c a l c h e m i c a l c h a n g e s i n b a r l e y and w i l d o a t s treated with d i c l o f o p methyl herbicide i n r e l a t i o n to plant t o l e r a n c e . J . A g r i c . F o o d Chem. 2 6 : 1 1 3 4 - 1 1 3 7 . C h r o w l e y , J . and G.N. P r e n d e v i l l e . 1 9 7 9 . E f f e c t s o f d i c l o f o p m e t h y l on l e a f c e l l p e r m e a b i l i t y i n w i l d o a t s , b a r l e y and wheat. Can. J . P l a n t S c i . 59:275-278. C h r o w l e y , J . and G.N. P r e n d e v i l l e 1 9 8 0 . E f f e c t o f h e r b i c i d e s on d i f f e r e n t modes o f a c t i o n o f l e a f c e l l membrane p e r m e a b i l t y in P h a s e o l u s v u l g a r i s . Can J . P l a n t S c i 6 0 : 6 1 3 - 6 2 0 . C o l l a n d e r , R. 1 9 5 4 . The p e r m e a b i l i t y o f n o n - e l e c t r o l y t e s . P h y s i o l . P l a n t . 7:420.  Nitella  c e l l s to  C r i s p , C.E. 1 9 7 2 . The m o l e c u l a r d e s i g n o f s y s t e m i c i n s e c t i c i d e s and o r g a n i c f u n c t i o n a l g r o u p s i n t r a n s l o c a t i o n . P r o c . Sec Internat. IUPAC C o n g r . P e s t . Chem. v o l 1. e d . A.S. T a h o r i . G o r d o n Nd B r e a c h . L o n d o n .  D a v i s , D.G. and A . B r e z e a n u . 1 9 7 9 . The u l t r a s t r u c t u r a l s t u d y o f T r i t i c u m monococcum c e l l s u s p e n s i o n c u l t u r e s d u r i n g a g i n g and a f t e r treatment with the h e r b i c i d e d i c l o f o p methyl (methyl-2-(4-(2,4-diclorophenoxy)phenoxy)propanoate). Can.J.Bot. 57:2006-2020. D e v l i n , R.M. and R.W. Y a h l i c h . 1 9 7 2 . I n f l u e n c e o f two p h e n o x y g r o w t h r e g u l a t o r s on u p t a k e and a c c u m u l a t i o n o f n a p t h a l a m by bean p l a n t s . P h y s i o l . Plant.27:317-320 .  - 8 1 -  D o n a l d , W.W. a n d R.H. S h i m a b u k u r o . 1 9 8 1 . S e l e c t i v i t y o f d i c l o f o p m e t h y l between wheat and w i l d o a t g r o w t h and h e r b i c i d e m e t a b o l i s m . P h y s i o l . P l a n t . 49:459-464. D u s k y , J . A . , D .G. D a v i s a n d R.H. S h i m a b u k u r o . 1 9 8 0 . M e t a b o l i s m of d i c l o f o p methyl (methyl-2-(4-(24 -dichlorophenoxy)phenoxy)propanoate) i n c e l l s u s p e n s i o n s o f d i p l o i d wheat ( T r i t i c u m monococcum ) . P h y s i o l . P l a n t . 49:151-156. 1  Eames, A . J . 1950. D e s t r u c t i o n o f phloem i n young bean a f t e r t r e a t m e n t w i t h 2,4-D. Am. J . b o t . 3 7 : 8 4 0 - 8 4 7 .  plants  E d w a r d s , G.E., R. McC. L i l l e y , S. C r a i g , a n d M.D. H a t c h . 1 9 7 9 . I s o l a t i o n o f i n t a c t and f u n c t i o n a l c h l o r o p l a s t s f r o m m e s o p h y l l a n d b u n d l e s h e a t h p r o t o p l a s t s o f C4 p l a n t Panicum miliaceum . P l a n t P h y s i o l . 63:821-827. F i s k e , C.H., a n d Y S u b b a R o w . 1 9 2 5 . T h e c o l o r i m e t r i c d e t e r m i n a t i o n o f Phosphorus. J.Biol.chem 66:375-400. F r e i s e n , H.A., P.A. O ' S u l l i v a n a n d W.H. V a n d e n B o r n . 1976 . HOE 2 3 4 0 8 , A new s e l e c t i v e h e r b i c i d e f o r w i l d o a t s a n d g r e e n f o x t a i l i n wheat and b a r l e y . Can. J . P l a n t . S c i . 5 6 : 5 6 7 - 5 7 8 . G a f f , D.F. a n d 0. O k o n g ' 0 - o g o l a . 1 9 7 0 . The u s e o f non-permeating pigments f o r t e s t i n g the s u r v i v a l of c e l l s . Exp. B o t . 22:756-757.  J.  G e i g e r , D.R., J . M a l o n e a n d D.A. C a t a l d o . 1 9 7 1 . S t r u c t u r a l e v i d e n c e f o r a t h e o r y o f v e i n l o a d i n g o f t r a n s l o c a t e . Amer. J . Bot. 58:672-675. G i a q u i n t a , R. 1 9 7 6 . Evidence f o r phloem l o a d i n g from the a p o p l a s t : C h e m i c a l m o d i f i c a t i o n o f membrane s u l f h y d r y l g r o u p s . P l a n t P h y s i o l . 57. 872-875. G o r b a c h , S.G., K. m e t a b o l i s m o f Hoe 25:507-51 1 .  Kuenzler 2 3 4 0 8 OH  a n d J . A s s h a u e r . 1 9 7 7 . On t h e i n w h e a t . J . A g r i c . F o o d Chem.  G o r e c k a , K. ,R.H. S h i m a b u k u r o a n d W.C. Walsh. 1981. A r y l h y d r o x y l a t i o n : A s e l e c t i v e mechanism f o r the h e r b i c i d e s d i c l o f o p - m e t h y l and c l o f o p - i s o b u t y l , i n g r a m i n a c e o u s s p e c i e s . P h y s i o l . P l a n t . 53:55-63.  -82-  Guy, M., L . R e i n h o l d , and G.C. L a t i e s . 1 9 7 8 . Membrane t r a n s p o r t o f s u g a r s and amino a c i d s i n i s o l a t e d p r o t o p l a s t s . P l a n t P h y s i o l . 61: 5 9 3 - 5 9 6 . Guy, M., L . R e i n h o l d , and M. R a h a t . 1 9 8 0 . E n e r g i z a t i o n o f s u g a r t r a n s p o r t m e c h a n i s m i n t h e plasmalemma o f i s o l a t e d mesophyll protoplasts. P l a n t . P h y s i o l . 65:550-553H a l l , C. 1 9 8 1 . M.Sc. t h e s i s . U n i v e r s i t y o f G u e l p h . G u e l p h , Ontario . H i l l , B.D., B.G. Todd and E.H. S t o b b e . 1 9 8 0 . E f f e c t o f 2,4-D on t h e h y d r o l y s i s o f d i c l o f o p m e t h y l i n w i l d o a t s ( Avena f a t u a ) . Weed S c i . 28: 7 2 5 - 7 2 9 . H o e r a o u f , R.A. and R.H. S h i m a b u k u r o . 1 9 7 9 . The r e s p o n s e o f r e s i s t a n t and s u s c e p t i b l e p l a n t s t o d i c l o f o p - m e t h y l . Weed R e s . 19:293-299. Hoppe, H.H. 1 9 8 0 . E f f e c t s o f d i c l o f o p - m e t h y l on g r o w t h and d e v e l o p m e n t o f Zea mays L . s e e d l i n g s . Weed R e s . 20:371-376.Abstr . Hoppe, H.H. 1 9 8 1 . E f f e c t s o f d i c l o f o p m e t h y l on p r o t e i n s y n t h e s i s , n u c l e i c a c i d and l i p i d b i o s y n t h e s i s i n t i p s o f r a d i c l e s o f Zea mays Z. P l a n z e n p h y s i o l B d . 1 0 2 . s . 189-197. Abstr .  Kao . K.N., F. C o n s t a b e l , M.R. M i c h a y l u k and 0 .L . Gamborg. 1 9 7 4 . P l a n t p r o t o p l a s t f u s i o n and g r o w t h o f i n t e r g e n e r i c h y b r i d c e l l s . P l a n t a 120:215-227. K e n n e d y , C.D. and R . A . S t e w a r t . 1 9 7 9 . E f f e c t s o f 2 , 4 - d i c h l o r o p h e n o x y a c e t i c a c i d on i o n u p t a k e by m a i z e J . o f Exp.Bot. 31:135-150.  roots.  K i r k w o o d , R.C., J . D a l z i e l , B . M a t l i b and L . S o m e r v i l l e . 1 9 7 2 . The r o l e o f t r a n s l o c a y t i o n i n s e l e c t i v i t y o f h e r b i c i d e s w i t h r e f e r e n c e t o MCPA and MCPB. P e s t i c . S c i 2:307-321 . K i r k w o o d R.C. 1 9 7 6 . Some c r i t e r i a d e t e r m i n i n g p e n e t r a t i o m and t r a n s l o c a t i o n o f f o l i a g e a p p l i e d h e r b i c i d e s . I n H e r b i c i d e s and f u n g i c i d e s : F a c t o r s a f f e c t i n g t h e a c t i v i t y . E d t . N.R. McFarlane .  -83K i r k w o o d , R.C. 1978. The u p t a k e and t r a n s l o c a t i o n o f foliar a p p l i e d h e r b i c i d e s u s i n g an e x p l a n t s y s t e m . JEn Advances i n p e s t i c i d e s c i e n c e . P a r t 3. p 4 1 0 - 4 1 5 . E d . H . G e i s s b u h l e r . Lehninger, N .Y ., N .Y .  Lillie, Wilkins  R.D. Co.  A .L.  1976  .  Biochemistry  Worth  1969. H.J. Conn's B i o l o g i c a l Baltimore.  L i n , W. 1 9 8 0 . Corn r o o t c h a r a c t e r i z a t i o n of ion  Publishing,  Stains.  p r o t o p l a s t s , i s o l a t i o n and transport. Plant Physiol.  Inc.  Williams  and  general 66: 550-554  M a r r e , E., P. L a d o , F. R a s i c a l d o g n o a n d R. C o l u m b o . 1973. C o r r e l a t i o n b e t w e e n c e l l e n l a r g e m a n t i n pea i n t e r n o d e s e g m e n t s a n d d e c r e a s e s i n pH o f t h e m e d i u m o f i n c u b a t i o n . Plant. Sci. L e t t e r s 1 :179-184 .  M a r t i n , R.A. and L.V. E d g i n g t o n . 1981. Comparative systemic t r a n s l o c a t i o n of several xenobiotics and s u c r o s e . P e s t . Biochem. P h y s i o l . 16:87-96.  M e t t l e r , I . J . and R. T. L e o n a r d . 1 9 7 9 . Ion t r a n s p o r t in isolated protoplasts f r o m t o b a c c o s u s p e n s i o n c e l l s . 1. G e n e r a l c h a r a c t e r i s t i c s . P l a n t . P h y s i o l . 63: 183-190. N e s t l e r , H.J, P. L a n g e l u d d e k e , H. S c h o n o w s k y a n d F. S c h w e r d t l e 1978. P h e n o x y - p h e n o x y - p r o p i o n i c a c i d s and d e r i v a t i v e s as g r a s s h e r b i c i d e s . I n A d v a n c e s i n P e s t i c i d e S c i e n c e P a r t 2. p. 2 4 8 - 2 5 5 . E d t . H. G e i s s b u h l e r . P e r g a m o n P r e s s . O ' S u l l l i v a n , P .A .and V a n d e n B o r n . 1975 . Wild oat herbicide c o m b i n a t i o n s . Dept. P l a n t S c i . , U n i v . A l b e r t a , Edmonton, A l b e r t a . 97pp. O l s o n , W.A. and diclofop methyl  J.D. N a l e w a j a . 1982. E f f e c t s o f MCPA on 14C u p t a k e a n d t r a n s l o c a t i o n . Weed S c i . 3 0 : 5 9 - 6 3  O w i n o , M.G. 1977. E f f e c t o f d i c l o f o p m e t h y l on t h e d e v e l o p m e n t o f w h e a t , b a r l e y , w i l d o a t s , and g r e e n M.Sc. T h e s i s . U n i v e r s i t y of M a n i t o b a . 120pp.  growth and foxtail.  -84-  P e n n i s t o n , J . J . , L . B e c k e t , D.L. B e n t l e y and C.Hansen. 1 9 6 9 . P a s s i v e p e r m e a t i o n o f o r g a n i c compounds t h r o u g h b i o l o g i c a l t i s s u e : a non s t e a d y s t a t e t h e o r y . M o l . P h a r m a c o l . 5: 3 3 3 - 3 4 1 . P e t e r s o n , C.A. and L.V. E d g i n g t o n 1 9 7 6 . E n t r y o f p e s t i c i d e s i n t o t h e p l a n t s y m p l a s t a s m e a s u r e d by t h e i r l o s s f r o m an ambient s o l u t i o n . P e s t i c . S c i . 7:483-491. P o o l e . R . J . 1978. Energy c o u p l i n g Rev. P l a n t P h y s i o l . 29: 4 3 7 - 4 6 0 .  f o r membrane t r a n s p o r t . A n n .  P r i c e , C.E. 1 9 7 6 . P e n e t r a t i o n and t r a n s l o c a t i o n o f h e r b i c i d e s and f u n g i c i d e s i n p l a n t s . I n H e r b i c i d e s and f u n g i c i d e s : F a c t o r s a f f e c t i n g t h e i r a c t i v i t y . E d . N.R . M c F a r l a n e . p 4 2 - 6 6 . P r i c e , C.E. 1 9 7 8 . Movement o f x e n o b i o t i c s i n p l a n t s - P e r s p e c t i v e s . I n advances i n p e s t i c i d e s c i e n c e . p. 401-409- E d . H. G e i s s b u h l e r .  Part  3.  Q u e r e s h i , F.A. and W.H. Vanden B o r n . 1 9 7 9 . I n t e r a c t i o n o f d i c l o f o p m e t h y l and MCPA on w i l d o a t s ( Avena f a t u a ) Weed S c i 27: 2 0 2 - 2 0 5 . R o b e r t s o n , M. M. and R.C. K i r k w o o d . 1 9 7 0 . Mode o f a c t i o n o f f o l i a g e applied translocated herbicides with particular e v i d e n c e t o p h e n o x y - a c i d compounds. Weed R e s . 10. 9 4 - 1 2 0 . R u b e r y , P.H. 1 9 7 8 . H y d r o g e n i o n d e p e n d e n c e o f c a r r i e r m e d i a t e d a u x i n u p t a k e by s u s p e n s i o n c u l t u r e d g a l l c e l l s . P l a n t a 142:203-206 . R u b i s t e i n , B. and T.A. T a t t a r . 1 9 8 0 . R e g u l a t i o n o f amino a c i d u p t a k e i n t o o a t m e s o p h y l l c e l l s : A c o m p a r i s o n between p r o t o p l a s t s and l e a f s e g m e n t s . J . E x p . B o t . 31 : 2 6 9 - 2 7 1 . S h i m a b u k u r o , M.A., R.H. S h i m a b u k u r o , W.S. N o r d . a n d R.A. H o e r a u f . 1978. P h y s i o l o g i c a l e f f e c t s o f m e t h y l 2 - ( 4 - ( 2 , 4 - d i c h l o r o p h e n o x y ) p h e n o x y ) p r o p a n o a t e on o a t w i l d o a t and w h e a t . P e s t i c . B i o c h e m . P h y s i o l . 8:199-207. S h i m a b u k u r o , R.H., W.O. Walsh and R.A. H o e r a u f . 1 9 7 9 . M e t a b o l i s m and s e l e c t i v i t y o f d i c l o f o p m e t h y l i n w i l d o a t and w h e a t . J . A g r i c . F o o d . Chem. 2 7 : 6 1 5 - 6 2 3 .  -85S m e j t e k , p . a n d M. P a u l i s - I l l a n g a s e k a r e . 1979. M o d i f i c a t i o n ion transport i n l i p i d b i l a y e r membranes i n t h e p r e s e n c e o f 2,4-dichlorophenoxyacetic acid. Biophys, J . 26:441. Smith, A.E. 1977. D e g r a d a t i o n o f t h e h e r b i c i d e d i c l o f o p in prairie soils. J . A g r i c . F o o d Chem. 25:893-899.  of  methyl  T o d d , B.G.and E.H. S t o b b e . 1 9 7 7 . S e l e c t i v i t y o f d i c l o f o p m e t h y l among w h e a t , b a r l e y w i l d o a t a n d g r e e n f o x t a i l . Weed S c i . 25:382-385 . T o d d , B.G. 1 9 7 9 . s e l e c t i v i t y a n d m e t a b o l i s m o f d i c l o f o p m e t h y l i n w h e a t , b a r l e y , w i l d o a t , a n d g r e e n f o x t a i l . PhD. T h e s i s . U n i v e r s i t y o f Manitoba, Winnipeg. 187pp.  Wu, G.H. activity  a n d P.W. S a n t e l m a n n . 1 9 7 6 . P h y t o t o x i c i t y o f Hoe 2 3 4 0 8 . Weed S c i 2 4 : 6 0 1 - 6 0 4 .  and  soil  -86-  Appendix  1.  Analysis of variance with i n d i v i d u a l degree of freedom contrasts f o r data presented i n Table l a . The e f f e c t of ethanol on the leakage of neutral red dye from oat protoplasts Treatments 1-5 represent ethanol concentrations of 0.0% 0.1%, 1.0%, 5.0% and 10.0% respectively  A N A L Y S I S OF VARIANCE SOURCE  DF  BLOCK TREAT BXT ERROR TOTAL ********  2 4 8 30 44  SUM SO  »•******<  TREAT  1 2 3 4  BXT  .3.  .2.  0.3140 CONTRAST 1 2  -0.00000 -0.00001  0.36005 0.60956E-01 0.51557E-02 0.40933E-03  0.23280E-26 0.19376E-02 O.10141E-06  1 - OOOOO  -o.OOOOO  1 .OOOOO  0-SOUARED 0.243636 0.100932E-03 0.621993E-04 0.239995E-04  IS  DUNCAN'S M U L T I P L E RANGE 3.2570 3.3960  0.8945E-02 TEST.  0.5023  4  3  CONTRAST LINEAR QUAD CUB DEV  .5.  0.4119  0.3303  0.3133  1.OOOOO -0.OOOOO -O.OOOOO  FOR CONTRASTS  PR0B  879.59 11.823 12.595  .4 .  F-VALUE 47.255 0.19577E-01 0.12064E-01 0.46549E-02  TREATMENT SUM OF SQUARES 0.243824 SUM OF Q SQUARED 0.243823 D I F F E R E N C E OR RESIDUAL 0.279808E-06 AVERAGE F-RATIO 11.8229 D I F F E R E N C E IS 0 . 1 1 4 7 6 E - 0 3 PERCENT OF TREATMENT TIME  F-VALUE  .**************************************************  MN DENSITY  1.OOOOO -0.OOOOO  ERROR  MEAN SO  0.72009 0.24382 0.41246E-01 0.12280E-01 1 .0174  1 .  CONTRAST  - DENSITY  SUM OF  PROBABILITY 0.00013 0.89218 0.91524 0.94728  SQUARES  SECONDS.  RANGES 3.4775  FOR  ALPHA=0.05 3.5179  THERE ARE 3 HOMOGENEOUS SUBSETS ( S U B S E T S OF ELEMENTS. NO PAIR OF WHICH D I F F E R BY MORE THAN THE SHORTEST S I G N I F I C A N T FOR A SUBSET OF THAT S I Z E ) WHICH ARE L I S T E D AS FOLLOWS ( 2, 1. 3) ( 4) ( 5)  RANGE  *********  -67-  Appendix  2.  A n a l y s i s o f v a r i a n c e w i t h i n d i v i d u a l degree of freedom c o n t r a s t s f o r data presented i n Table l b . The e f f e c t s o f e t h a n o l on the leakage o f K from o a t p r o t o p l a s t s . Treatment numbers 1 - 4 r e p r e s e n t e t h a n o l concent r a t i o n s o f 0.0%, 0.1%, 1.0% and 10.0% respectively.  ANALYSIS OF VARIANCE SOURCE  OF  SUM SO  - DENSITY  MEAN SO  ERROR  F-VALUE  PROB  0.29690E-11 97.596 0.91727 1 .8345 BLOCK 2 0.38821E-03 33.333 BXT 2.5236 7.5708 TREAT 3 0.78078E-04 8.0552 0.75708E-01 BXT 0.45425 6 0.93986E-02 ERROR 0.22557 24 TOTAL 35 10.085 ************ *************.****************************************************** TREAT  FREQUENCIES 9 MN DENSITY 0.9050 CONTRAST 1 2 CONTRAST 1 1.00000 2 3  1 2 3  0.OOOOO O.OOOOO  .4.  .2.  1 .  1•OOOOO O.OOOOO  CONTRAST LINEAR QUAD DEV  9 0.9114  9 0.9054  9 1.966  1.OOOOO  Q-SQUARED 7.51287 0.578681E-01 0.316594E-05  PROBABILITY F-VALUE 0.OOOO6 99.235 0.41559 0.76436 0.99505 0.41818E-04  TREATMENT SUM OF SQUARES 7.57076 SUM OF 0 SQUARED 7.57074 DIFFERENCE OR RESIDUAL 0.168373E-04 AVERAGE F-RATIO 33.3331 DIFFERENCE IS 0.22240E-03 PERCENT OF TREATMENT SUM OF SQUARES TIME FOR CONTRASTS IS  0.8633E-02 SECONDS.  DUNCAN'S MULTIPLE RANGE TEST, RANGES FOR ALPHA«0.05 3.4605 3.5867 3.6452 THERE ARE 2 HOMOGENEOUS SUBSETS (SUBSETS OF ELEMENTS. NO PAIR OF WHICH DIFFER BY MORE THAN THE SHORTEST SIGNIFICANT RANGE FOR A SUBStT OF THAT SIZE) WHICH ARE LISTED AS FOLLOWS ' ( 1. 2. 3) ( 4 )  -88-  Am^ndix 3 Appendix 3.  Analysis of variance with i n d i v i d u a l degree An * t a presented i n ~ + « «-p nwt on r>n leakage 1 palraffe of of Table 2a. The e f f e c t s of DM neutral red dye from oat protoplasts. c  _  .  .  -  o  n  t  r  a  s  t  s  f  o  r  d a  r r , i . .  Treatment numbers 1-4 represent DM concentrations of O.OuM, lOuM, 50uM, and lOOuM r e s p e c t i v e l y .  ANALYSIS OF VARIANCE DF  SOURCE BLOCK TREAT BXT ERROR TOTAL  5 3 15 44 67  TREAT  1 .  SUM SO  - DENSITY  MEAN SO  0.76937 0.15292E-01 0.95965E-02 0.86710E-02 0.80293  ERROR  0. 15387 0.50973E-02 0.63977E-03 0.19707E-03  BXT  F-VALUE 780.82 7.9675 3.2464  PROB 0.27756E-16 0.20709E-02 O.11854E-02  *  .****»»*******»».  0.2996 CONTRAST 1 2  MN DENSITY  CONTRAST 1 1.OOOOO 2 -O.OOOOO 3 -O.OOOOO  1 2 3  0.3179  0.3029  0.3375  3  1.OOOOO -O.OOOOO  CONTRAST LINEAR OUAD DEV  .4.  .3.  2.  1.OOOOO Q-SQUARED 0.152867E-01 0.442057E-05 0.791969E-06  PROBABILITY F-VALUE 0.00020 23.894 0.93485 069097E-02 0.97240 O.12379E-02  O.152919E-01 TREATMENT SUM OF SQUARES O.152919E-01 SUM OF 0 SQUARED 0.307532E-07 DIFFERENCE OR RESIDUAL 7.96744 AVERAGE F-RATIO DIFFERENCE IS 0.20111E-03 PERCENT OF TREATMENT SUM OF SQUARES TIME FOR CONTRASTS IS  0.8789E-02 SECONDS.  DUNCAN'S MULTIPLE RANGE TEST. RANGES 3.0090 3.1564 3.2575 THERE ARE 2 HOMOGENEOUS SUBSETS NO PAIR OF WHICH DIFFER BY MORE THAN FOR A SUBSET OF THAT SIZE) WHICH ARE ' ( (  1 . 2 . 3 ) 4)  FOR  ALPHA-0.05  (SUBSETS OF ELEMENTS. THE SHORTEST SIGNIFICANT RANGE LISTED AS FOLLOWS  -89-  Appendix  4.  A n a l y s i s o f v a r i a n c e w i t h i n d i v i d u a l degree o f freedom c o n t r a s t s f o r data presented i n Table 2b. The e f f e c t s o f DM on leakage o f K from oat p r o t o p l a s t s . Treatments numbered 1-4 r e p r e s e n t DM c o n c e n t r a t i o n s o f O.OuM, 10uM, 50uM and lOOuM r e s p e c t i v e l y . ANALYSIS  SOURCE BLOCK TREAT BXT ERROR TOTAL  DF  SUM SO  2 3 6 24 35  18332 04 4 0.11878 0.23916  «*»«*»***»***•  TREAT  VARIANCE  DENSITY  M E A N SO  ERROR  0 91659 0.15075 0.19797E-01 0.99651E-02  0.8990 CONTRAST  BXT  1 .OOOOO 0-SOUARED 0.430388 0.342104E-02 0.184268E-01  IS  0.8359E-02  D U N C A N ' S M U L T I P L E RANGE 3.4605 3.5867 THERE  ARE  NO P A I R FOR ( ( '(  OF  3  TEST.  HOMOGENEOUS  WHICH D I F F E R  A S U B S E T OF T H A T 2. 1) 1 . 3 ) 3. 4)  *«**  3  2  1.OOOOO -O.OOOOO  CONTRASTS  0.55820E-11 °-^OBSE-Ol 0.10744  1 . 149  BY  SIZE)  SUM OF  SQUARES  SECONDS.  RANGES 3.6452  SUBSETS MORE  PROBABILITY 0.00346 0.69209 0.37191  F-VALUE 21.740 0 . 17281 0.93080  0.452237 T R E A T M E N T SUM OF S Q U A R E S 0.452236 SUM OF 0 S Q U A R E D 0.550734E-06 D I F F E R E N C E OR R E S I D U A L 7.61467 AVERAGE F-RATIO DIFFERENCE IS 0 . 1 2 1 7 8 E - 0 3 P E R C E N T OF T R E A T M E N T FOR  91.980 7.6147 1.9866  ......».»**»  1 .049  0.8749  CONTRAST LINEAR QUAD DE V  TIME  PROB  F-VALUE  .3.  .2.  1 CONTRAST 1 1.OOOOO 2 O.OOOOO 3 0.0  1 2 3  -  ,.,*•?:??*?•*•••••**•••*  . 1  MN D E N S I T Y  OF  FOR  ( S U B S E T S OF  T H A N THE  WHICH ARE  ALPHA=0.05  SHORTEST  LISTED  AS  ELEMENTS. SIGNIFICANT  FOLLOWS  RANGE  *  -905« A n a l y s i s o f v a r i a n c e w i t h i n d i v i d u a l degree o f freedom c o n t r a s t s f o r data presented i n Table 3a. The e f f e c t s o f d i c l o f o p on leakage o f n e u t r a l r e d dye from o a t p r o t o p l a s t s .  Appendix  Treatment numbers 1-4 r e p r e s e n t d i c l o f o p c o n c e n t r a t i o n s o f O.OuM, 10uM,50uM and lOOuM respectively. t  ANALYSIS OF VARIANCE  MEAN SO  SUM S O  DF  SOURCE  - DENSITY  0.64444E-01 0.87679E-03 026229E-03 O.14537E-03  O.19333 3 O.26304E-02 3 TREAT 0.23G06E-02 9 BXT 0.40703E-02 28 ERROR 0.20239 43 TOTAL „».*,*.***************** BLOCK  0.2198 CONTRAST 1 2  MN DENSITY  CONTRAST 1 .OOOOO 1 0.OOOOO 2 0.OOOOO 3  .3 .  .2.  .1 .  1•OOOOO  -0.00000  CONTRAST LINEAR QUAD DEV TREATMENT SUM OF SQUARES SUM OF Q SQUARED DIFFERENCE OR RESIDUAL  0.2341  0.2285  443.32 3.3428 1.8043  BXT  »...».»...**..***************  TREAT  F-VALUE  ERROR  TI  0.69389E-16 0.69599E-01 O.11203  *»*•**********************'  .4 .  0.2409  3  1.ooooo 0-SOUARED 0.234263E-02 0.108916E-03 O. 178784E-03  F-VALUE 8.9313 0.41525 0.68162  PROBABILITY 0.01524 0.53539 0.43037  g^gg"^ °/SioE-07 VXSK  ^ FFERlNCE"rS S.13171E-02 PERCENT OF TREATMENT SUM OF SQUARES E  PROB  -91-  Appendix  6.  Analysis of variance with i n d i v i d u a l degree of freedom contrasts f o r data presented i n Table 3b. The e f f e c t s of d i c l o f o p on leakage of K from oat protoplasts. Treatment numbers represent d i c l o f o p concentrations of O.OuM, lOuM, 50uM, and lOOuM respectively.  A N A L Y S I S OF VARIANCE SOURCE  .  BLOCK TREAT BXT ERROR TOTAL  TREAT  DF 2 3 € 24 35  .1  SUM SO  MEAN SO  1 .5869 0.28757E-01 0.20840E-01 0.20526 1 .8417  92.775 2.7598 0.40613  BXT  0.50944E-1 1 O . 13407 0.86760  .*.  .3. 9  TREATMENT SUM OF SOUARES SUM OF Q SQUARED D I F F E R E N C E OR RESIDUAL AVERAGE F-RATIO D I F F E R E N C E IS 0.49558E-03  PROB  F-VALUE  .«.»..*.*....  .2.  CONTRAST LINEAR QUAD DEV  ERROR  0.79345 0.95858E-02 0.34734E-02 0.85524E-02  FREQUENCIES 9 MN DENSITY 0.7809 0.7968 CONTRAST 1 2 3 CONTRAST 1 1.OOOOO 2 O.OOOOO 1.OOOOO 3 o.O -0.OOOOO 1.OOOOO  1 2 3  - DENSITY  9  9  0.8531  0.7898  Q-SQUARED 0.225978E-01 O.430441E-02 0.185507E-02  F-VALUE 6 .5059 1 .2392 0.53408  0.287574E-01 0.287573E-01 O. 142516E-06 2 .75975 PERCENT OF TREATMENT  PROBABILITY 0.04345 0.30822 0.49244  SUM OF SQUARES  -92-  Appendix  ?.  A n a l y s i s o f v a r i a n c e w i t h i n d i v i d u a l degree o f freedom c o n t r a s t s f o r data presented i n Table 4. Time p r o f i l e o f DM uptake. Treatment numbers 1-7 r e p r e s e n t c o n t r o l s , 5.0 seconds, 10 seconds, 3 0 seconds, 1.0 minute, 10 minutes, and 60 minutes r e s p e c t i v e l y .  ANALYSIS SOURCE DATS TREAT D X T ERROR TOTAL STANDARD  OF VARIANCE  SUM SO  DF  -  YIELD ERROR  MEAN SO  476.27 2 O.16339E+06 6 25682. 12 37499. 42 0.22705E+06 62 D E V I A T I O N OF VARIABLE  238.14 27232. 2140.2 892.84 1 IS  D X T  F-VALUE 0.26672 12.724 2.3970  PROB 0.76718 . 0.13735E-03 O.18272E-01  60.515  * TREAT  9 198 .9 32.39  FREQUENCIES 9 MN Y I E L D 1 48.42 SD Y I E L D 1 16.12 CONTRAST 1 CONTRAST 1 1.OOOOO  CONTRAST CONTROL / TREAT  TREATMENT SUM OF SQUARES SUM OF Q SQUARED DIFFERENCE OR RESIDUAL AVERAGE F-RATIO DIFFERENCE I S 4.9615 TEST OF DIFFERENCE  .4.  .2.  1.  9 181.5 31 . 9 5  9 202.6 29.56  Q-SQUARED 155283.  F-VALUE 72.556  163390. 155283. 8106.52 72.5562 PERCENT OF TREATMENT  G I V E S AN F - V A L U E OF  0.75756  9 199.0 40.84  PROBABILITY 0.OOOOO  SUM OF SQUARES AND FPROB OF 0 . 5 9 6 9 1  9 192.2 39.01  9 167.5 39 .55  -93-  Appendix  8.  Analysis of variance with i n d i v i d u a l degree of freedom contrasts f o r data presented i n Table 5. Time p r o f i l e of d i c l o f o p uptake. Treatment numbers 1-7 represent c o n t r o l , 5 seconds, 10 seconds, 30 seconds, 1.0 minute 10 minutes, and 60 minutes respectively.  A N A L Y S I S OF VARIANCE SOURCE DAYS TREAT D X T ERROR TOTAL  FREQUENCIES. ************ TREAT  PICAMOLE  DF  SUM SO  MEAN SO  2 6 12 35 55  5388.1 2095.9 1377.9 3221 . 5 12083.  2694.0 349.32 114 . 8 2 92.044  ERROR  PROB  F-VALUE 29.269 3.0422 1 .2475  D X T  0.33799E-07 0.47765E-01 0.29198  MEANS. STANDARD D E V I A T I O N S ****************** ,**************************' ********************** .6. .5. .4 . .3. . 1.  Q-SQUARED 1787.94  CONTRAST TREAT/CONT  TREATMENT SUM OF SQUARES SUM OF Q SQUARED D I F F E R E N C E OR RESIDUAL AVERAGE F-RATIO DIFFERENCE IS 14.694 OF D I F F E R E N C E  GIVES  8 23.54 17.25  8 19.87 12.60  8 23.87 18.91  FREQUENCIES 8 MN PICAMOLE 5.772 SD PICAMOLE 2.291 CONTRAST 1 CONTRAST 1 1.OOOOO  TEST  -  F-VALUE 15.571  2095.93 1787.94 307.986 15.5710 PERCENT OF TREATMENT AN F - V A L U E  OF  0.53644  8 21 . 6 0 1 1 .49  PROBABILITY 0.00194  SUM OF SQUARES AND  FPROB OF 0 . 7 4 5 3 3  8 17 .64 8.64 1  .7.  -9k~  Appendix  9.  Analysis of variance with i n d i v i d u a l degree of freedom contrasts f o r the DM concentration p r o f i l e shown i n Figure 7* Treatment numbers 1-6 represent DM concentrations of 2.0, k.0, 8.0, 15.0, 25.0, and 50.0uM DM respectively. A N A L Y S I S OF VARIANCE SUM  DF  SOURCE  PICAMOLE  MEAN SO  SO  0.43989E+07 0.75B59E+08 0.86616E+07 0.46198E+07 0.93539E+08  4 5 20 54 83  BLOCK TREAT BXT ERROR TOTAL  -  O.10997E+07 0 . 15172E+08 0.43308E+06 85551.  ERROR  PROB  F-VALUE 12.854 35.032 5.0622  BXT  0.20291E-06 0.31295E-08 0.99872E-06  F R E Q U E N C I E S . MEANS. STANDARD D E V I A T I O N S «»»»»»«*»* *»»»*«»•»***•**»***»*•••***************' »»*«»•*•*•»•***•**************  FREQUENCIES MN PICAMOLE SD PICAMOLE  14 115.1 31 .94  .6.  .3.  .2.  . 1 .  TREAT  14 894 .4 209.7  14 479.7 138.8  14 221 . 0 71 . 16  14 1493 . 397.8  CONTRAST 1  CONTRAST 1 1.OOOOO 2 O OOOOO 3 0 OOOOO  1•OOOOO -O.OOOOO  -O.OOOOO  O.OOOOO  4 5  -0  ooooo  CONTRAST LINEAR OUAD CUB QUART DEV  -0.00000  1•OOOOO  -0.00000 O.OOOOO  1 .OOOOO -O.OOOOO  Q-SQUARED 0.758166E+08 39214.4 851 .021 6.84923 2390.84  1.OOOOO PROBABILITY F-VALUE O.OOOOO 175.06 0.76659 0.9054BE-01 0.96508 0 . 19650E-02 0.99687 O.15815E-04 0.94151 0.55206E-02  0.758591E+08 TREATMENT SUM OF SQUARES 0.758590E+08 SUM OF 0 SQUARED 66.3195 D I F F E R E N C E OR R E S I D U A L 35.0324 AVERAGE F-RATIO D I F F E R E N C E IS 0 . 8 7 4 2 5 E - 0 4 PERCENT OF TREATMENT  SUM OF  SQUARES  14 2872. 1064.  -95Analysis of variance with i n d i v i d u a l degree of freedom contrasts f o r data presented i n Table 6. Uptake of DM and d i c l o f o p by burst and i n t a c t protoplasts.  Appendix 10,  Treatment numbers 1-3 represent DM uptake i n control, burst and i n t a c t protoplasts at  2.OuM DM, 4-6 at 4.OuM DM and 7-9 at 8.OuM  DM.  ANALYSIS DF  SOURCE BLOCK TREAT BXT ERROR TOTAL  SUM SO  MEANS,  -  PICAMOLE  MEAN SO  O. 33825E+06 0.56107E+07 85657. 0.78791E+06 0.68225E+07  2 8 16 49 75  FREQUENCIES.  OF VARIANCE  STANDARD  ERROR  0.16912E+06 0.70134E+06 5353.6 16O80.  PROB  F-VALUE  O.15830E-03 0.41428E-12 0.99054  10.518 131.00 0.33294  BXT  DEVIATIONS  ,..........**.*.*•*«»**»»»**.»*»»*»•**«***•*«*****»*******«******•** ******' TREAT  .1.  MN PICAMOLE  .2.  62.42  CONTRAST 1 CONTRAST 1 1.OOOOO 2 O.O  1 2  .9.  201.2  210.4  1.OOOOO Q-SQUARED 0.281901E+07 8309.59  CONTRAST CONTROL/TREAT BURST/INTACT  OF D I F F E R E N C E  753.3  2  TREATMENT SUM OF SQUARES SUM OF Q SQUARED D I F F E R E N C E OR RESIDUAL AVERAGE F-RATIO D I F F E R E N C E IS 49.60B TEST  67.02  432.6  .8.  40.99  .5.  .3.  396.9  .7.  ,,  F-VALUE 526.57 1.5522  0.561070E+07 0.282732E+07 0.27833BE+07 264.059 PERCENT OF TREATMENT  G I V E S AN F - V A L U E  OF  86.652  PROBABILITY O.OOOOO 0.23075  SUM OF SQUARES , AND FPROB OF 0.OOOOO  785.9  -96-  Treatment numbers 1-3 represent uptake of diclofop by control, burst and i n t a c t at 2.OuM diclofop, 4-6 a t 4.OuM diclofop and 7-9 a t 8.OuM diclofop. ANALYSIS SOURCE BLOCK TREAT BXT ERROR TOTAL  FREQUENCIES .  OF VARIANCE  DF  SUM SO  MEAN SQ  2 8 16 54 80  1929.3 14804. 3060.8 4928.4 24722.  964.66 1850.5 191.30 91.266  MEANS. STANDARD  ****************************  TREAT MN  . 1 .  PICAMOLE  7.338  CONTRAST 1 .OOOOO 1 0.0 2  ERROR  PROB  F-VALUE  BXT  10.570 9.6729 2.0961  0. 13371E-03 0.74740E-04 0.22311E-01  DEVIATIONS .4.  .3. 14 . 2 3  24  9.667  .5. 28.09  .9.  .8. 36 . 19  1 1 . 16 CONTRAST 1  PICAMOLE  »•««*»»*»»»*  .2.  .7 .  1 2  -  47  2  1.00000  CONTRAST CONTROL/TREAT BURST/INTACT  TREATMENT SUM OF SQUARES SUM OF Q SQUARED D I F F E R E N C E OR RESIDUAL AVERAGE F-RATIO D I F F E R E N C E IS 32.667  Q-SQUARED 8585.36 13B2.41  F-VALUE 44.878 7.2263  14803.6 9967.77 4835.84 26.0524 PERCENT OF TREATMENT  PROBABILITY 0.00001 0.01616  SUM OF SQUARES  .6. 36.80  -97-  Analysis o f variance with i n d i v i d u a l degree of freedom contrasts f o r data presented i n Table 11. The e f f e c t of 2,4-D on DM uptake.  Appendix 11  Treatment numbers 1-5 represent control, O.OuM, lOuM, 25uM, and 50uM 2,4-D respectively. ANALYSIS DF  SOURCE TREAT BLOCK ERROR TOTAL STANDARD  OF VARIANCE  -  PICAMOLE ERROR  MEAN SO  SUM SO  63295. 4 949.76 2 4177.2 8 68422 . 14 D E V I A T I O N OF V A R I A B L E  30.305 0.90947  15824. 474.88 5 2 2 . 15 1 IS  PROB  F-VALUE  0.69818E-04 0.44066  69.909  »*****«•*•**' TREAT  FREQUENCIES 3 MN PICAMOLE 34 . 13 SD PICAMOLE 11.04 CONTRAST 1 2 CONTRAST 1 1.OOOOO 2 -O.OOOOO  Q-SQUARED  OF D I F F E R E N C E  115.25 2.2444  63294.8 61351.3 1943.55 58.7483 PERCENT OF TREATMENT  G I V E S AN F - V A L U E  OF  PROBABILITY  F-VALUE  60179.4 1171.93  TREAT/CONT 24D  TREATMENT SUM OF SQUARES SUM OF Q SQUARED D I F F E R E N C E OR RESIDUAL AVERAGE F-RATIO DIFFERENCE IS 3.0706 TEST  3 206.0 2.603  3 177 . 6 12.04  3 210.9 26.56  3 175.4 39.80  1.OOOOO  CONTRAST 1 2  5.  4.  1.  1.8611  O.OOOOO 0 . 17248  SUM OF SQUARES AND FPROB OF 0 . 2 1 6 9 3  -98-  A.ll.  cont'd  Treatment numbers 1-6 represent control, O.OuM, lOOuM, 500uM, l.OmM, and 2.OmM 2,4-D. respectively. ANALYSIS  SOURCE TREAT BLOCK ERROR TOTAL  FREQUENCIES,  OF  OF  SUM  5 2 10 17  70769. 1631.0 8355.0 80755.  VARIANCE  - PERCENT  MEAN  SQ  ERROR  SO  16.941 0.97608  14154. 815.51 835.50  MEANS, STANDARD  PROB  F -VALUE  0. 13413E-03 0.40999  DEVIATIONS  * * * * » » » » » » • » * * » » » * * * » » » » » • * * * * * * » * * * * * * » * * » * * * * * » * * * * * * * ft*****.***************** TREAT  1 .  1 2  Q-SQUARED 69544.4 342.249  CONTRAST CONT/TREAT 24D  TREATMENT SUM OF SQUARES SUM OF 0 SQUARED D I F F E R E N C E OR RESIDUAL AVERAGE F-RATI0 D I F F E R E N C E IS 1.2469 TEST  OF  DIFFERENCE  G I V E S AN  OF  PROBABILITY 0. OOOOO 0.53654  F-VALUE .83.237 0.40964  70769.0 69886.6 882.419 41 .8235 PERCENT OF TREATMENT SUM F-VALUE  3 181.1 14.71  3 194.3 6. 162  3 203.4 17.55  3 3 FREQUENCIES 179. 1 MN PERCENT 21 .83 29.07 SD PERCENT 6.673 CONTRAST 1 2 CONTRAST 1 1.OOOOO 2 O.OOOOO 1.OOOOO  0.35205  6.  5.  4 .  3.  2.  OF  SQUARES  . AND  FPROB OF  0.78872  3 185.3 59.50  -99-  Appendix 12.  Analysis of variance with i n d i v i d u a l degre< of freedom contrasts f o r data presented i n Table 12. The e f f e c t of 2,4-D on diclofop uptake. Treatment numbers 1-5 represent control, O.OuM, lOuM, 25uM, and 50uM 2,4-D respectively.  ANALYSIS OF VARIANCE SOURCE TREAT BLOCK ERROR TOTAL  - PICAMOLE  DF  SUM SO  MEAN SO  4 2 8 14  183.29 27.604 327.71 538.60  45.823 13.802 40.964  ERROR  PROB  F-VALUE 1 . 1186 0.33693  0.41184 0.72362  FREQUENCIES. MEANS. STANDARD DEVIATIONS TREAT  1.  2.  3.  FREQUENCIES 3 3 MN PICAMOLE 9.833 16.37 SD PICAMOLE 0.7505 3.798 CONTRAST 1 2 CONTRAST 1 1.OOOOO 2 -O.OOOOO 1.OOOOO  1 1  CONTRAST TREAT/CONT TREAT/CONT  TREATMENT SUM OF SQUARES SUM OF Q SQUARED DIFFERENCE OR RESIDUAL A V F R A G E F-RATIO DIFFERENCE IS 9.8969  4.  3 17.07 5.636  Q-SQUARED 155.848 9.30257  5.  3 20.37 9.075  ^y/LUE 3.8045 0.22709  3 17.77 6.967  P  R  ^ ^ 0.08692 0.64643 0  Y  183.291 165.151 18.1401 2.01582 PERCENT OF TREATMENT SUM OF SQUARES  TEST OF DIFFERENCE GIVES AN F-VALUE OF  0.22142  . AND FPROB OF 0.80613  -100-  .12. cont'd  Treatment numbers 1-6 represent control, O.OuM, lOOuM, 500uM, l.OmM, and 2.OmM 2,4-D respectively. ANALYSIS OF VARIANCE  SOURCE TREAT BLOCK ERROR TOTAL  FREQUENCIES,  DF  SUM  5 2 10 17  180.04 8. 1674 67.395 255.61  -  MEAN  SO  PERCENT ERROR  SO  PROB  F-VALUE  0. 11962E-01 0.56443  5.3430 0.60593  36.009 4.0837 6.7395  MEANS, STANDARD  «.».*•»..***.*»»»»»*»»»»**»* TREAT  1 .  CONTRAST CONT/TREAT 24D  TREATMENT SUM OF SQUARES SUM OF 0 SOUARED D I F F E R E N C E OR RESIDUAL AVERAGE F-RATIO DIFFERENCE IS 7.4209 T E S T OF  D I F F E R E N C E G I V E S AN  Q-SQUARED 149.872 16.8116  OF  PROBABILITY 0.00082 O.14533  F-VALUE 22.238 2.4945  180.045 166.684 13.3609 12 3662 PERCENT OF TREATMENT SUM F-VALUE  3 10.58 4 .605  3 9.903 1 .887  3 12.22 1 .683  3 FREQUENCIES 13 4.053 MN PERCENT 1.1 0.7563 SD PERCENT CONTRAST 1 2 CONTRAST 1 1.00000 2 0.OOOOO 1.OOOOO  1 2  6.  4 .  2.  0.66082  OF  SQUARES  AND  FPROB OF  0.59468  3 12.37 2.451  -101-  Appendix 13. Appendix ^  Analysis of variance with i n d i v i d u a l degree freedom contrasts f o r data presented i n Table 13. The effects of 2,4-D on the conversion of DM to diclofop. Treatment numbers 1-4 represent the percent radiolabel as diclofop i n the external solution i n O.OuM, lOuM, 25uM and 50uM 2,4-D respectively.  ANALYSIS OF VARIANCE - PERCENT DF  SOURCE  3 2 6 11  TREAT BLOCK ERROR TOTAL FREQUENCIES,  SUM SO  MEAN SO  5.7339 20.520 8.8621 35.116  1 .9113 10.260 1.4770  MEANS. STANDARD DEVIATIONS *  TREAT FREQUENCIES MN PERCENT SD PERCENT  3 81.51 1 .889  PROB 0.35935 0.27438E-01  ***»**•*.»**»»*****'  3 82.01 1 .676  3 83 .00 2.294  F-VALUE 1 .2940 6.9465  3.  2.  1 .  ERROR  4. 3 81 . 16 1 .744  CONTRAST 1 CONTRAST 1 1.OOOOO 2 -0.00000 3 -0.00000  1 2 3  CONTRAST LINEAR OUAD CUB  1 . OOOOO 0. OOOOO  1.OOOOO Q-SQUARED 1 .29355 2.14727 2.29316  F-VALUE 0.87578 1.4538 1 .5526  PROBABILITY 0.38548 0.27331 O. 25920  5.73393 TREATMENT SUM OF SQUARES 5.73397 SUM OF 0 SQUARED -0.401697E-04 DIFFERENCE OR RESIDUAL 1 .29404 AVERAGE F-RATIO 0.70056E-03 PERCENT OF TREATMENT SUM OF SQUARES . DIFFERENCE IS  -10213. cont'd  Treatment numbers 1-5  " V " " ^ . ^ ^ ^  S a ^ J » " o . S » ^ SoJSTKS- an, 2.OmM 2,4-D respectively. ANALYSIS SUM  DF  SOURCE  9  2  BLOCK  PERCENT ERROR  SO  0.374G3E-01  4.3194  0.78463  0.27757  4.86G7  .7333  PROB  F-VALUE  17.533  452.93  14  TOTAL  -  75.733  140.27  8  ERROR  MEAN  SO  302.93  4  TREAT  OF VARIANCE  .«**»«•»*<  MN  PERCENT  SD  PERCENT  77  78.33  79.00  66.67  .OO  6.658  1 .732  2.517  4.359  3  3  3  3  FREQUENCIES  4 .  3.  2.  TREAT  3 74  .33  1 .528  CONTRAST 1 CONTRAST  1.00000  1.OOOOO  0.OOO01  -0.OOOOO  0.00000  -0.OOOOO  0.00000  1 .OOOOO 0.OOOOO  Q-SQUARED  CONTRAST  142.483  QUAD  73.4304  CUB  4.37063  DEV  SUM  OF  SUM OF  SQUARES  Q SQUARED  DIFFERENCE  —VALUE  82.6479  LINEAR  TREATMENT  1.OOOOO  OR R E S I D U A L  SIFFERENCE"IS  T I  0.63936E-03  4.7138 8.  1264  4 . 1880 0.24928  PROBABILITY 0.06171 0.02146 0.07492 0.63102  ^ 2 " 9 3 1 n  1Q3685E-02  VSSaS  PERCENT  OF TREATMENT  SUM OF  SQUARES  ]03> -104-  A.13- cont'd  Treatment numbers 1-4 r e p r e s e n t the percent r a d i o l a b e l as d i c l o f o p i n the P r o t o p l a s t p e l l e t i n O.OuM, 10uM, 25uM and 50uM 2,4-D respectively.  ANALYSIS OF VARIANCE - PERCENT SUM SO MEAN SO ERROR F-VALUE 0.74598 0.58997 1.7699 0.35260 0.27886 0.55772 0.79086 4.7452 7.0728  SOURCE DF 3 TREAT 2 BLOCK 6 ERROR 11 TOT»A L •*»****»*»**»*»***  ......................  3. TREAT 3 3 FREQUENCIES 3 11 .29 MN PERCENT 10.86 10.47 0.5220 SD PERCENT 0.6902 1 .320 CONTRAST 1 2 CONTRAST 1 1.00000 2 -O.OOOOO 1.00000 3 -O.OOOOO O.OOOOO 1.OOOOO CONTRAST 1 LINEAR 2 QUAD 3 CUB  O-SQUARED 0.229803 0.573051 0.967047  3 10.29 0.3999  F-VALUE  0.29057 0.72459 1 .2228  PROBABILITY 0.60926 0.42730 0.31117  TREATMENT SUM OF SOUARES 1 • 76990 SUM OF Q SQUARED 1.76990 DIFFERENCE OR RESIDUAL ~°™1QOO DIFFERENCE-IS-0.18191E-04 PERCENT^ TREATMENT SUM OF SOUARES T  PROB 0.56288 0.71650  -105A.13. cont'd ^ , Treatment numbers 1-5 represent the percent radiolabel as d i c l o f o p i n the protoplast p e l l e t i n O.OuM, lOOuM, 500uM, l.OmM and 2.OmM 2,4-D respectively.  ANALYSIS SOURCE TREAT BLOCK ERROR TOTAL  FREQUENCIES.  TREAT  OF VARIANCE  DF  SUM SO  4 2 8 14  38.631 O. 18545 18.656 57.472  CONTRAST 1 1 .OOOOO 2 0.O0001 3 -O.OOOOO 4 -O.OOOOO  ERROR  CONTRAST LINEAR QUAD CUB DEV  TREATMENT SUM OF SQUARES SUM OF Q SQUARED D I F F E R E N C E OR RESIDUAL AVERAGE F-RATIO DIFFERENCE I S 0.37474E-03  PROB 0.41588E-01 0.96121  DEVIATIONS  3.  3 10.37 2.470  1.OOOOO -0.OOOOO -O.OOOOO  F-VALUE 4. 1413 0.39762E-01  9.6577 0.92727E- 01 2.3320  2.  FREQUENCIES 3 MN PERCENT 12.44 SD PERCENT 1.744 CONTRAST 1 2  PERCENT  MEAN SO  MEANS, STANDARD  1.  -  3 9.520 0.2777  3  3 8.320 0.2858  3 7.967 0.3453  4  1.OOOOO O.OOOOO  1.ooooo  O-SQUARED 27.5656 7.32224 0.718416 3.02438  F-VALUE 11.820 3. 1398 0.30806 1.2969  PROBABILITY 0.00885 0.11434 0.59405 0.28773  38.6308 38.6306 0.144766E-03 4.14130 PERCENT OF TREATMENT SUM OF SQUARES  -106-  Appendix  14.  ANALYSIS  R e g r e s s i o n a n a l y s i s o f p r o t e i n content and p r o t o p l a s t s number.  OF VARIANCE  OF 2.ABSORBAN DF  SOURCE  1 2 3  REGRESSION ERROR TOTAL MULT  R=  .99450  R-SQR=  VARIABLE  PARTIAL  CONSTANT CONC .  .99450  N= 4 OUT OF 4  SUM SQRS  MEAN SOR  F-STAT  SIGNIF  4084.1 45.259 4129.4  4084.1 22.630  180.48  .0055  T-STAT  SIGNIF  .98904 SE= 4.7571  COEFF . 13853 -1 .86695  STD  ERROR  .36751 13.434  3.7694 .64533 -1  -2  .9974 .0055  Standard curve o f p r o t e i n content and p r o t o p l a s t number.  1.0  0.8  E 0.6 J  o u p.  O.k A  0.2  4  i  0  2.0  i  1  4.0  6.0  i  8.0  i  10.0  protoplasts (x 10^) per ml  R e g r e s s i o n equations Y 0.86?X  0.014,  -107-  Appendix 15.  A n a l y s i s o f v a r i a n c e w i t h i n d i v i d u a l degree o f freedom c o n t r a s t s showing a l i n e a r r e l a t i o n s h i p between K c o n c e n t r a t i o n and atomic a b s o r p t i o n a t nM. Treatment numbers r e p r e s e n t K c o n c e n t r a t i o n 0.0, 1.0, 2.0, 3.0, and k.0 ppm K .  ANALYSIS SOURCE  DF  BLOCK TREAT BXT ERROR TOTAL »»»•*»*»»»*'  2 4 8 30 44  TREAT  SUM  OF VARIANCE  SO  0.25973E-01 0.62300 O.11416E-01 0.63333E-03 0.66102  MEAN SO  .  PROB  F-VALUE  BXT  615.16 109.15 67.592  0.44630E-24 0.51662E-06 0.45305E-16  .5.  .2.  9 9 FREQUENCIES O. 1011 MN DENSITY 0.0 CONTRAST 1 2 CONTRAST 1 .OOOOO 1 1.00000 0.0 2 1 .OOOOO 0.0 0.0 3 0.0 O.OOOOO 0.0 4  CONTRAST LINEAR QUAD CUB DEV  ERROR  0.12987E-01 O.15575 0.14269E-02 0.21111E-04  ».»«»**»**«»*»*»*»..•** . 1 .  - DENSITY  9 0.1822  9 0.2656  9 0.3328  1.ooooo  0-SOUARED 0.620010 0.276269E-02 O.136108E-04 0.211470E-03  PROBABILITY F-VALUE O.OOOOO 434.50 0.20156 1.9361 0.92460 0.95384E-02 0.71030 O. 14820  0.622998 TREATMENT SUM OF SQUARES 0-622998 SUM OF 0 SQUARED 0.766097E-07 D I F F E R E N C E OR RESIDUAL 109.149 AVERAGE F-RATIO PERCENT OF TREATMENT SUM OF SQUARES DIFFERENCE IS 0.12297E-04  -108-  A.15.  cont'd  Standard curve f o r atomic absorption of potassium.  - 1 0 9 -  Appendix 1 6 .  Glossary  Herbicides amitrole:  3-amino-l,2,4-triazole  chloramben:  3-amino-2,5-dichlorobenzoic  cycluron:  N-cyclo-octyl-N,N-dimethylurea  DNOC:  2-methyl-4,6-dinitrophenol  dalapon:  2,2-dichloropropionic  desmetryne:  4-isopropylamino-6-methylamino-2-methylthio 1,3,5-triazine  dicamba:  3,6-dichloro-2-methoxybenzoic  2,4-D  2,4-dichlorophenoxy acetic  acid  acid  acid  methyl(2-(4-(2,4-dichlorophenoxy)phenoxy) propanoate  d i c l o f o p methyl: dinoseb:  acid  2- ( 1 - m e t h y l p r o p y l ) - 4 , 6 - d i n i t r o p h e n o l  fluoronitrofen: 2,4-dichloro-6-fluorophenyl-4-nitrophenyl ether glyphosate:  N-(phosphonomethyl)plycine  ioxynil:  4-hydroxy-3,5-di-iodobenzonitrile  isoproturon:  N'-(4-isopropylphenyl)-N,N-dimethyl  linuron:  N«  MCPA:  4-chloro-2-methylphenoxy  monolinuron:  N'-(4-chlorophenyl)-N-methoxy-N-methylurea  monuron:  N'-(4-chlorophenyl)-N;N-dimethylurea  napthalam:  N-l-naphthylphtalamic  picloram:  4-amino-3,5,6-trichloropicolinic  prometryne:  4,6-bisisopropylamino-2-methylthio -1,3,5-triazine  TCA:  trichloroacetic  urea  -3,Zj.-dichlorophenyl)-N-methoxy-N-methylurea  acid  acetic  acid  acid acid  -110-  Buffers Ches:  2-(Cyclohexylamino)ethanesulphonic  Hepes:  N-2 H y d r o x y e t h y l p i p e r a z i n e - N ' - 2 - e t h a n e acid  Mes:  2(N-Morpholinino)ethane  Mops:  3-(N-Morpholino)propane s u l p h o n i c a c i d  Tricine:  N-(Tris-hydroxymethyl)methylglycine  acid  sulphonic acid  sulphonic  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0095829/manifest

Comment

Related Items